U.S. patent application number 13/946711 was filed with the patent office on 2014-06-05 for apparatuses, compositions, and methods for prolonging survival of platelets.
This patent application is currently assigned to Velico Medical, Inc.. The applicant listed for this patent is Velico Medical, Inc.. Invention is credited to Henrik Clausen, Keith Rosiello.
Application Number | 20140154665 13/946711 |
Document ID | / |
Family ID | 35759421 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154665 |
Kind Code |
A1 |
Rosiello; Keith ; et
al. |
June 5, 2014 |
Apparatuses, Compositions, and Methods for Prolonging Survival of
Platelets
Abstract
The present invention provides modified platelets having a
reduced platelet clearance and methods for reducing platelet
clearance. Also provided are compositions for the preservation of
platelets. The invention also provides methods for making a
pharmaceutical composition containing the modified platelets and
for administering the pharmaceutical composition to a mammal to
mediate hemostasis.
Inventors: |
Rosiello; Keith;
(Shrewsbury, MA) ; Clausen; Henrik; (Holte,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Velico Medical, Inc. |
Beverly |
MA |
US |
|
|
Assignee: |
Velico Medical, Inc.
Beverly
MA
|
Family ID: |
35759421 |
Appl. No.: |
13/946711 |
Filed: |
July 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13291402 |
Nov 8, 2011 |
8517967 |
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13946711 |
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11574857 |
Dec 31, 2007 |
8052667 |
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PCT/US2005/031921 |
Sep 7, 2005 |
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13291402 |
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60678724 |
May 6, 2005 |
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60619176 |
Oct 15, 2004 |
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60607600 |
Sep 7, 2004 |
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Current U.S.
Class: |
435/2 ;
435/307.1 |
Current CPC
Class: |
A61P 7/02 20180101; A01N
1/00 20130101; A61P 7/04 20180101; A01N 1/02 20130101; Y10T
436/108331 20150115; A01N 1/0226 20130101 |
Class at
Publication: |
435/2 ;
435/307.1 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1. An apparatus for processing a sample of platelets, the apparatus
comprising a first sterile container having one or more ports
including a first container port and containing a preparation of
blood cells comprising platelets, a second sterile container having
one or more ports including a second container port and containing
a blood cell modifying agent which comprises cytidine
5'-monophospho-N-acetylneuraminic acid, the first container being
adapted to the second container through a first sterile conduit
reversibly attachable to the first container port and the second
container port, the first sterile conduit further comprising a
valve, wherein the blood cell modifying agent present in the second
sterile container is introduced into the first sterile container
and the platelets in the preparation of blood cells therein are
rendered cold storage competent after the platelets are contacted
with the blood cell modifying agent.
2. The apparatus of claim 1, further comprising a third sterile
container having one or more ports including a third container port
adapted to the first container through a second sterile conduit
reversibly attachable to the first container port and the third
container port, the second sterile conduit further comprising a
valve.
3. The apparatus of claim 1, wherein the first sterile conduit is
adapted to an in-line filter having a median pore diameter small
enough to substantially prevent the flow of bacteria through the
in-line filter.
4. The apparatus of claim 1, wherein the first container, second
container, the third container, the first container and the second
container, the first container and the third container, the second
container and the third container, or the first container, the
second container, and the third container are blood bags.
5. The apparatus of claim 1, wherein the blood cells further
comprise a population of platelets obtained from individual random
donor blood, pooled random donor blood, or single donor blood.
6. The apparatus of claim 1, wherein the second container port has
a frangible barrier.
7. The apparatus of claim 1, wherein the first conduit or the
second conduit reversibly attaches to the first container port, the
second container port, or the third container port through a
sterile dock.
8. An apparatus for processing a sample of platelets, the apparatus
comprising a first sterile container having one or more ports and
containing a blood cell modifying agent which comprises cytidine
5'-monophospho-N-acetylneuraminic acid, and an array comprising a
first conduit and a plurality of sterile docks, wherein each of the
sterile docks is reversible adaptable to blood storage containers,
the blood storage containers having a sample of blood cells
comprising platelets and further comprising at least one port for
connecting to the sterile docks of the array, wherein the platelets
are introduced into the first sterile container through the conduit
and are rendered cold storage competent by contacting the platelets
with the blood cell modifying agent present in the sterile first
container.
9. The apparatus of claim 8, wherein the blood cells further
comprise a population of platelets obtained from individual random
donor blood, pooled random donor blood, or single donor blood.
10. The apparatus of claim 8, further comprising a second container
having one or more ports and containing a blood cell modifying
agent that modifies the platelet cell surface, the first container
adapted to the second container through a second sterile conduit
reversibly attachable to the first container port and the second
container port.
11. The apparatus of claim 10, wherein the second sterile conduit
is adapted to an in-line filter having a median pore diameter small
enough to substantially prevent the flow of bacteria through the
in-line filter.
12. The apparatus of claim 11, wherein the second container port
has a frangible barrier.
13. An apparatus for processing a sample of platelets, the
apparatus comprising a first sterile container having one or more
ports, the first container further comprising a subcontainer
disposed therein, the subcontainer having a port and a frangible
barrier and containing a blood cell modifying agent which comprises
cytidine 5'-monophospho-N-acetylneuraminic acid, and an array
comprising a conduit and a plurality of sterile docks, wherein each
of the sterile docks is reversible adaptable to blood storage
containers, the blood storage containers having a sample of blood
cells comprising platelets and further comprising at least one port
for connecting to the sterile docks of the array, wherein the
platelets are introduced into the first sterile container through
the conduit and are rendered cold storage competent after the
platelets are contacted with the blood cell modifying agent present
in the first sterile container.
14. The apparatus of claim 13, wherein the blood cells further
comprise a population of platelets obtained from individual random
donor blood, pooled random donor blood, or single donor blood.
15. A method for treating a platelet using an apparatus, wherein
the apparatus is selected from the group consisting of: a. an
apparatus comprising a first sterile container having one or more
ports including a first container port and containing a preparation
of blood cells comprising platelets, a second sterile container
having one or more ports including a second container port and
containing a blood cell modifying agent, the first container being
adapted to the second container through a first sterile conduit
reversibly attachable to the first container port and the second
container port, the first sterile conduit further comprising a
valve; b. an apparatus comprising a first sterile container having
one or more ports and containing a blood cell modifying agent, and
an array comprising a first conduit and a plurality of sterile
docks, wherein each of the sterile docks is reversible adaptable to
blood storage containers, the blood storage containers having a
sample of blood cells comprising platelets and further comprising
at least one port for connecting to the sterile docks of the array;
and c. an apparatus comprising a first sterile container having one
or more ports, the first container further comprising a
subcontainer disposed therein, the subcontainer having a port and a
frangible barrier and containing a blood cell modifying agent, and
an array comprising a conduit and a plurality of sterile docks,
wherein each of the sterile docks is reversible adaptable to blood
storage containers, the blood storage containers having a sample of
blood cells comprising platelets and further comprising at least
one port for connecting to the sterile docks of the array; wherein
the steps of the method comprise contacting the platelets with the
one or more blood cell modifying agents, wherein the one or more
blood cell modifying agents comprise cytidine
5'-monophospho-N-acetylneuraminic acid or both UDP-galactose and
cytidine 5'-monophospho-N-acetylneuraminic acid, to thereby obtain
treated platelets.
16. The method of claim 15, wherein the conduit further comprises a
valve, and wherein the platelets, after being processed using the
apparatus such that the platelets are contacted with the one or
more blood cell modifying agents, are rendered cold-storage
competent.
17. The method of claim 15, wherein the blood cells are contacted
with the one or more blood cell modifying agents before infusion of
the treated blood cells into a patient.
18. The method of claim 15, wherein the blood cells are contacted
with the one or more blood cell modifying agents before cold
storage of the blood cells.
19. The method of claim 15, wherein the blood cells are contacted
with the one or more blood cell modifying agents at the time of
blood collection from a blood donor.
20. The method of claim 15, further including separating the blood
cells into subpopulations of platelets, plasma, red blood cells,
and white blood cells.
21. The method of claim 15, wherein the blood cells are contacted
with the one or more blood cell modifying agents after the blood
cells have been separated by apheresis.
22. The method of claim 15, wherein an amount of each of the one or
more blood cell modifying agents is between about 1 micromolar and
about 10 millimolar.
23. The method of claim 15, further comprising storing the treated
platelets for a period of time ranging between about 24 hours and
about 20 days.
24. The method of claim 15, further comprising storing the treated
platelets at a temperature ranging between about 0.degree. C. and
about 4.degree. C.
25. A treated blood cell obtained through a method for treating a
platelet using an apparatus, wherein the apparatus is selected from
the group consisting of: a. an apparatus comprising a first sterile
container having one or more ports including a first container port
and containing a preparation of blood cells comprising platelets, a
second sterile container having one or more ports including a
second container port and containing a blood cell modifying agent,
the first container being adapted to the second container through a
first sterile conduit reversibly attachable to the first container
port and the second container port, the first sterile conduit
further comprising a valve; b. an apparatus comprising a first
sterile container having one or more ports and containing a blood
cell modifying agent, and an array comprising a first conduit and a
plurality of sterile docks, wherein each of the sterile docks is
reversible adaptable to blood storage containers, the blood storage
containers having a sample of blood cells comprising platelets and
further comprising at least one port for connecting to the sterile
docks of the array; and c. an apparatus comprising a first sterile
container having one or more ports, the first container further
comprising a subcontainer disposed therein, the subcontainer having
a port and a frangible barrier and containing a blood cell
modifying agent, and an array comprising a conduit and a plurality
of sterile docks, wherein each of the sterile docks is reversible
adaptable to blood storage containers, the blood storage containers
having a sample of blood cells comprising platelets and further
comprising at least one port for connecting to the sterile docks of
the array; wherein the steps of the method comprise processing the
platelets using the apparatus such that the platelets are exposed
to the two or more blood cell modifying agents that include
cytidine 5'-monophospho-N-acetylneuraminic acid or both
UDP-galactose and cytidine 5'-monophospho-N-acetylneuraminic
acid.
26. The treated blood cell of claim 25, wherein the blood cells,
following cold storage, are suitable for transfusion into a
patient.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/291,402, filed Nov. 8, 2011, which is a
continuation of U.S. patent application Ser. No. 11/574,857, filed
Mar. 7, 2007 (371 date Dec. 31, 2007), now U.S. Pat. No. 8,052,667,
issued Nov. 8, 2011, which is the U.S. National stage of
International Application No. PCT/US2005/031921, filed on Sep. 7,
2005, which claims the benefit of U.S. Provisional Application No.
60/678,724, filed May 6, 2005, U.S. Provisional Application No.
60/619,176, filed on Oct. 15, 2004, and U.S. Provisional
Application No. 60/607,600, filed on Sep. 7, 2004. The entire
teachings of the above applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The inventions relate to compositions and methods for
reducing the clearance of platelets and prolonging the survival of
platelets.
BACKGROUND OF THE INVENTION
[0003] Platelets are anucleate bone marrow-derived blood cells that
protect injured mammals from blood loss by adhering to sites of
vascular injury and by promoting the formation of plasma fibrin
clots. Humans depleted of circulating platelets by bone marrow
failure suffer from life threatening spontaneous bleeding, and less
severe deficiencies of platelets contribute to bleeding
complications following trauma or surgery.
[0004] A reduction in the number of circulating platelets to below
-70,000 per uL reportedly results in a prolongation of a
standardized cutaneous bleeding time test, and the bleeding
interval prolongs, extrapolating to near infinity as the platelet
count falls to zero. Patients with platelet counts of less than
20,000 per uL are thought to be highly susceptible to spontaneous
hemorrhage from mucosal surfaces, especially when the
thrombocytopenia is caused by bone marrow failure and when the
affected patients are ravaged with sepsis or other insults. The
platelet deficiencies associated with bone marrow disorders such as
a plastic anemia, acute and chronic leukemias, metastatic cancer
but especially resulting from cancer treatment with ionizing
radiation and chemotherapy represent a major public health problem.
Thrombocytopenia associated with major surgery, injury and sepsis
also eventuates in administration of significant numbers of
platelet transfusions.
[0005] A major advance in medical care half a century ago was the
development of platelet transfusions to correct such platelet
deficiencies, and over 9 million platelet transfusions took place
in the United States alone in 1999 (Jacobs et al., 2001).
Platelets, however, unlike all other transplantable tissues, do not
tolerate refrigeration, because they disappear rapidly from the
circulation of recipients if subjected to even very short periods
of chilling, and the cooling effect that shortens platelet survival
is irreversible (Becker et al., 1973; Berger et al., 1998).
[0006] The resulting need to keep these cells at room temperature
prior to transfusion has imposed a unique set of costly and complex
logistical requirements for platelet storage. Because platelets are
actively metabolic at room temperature, they require constant
agitation in porous containers to allow for release of evolved
CO.sub.2 to prevent the toxic consequences of metabolic acidosis.
Room temperature storage conditions result in macromolecular
degradation and reduced hemostatic functions of platelets, a set of
defects known as "the storage lesion" (Chemoff and Snyder, 1992).
But the major problem with room-temperature storage, leading to its
short (5-day) limitation, is the higher risk of bacterial
infection. Bacterial contamination of blood components is currently
the most frequent infectious complication of blood component use,
exceeding by far that of viral agents (Engelfriet et al., 2000). In
the USA, 3000-4500 cases yearly of bacterial sepsis occur because
of bacterially contaminated blood components (Yomtovian et al.,
1993).
[0007] The mechanism underlying the unique irreversible cold
intolerance of platelets has been a mystery as has its
physiological significance. Circulating platelets are
smooth-surfaced discs that convert to complex shapes as they react
to vascular injury. Over 40 years ago investigators noted that
discoid platelets also change shape at refrigeration temperatures
(Zucker and Borrelli, 1954). Subsequent evidence that a discoid
shape was the best predictor of viability for platelets stored at
room temperature (Schlichter and Harker, 1976) led to the
conclusion that the cold-induced shape change per se was
responsible for the rapid clearance of chilled platelets.
Presumably irregularly-shaped platelets deformed by cooling became
entrapped in the microcirculation.
[0008] Based on our studies linking signaling to the mechanisms
leading to platelet shape changes induced by ligands (Hartwig et
al., 1995), we predicted that chilling, by inhibiting calcium
extrusion, could elevate calcium levels to a degree consistent with
the activation of the protein gelsolin, which severs actin
filaments and caps barbed ends of actin filaments. We also reasoned
that a membrane lipid phase transition at low temperatures would
cluster phosphoinositides. Phosphoinositide clustering uncaps actin
filament barbed ends (Janmey and Stossel, 1989) to create
nucleation sites for filament elongation. We produced experimental
evidence for both mechanisms, documenting gelsolin activation,
actin filament barbed end uncapping, and actin assembly in cooled
platelets (Hoffmeister et al., 2001; Winokur and Hartwig, 1995).
Others have reported spectroscopic changes in chilled platelets
consistent with a membrane phase transition (Tablin et al., 1996).
This information suggested a method for preserving the discoid
shape of chilled platelets, using a cell-permeable calcium chelator
to inhibit the calcium rise and cytochalasin B to prevent barbed
end actin assembly. Although addition of these agents retained
platelets in a discoid shape at 4.degree. C. (Winokur and Hartwig,
1995), such platelets also clear rapidly from the circulation, as
we report here. Therefore, the problem of the rapid clearance of
chilled platelets remains, and methods of increasing circulation
time as well as storage time for platelets are needed.
SUMMARY OF THE INVENTION
[0009] The present invention provides modified platelets having a
reduced platelet clearance and methods for reducing platelet
clearance. Also provided are compositions and methods for the
preservation and storage of platelets, such as mammalian platelets,
particularly human platelets. The invention also provides methods
for making a pharmaceutical composition containing the modified
platelets and for administering the pharmaceutical composition to a
mammal to mediate hemostasis.
[0010] It has now been discovered that cooling of human platelets
causes clustering of the von Willebrand factor (vWf) receptor
complex .alpha. subunit (GP1b.alpha.) complexes on the platelet
surface. The clustering of GP1b.alpha. complexes on the platelet
surface elicits recognition by macrophage complement type three
receptors (.alpha.M.beta.2, CR3) in vitro and in vivo. CR3
receptors recognize N-linked sugars with terminal .beta.GlcNAc on
the surface of platelets, which have formed GP1b.alpha. complexes,
and phagocytose the platelets, clearing them from the circulation
and resulting in a concomitant loss of hemostatic function.
[0011] Applicants have discovered that treatment of platelets with
an effective amount of a glycan modifying agent such as
N-acetylneuraminic acid (sialic acid), or certain nucleotide-sugar
molecules, such as CMP-sialic acid or UDP-galactose leads to
sialylation or glycation of the exposed .beta.GlcNAc residues on
GP1b.alpha.. Effective amounts of a glycan modifying agent range
from about 1 micromolar to about 10 millimolar, about 1 micromolar
to about 1 millimolar, and most preferably about 200 micromolar to
about 600 micromolar of the glycan modifying agent. This has the
functional effect of reducing platelet clearance, blocking platelet
phagocytosis, increasing platelet circulation time, and increasing
both platelet storage time and tolerance for temperature changes.
Additionally, platelets removed from a mammal may be stored cold
for extended periods, i.e., at 4 degrees C. for 24 hours, 2 days, 3
days, 5 days, 7 days, 12 days or 20 days or more, without
significant loss of hemostatic function following transplantation.
Cold storage provides an advantage that it inhibits the growth of
contaminating microorganisms in the platelet preparation, important
as platelets are typically given to cancer patients and other
immunocompromised patients.
[0012] According to one aspect of the invention, methods for
increasing the circulation time of a population of platelets is
provided. The method comprises contacting an isolated population of
platelets with at least one glycan modifying agent in an amount
effective to reduce the clearance of the population of platelets.
In some embodiments, the glycan modifying agent is selected from
the group consisting UDP-galactose and UDP-galactose precursors. In
some preferred embodiments, the glycan modifying agent is
UDP-galactose.
[0013] In some embodiments, the method further comprises adding an
enzyme that catalyzes the modification of a glycan moiety on the
platelet. One example of an enzyme that catalyzes the modification
of the glycan moiety is galactosyl transferase, particularly a
beta-1-4-galactosyl transferase. Another example of an enzyme that
catalyzes the modification of a glycan moiety is a sialyl
transferase, which adds sialic acid to the terminal galactose on
the glycan moiety of the platelet.
[0014] In one of the preferred embodiments, the glycan modifying
agent is UDP-galactose and the enzyme that catalyzes the
modification of the glycan moiety is galactosyl transferase. In
certain aspects, the glycan modifying agent further includes a
second chemical moiety, which is added to the glycan on the
platelet in a directed manner. An example of this second chemical
moiety is polyethylene glycol (PEG), which when coupled to the
glycan modifying agent such as UDP-galactose as UDP-galactose-PEG,
in the presence of an enzyme such as galactosyl transferase, will
catalyze the addition of PEG to the platelet at the terminus of the
glycan moiety. Thus in certain embodiments, the invention provides
for compositions and methods for the targeted addition of compounds
to the sugars and proteins of cells.
[0015] In some embodiments, the method for increasing the
circulation time of a population of platelets further comprises
chilling the population of platelets prior to, concurrently with,
or after contacting the platelets with the at least one glycan
modifying agent.
[0016] In some embodiments, the population of platelets retains
substantially normal hemostatic activity.
[0017] In some embodiments, the step of contacting the population
of platelets with at least one glycan modifying agent is performed
in a platelet bag.
[0018] In some embodiments, the circulation time is increased by at
least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%,
150%, 200%, 500% or more.
[0019] According to another aspect of the invention, a method for
increasing the storage time of platelets is provided. The method
comprises contacting an isolated population of platelets with an
amount of at least one glycan modifying agent effective to reduce
the clearance of the population of platelets, and storing the
population of platelets. Effective amounts of a glycan modifying
agent range from about 1 micromolar to about 1200 micromolar, and
most preferably about 200 micromolar to about 600 micromolar of the
glycan modifying agent. In certain aspects the platelet preparation
is stored at cold temperatures, i.e., frozen or refrigerated.
[0020] In some embodiments, the glycan modifying agent is selected
from the group consisting of: a sugar, a monosaccharide sugar, a
nucleotide sugar, sialic acid, sialic acid precursors, CMP-sialic
acid, UDP-galactose, and UDP-galactose precursors. In some
embodiments, the glycan modifying agent is preferably
UDP-galactose.
[0021] In some embodiments, the method further comprises adding an
effective amount of an enzyme that catalyzes the addition of the
glycan modifying agent to a glycan on the surface of the platelets.
In one of the preferred embodiments, the glycan modifying agent is
UDP-galactose and the enzyme that catalyzes the addition of the
glycan modifying agent to a glycan on the surface of the platelets
is galactosyl transferase, preferably a beta-1-4-galactosyl
transferase. In another preferred embodiment, the glycan modifying
agent is CMP-sialic acid and the enzyme that catalyzes the addition
of the glycan modifying agent to a glycan on the surface of the
platelets is sialyl transferase.
[0022] In some embodiments, the method further comprises chilling
the population of platelets prior to, concurrently with, or after
contacting the platelets with the at least one glycan modifying
agent.
[0023] In some embodiments, the population of platelets retains
substantially normal hemostatic activity when transplanted in a
mammal. Prior to transplantation the glycan modifying agent is
preferably diluted or reduced to concentrations of about 50
micromolar or less.
[0024] In certain embodiments, the step of contacting the
population of platelets with at least one glycan modifying agent is
performed during collection of whole blood or collection of the
platelets. In certain embodiments, the glycan modifying agent is
introduced into a platelet bag prior to, concurrently with, or
after collection of the platelets.
[0025] The platelets are capable of being stored at reduced
temperatures, for example, frozen, or chilled, and can be stored
for extended periods of time, such as at least about 3 days, at
least about 5 days, at least about 7 days, at least about 10 days,
at least about 14 days, at least about 21 days, or at least about
28 days.
[0026] According to another aspect of the invention, a modified
platelet is provided. The modified platelet comprises a plurality
of modified glycan molecules on the surface of the platelet. The
modified glycan molecules include sialic acid additions to the
terminal sugar residues, or galactosylation of the terminal sugar
residues.
[0027] In some embodiments, the modified glycan molecules are
moieties of GP1b.alpha. molecules. The modified glycan molecules
comprise sialic acid or at least one added sugar molecule. The
added sugar may be a natural sugar or may be a non-natural sugar.
Examples of added sugars include but are not limited to: nucleotide
sugars such as UDP-galactose and UDP-galactose precursors. In one
of the preferred embodiments, the added nucleotide sugar is
CMP-sialic acid or UDP-galactose.
[0028] In another aspect, the invention provides a platelet
composition comprising a plurality of modified platelets. In some
embodiments, the platelet composition further comprises a storage
medium. In some embodiments, the platelet composition further
comprises a pharmaceutically acceptable carrier.
[0029] According to yet another aspect of the invention, a method
for making a pharmaceutical composition for administration to a
mammal is provided. The method comprises the steps of:
[0030] (a) contacting a population of platelets contained in a
pharmaceutically-acceptable carrier with at least one glycan
modifying agent to form a treated platelet preparation,
[0031] (b) storing the treated platelet preparation, and
[0032] (c) warming the treated platelet preparation.
[0033] In some embodiments, the step of warming the treated
platelet preparation is performed by warming the platelets to
37.degree. C.
[0034] In some embodiments, the step of contacting a population of
platelets contained in a pharmaceutically-acceptable carrier with
at least one glycan modifying agent comprises contacting the
platelets with at least one glycan modifying agent, alone or in the
presence of an enzyme that catalyzes the modification of a glycan
moiety. The glycan modifying agent is preferably added at
concentrations of about 1 micromolar to about 1200 micromolar, and
most preferably about 200 micromolar to about 600 micromolar. In
some embodiments, the method further comprises reducing the
concentration of, or removing or neutralizing the glycan modifying
agent or the enzyme in the platelet preparation. Methods of
reducing the concentration of removing or neutralizing the glycan
modifying agent or enzyme include, for example, washing the
platelet preparation or dilution of the platelet preparation. The
glycan modifying agent is preferably diluted to about 50 micromolar
or less prior to transplantation of the platelets into a human
subject.
[0035] Examples of glycan modifying agents are listed above. In one
of the preferred embodiments, the glycan modifying agent is
CMP-sialic acid or UDP-galactose. In some embodiments, the method
further comprises adding an exogenous enzyme that catalyzes the
addition of the glycan modifying agent to a glycan moiety, such as
a beta-1-4 galactosyl transferase.
[0036] In one of the preferred embodiments, the glycan modifying
agent is UDP-galactose and the enzyme is galactosyl
transferase.
[0037] In some embodiments, the population of platelets demonstrate
substantially normal hemostatic activity, preferably after
transplantation into a mammal.
[0038] In certain embodiments, the step of contacting the
population of platelets with at least one glycan modifying agent is
performed during the collection process on whole blood or
fractionated blood, such as on platelets in a platelet bag.
[0039] In some embodiments, the platelet preparation is stored at a
temperature of less than about 15.degree. C., preferably less than
10.degree. C., and more preferably less than 5.degree. C. In some
other embodiments, the platelet preparation is stored at room
temperature. In other embodiments, the platelets are frozen, e.g.,
0.degree. C., -20.degree. C., or -80.degree. C. or cooler.
[0040] According to yet another aspect of the invention, a method
for mediating hemostasis in a mammal is provided. The method
comprises administering a plurality of modified platelets or a
modified platelet composition to the mammal. The platelets are
modified with the glycan modifying agent prior to administration,
such as during collection, prior to storing, after storage and
during warming, or immediately prior to transplantation.
[0041] According to still yet another aspect of the invention, a
storage composition for preserving platelets is provided. The
composition comprises at least one glycan modifying agent, added to
the platelets in an amount sufficient to modify platelets glycans,
thereby increase the storage time and/or the circulation time of
platelets added to the storage composition by reducing platelet
clearance.
[0042] In some embodiments the composition further comprises an
enzyme that catalyzes the modification of a glycan moiety. The
enzyme may be exogenously added. A beta-1-4 galatosyl transferase
or a sialyl transferase, or both, exemplify preferred enzymes for
catalyzing the modification of the glycan moieties on the
platelets.
[0043] According to another aspect of the invention, a container
for collecting (and optionally processing) platelets is provided.
The container comprises at least one glycan modifying agent in an
amount sufficient to modify glycans of platelets contained therein.
The container is preferably a platelet bag, or other blood
collection device.
[0044] In some embodiments, the container further comprises an
enzyme that catalyzes the modification of a glycan moiety with the
glycan modifying agent, such as a beta-1-4 galatosyl transferase or
a sialyl transferase.
[0045] In some embodiments the container further comprises a
plurality of platelets or plasma comprising a plurality of
platelets.
[0046] In some embodiments, the glycan modifying agent is present
at a concentration higher than it is found in naturally occurring
platelets or in serum. In certain aspects these concentrations are
1 micromolar to 1200 micromolar, and most preferably about 200
micromolar to about 600 micromolar. In other embodiments, the
beta-1-4 galatosyl transferase or a sialyl transferase is at a
concentration higher than it is found in naturally occurring
platelets or in serum, such as concentrations that would be
observed if the enzyme were added exogenously to the platelets.
[0047] According to still yet another aspect of the invention, a
device for collecting and processing platelets is provided. The
device comprises: a container for collecting platelets; at least
one satellite container in fluid communication with said container;
and at least one glycan modifying agent in the satellite container.
The container optionally includes an enzyme such as a beta-1-4
galatosyl transferase or a sialyl transferase.
[0048] In some embodiments, the glycan modifying agent in the
satellite container is present in sufficient amounts to preserve
the platelets in the container, for example from concentrations of
about 1 micromolar to about 1200 micromolar.
[0049] In some embodiments, the glycan modifying agent in the
satellite container is prevented from flowing into the container by
a breakable seal.
[0050] In other aspects, the invention includes a kit having a
sterile container capable of receiving and containing a population
of platelets, the container substantially closed to the
environment, and a sterile quantity of a glycan modifying agent
sufficient to modify a volume of platelets collected and stored in
the container, the kit further includes suitable packaging
materials and instructions for use. Glycan modifying agents in the
kit include CMP-sialic acid, UDP-galactose, or sialic acid. The
container is suitable for cold-storage of platelets.
[0051] The invention also includes, in certain aspects, a method of
modifying a glycoprotein comprising, obtaining a plurality of
platelets having GP1b.alpha. molecules, and contacting the
platelets with a glycan modifying agent, wherein the glycan
modifying agent galactosylates or sialylates the terminus of a
GP1b.alpha. molecule on the platelets.
[0052] The invention further includes a method of modifying a blood
constituent comprising, obtaining a sample of blood having
platelets, and contacting at least the platelets with a glycan
modifying agent, wherein the glycan modifying agent galactosylates
or sialylates the terminus of a GP1b.alpha. molecule on the
platelets.
[0053] In other aspects, the invention includes a method of
reducing pathogen growth in a blood sample comprising, obtaining a
sample of blood having platelets, contacting at least the platelets
with a glycan modifying agent, wherein the glycan modifying agent
galactosylates or sialylates the terminus of a GP1b.alpha. molecule
on the platelets, and storing the blood sample having modified
platelets at a temperature of about 2 degrees C. to about 18
degrees C. for at least three days, thereby reducing pathogen
growth in the blood sample.
[0054] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first
container having one or more ports and containing a preparation of
blood cells, a second sterile container having one or more ports
and containing a blood cell modifying agent, (also referred to as a
platelet solution or a glycan modifying agent) the first container
adapted to the second container through a sterile conduit
reversibly attachable to the first container port and the second
container port, the conduit further comprising a valve, wherein the
blood cell modifying agent is introduced into the first container
and the preparation of blood cells therein is rendered cold storage
competent after the blood cells are contacted with the blood cell
modifying agent. In one embodiment, the invention includes a
sterile third container having one or more ports adapted to the
first container through a second sterile conduit reversibly
attachable to the first container port and the third container
port, the conduit further comprising a valve. In another
embodiment, the invention includes a leukocyte filter. In various
embodiments, some shown in the figures, the first container, second
container or the third container are blood bags or a syringe. In
other embodiments, the blood cell modifying agent is a nucleoside
sugar such as UDP galactose, or cytidine
5'monophospho-N-acetylneuraminic acid. The blood cells suitable for
modification in the bioprocess include a population of platelets
obtained from individual random donor blood, pooled random donor
blood, or single donor blood. In various other embodiments, the
conduit is adapted to an in-line filter having a median pore
diameter small enough to substantially prevent the flow of bacteria
through the in-line filter. Preferred median pore diameters for the
in-line filter are less than about 1 micron, more preferably less
than about 0.50 microns and most preferably about 0.22 microns. In
yet another embodiment, the second container port has a frangible
barrier. In even another embodiment, the first conduit or the
second conduit reversibly attaches to the first container port, the
second container port or the third container port through a sterile
dock.
[0055] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first
container having one or more ports, and an array having a conduit
and a plurality of sterile docks, wherein each of the sterile docks
are reversible adaptable to blood storage containers, the blood
storage containers having a sample of blood cells and further
comprising at least one port for connecting to the sterile docks of
the array, wherein the blood cells are introduced into the sterile
first container through the conduit and are rendered cold storage
competent after the blood cells are contacted with a blood cell
modifying agent introduced into the first container. In some
embodiments, the blood cell modifying agent is a sterile nucleoside
sugar such as UDP galactose or a sterile preparation of cytidine
5'monophospho-N-acetylneuraminic acid. In various other
embodiments, the invention provides an in-line filter having a
median pore diameter small enough to substantially prevent the flow
of bacteria through the in-line filter. Preferred median pore
diameters for the in-line filter are less than about 1 micron, more
preferably less than about 0.50 microns and most preferably about
0.22 microns. In one embodiment, the blood cells further comprise a
population of platelets obtained from individual random donor
blood, pooled random donor blood, or single donor blood. In another
embodiment, the array further comprises a leukocyte filter proximal
to the first container. In even another embodiment, the blood cell
modifying agent is contained in the first container. In another
embodiment, the invention includes a second container having one or
more ports and containing a blood cell modifying agent, the first
container adapted to the second container through a sterile conduit
reversibly attachable to the first container port and the second
container port. In still yet another embodiment, the second
container is a syringe. In one embodiment, the conduit is adapted
to an in-line filter having a median pore diameter small enough to
substantially prevent the flow of bacteria through the in-line
filter. In another embodiment median pore diameters for the in-line
filter are less than about 1 micron, more preferably less than
about 0.50 microns and most preferably about 0.22 microns. In
another embodiment, the second container port has a frangible
barrier.
[0056] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first
container having one or more ports the first container further
comprising a subcontainer disposed therein, the subcontainer having
a port and a frangible barrier and containing a blood cell
modifying agent, and an array comprising a conduit and a plurality
of sterile docks, wherein each of the sterile docks are reversible
adaptable to blood storage containers, the blood storage containers
having a sample of blood cells and further comprising at least one
port for connecting to the sterile docks of the array, wherein the
blood cells are introduced into the sterile first container through
the conduit and are rendered cold storage competent after the blood
cells are contacted with a blood cell modifying agent introduced
into the first container. In one embodiment, the blood cell
modifying agent is a sterile nucleoside sugar such as UDP galactose
or a sterile preparation of cytidine
5'monophospho-N-acetylneuraminic acid. In another embodiment median
pore diameters for the in-line filter are less than about 1 micron,
more preferably less than about 0.50 microns and most preferably
about 0.22 microns. In another embodiment, the second container
port has a frangible barrier. In another embodiment, the blood
cells further comprise a population of individual random donor
blood, pooled random donor blood, or single donor blood. In another
embodiment, the array further comprises a leukocyte filter proximal
to the first container.
[0057] In one aspect, the invention provides a method for treating
a blood cell, including obtaining an apparatus as described,
obtaining a sample of blood cells including a subpopulation of
platelets, and exposing the blood cells to the blood cell modifying
agent in the apparatus thereby rendering the subpopulation of
platelets cold-storage competent. In one embodiment, the method
includes separating the leukocytes from the blood cells prior to
exposing the blood cells to the blood cell modifying agent. In one
embodiment, the blood cell modifying agent is a sterile nucleoside
sugar such as UDP galactose or a sterile preparation of cytidine to
5'monophospho-N-acetylneuraminic acid. In another embodiment median
pore diameters for the in-line filter are less than about 1 micron,
more preferably less than about 0.50 microns and most preferably
about 0.22 microns. In another embodiment, the second container
port has a frangible barrier. In another embodiment, the blood
cells further comprise a population of individual random donor
blood, pooled random donor blood, or single donor blood. In another
embodiment, the method provides that the blood cells are contacted
with the blood cell modifying agent before infusion of the treated
blood cells into a patient. In another embodiment, the method
provides that the blood cells are contacted with the blood cell
modifying agent before cold storage of the blood cells. In another
embodiment, the method provides that the blood cells are contacted
with the blood cell modifying agent at the time of blood collection
from a blood donor. In another embodiment, the method provides for
separating the blood cells into subpopulations of platelets,
plasma, red blood cells and white blood cells. In another
embodiment, the blood cells are contacted with the blood cell
modifying agent after the blood cells have been separated by
apheresis.
[0058] In another aspect, the invention provides for a treated
blood cell obtained through the methods described. The treated
blood cells, following cold storage, are suitable for transfusion
into a patient. These and other aspects of the invention, as well
as various advantages and utilities, will be more apparent in
reference to the following detailed description of the invention.
Each of the limitations of the invention can encompass various
embodiments of the invention. It is therefore, anticipated that
each of the limitations involving any one element or combination of
elements can be included in each aspect of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1A shows circulation time in mice of room temperature
platelets and of platelets chilled and rewarmed in the presence or
absence of EGTA-AM and Cytochalasin B. The curves depict the
survival of 5-chloromethylfluorescein diacetate (CMFDA) labeled,
room temperature (RT) platelets, platelets chilled at ice-bath
temperature (Cold) and rewarmed to room temperature before
injection and chilled and rewarmed platelets treated with EGTA-AM
and cytochalasin B (Cold+CytoB/EGTA) to preserve their discoid
shape. Each curve represents the mean.+-.SD of 6 mice. Identical
clearance patterns were observed with .sup.111Indium-labeled
platelets.
[0060] FIG. 1B shows that chilled platelets aggregate normally in
vitro. Washed, chilled-rewarmed (Cold) or room temperature (RT)
wild type platelets were stimulated by the addition of the
indicated agonists at 37.degree. C. and light transmission was
recorded on a standard to aggregometer. Aggregation responses of
chilled platelets treated with EGTA-AM and cytochalasin B were
identical to untreated chilled platelets.
[0061] FIG. 1C shows that cold induced clearance occurs
predominantly in the liver of mice. The liver is the primary
clearance organ of chilled platelets, containing 60-90% of injected
platelets. In contrast, RT platelets are cleared more slowly in the
spleen. .sup.111Indium labeled platelets were injected into
syngeneic mice and tissues were harvested at 0.5, 1 and 24 hours.
Data are expressed per gram of tissue. Each bar depicts the mean
values of 4 animals analyzed.+-.SD.
[0062] FIG. 1D shows that chilled platelets co-localize with
hepatic sinusoidal macrophages (Kupffer cells). This representative
confocal-micrograph shows the hepatic distribution of
CMFDA-labeled, chilled-rewarmed platelets (green) after 1 hour of
transfusion, which preferentially accumulate in periportal and
midzonal fields of liver lobules. Kupffer cells were visualized
after injection of nile red-labeled spheres. The merged micrograph
that shows co-localization of chilled platelets and macrophages in
yellow. The lobule organization is indicated (CV: central vein; PV:
portal vein, bar: 100 .mu.M).
[0063] FIG. 2 shows that chilled platelets circulate normally in
CR3-deficient mice, but not in complement 3 (C3) or vWf deficient
mice. CMFDA-labeled chilled-rewarmed (Cold) and room temperature
(RT) wild type platelets were transfused into six each of syngeneic
wild type (WT), CR3-deficient (A), vWf-deficient (B) and
C3-deficient (C) recipient mice and their survival times
determined. Chilled platelets circulate in CR3-deficient animals
with the same kinetics as room-temperature platelets, but are
cleared rapidly from the circulation of C3- or vWf-deficient mice.
Data are mean.+-.SD for 6 mice.
[0064] FIG. 3A-3C show that chilled platelets adhere tightly to
CR3-expressing mouse macrophages in vivo. FIG. 3A--Chilled-rewarmed
TRITC-labeled platelets (left panel) adhere with a 3-4.times.
higher frequency to liver sinusoids than room temperature
CMFDA-labeled platelets (right panel). The intravital fluorescence
micrographs were obtained 30 min after the infusion of the
platelets. FIG. 3B-Chilled-rewarmed (Cold, open bars) and room
temperature platelets (RT, filled bars) adhere to sinusoidal
regions with high macrophage density (midzonal) with similar
distributions in wild type mice. FIG. 3C--Chilled-rewarmed
platelets adhere 3-4.times. more than room temperature platelets to
macrophages in the wild type liver (open bars). In contrast,
chilled-rewarmed or room temperature platelets have identical
adherence to macrophages in CR3-deficient mice (filled bars). 9
experiments with wild type mice and 4 experiments with
CR3-deficient mice are shown (mean.+-.SEM, *P<0.05:
**P<0.01).
[0065] FIG. 4A-4C show that GP1b.alpha. mediates chilled platelet
clearance, aggregates in the cold, but to binds activated vWf
normally on chilled platelets. FIG. 4A--CMFDA-labeled platelets
enzymatically cleared of the GP1b.alpha. extracellular domain (left
panel, inset, filled area) or control platelets were kept at room
temperature (left panel) or chilled-rewarmed (right panel) infused
into syngeneic wild type mice, and platelet survivals were
determined. Each survival curve represents the mean values 3 SD for
6 mice. FIG. 4B--Chilled, or RT platelet rich plasma was treated
with (shaded area) or without (open area) botrocetin. vWf bound was
detected using FITC labeled anti-vWf antibody. FIG. 4C--The vWf
receptor redistributes from linear arrays (RT) into aggregates
(Chilled) on the surface of chilled murine platelets. Fixed,
chilled-rewarmed, or room temperature platelets (RT) were incubated
with monoclonal rat anti-mouse GP1b.alpha. antibodies followed by
10 nm colloidal gold particles coated with goat anti-rat IgG. The
bars are 100 nm. Inset: low magnification of platelets.
[0066] FIG. 5A-5C show GP1b.alpha.-CR3 interaction mediates
phagocytosis of chilled human platelets in vitro. FIGS. 5A and 5B
show a representative assay result of THP-1 cells incubated with
room temperature (RT) (FIG. 5A) or chilled-rewarmed (Cold)
platelets (FIG. 5B). CM-Orange-labeled platelets associated with
macrophages shift in orange fluorescence up the y axis. The mean
percentage of the CM-Orange positive native macrophages incubated
with platelets kept at room temperature was normalized to 1.
Chilling of platelets increases this shift from .about.4% to 20%.
The platelets are predominantly ingested, because they do not dual
label with the FITC-conjugated mAb to CD61. FIG. 5C
Undifferentiated (open bars) THP-1 cells express .about.50% less
CR3, and ingest half as many chilled-rewarmed platelets.
Differentiation (filled bars) of CR3 expression however, had no
significant effect on the uptake of RT platelets. Treatment of
human platelets with the snake venom metalloprotease, mocarhagin
(Moc), which removes the N-terminus of GP1b.alpha. from the surface
of human platelets (inset; control: solid line, mocarhagin treated
platelets: shaded area), reduced phagocytosis of chilled platelets
by .about.98%. Data shown are means.+-.SD of 5 experiments.
[0067] FIG. 6A-6E show circulating, chilled platelets have
hemostatic function in CR3 deficient mice. Normal in vivo function
of room temperature (RT) platelets transfused into wild type mice
(FIGS. 6A and 6B) and of chilled (Cold) platelets transfused into
CR3 deficient mice (FIGS. 6C and 6D), as determined by their
equivalent presence in platelet aggregates emerging from the wound
24 hrs after infusion of autologous CMFDA labeled platelets.
Peripheral blood (FIGS. 6A and 6C) and the blood emerging from the
wound (shed blood, FIGS. 6B and 6D) were analyzed by whole blood
flow cytometry. Platelets were identified by forward light scatter
characteristics and binding of the PE-conjugated anti-GP1b.alpha.
mAb (pOp4). The infused platelets (dots) were identified by their
CMFDA fluorescence and the non-infused platelets (contour lines) by
their to lack of CMFDA fluorescence. In the peripheral whole blood
samples, analysis regions were plotted around the
GP1b.alpha.-positive particles to include 95% of the population on
the forward scatter axis (region 1) and the 5% of particles
appearing above this forward light scatter threshold were defined
as aggregates (region 2). The percentages refer to the number of
aggregates formed by CMFDA-positive platelets. This shown result is
representative of 4 experiments. FIG. 6E shows ex vive function of
CM-Orange, room temperature (RT) platelets transfused into wild
type mice and CM-Orange, chilled-rewarmed (Cold) platelets
transfused into CR3 deficient mice, as determined by exposure of
P-selectin and fibrinogen binding following thrombin (1 U/ml)
activation of blood drawn from the mice after 24 hours post
infusion. CM-Orange labeled platelets have a circulation half-life
time comparable to that of CMFDA labeled platelets (not shown).
Transfused platelets were identified by their CM-Orange
fluorescence (filled bars). Non-transfused (non-labeled) analyzed
platelets are represented as open bars. Results are expressed as
the percentage of cells present in the P-selectin and fibrinogen
positive regions (region 2). Data are mean.+-.SD for 4 mice.
[0068] FIG. 7 is a schematic depicting two platelet clearance
pathways. Platelets traverse central and peripheral circulations,
undergoing reversible priming at lower temperatures at the body
surface. Repeated priming leads to irreversible GP1b-X-V (vWfR)
receptor complex reconfiguration and clearance by complement
receptor type 3 (CR3) bearing hepatic macrophages. Platelets are
also cleared after they participate in microvascular
coagulation.
[0069] FIG. 8A-8D show the effect of monosaccharides on
phagocytosis of chilled platelets.
[0070] FIG. 9A-9F show the dot plots of binding of WGA lectin to
room temperature platelets or chilled platelets.
[0071] FIG. 10 shows the analysis of various FITC labeled lectins
bond to room temperature or chilled platelets.
[0072] FIG. 11A shows the summary of FITC-WGA binding to the
surface of room temperature or chilled platelets obtained by flow
cytometry before and after 3-hexosaminidase treatment.
[0073] FIG. 11B shows that GP1b.alpha. removal from the platelet
surface reduced FITC-WGA binding to chilled platelets.
[0074] FIG. 12 shows that galactose transfer onto platelet
oligosaccharides reduces chilled platelet (Cold) phagocytosis, but
does not affect the phagocytosis of room temperature (RT)
platelets.
[0075] FIG. 13 shows the survival of chilled, galactosylated murine
platelets relative to untreated platelets.
[0076] FIG. 14A-14C show that platelets containing galactose
transferases on their surface transfer galactose without the
addition of external transferases as judged by WGA binding (FIG.
14A) and in vitro phagocytosis results for human platelets (FIG.
14B). FIG. 14C shows that of UDP-galactose with or without
Galactose transferase (GalT) on survival of murine platelets.
UDP-galactose with or without GalT was added to murine platelets
before chilling for 30 min at 37.degree. C. The platelets were
chilled for 2 hours in an ice bath and then transfused (10
platelets/mouse) into mice and their survival determined.
[0077] FIG. 15 shows the time course of .sup.14C-labeled
UDP-galactose incorporation into human platelets.
[0078] FIG. 16 shows galactosylation of platelets in four platelet
concentrate samples at different concentrations of
UDP-galactose.
[0079] FIG. 17 shows the complement receptor mediates phagocytosis
and clearance of chilled platelets.
[0080] FIG. 18 shows the GP1b.alpha. subunit of platelet von
Willebrand factor receptor binds the I-domain of .alpha.M of
.alpha.M/.beta.2 integrin.
[0081] FIG. 19 shows that chilled platelets circulate and function
normally in .alpha.M knockout mice.
[0082] FIG. 20 illustrates vWf receptor inactivation.
[0083] FIG. 21 shows that .alpha.M/.beta.2 recognizes the outer tip
of GP1b.alpha. and mediates clearance of chilled platelets, thus
demonstrating that GP1b.alpha. has coagulant (vWf binding) and
non-coagulant (clearance) functions.
[0084] FIG. 22 illustrates the primary structure of .alpha.M
(CD11b).
[0085] FIG. 23 shows that .alpha.M has a lectin affinity site.
[0086] FIG. 24 shows that the lectin domain of macrophage
.alpha..alpha.M/.beta.2 receptors recognizes .beta.GlcNAc residues
on clustered GP1b.alpha..
[0087] FIG. 25 shows that a soluble .alpha.M-lectin domain inhibits
chilled human platelet phagocytosis by macrophages.
[0088] FIG. 26 shows the construction of CHO cells expressing
.alpha.M.alpha.X chimeric proteins.
[0089] FIG. 27 illustrates a phagocytic assay for altered platelet
surface induced by chilling.
[0090] FIG. 28 shows that the .alpha.M-lectin domain mediates
chilled human platelet phagocytosis.
[0091] FIG. 29 shows that macrophage .alpha.M/.beta.32 receptors
recognize .beta.GlcNAc residues on clustered GP1b.alpha. receptors
of chilled platelets.
[0092] FIG. 30 illustrates the galactosylation of platelets through
GP1b.alpha..
[0093] FIG. 31 shows expression of .beta.4GalT1 on the platelet
surface.
[0094] FIG. 32 illustrates that galatosylated chilled murine
platelets can circulate in vivo.
[0095] FIG. 33 illustrates that galatosylated chilled murine
platelets can function normally in murine models.
[0096] FIG. 34 shows that human platelet concentrates can be
galactosylated, which preserves platelet function.
[0097] FIG. 35 illustrates a method for galactosylation of human
platelet concentrates.
[0098] FIG. 36 shows surface galactose on platelet concentrates is
stable.
[0099] FIG. 37 shows that galactosylation inhibits phagocytosis by
THP-1 macrophages of human chilled platelets.
[0100] FIG. 38 shows that platelet counts and pH remain unchanged
in refrigerated platelet concentrates.
[0101] FIG. 39 shows the effects of refrigeration and
galactosylation on retention of platelet responses to agonists
during storage of concentrates.
[0102] FIG. 40 shows the effect of storage conditions on shape
change (spreading) and clumping of platelets in concentrates.
[0103] FIG. 41 illustrates an embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is
described. Platelets are derived from a variety of blood sources,
including IRDP--Individual Random Donor Platelets, PRDP--Pooled
Random Donor Platelets and SDP--Single Donor Platelets. The
container having the glycan modifying agent, e.g., a solution of
UDP-Gal and/or CMP-NeuAc is sterile docked to the bag containing
the platelets. A sterile dock is also referred to as a sterile
connection device (SCD) or a total containment device (TCD). The
sterile dock permits connection of two pieces of conduit while
maintaining sterility of the system. The glycan modifying agent is
mixed with the platelets and then the modified platelets are
transferred to a non-breathable bag. The glycan modifying agent can
be introduced to the platelets at a variety of times, e.g., before
infusion, before storage, after componentization or directly to
whole blood, or during the platelet apheresis procedure at the time
of donation. Likewise, the glycan modifying solution may be
provided in a variety of forms, such as full strength concentration
liquid, concentrated liquid--diluted before use, dehydrated, freeze
dried, lyophilisized, powder, frozen, viscous fluid, suspension,
base and activator, or reactant and catalyst. In this embodiment,
the blood is passed through a leukocyte filter. Various methods of
leukocyte depletion are known in the art, e.g., glass wool or other
affinity separation methods for removing leukocyte fractions from
whole blood, and provide examples of means for filtering the
leukocytes from the rest of the blood and specifically the
platelets.
[0104] FIG. 42 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. This illustration is similar to FIG. 41 but does not
include a leukocyte filter.
[0105] FIG. 43 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The bag containing the platelets is sterile docked to
the bag containing the platelet solution. The glycans modifying
solution, also called a platelet solution, is mixed with the
platelets and then transferred to a non-breathable bag and thru a
leukocyte filter.
[0106] FIG. 44 illustrates a variation of FIG. 43, that does not
include a leukocyte filter.
[0107] FIG. 45 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The syringe containing the platelet solution (UDP-Gal
and/or CMP-NeuAc) is sterile docked to the bag containing the
platelets. The platelet solution is mixed with the platelets and
then transferred to a non-breathable bag and thru a leukocyte
filter.
[0108] FIG. 46 illustrates a variation of FIG. 45, that does not
include a leukocyte filter.
[0109] FIG. 47 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The bag containing the platelet solution (UDP-Gal
and/or CMP-NeuAc) is connected to the container port using a bag
spike thru a 0.22 micron filter to the bag containing the
platelets. The platelet solution is mixed with the platelets and
then transferred to a non-breathable bag and thru a leukocyte
filter. A 0.22 micron filter is illustrated, but larger pore
diameter filters are suitable to provide increased flow rate.
Median pore sizes greater than about 1 micron are not suitable for
sterile filtration. Preferred sizes are less than about 0.75
microns, more preferably less than about 0.5 microns, and most
preferably about 0.22 microns.
[0110] FIG. 48 illustrates a variation of FIG. 47, that does not
include a leukocyte filter.
[0111] FIG. 49 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The bag containing the platelet solution (either
single dose or bulk) is connected using a luer lock thru a 0.22
micron filter to the bag containing the platelets. The platelet
solution is mixed with the platelets and then transferred to a
non-breathable bag and thru a leukocyte filter.
[0112] FIG. 50 illustrates a variation of FIG. 49, that does not
include a leukocyte filter.
[0113] FIG. 51 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The syringe containing the platelet solution is
connected using a luer lock thru a 0.22 micron filter to the bag
containing the platelets. The platelet solution is mixed with the
platelets and then transferred to a non-breathable bag and thru a
leukocyte filter. Also shown, IRDP can be pooled to form PRDP.
[0114] FIG. 52 illustrates a variation of FIG. 51, that does not
include a leukocyte filter.
[0115] FIG. 53 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The syringe containing the platelet solution is
connected using a luer lock thru a 0.22 micron filter to the bag
containing the platelets. The platelet solution is mixed with the
platelets and then transferred to a non-breathable bag and thru a
leukocyte filter. The syringe can be aseptically refilled from the
bulk platelet solution because of the in-line filtration
device.
[0116] FIG. 54 illustrates a variation of FIG. 53, that does not
include a leukocyte filter.
[0117] FIG. 55 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The large non-breathable bag (final storage bag)
containing the platelet solution includes an array comprising long
piece of conduit and a plurality of ports to allow the sterile
docking of multiple IRDP bags sequentially from the distal end of
the tube (denoted #8) to the proximal end (denoted #1) thru a 0.22
micron filter to the bag containing the platelet solution. The
platelet solution is mixed with the pooled platelets.
[0118] FIG. 56 illustrates a variation of FIG. 55, that does not
include a leukocyte filter.
[0119] FIG. 57 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. Platelet solution delivery to the containment bag is
facilitated by an SCD on the bag.
[0120] FIG. 58 illustrates a variation of FIG. 57, that does not
include a leukocyte filter.
[0121] FIG. 59 illustrates a variation of FIG. 57. The large
non-breathable bag (final storage bag) has the platelet solution,
stored in a syringe, aseptically connected and added thru a 0.22
micron filter.
[0122] FIG. 60 illustrates a variation of FIG. 59, that does not
include a leukocyte filter.
[0123] FIG. 61 illustrates a variation of the invention, wherein a
container having platelet solution is adapted to the container
having blood cells through a conduit attachable via a luer lock
connection. The conduit has a bag spike to puncture a barrier in
the container, thereby permitting withdrawal of the glycans
modifying solution.
[0124] FIG. 62 illustrates a variation of FIG. 61, that does not
include a leukocyte filter.
[0125] FIG. 63 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The large non-breathable bag (final storage bag) has
the platelet solution, stored in a bag, connected with a frangible
plug that can be opened to deliver the platelet solution.
[0126] FIG. 64 illustrates a variation of FIG. 63, that does not
include a leukocyte filter.
[0127] FIG. 65 illustrates another embodiment of the invention
wherein a bioprocess for collecting, treating and storing platelets
is described. The large non-breathable bag (final storage bag)
includes an integrated bag of platelet solution having a frangible
plug that can be opened to deliver the platelet solution directly
into the platelet storage container.
[0128] FIG. 66 illustrates a variation of the embodiment
illustrated as FIG. 65. The bag having the platelet modifying
solution is integrated within the storage bag. The platelet
solution is released upon breaking of the frangible plug or
separation membrane.
DETAILED DESCRIPTION OF THE INVENTION
[0129] The invention provides a population of modified platelets
that have enhanced circulation properties and that retain
substantially normal in vive hemostatic activity. Hemostatic
activity refers broadly to the ability of a population of platelets
to mediate bleeding cessation. Various assays are available for
determining platelet hemostatic activity (Bennett, J. S. and
Shattil, S. J., 1990, "Platelet function," Hematology, Williams, W.
J., et al., Eds. McGraw Hill, pp 1233-12250). However,
demonstration of "hemostasis" or "hemostatic activity" ultimately
requires a demonstration that platelets infused into a
thrombocytopenic or thrombopathic (i.e., non-functional platelets)
animal or human circulate and stop natural or
experimentally-induced bleeding.
[0130] Short of such a demonstration, laboratories use in vitro
tests as surrogates for determining hemostatic activity. These
tests, which include assays of aggregation, secretion, platelet
morphology and metabolic changes, measure a wide variety of
platelet functional responses to activation. It is generally
accepted in the art that the in vitro tests are reasonably
indicative of hemostatic function in vivo.
[0131] Substantially normal hemostatic activity refers to an amount
of hemostatic activity seen in the modified platelets, that is
functionally equivalent to or substantially similar to the
hemostatic activity of untreated platelets in vivo, in a healthy
(non-thrombocytopenic or non-thrombopathic mammal) or functionally
equivalent to or substantially similar to the hemostatic activity
of a freshly isolated population of platelets in vitro.
[0132] The instant invention provides methods for reduced
temperature storage of platelets which increases the storage time
of the platelets, as well as methods for reducing clearance of or
increasing circulation time of a population of platelets in a
mammal. Also provided are platelet compositions methods and
compositions for the preservation of platelets with preserved
hemostatic activity as well as methods for making a pharmaceutical
composition containing the preserved platelets and for
administering the pharmaceutical composition to a mammal to mediate
hemostasis. Also provided are kits for treating a platelet
preparation for storage, and containers for storing the same.
[0133] In one aspect of the invention, the method for increasing
circulation time of an isolated population of platelets involves
contacting an isolated population of platelets with at least one
glycan modifying agent in an amount effective to reduce the
clearance of the population of platelets. As used herein, a
population of platelets refers to a sample having one or more
platelets. A population of platelets includes a platelet
concentrate. The term "isolated" means separated from its native
environment and present in sufficient quantity to permit its
identification or use. As used herein with respect to a population
of platelets, isolated means removed or cleared from the blood
circulation of a mammal. The circulation time of a population of
platelets is defined as the time when one-half of the platelets in
that population are no longer circulating in a mammal after
transplantation into that mammal. As used herein, "clearance" means
removal of the modified platelets from the blood circulation of a
mammal (such as but not limited to by macrophage phagocytosis). As
used herein, clearance of a population of platelets refers to the
removal of a population of platelets from a unit volume of blood or
serum per unit of time. Reducing the clearance of a population of
platelets refers to preventing, delaying, or reducing the clearance
of the population of platelets. Reducing clearance of platelets
also may mean reducing the rate of platelet clearance.
[0134] A glycan modifying agent refers to an agent that modifies
glycan residues on the platelet. As used herein, a "glycan" or
"glycan residue" is a polysaccharide moiety on surface of the
platelet, exemplified by the GP1b.alpha. polysaccharide. A
"terminal" glycan or glycan residue is the glycan at the distal
terminus of the polysaccharide, which typically is attached to
polypeptides on the platelet surface. Preferably, the glycan
modifying agent alters GP1b.alpha. on the surface of the
platelet.
[0135] The glycan modifying agents suitable for use as described
herein, includes monosaccharides such as arabinose, fructose,
fucose, galactose, mannose, ribose, gluconic acid, galactosamine,
glucosamine, N-acetylgalactosamine, muramic acid, sialic acid
(N-acetylneuraminic acid), and nucleotide sugars such as cytidine
monophospho-N-acetylneuraminic acid (CMP-sialic acid), uridine
diphosphate galactose (UDP-galactose) and UDP-galactose precursors
such as UDP-glucose. In some preferred embodiments, the glycan
modifying agent is UDP-galactose or CMP-sialic acid.
[0136] UDP-galactose is an intermediate in galactose metabolism,
formed by the enzyme UDP-glucose-.alpha.-D-galactose-1-phosphate
uridylyltransferase which catalyzes the release of
glucose-1-phosphate from UDP-glucose in exchange for
galactose-1-phosphate to make UDP-galactose. UDP-galactose and
sialic acid are widely available from several commercial suppliers
such as Sigma. In addition, methods for synthesis and production of
UDP-galactose are well known in the art and described in the
literature (see for example, Liu et al, ChemBioChem 3, 348-355,
2002; Heidlas et al, J. Org. Chem. 57, 152-157; Butler et al, Nat.
Biotechnol. 8, 281-284, 2000; Koizumi et al, Carbohydr. Res. 316,
179-183, 1999; Endo et al, Appl. Microbiol., Biotechnol. 53,
257-261, 2000). UDP-galactose precursors are molecules, compounds,
or intermediate compounds that may be converted (e.g.,
enzymatically or biochemically) to UDP-galactose. One non-limiting
example of a UDP-galactose precursor is UDP-glucose. In certain
embodiments, an enzyme that converts a UDP-galactose precursor to
UDP-galactose is added to a reaction mixture (e.g. in a platelet
container).
[0137] An effective amount of a glycan modifying agent is that
amount of the glycan modifying agent that alters a sufficient
number of glycan residues on the surface of platelets, that when
introduced to a population of platelets, increases circulation time
and/or reduces the clearance of the population of platelets in a
mammal following transplantation of the platelets into the mammal.
An effective amount of a glycan modifying agent is a concentration
from about 1 micromolar to about 1200 micromolar, preferably from
about 10 micromolar to about 1000 micromolar, more preferably from
about 100 micromolar to about 750 micromolar, and most preferably
from about 200 micromolar to about 600 micromolar.
[0138] Modification of platelets with glycan modifying agents can
be preformed as follows. The population of platelets is incubated
with the selected glycan modifying agent (concentrations of 1-1200
.mu.M) for at least 1, 2, 5, 10, 20, 40, 60, 120, 180, 240, or 300
min. at 22.degree. C.-37.degree. C. Multiple glycan modifying
agents (i.e., two, three four or more) may be used simultaneously
or sequentially. In some embodiments 0.1-500 mU/ml galactose
transferase or sialyl transferase is added to the population of
platelets. Galactose transfer can be monitored functionally using
FITC-WGA (wheat germ agglutinin) binding. The goal of the glycan
modification reaction is to reduce WGA binding to resting room
temperature WGA binding-levels. Galactose transfer can be
quantified using .sup.14C-UDP-galactose. Non-radioactive
UDP-galactose is mixed with .sup.14C-UDP-galactose to obtain
appropriate galactose transfer. Platelets are extensively washed,
and the incorporated radioactivity measured using a
.gamma.-counter. The measured cpm permits calculation of the
incorporated galactose. Similar techniques are applicable to
monitoring sialic acid transfer.
[0139] Reducing the clearance of a platelet encompasses reducing
clearance of platelets after storage at room temperature, or after
chilling, as well as "cold-induced platelet activation".
Cold-induced platelet activation is a term having a particular
meaning to one of ordinary skill in the art. Cold-induced platelet
activation may manifest by changes in platelet morphology, some of
which are similar to the changes that result following platelet
activation by, for example, contact with glass. The structural
changes indicative of cold-induced platelet activation are most
easily identified using techniques such as light or electron
microscopy. On a molecular level, cold-induced platelet activation
results in actin bundle formation and a subsequent increase in the
concentration of intracellular calcium. Actin-bundle formation is
detected using, for example, electron microscopy. An increase in
intracellular calcium concentration is determined, for example, by
employing fluorescent intracellular calcium chelators. Many of the
above-described chelators for inhibiting actin filament severing
are also useful for determining the concentration of intracellular
calcium (Tsien, R., 1980, supra.). Accordingly, various techniques
are available to determine whether or not platelets have
experienced cold-induced activation.
[0140] The effect of galactose or sialic acid addition to the
glycan moieties on platelets, resulting in diminished clearance of
modified platelets, can be measured for example using either an in
vitro system employing differentiated THP-1 cells or murine
macrophages, isolated from the peritoneal cavity after
thioglycolate injection stimulation. The rate of clearance of
modified platelets compared to unmodified platelets is determined.
To test clearance rates, the modified platelets are fed to the
macrophages and ingestion of the platelets by the macrophages is
monitored. Reduced ingestion of modified platelets relative to
unmodified platelets (twofold or greater) indicates successful
modification of the glycan moiety for the purposes described
herein.
[0141] In accordance with the invention, the population of modified
platelets can be chilled without the deleterious effects
(cold-induced platelet activation) usually experienced on chilling
of untreated platelets. The population of modified platelets can be
chilled prior to, concurrently with, or after contacting the
platelets with the at least one glycan modifying agent. The
selective modification of glycan moieties reduces clearance,
following chilling (also if not chilled), thus permitting
longer-term storage than is presently possible. As used herein,
chilling refers to lowering the temperature of the population of
platelets to a temperature that is less than about 37.degree. C. In
some embodiments, the platelets are chilled to a temperature that
is less than about 15.degree. C. In some preferred embodiments, the
platelets are chilled to a temperature ranging from between about
0.degree. C. to about 4.degree. C. Chilling also encompasses
freezing the platelet preparation, i.e., to temperatures less than
0.degree. C., -20.degree. C., -50.degree. C., and -80.degree. C. or
cooler. Process for the cryopreservation of cells are well known in
the art.
[0142] In some embodiments, the population of platelets is stored
chilled for at least 3 days. In some embodiments, the population of
platelets is stored chilled for at least 5, 7, 10, 14, 21, and 28
days or longer.
[0143] In some embodiments of the invention, the circulation time
of the population of platelets is increased by at least about 10%.
In some other embodiments, the circulation time of the population
of platelets is increased by at least about 25%. In yet some other
embodiments, the circulation time of the population of platelets is
increased by at least about 50% to about 100%. In still yet other
embodiments, the circulation time of the population of platelets is
increased by about 150% or greater.
[0144] The invention also embraces a method for increasing the
storage time of platelets. As used herein the storage time of
platelets is defined as the time that platelets can be stored
without substantial loss of platelet function or hemostatic
activity such as the loss of the ability to circulate or increased
platelet clearance.
[0145] The platelets are collected from peripheral blood by
standard techniques known to those of ordinary skill in the art,
for example by isolation from whole blood or by apheresis
processes. In some embodiments, the platelets are contained in a
pharmaceutically-acceptable carrier prior to treatment with a
glycan modifying agent.
[0146] According to another aspect of the invention, a modified
platelet or a population of modified platelets is provided. The
modified platelet comprises a plurality of modified glycan
molecules on the surface of the platelet. In some embodiments, the
modified glycan moieties are GP1b.alpha. molecules. The invention
also encompasses a platelet composition in a storage medium. In
some embodiments the storage medium comprises a pharmaceutically
acceptable carrier.
[0147] The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the platelets and that is a non-toxic
material that is compatible with a biological system such as a
cell, cell culture, tissue, or organism. Pharmaceutically
acceptable carriers include diluents, fillers, salts, buffers,
stabilizers, solubilizers, and other materials which are well known
in the art, for example, a buffer that stabilizes the platelet
preparation to a pH of 7.4, the physiological pH of blood, is a
pharmaceutically acceptable composition suitable for use with the
present invention.
[0148] The invention further embraces a method for making a
pharmaceutical composition for administration to a mammal. The
method comprises preparing the above-described platelet
preparation, and warming the platelet preparation. In some
embodiments, the method comprises neutralizing, removing or
diluting the glycan modifying agent(s) and/or the enzyme(s) that
catalyze the modification of the glycan moiety, and placing the
modified platelet preparation in a pharmaceutically acceptable
carrier. In a preferred embodiment, the chilled platelets are
warmed to room temperature (about 22.degree. C.) prior to
neutralization or dilution. In some embodiments, the platelets are
contained in a pharmaceutically acceptable carrier prior to contact
with the glycan modifying agent(s) with or without the enzyme(s)
that catalyze the modification of the glycan moiety and it is not
necessary to place the platelet preparation in a pharmaceutically
acceptable carrier following neutralization or dilution.
[0149] As used herein, the terms "neutralize" or "neutralization"
refer to a process by which the glycan modifying agent(s) and/or
the enzyme(s) that catalyze the modification of the glycan moiety
are rendered substantially incapable of glycan modification of the
glycan residues on the platelets, or their concentration in the
platelet solution is lowered to levels that are not harmful to a
mammal, for example, less that 50 micromolar of the glycan
modifying agent. In some embodiments, the chilled platelets are
neutralized by dilution, e.g., with a suspension of red blood
cells. Alternatively, the treated platelets can be infused into the
recipient, which is equivalent to dilution into a red blood cell
suspension. This method of neutralization advantageously maintains
a closed system and minimizes damage to the platelets. In a
preferred embodiment of glycan modifying agents, no neutralization
is required.
[0150] An alternative method to reduce toxicity is by inserting a
filter in the infusion line, the filter containing, e.g. activated
charcoal or an immobilized antibody, to remove the glycan modifying
agent(s) and/or the enzyme(s) that catalyze the modification of the
glycan moiety.
[0151] Either or both of the glycan modifying agent(s) and the
enzyme(s) that catalyze the modification of the glycan moiety also
may be removed or substantially diluted by washing the modified
platelets in accordance with standard clinical cell washing
techniques.
[0152] The invention further provides a method for mediating
hemostasis in a mammal. The method includes administering the
above-described pharmaceutical preparation to the mammal.
Administration of the modified platelets may be in accordance with
standard methods known in the art. According to one embodiment, a
human patient is transfused with red blood cells before, after or
during administration of the modified platelets. The red blood cell
transfusion serves to dilute the administered, modified platelets,
thereby neutralizing the glycan modifying agent(s) and the
enzyme(s) that catalyze the modification of the glycan moiety.
[0153] The dosage regimen for mediating hemostasis using the
modified platelets is selected in accordance with a variety of
factors, including the type, age, weight, sex and medical condition
of the subject, the severity of the disease, the route and
frequency of administration. An ordinarily skilled physician or
clinician can readily determine and prescribe the effective amount
of modified platelets required to mediate hemostasis.
[0154] The dosage regimen can be determined, for example, by
following the response to the treatment in terms clinical signs and
laboratory tests. Examples of such clinical signs and laboratory
tests are well known in the art and are described, see, Harrison's
Principles of Internal Medicine, 15th Ed., Fauci A S et al., eds.,
McGraw-Hill, New York, 2001.
[0155] Also within the scope of the invention are storage
compositions and pharmaceutical compositions for mediating
hemostasis. In one embodiment, the compositions comprise a
pharmaceutically-acceptable carrier, a plurality of modified
platelets, a plurality of glycan modifying agent(s) and optionally
the enzyme(s) that catalyze the modification of the glycan moiety.
The glycan modifying agent(s) and the enzyme(s) that catalyze the
modification of the glycan moiety are present in the composition in
sufficient amounts so as to reduce platelet clearance. Preferably,
glycan modifying agent(s) (and optionally the enzyme(s) that
catalyze the modification of the glycan moiety) are present in
amounts whereby after chilling and neutralization, the platelets
maintain substantially normal hemostatic activity. The amounts of
glycan modifying agent(s) (and optionally the enzyme(s) that
catalyze the modification of the glycan moiety) which reduce
platelet clearance can be selected by exposing a preparation of
platelets to increasing amounts of these agents, exposing the
treated platelets to a chilling temperature and determining (e.g.,
by microscopy) whether or not cold-induced platelet activation has
occurred. Preferably, the amounts of glycan modifying agent(s) and
the enzyme(s) that catalyze the modification of the glycan moiety
can be determined functionally by exposing the platelets to varying
amounts of glycan modifying agent(s) and the enzyme(s) that
catalyze the modification of the glycan moiety, chilling the
platelets as described herein, warming the treated (chilled)
platelets, optionally neutralizing the platelets and testing the
platelets in a hemostatic activity assay to determine whether the
treated platelets have maintained substantially normal hemostatic
activity.
[0156] For example, to determine the optimal concentrations and
conditions for preventing cold-induced activation of platelets by
modifying them with a glycan modifying agent(s) (and optionally the
enzyme(s) that catalyze the modification of the glycan moiety),
increasing amounts of these agents are contacted with the platelets
prior to exposing the platelets to a chilling temperature. The
optimal concentrations of the glycan modifying agent(s) and the
enzyme(s) that catalyze the modification of the glycan moiety are
the minimal effective concentrations that preserve intact platelet
function as determined by in vitro tests (e.g., observing
morphological changes in response to glass, thrombin,
cryopreservation temperatures; ADP-induced aggregation) followed by
in vivo tests indicative of hemostatic function (e.g., recovery,
survival and shortening of bleeding time in a thrombocytopenic
animal or recovery and survival of .sup.51Cr-labeled platelets in
human subjects).
[0157] According to yet another aspect of the invention, a
composition for addition to platelets to reduce platelet clearance
or to increase platelet storage time is provided. The composition
includes one or more glycan modifying agents. In certain
embodiments, the composition also includes an enzyme(s) that
catalyze the modification of the glycan moiety. The glycan
modifying agent and the enzyme(s) that catalyzes the modification
of the glycan moiety are present in the composition in amounts that
prevent cold-induced platelet activation.
[0158] The invention also embraces a storage composition for
preserving platelets. The storage composition comprises at least
one glycan modifying agent in an amount sufficient to reduce
platelet clearance. In some embodiments the storage composition
further comprises an enzyme that catalyzes the modification of a
glycan moiety on the platelet. The glycan modifying agent is added
to the population of platelets that are preferably kept between
about room temperature and 37.degree. C. In some embodiments,
following treatment, the population of platelets is cooled to about
4.degree. C. In some embodiments, the platelets are collected into
a platelet pack, bag, or container according to standard methods
known to one of skill in the art. Typically, blood from a donor is
drawn into a primary container which may be joined to at least one
satellite container, all of which containers are connected and
sterilized before use. In some embodiments, the satellite container
is connected to the container for collecting platelets by a
breakable seal. In some embodiments, the primary container further
comprises plasma containing a plurality of platelets.
[0159] In some embodiments, the platelets are concentrated (e.g. by
centrifugation) and the plasma and red blood cells are drawn off
into separate satellite bags (to avoid modification of these
clinically valuable fractions) prior to adding the glycan modifying
agent with or without the enzyme that catalyzes the modification of
a glycan moiety on the platelet. Platelet concentration prior to
treatment also may minimize the amounts of glycan modifying agents
required for reducing the platelet clearance, thereby minimizing
the amounts of these agents that are eventually infused into the
patient.
[0160] In one embodiment, the glycan modifying agent(s) are
contacted with the platelets in a closed system, e.g. a sterile,
sealed platelet pack, so as to avoid microbial contamination.
Typically, a venipuncture conduit is the only opening in the pack
during platelet procurement or transfusion. Accordingly, to
maintain a closed system during treatment of the platelets with the
glycan modifying agent(s), the agent(s) is placed in a relatively
small, sterile container which is attached to the platelet pack by
a sterile connection tube (see e.g., U.S. Pat. No. 4,412,835, the
contents of which are incorporated herein by reference). The
connection tube may be reversibly to sealed, or have a breakable
seal, as will be known to those of skill in the art. After the
platelets are concentrated, e.g. by allowing the platelets to
settle and squeezing the plasma out of the primary pack and into a
second bag according to standard practice, the seal to the
container(s) including the glycan modifying agent(s) is opened and
the agents are introduced into the platelet pack. In one
embodiment, the glycan modifying agents are contained in separate
containers having separate resealable connection tubes to permit
the sequential addition of the glycan modifying agents to the
platelet concentrate.
[0161] Following contact with the glycan modifying agent(s), the
treated platelets are chilled. In contrast to platelets stored at,
for example, 22.degree. C., platelets stored at cryopreservation
temperatures have substantially reduced metabolic activity. Thus,
platelets stored at 4.degree. C. are metabolically less active and
therefore do not generate large amounts of CO.sub.2 compared with
platelets stored at, for example, 22.degree. C. (Slichter, S. J.,
1981, Vox Sang 40 (Suppl 1), pp 72-86, Clinical Testing and
Laboratory-Clinical correlations.). Dissolution of CO.sub.2 in the
platelet matrix results in a reduction in pH and a concomitant
reduction in platelet viability (Slichter, S., 1981, supra.). This
can be resolved by adding buffers to the platelet population, the
buffers selected to keep the platelet population at or near the
physiological pH of blood. Likewise, conventional platelet packs
are formed of materials that are designed and constructed of a
sufficiently permeable material to maximize gas transport into and
out of the pack (O.sub.2 in and CO.sub.2 out). The prior art
limitations in platelet pack design and construction are obviated
by the instant invention, which permits storage of platelets at
cryopreservation temperatures, thereby substantially reducing
platelet metabolism and diminishing the amount of CO.sub.2
generated by the platelets during storage. Accordingly, the
invention further provides platelet containers that are
substantially non-permeable to CO.sub.2 and/or O.sub.2, which
containers are useful particularly for cold storage of platelets.
In both the gas permeable and non-gas permeable embodiments, the
invention provides for a blood storage container having therein, a
quantity of a glycan modifying agent sufficient to substantially
modify the carbohydrates of the platelets introduced therein, such
that the platelets become capable of cold storage and subsequent in
vivo circulation.
[0162] The present invention also provides for kits that are used
for platelet collection, processing and storage, further including
suitable packaging materials and instructions for using the kit
contents. It is preferred that all reagents and supplies in the kit
be sterile, in accordance with standard medical practices involving
the handling and storage of blood and blood products. Methods for
sterilizing the kit contents are known in the art, for example,
ethylene gas, irradiation and the like. In certain embodiments, the
kit may include venipuncture supplies and/or blood collection
supplies, for example a needle set, solution for sterilizing the
skin of a platelet donor, and a blood collection bag or container.
Preferably the container is "closed", i.e., substantially sealed
from the environment. Such closed blood collection containers are
well known in the art, and provide a means of preventing microbial
contamination of the platelet preparation contained therein. Other
embodiments include kits containing supplies for blood collection
and platelet apheresis. The kits may further include a quantity of
the glycan modifying agent, sufficient to modify the volume of
platelets collected and stored in the container. In certain
embodiments, the kit includes reagents for modifying the terminal
glycan of platelets with a second or third chemical moiety, for
example to PEGylate collected platelets. In other embodiments, the
kit includes a blood collection system having a blood storage
container wherein the glycan modifying agent is provided within the
container in an amount sufficient to treat the volume of blood or
platelets held by the container. The quantity of glycan modifying
agent will depend on the volume of the container. It is preferred
the glycan modifying agent be provided as a sterile non-pyogenic
solution, but it may also be supplied as a lyophilized powder. For
example, a blood bag is provided having a capacity of 250 ml.
Contained in the blood bag is a quantity of UDP-Gal such that when
250 ml of blood is added, the final concentration of the UDP-Gal is
approximately 200 micromolar. Other embodiments contain different
concentrations of glycan modifying agents, for example but not
limited to quantities resulting in final concentrations of 10
micromolar to 10 millimolar, and preferably 100 micromolar to 1
millimolar of the glycan modifying agents. Other embodiments use
combinations of glycan modifying agents, e.g., to effect sialyation
or galactosylation of N-linked glycoproteins on blood products
introduced into the container.
[0163] The invention will be more fully understood by reference to
the following examples. These examples, however, are merely
intended to illustrate the embodiments of the invention and are not
to be construed to limit the scope of the invention.
EXAMPLES
Example 1
Introduction
[0164] Modest cooling primes platelets for activation, but
refrigeration causes shape changes and rapid clearance,
compromising storage of platelets for therapeutic transfusions. We
found that shape change inhibition does not normalize cold-induced
clearance. We also found that cooling platelets rearranges the
surface configuration of the von Willebrand factor (vWf) receptor
complex .alpha. subunit (GP1b.alpha.) such that it becomes targeted
for recognition by complement receptor 3 receptors (CR3)
predominantly expressed on liver macrophages, leading to platelet
phagocytosis and clearance. GP1b.alpha. removal prolongs survival
of unchilled platelets. Chilled platelets bind vWf and function
normally in vitro and ex vivo after transfusion into CR3-deficient
mice. Cooled platelets, however, are not "activated" like platelets
exposed to thrombin or ADP, and their vWf-receptor complex reacts
normally with activated vWf.
[0165] As the temperature falls below 37.degree. C. platelets
become more susceptible to activation by thrombotic stimuli, a
phenomenon known as "priming" (Faraday and Rosenfeld, 1998;
Hoffmeister et al., 2001). Priming may be an adaptation to limit
bleeding at lower temperatures of body surfaces where most injuries
occur. We propose that the hepatic clearance system's purpose is to
remove repeatedly primed platelets, and that conformational changes
in GP1b.alpha. that promote this clearance do not affect
GP1b.alpha.'s hemostatically important binding to vWf. Therefore,
selective modification of GP1b.alpha. may accommodate cold storage
of platelets for transfusion.
Materials and Methods
[0166] We obtained fluorescein isothiocyanate (FITC)-conjugated
annexin V, phycoerythrin (PE)-conjugated anti-human CD11b/Mac-1
monoclonal antibodies (mAb), FITC-conjugated anti-mouse and
anti-human IgM mAb, FITC-conjugated anti-mouse and anti-human
CD62P-FITC mAb from Pharmingen (San Diego, Calif.); FITC-conjugated
rat anti-mouse anti-human IgG mAb from Santa Cruz Biotechnology,
Inc. (Santa Cruz, Calif.); FITC-conjugated anti-human CD61 mAbs
(clone BL-E6) from Accurate Scientific Corp. (Westbury, N.Y.);
FITC-conjugated anti-human GP1b.alpha. mAb (clone SZ2) from
Immunotech (Marseille, France); and FITC-conjugated polyclonal
rabbit anti-vWf antibody from DAKOCytomation (Glostrup, Denmark).
We purchased EGTA-acetoxymethylester (AM), Oregon Green coupled
fibrinogen from human plasma, CellTracker.TM. Orange CMTMR;
CellTracker Green CMFDA, Nile-red (535/575) coupled and
carboxylate-modified 1 .mu.m microspheres/FluoSpheres from
Molecular Probes, Inc. (Eugene, Oreg.) and .sup.111Indium from NEN
Life Science Products (Boston, Mass.). We purchased Cytochalasin B,
dimethyl sulfoxide (DMSO), trisodium isothiocyanate (TRITC), human
thrombin, prostaglandin E1 (PGE.sub.1), phorbol ester
12-tetradecanoylphorbol-13 acetate (PMA), A23187 ionophore from
Sigma (St. Louis, Mo.); botrocetin from Centerchem Inc. (Norwalk,
Conn.); and O-sialoglycoprotein-endopeptidase from Cerladane
(Hornby, Canada). HBSS containing Ca.sup.2+ and Mg.sup.2+, pH 6.4;
RPMI 1640; 0.05% Trypsin-EDTA (0.53 mM) in HBSS without Ca.sup.2+
and Mg.sup.2+; and other supplements (penicillin, streptomycin and
fetal bovine serum) were from GIBCO Invitrogen Corp. (Grand Island,
N.Y.). TGF-.beta.1 from Oncogene to Research Products (Cambridge,
Mass.); 1,25-(OH).sub.2 vitamin D3 from Calbiochem (San Diego,
Calif.); and Adenosine-5'-Diphosphate (ADP) were from USB
(Cleveland, Ohio). Avertin (2,2,2-tribromoethanol) was purchased
from Fluka Chemie (Steinheim, Germany). Collagen related peptide
(CRP) was synthesized at the Tufts Core Facility, Physiology Dept.
(Boston, Mass.) and cross-linked as previously described (Morton et
al., 1995). Mocarhagin, a snake venom metalloprotease, was provided
by Dr. M. Berndt, Baker Medical Research Institute, Melbourne
Victoria 318 1, Australia. Additional unconjugated anti mouse
GP1b.alpha. mAbs and a PE-conjugated anti-mouse GP1b.alpha. mAb
pOp4 were provided by Dr. B. Nieswandt (Witten/Herdecke University,
Wuppertal, Germany). We obtained THP-1 cells from the American Type
Culture Collection (Manassas, Va.).
Animals
[0167] For assays of clearance and survival studies, we used age-,
strain- and sex-matched C57BL/6 and C57BL/6.times.129/sv wild type
mice obtained from Jackson Laboratory (Bar Harbor, Me.).
C57BL/6.times.129/sv mice deficient in complement component C3
(Wessels et al., 1995) were provided by Dr. M. C. Carroll (Center
for Blood Research and Department of Pediatrics, Harvard Medical
School, Boston, Mass.). C57BL/6 mice deficient in CR3 (Coxon et
al., 1996) were provided by Dr. T Mayadas and C57BL/6 mice
deficient in vWf (Denis et al., 1998) were provided by Dr. D.
Wagner. Mice were maintained and treated as approved by Harvard
Medical Area Standing Committee on Animals according to NIH
standards as set forth in The Guide for the Care and Use of
Laboratory Animals.
Human Platelets
[0168] Blood was drawn from consenting normal human volunteers
(approval was obtained from the Institutional Review Boards of both
Brigham and Women's Hospital and the Center for Blood Research
(Harvard Medical School)) by venipuncture into 0.1 volume of
Aster-Jandl citrate-based anticoagulant (Hartwig and DeSisto, 1991)
and platelet rich plasma (PRP) was prepared by centrifugation of
the anticoagulated blood at 300.times.g for 20 min at room
temperature. Platelets were separated from plasma proteins by
gel-filtration at room temperature through a small Sepharose 2B
column (Hoffmeister et al., 2001). Platelets used in the in vitro
phagocytosis assay described below were labeled with 1.8 .mu.M
CellTracker.TM. Orange CMTMR (CM-Orange) for 20 min at 37.degree.
C. (Brown et al., 2000), and unincorporated dye was removed by
centrifugation (850.times.g, 5 min.) with 5 volumes of washing
buffer containing 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate,
10 mM glucose, and 12.5 mM sucrose, 1 .mu.g/ml PGE.sub.1, pH 6.0
(buffer A). Platelets were resuspended at 3.times.10.sup.8/ml in a
solution containing 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl.sub.2, 5 mM
NaHCO.sub.3, 10 mM glucose and 10 mM Hepes, pH 7.4 (buffer B).
[0169] The N-terminus of GP1b.alpha. was enzymatically removed from
the surface of chilled or room temperature maintained and labeled
platelets in buffer B, also containing 1 mM Ca.sup.2+ and 10
.mu.g/ml of the snake venom metalloprotease mocarhagin (Ward et
al., 1996). After the enzymatic digestion, the platelets were
washed by centrifugation with 5.times. volume of buffer A and
routinely checked by microscopy for aggregates.
GP1b.alpha.-N-terminus removal was monitored by incubating platelet
suspensions with 5 .mu.g/ml of FITC-conjugated anti-human
GP1b.alpha. (SZ2) mAb for 10 min at room temperature and followed
by immediate flow cytometry analysis on a FACScalibur Flow
Cytometer (Becton Dickinson Biosciences, San Jose, Calif.).
Platelets were gated by forward/side scatter characteristics and
50,000 events acquired.
Murine Platelets
[0170] Mice were anesthetized with 3.75 mg/g (2.5%) of Avertin, and
1 ml blood was obtained from the retroorbital eye plexus into 0.1
volume of Aster-Jandl anticoagulant. PRP was prepared by
centrifugation of anticoagulated blood at 300.times.g for 8 min at
room temperature. Platelets were separated from plasma proteins by
centrifugation at 1200.times.g for 5 min and washed two times by
centrifugation (1200.times.g for 5 min) using 5.times. volumes of
washing buffer (buffer A). This procedure is meant by subsequent
use of the term "washed". Platelets were resuspended at a
concentration of 1.times.10.sup.9/ml in a solution containing 140
mM NaCl, 3 mM KCl, 0.5 mM MgCl.sub.2, 5 mM NaHCO.sub.3, 10 mM
glucose and 10 mM Hepes, pH 7.4 (buffer B). Platelet count was
determined using a Bright Line Hemocytometer (Hausser Scientific,
Horsham, Pa.) under a phase-contrast microscope at 400.times.
magnification. Some radioactive platelet clearance studies were
performed with .sup.111Indium, and we labeled mouse platelets using
a method described for primate platelets (Kotze et al., 1985).
Platelets were resuspended at a concentration of
2.times.10.sup.9/ml in 0.9% NaCl, pH 6.5 (adjusted with 0.1 M
sodium citrate), followed by the addition of 500 .mu.Ci
.sup.111Indium chloride for 30 min at 37.degree. C. and washed as
described above and suspended in buffer B at a concentration of
1.times.10.sup.9/ml.
[0171] For intravital microscopy or other platelet survival
experiments, washed platelets were labeled either with 2.5 .mu.M
CellTracker Green CMFDA (5-chloromethyl fluorescein diacetate)
(CMFDA) for 20 min at 37.degree. C. (Baker et al., 1997) or with
0.15 .mu.M TRITC for 20 min at 37.degree. C. in buffer B also
containing 0.001% DMSO, 20 mM HEPES. Unincorporated dye was removed
by centrifugation as described above, and platelets were suspended
at a concentration of 1.times.10.sup.9/ml in buffer B.
[0172] The N-terminus of GP1b.alpha. was enzymatically removed from
the surface of chilled or room temperature labeled platelets with
100 .mu.g/ml O-sialoglycoprotein endopeptidase in buffer B
containing 1 mM Ca.sup.2+ for 20 min at 37.degree. C. (Bergmeier et
al., 2001). After enzymatic digestion, platelets were washed by
centrifugation and checked by light microscopy for aggregates.
Enzymatic removal of the GP1b.alpha.-N-terminus removal was
monitored by incubating the platelet suspensions with 5 .mu.g/ml of
PE-conjugated anti-mouse GP1b.alpha. mAb pOp4 for 10 min at room
temperature, and bound PE analyzed by flow cytometry.
[0173] To inhibit cold-induced platelet shape changes, 10.sup.9/ml
platelets in buffer B were loaded with 2 .mu.M EGTA-AM followed by
2 .mu.M cytochalasin B as previously described (Winokur and
Hartwig, 1995), labeled with 2.5 .mu.M CMFDA for 30 min at
37.degree. C. and then chilled or maintained at room temperature.
The platelets were subjected to standard washing and suspended at a
concentration of 1.times.10.sup.9/ml in buffer B before injection
into mice.
Platelet Temperature Protocols
[0174] To study the effects of temperature on platelet survival or
function, unlabeled, radioactively labeled, or
fluorescently-labeled mouse or human platelets were incubated for 2
hours at room temperature (25-27.degree. C.) or else at ice bath
temperatures and then rewarmed for 15 minutes at 37.degree. C.
before transfusion into mice or in vitro analysis. Platelets
subjected to these treatments are designated cooled or chilled (or
chilled, rewarmed) and room temperature platelets respectively.
Murine Platelet Recovery, Survival and Fate
[0175] CMFDA labeled chilled or room temperature murine platelets
(10.sup.8) were injected into syngeneic mice via the lateral tail
vein using a 27-gauge needle. For recovery and survival
determination, blood samples were collected immediately (<2 min)
and 0.5, 2, 24, 48, 72 hours after transfusion into 0.1 volume of
Aster-Jandl anticoagulant. Whole blood analysis using flow
cytometry was performed and the percentage of CMFDA positive
platelets determined by gating on all platelets according to their
forward and side scatter characteristics (Baker et al., 1997).
50,000 events were collected in each sample. CMFDA positive
platelets measured at a time <2 min was set as 100%. The input
of transfused platelets per mouse was .about.2.5-3% of the whole
platelet population.
[0176] To evaluate the fate of platelets, tissues (heart, lung,
liver, spleen, muscle, and femur) were harvested at 0.5, 1 and 24
hours after the injection of 10.sup.8 chilled or room temperature
.sup.111Indium labeled platelets into mice. The organ-weight and
their radioactivity were determined using a Wallac 1470 Wizard
automatic gamma counter (Wallac Inc., Gaithersburg, Md.). The data
were expressed as gamma count per gram organ. For recovery and
survival determination of radioactive platelets, blood samples were
collected immediately (<2 min) and 0.5 and hours after
transfusion into 0.1 volume of Aster-Jandl anticoagulant and their
gamma counts determined (Kotze et al., 1985).
Platelet Aggregation
[0177] Conventional tests were performed and monitored in a
Bio/Data aggregometer (Horsham, Pa.). Samples of 0.3-ml murine
washed and stirred platelets were exposed to 1 U/ml thrombin, 10
.mu.M ADP, or 3 .mu.g/ml CRP at 37.degree. C. Light transmission
was recorded over 3 min.
Activated VWf Binding
[0178] Platelet rich plasma was treated with or without 2 U/ml
botrocetin for 5 min at 37.degree. C. (Bergmeier et al., 2001).
Bound vWf was detected by flow cytometry using FITC conjugated
polyclonal rabbit anti-vWf antibody.
Surface labeling of platelet GP1b.alpha.
[0179] Resting mouse platelets maintained at room temperature or
chilled 2 hrs were diluted to a concentration of
2.times.10.sup.6/ml in phosphate buffered saline (PBS) containing
0.05% glutaraldehyde. Platelet solutions (200 .mu.l) were placed on
a polylysine-coated glass coverslip contained in wells of 96-well
plate, and the platelets were adhered to each coverslip by
centrifugation at 1,500.times.. g for 5 min at room temperature.
The supernatant fluid was then removed, and platelets bound to the
coverslip were fixed with 0.5% glutaraldehyde in PBS for 10 min.
The fixative was removed, unreacted aldehydes quenched with a
solution containing 0.1% sodium borohydride in PBS followed by
washing with PBS containing 10% BSA. GP1b.alpha. on the platelet
surface was labeled with a mixture of three rat anti-mouse
GP1b.alpha. monoclonal antibodies, each at 10 .mu.g/ml (Bergmeier
et al., 2000) for 1 hr followed by 10 nm gold coated with goat
anti-rat IgG. The coverslips were extensively washed with PBS,
post-fixed with 1% glutaraldehyde, washed again with distilled
water, rapidly frozen, freeze-dried, and rotary coated with 1.2 nm
of platinum followed by 4 nm of carbon without rotation in a
Cressington CFE-60 (Cressington, Watford, UK). Platelets were
viewed at 100 kV in a JEOL 1200-EX electron microscope (Hartwig et
al., 1996; Kovacsovics and Hartwig, 1996)
In Vitro Phagocytic Assay
[0180] Monocytic THP-1 cells were cultured for 7 days in RPMI 1640
cell culture media supplemented with 10% fetal bovine serum, 25 mM
Hepes, 2 mM glutamine and differentiated using 1 ng/ml TGF.beta.
and 50 nM 1,25-(OH).sub.2 vitamin D3 for 24 hours, which is
accompanied by increased expression of CR3 (Simon et al., 2000).
CR3 expression was monitored by flow cytometry using a
PE-conjugated anti-human CD11b/Mac-1 mAb. Undifferentiated or
differentiated THP-1 cells (2.times.10.sup.6/ml) were plated onto
24-well plates and allowed to adhere for 45 minutes at 37.degree.
C. The adherent undifferentiated or differentiated macrophages were
activated by the addition of 15 ng/ml PMA for 15 min.
CM-range-labeled, chilled or room temperature platelets
(10.sup.7/well), previously subjected to different treatments were
added to the undifferentiated or differentiated phagocytes in
Ca.sup.2+- and Mg.sup.2+-containing HBSS and incubated for 30 min
at 37.degree. C. Following the incubation period, the phagocyte
monolayer was washed with HBSS for 3 times, and adherent platelets
were removed by treatment with 0.05% trypsin/0.53 mM EDTA in HBSS
at 37.degree. C. for 5 min followed by 5 mM EDTA at 4.degree. C. to
detach the macrophages for flow cytometric analysis of adhesion or
ingestion of platelets (Brown et al., 2000). Human
CM-Orange-labeled, chilled or room temperature platelets all
expressed the same amount of the platelet specific marker CD61 as
freshly isolated unlabeled platelets (not shown). CM-Orange-labeled
platelets incubated with macrophages were resolved from the
phagocytes according to their forward and side scatter properties.
The macrophages were gated, 10,000 events acquired for each sample,
and data analyzed with CELLQuest software (Becton Dickenson).
CM-Orange-labeled platelets that associate with the phagocyte
population have a shift in orange fluorescence (FIG. 6a and FIG.
6b, ingested, y axis). These platelets were ingested rather than
merely adherent, because they failed to dual label with the
FITC-conjugated mAb to CD61.
Immunolabeling and Flow Cytometry of Platelets
[0181] Washed murine or human platelets (2.times.10.sup.6) were
analyzed for surface expression of CD62P, CD61, or surface bound
IgM and IgG after chilling or room temperature storage by staining
with fluorophore-conjugated Abs (5 .mu.g/ml) for 10 min at
37.degree. C. Phosphatidylserine exposure by chilled or room
temperature platelets was determined by resuspending 5 .mu.l of
platelets in 400 .mu.l of HBSS containing 10 mM Ca.sup.2+ with 10
.mu.g/ml of FITC-conjugated annexin-V. As a positive control for PS
exposure, platelet suspensions were stimulated with 1 .mu.M A23187.
Fibrinogen binding was determined by the addition of Oregon
Green-fibrinogen for 20 min at room temperature. All platelet
samples were analyzed immediately by flow cytometry. Platelets were
gated by forward and side scatter characteristics.
Intravital Microscopy Experiments
[0182] Animal preparation, technical and experimental aspects of
the intravital video microscopy setup have been described (von
Andrian, 1996). Six to eight week-old mice of both sexes were
anesthetized by intraperitoneal injection of a mixture of Xylazine
and Ketamin. The right jugular vein was catheterized with PE-10
polyethylene tubing. The lower surface of the left liver lobe was
surgically prepared and covered by a glass cover slip for further
in vivo microscopy as described (McCuskey, 1986). 10.sup.8 chilled
platelets and room temperature platelets labeled with CMFDA and
TRITC respectively were mixed 1:1 and administered intravenously.
The circulation of labeled platelets in liver sinusoids was
followed by video triggered stroboscopic epi-illumination. Ten
video scenes were recorded from 3 centrilobular zones at each
indicated time point. The ratio of cooled (CMFDA)/RT (TRITC)
adherent platelets in the identical visualized field was
calculated. Confocal microscopy was performed using a Radiance 2000
MP confocal-multiphoton imaging system connected to an Olympus BX
50 WJ upright microscope (Biorad, Hercules, Calif.), using a
10.times. water immersion objective. Images were captured and
analyzed with Laser Sharp 2000 software (Biorad) (von Andrian,
2002).
Platelet Aggregation in Shed Blood
[0183] We used a flow cytometric method to analyze aggregate
formation by platelets in whole blood emerging from a wound as
described for primates (Michelson et al., 1994). We injected
10.sup.8 CMFDA labeled room temperature murine platelets into
syngeneic wild type mice and 10.sup.8 CMFDA labeled, chilled
platelets into CR3-deficient mice. Twenty-four hours after the
platelet infusion, a standard bleeding time assay was performed,
severing a 3-mm segment of a mouse tail (Denis et al., 1998). The
amputated tail was immersed in 100 .mu.l 0.9% isotonic saline at
37.degree. C. The emerging blood was collected for 2 min., and 0.1
volume of Aster-Jandl anticoagulant added and followed immediately
with 1% paraformaldehyde (final concentration). Peripheral blood
was obtained by retroorbital eye plexus bleeding in parallel as
described above and immediately fixed with 1% paraformaldehyde
(final concentration). To analyze the number of aggregates in vive
by flow cytometry, the shed blood emerging from the bleeding time
wound, as well as a peripheral whole blood sample, were diluted and
labeled with PE-conjugated anti-murine GP1b.alpha. mAb pOp4 (5
.mu.g/ml, 10 min.). Platelets were discriminated from red cells and
white cells by gating according to their forward scatter
characteristics and GP1b.alpha. positivity. A histogram of log
forward light scatter (reflecting platelet size) versus GP1b.alpha.
binding was then generated. In the peripheral whole blood samples,
analysis regions were plotted around the GP1b.alpha.-positive
particles to include 95% of the population on the forward scatter
axis (region 1) and the 5% of particles appearing above this
forward light scatter threshold (region 2). Identical regions were
used for the shed blood samples. The number of platelet aggregates
in shed blood as a percentage of the number of single platelets was
calculated from the following formula: [(number of particles in
region 2 of shed blood)-(number of particles in region 2 of
peripheral blood)]/(number of particles in region 1 of shed
blood).times.100%. The infused platelets were identified by their
CMFDA labeling and discriminated from the CMFDA negative
non-infused platelets.
Flow Cytometric Analysis of Murine Platelet Fibrinogen Binding and
P-Selectin Exposure of Circulating Platelets
[0184] Room temperature CM-Orange-labeled room temperature
platelets (10.sup.8) were injected into wild type mice and
CM-Orange-chilled labeled platelets (10.sup.8) into CR3 deficient
mice. Twenty-four hours after platelet infusion the mice were bled
and the platelets isolated. Resting or thrombin activated (1 U/ml,
5 min) platelet suspensions (2.times.10.sup.8) were diluted in PBS
and either stained with FITC-conjugated anti-mouse P-selectin mAb
or with 50 .mu.g/ml Oregon Green-conjugated fibrinogen for 20 min
at room temperature. Platelet samples were analyzed immediately by
flow cytometry. Transfused and non-transfused platelets were gated
by their forward scatter and CM-Orange fluorescence
characteristics. P-selectin expression and fibrinogen binding were
measured for each CM-Orange positive and negative population before
and after stimulation with thrombin.
Statistics
[0185] The intravital microscopy data are expressed as
means.+-.SEM. Groups were compared using the nonpaired t test. P
values<0.05 were considered significant. All other data are
presented as the mean.+-.SD.
Results
The Clearance of Chilled Platelets Occurs Predominantly in the
Liver and is Independent of Platelet Shape.
[0186] Mouse platelets kept at room temperature (RT) and infused
into syngeneic mice disappear at fairly constant rate over time for
about 80 hours (FIG. 1A). In contrast, approximately two-thirds of
mouse platelets chilled at ice-bath temperature and rewarmed (Cold)
before injection rapidly disappear from the circulation as observed
previously in humans and mice (Becker et al., 1973; Berger et al.,
1998). Chilled and rewarmed platelets treated with the
cell-permeable calcium chelator EGTA-AM and the actin filament
barbed end capping agent cytochalasin B (Cold+CytoB/EGTA) to
preserve their discoid shape (Winokur and Hartwig, 1995), left the
circulation as rapidly as chilled, untreated platelets despite the
fact that these platelets were fully functional as determined by
thrombin-, ADP- or collagen related peptide-(CRP) induced
aggregation in vitro (FIG. 1B). The recoveries of infused platelets
immediately following transfusion were 50-70%, and the kinetics of
platelet disappearance were indistinguishable whether we used
.sup.111Indium or CMFDA to label platelets. The relative survival
rates of room temperature and chilled mouse platelets resemble the
values reported previously for identically treated mouse (Berger et
al., 1998) and human platelets (Becker et al., 1973).
[0187] FIG. 1C shows that the organ destinations of room
temperature and chilled mouse platelets differ. Whereas
room-temperature platelets primarily end up in the spleen, the
liver is the major residence of chilled platelets removed from the
circulation. A greater fraction of radionuclide detected in the
kidneys of animals receiving .sup.111Indium-labeled chilled
compared with room-temperature platelets at 24 hours may reflect a
more rapid degradation of chilled platelets and delivery of free
radionuclide to the urinary system. One hour after injection the
organ distribution of platelets labeled with CMFDA was comparable
to that of platelets labeled with .sup.111Indium. In both cases,
60-90% of the labeled chilled platelet population deposited in the
liver, .about.20% in the spleen and .about.15% in the lung. In
contrast, a quarter of the infused room temperature platelets
distributed equally among the liver, spleen and lung.
Chilled Platelets Co-Localize with Liver Macrophages (Kupffer
Cells).
[0188] The clearance of chilled platelets by the liver and the
evidence for platelet degradation is consistent with recognition
and ingestion of chilled platelets by Kupffer cells, the major
phagocytic scavenger cells of the liver. FIG. 1D shows the location
of phagocytotic Kupffer cells and adherent chilled CMFDA-labeled
platelets in a representative confocal micrograph of a mouse liver
section 1 hour after transfusion. Sinusoidal macrophages were
visualized by the injection of 1 .mu.m carboxyl modified
polystyrene microspheres marked with Nile-red. Co-localization of
transfused platelets and macrophages is indicated in yellow in the
merged micrograph of both fluorescence emissions. The chilled
platelets localize with Nile-red-labeled cells preferentially in
the periportal and midzonal domains of liver acini, sites rich in
sinusoidal macrophages (Bioulac-Sage et al., 1996; MacPhee et al.,
1992).
CR3-Deficient Mice do not Rapidly Clear Chilled Platelets.
[0189] CR3 (.alpha..sub.M.beta..sub.2 integrin; CD11b/CD18; Mac-1)
is a major mediator of antibody independent clearance by hepatic
macrophages. FIG. 2a shows that chilled platelets circulate in
CR3-deficient animals with the same kinetics as room-temperature
platelets, although the clearance of both platelet populations is
shorter in the CR3-deficient mouse compared to that in wild-type
mice (FIG. 1a). The reason for the slightly faster platelet removal
rate by CR3-deficient mice compared to wild-type mice is unclear.
Chilled and rewarmed platelets also clear rapidly from complement
factor 3 C3-deficient mice (FIG. 2c), missing a major opsonin that
promotes phagocytosis and clearance via CR3 and from von Willebrand
factor (vWf) deficient mice (Denis et al., 1998) (FIG. 2b).
Chilled Platelets Adhere Tightly to Kupffer Cells In Vivo.
[0190] Platelet adhesion to wild-type liver sinusoids was further
investigated by intravital microscopy, and the ratio between
chilled and room temperature stored adherent platelets infused
together was determined. FIG. 3A-3C show that both chilled and room
temperature platelets attach to sinusoidal regions with high
Kupffer cell density (FIGS. 3a and 3b), but that 2.5 to 4-times
more chilled platelets attach to Kupffer cells in the wild-type
mouse than room-temperature platelets (FIG. 3c). In contrast, the
number of platelets adhering to Kupffer cells in CR3-deficient mice
was independent of chilling or room temperature exposure (FIG.
3c).
Chilled Platelets Lacking the N-Terminal Domain of GP1b.alpha.
Circulate Normally.
[0191] Because GP1b.alpha., a component of the GP1b-IX-V receptor
complex for vWf, can bind CR3 under certain conditions in vitro
(Simon et al., 2000), we investigated GP1b.alpha. as a possible
counter receptor on chilled platelets for CR3. The
0-sialoglycoprotein endopeptidase cleaves the 45-kDa N-terminal
extracellular domain of the murine platelet GP1b.alpha., leaving
other platelet receptors such as (.alpha..sub.IIb.beta..sub.3,
.alpha..sub.2.alpha..sub.1, GPVI/FcR.gamma.-chain and the
protease-activated receptors intact (Bergmeier et al., 2001).
Hence, we stripped this portion of the extracellular domain of
GP1b.alpha. from mouse platelets with 0-sialoglycoprotein
endopeptidase (FIG. 4A inset) and examined their survival in mice
following room temperature or cold incubation. FIG. 4A shows that
chilled platelets no longer exhibit rapid clearance after cleavage
of GP1b.alpha.. In addition, GP1b.alpha. depleted room
temperature-treated platelets have slightly elongated survival
times (.about.5-10%) when compared to the GP1b.alpha.-containing
room-temperature controls.
Chilling does not Affect Binding of Activated vWf to the Platelet
vWf-Receptor but Induces Clustering of GP1b.alpha. on the Platelet
Surface.
[0192] FIG. 4B shows that botrocetin-activated vWf binds
GP1b.alpha. equally well on room temperature as on cold platelets,
although chilling of platelets leads to changes in the distribution
of GP1b.alpha. on the murine platelet surface. GP1b.alpha.
molecules, identified by immunogold labeled monoclonal murine
anti-GP1b.alpha. antibodies, form linear aggregates on the smooth
surface of resting discoid platelets at room temperature (FIG. 4C,
RT). This arrangement is consistent with information about the
architecture of the resting blood platelet. The cytoplasmic domain
of GP1b.alpha. binds long filaments curving with the plane of the
platelet membrane through the intermediacy of filamin A molecules
(Hartwig and DeSisto, 1991). After chilling (FIG. 4C, Chilled) many
GP1b.alpha. molecules organize as clusters over the platelet
membrane deformed by internal actin rearrangements (Hoffmeister et
al., 2001; Winokur and Hartwig, 1995).
Recognition of Platelet GP1b.alpha. by CR3-Mediates Phagocytosis of
Chilled Human Platelets In Vitro.
[0193] Differentiation of human monocytoid THP-1 cells using
TGF-.beta.1 and 1,25-(OH).sub.2 Vitamin D3 increases expression of
CR3 by .about.2-fold (Simon et al., 1996). Chilling resulted in
3-fold increase of platelet phagocytosis by undifferentiated THP-1
cells and a -5-fold increase by differentiated THP-1 cells (FIGS.
5B and 5c), consistent with mediation of platelet uptake by CR3. In
contrast, the differentiation of THP-1 cells had no significant
effect on the uptake of room temperature stored platelets (FIGS. 5A
and 5c). To determine if GP1b.alpha. is the counter receptor for
CR3-mediated phagocytosis on chilled human platelets, we used the
snake venom metalloprotease mocarhagin, to remove the extracellular
domain of GP1b.alpha. (Ward et al., 1996). Removal of human
GP1b.alpha. from the surface of human platelets with mocarhagin
reduced their phagocytosis after chilling by .about.98% (FIG.
5C).
Exclusion of Other Mediators of Cold-Induced Platelet Clearance
[0194] Table 1 shows results of experiments that examined whether
cooling affected the expression of platelet receptors other than
GP1b.alpha. or their interaction with ligands. These experiments
revealed no detectable effects on the expression of P-selectin,
.alpha..sub.IIb.beta..sub.3-integrin density or on
.alpha..sub.IIb.beta..sub.3 fibrinogen binding, a marker of
.alpha..sub.IIb.beta..sub.3 activation. Chilling also did not
increase phosphatidylserine (PS) exposure, an indicator of
apoptosis, nor did it change platelet binding of IgG or IgM
immunoglobulins.
TABLE-US-00001 TABLE 1 Effect of chilling on binding of various
antibodies or ligands to platelet receptors. Binding ratio
4.degree. C.:22.degree. C. Platelet receptor (ligand) Human
platelets Murine platelets P-Selectin (anti-CD62P mAb) 1.01 .+-.
0.06 1.02 .+-. 0.03 Platelet associated IgGs 1.05 .+-. 0.14 1.06
.+-. 0.03 Platelet associated IgMS 0.93 .+-. 0.10 1.01 .+-. 0.02
Phosphatidylserine (annexin V) 0.95 .+-. 0.09 1.04 .+-. 0.02
.alpha..sub.IIb.beta..sub.3 anti-CD61 mAb) 1.03 .+-. 0.05 1.04 .+-.
0.10 .alpha..sub.IIb.beta..sub.3 (fibrinogen) 1.05 .+-. 0.10 1.06
.+-. 0.06
[0195] The binding of fluorescently labeled antibodies or ligands
against various receptors on chilled-rewarmed or room temperature
human and murine platelets was measured by flow cytometry. The data
are expressed as the ratio between the mean fluorophore bound to
the surface of chilled versus room temperature platelets
(mean.+-.SD, n=3-4).
Circulating Chilled Platelets have Hemostatic Function in
CR3-Deficient Mice.
[0196] Despite their rapid clearance in wild type mice, CM-Orange
or CMFDA labeled chilled platelets were functional 24 h after
infusion into CR3-deficient mice, as determined by three
independent methods. First, chilled platelets incorporate into
platelet aggregates in shed blood emerging from a standardized tail
vein bleeding wound (FIG. 6A-6E). CMFDA-positive room temperature
platelets transfused into wild type mice (FIG. 6b) and
CNIFDA-positive chilled platelets transfused into CR3-deficient
mice (FIG. 6d) formed aggregates in shed blood to the same extent
as CMFDA-negative platelets of the recipient mouse. Second, as
determined by platelet surface exposure of the fibrinogen-binding
site on .alpha..sub.IIb.beta..sub.3 24 hours after transfusion of
CM-Orange-labeled chilled and rewarmed platelets into CR3 deficient
mice following ex vivo stimulation by thrombin. Third, CM-Orange
platelets chilled and rewarmed were fully capable of upregulation
of P-selectin in response to thrombin activation (FIG. 6e).
Discussion
[0197] Cold-Induced Platelet Shape Change Alone does not Lead to
Platelet Clearance In Vivo
[0198] Cooling rapidly induces extensive platelet shape changes
mediated by intracellular cytoskeletal rearrangements (Hoffmeister
et al., 2001; White and Krivit, 1967; Winokur and Hartwig, 1995).
These alterations are partially but not completely reversible by
rewarming, and rewarmed platelets are more spherical than discoid.
The idea that preservation of platelet discoid shape is a major
requirement for platelet survival has been a dogma, despite
evidence that transfused murine and baboon platelets activated ex
vivo by thrombin circulate normally with extensive shape changes
(Berger et al., 1998; Michelson et al, 1996). Here we have shown
that chilling leads to specific changes in the platelet surface
that mediate their removal independently of shape change, and that
the shape change per se does not lead to rapid platelet clearance.
Chilled and rewarmed platelets, preserved as discs with
pharmacological agents, clear with the same speed as untreated
chilled platelets, and misshapen chilled and rewarmed platelets
circulate like room temperature maintained platelets in
CR3-deficient mice. The small size of platelets may allow them to
remain in the circulation, escaping entrapment despite these
extensive shape deformities.
Receptors Mediating Clearance of Chilled Platelets: CR3 and
GP1b.alpha.
[0199] The normal platelet life span in humans is approximately 7
days (Aas, 1958; Ware et 2000). The incorporation of platelets into
small blood clots engendered by continuous mechanical stresses
undoubtedly contributes to platelet clearance, because massive
clotting reactions, such as occur during disseminated intravascular
coagulation, cause thrombocytopenia (Seligsohn, 1995). The fate of
platelets in such clotting reactions differs from that of infused
ex vivo-activated platelets such as in the experiments of Michelson
et al (Michelson et al., 1996) and Berger et al (Berger et al.,
1998), because in vivo platelet stimulation occurs on injured
vessel walls, and the activated platelets rapidly sequester at
these sites.
[0200] Isoantibodies and autoantibodies accelerate the phagocytic
removal of platelets by Fc-receptor-bearing macrophages in
individuals sensitized by immunologically incompatible platelets or
in patients with autoimmune thrombocytopenia, but otherwise little
information exists regarding mechanisms of platelet clearance. We
showed, however, that the quantities of IgG or IgM bound to chilled
or room-temperature human platelets are identical, implying that
binding of platelet-associated antibodies to Fc-receptors does not
mediate the clearance of cooled platelets. We also demonstrated
that chilling of platelets does not induce detectable
phosphatidylserine (PS) exposure on the platelet surface in vitro
militating against PS exposure and the involvement of scavenger
receptors in the clearance of chilled platelets.
[0201] Although many publications have referred to effects of cold
on platelets as "activation", aside from cytoskeletally-mediated
shape changes, chilled platelets do not resemble platelets
activated by stimuli such as thrombin or ADP. Normal activation
markedly increases surface P-selectin expression, a consequence of
secretion from intracellular granules (Berman et al., 1986).
Chilling of platelets does not lead to up-regulation of P-selectin
(Table 1), but the clearance of chilled platelets isolated from
wild-type or P-selectin-deficient mice is equally rapid (Berger et
al., 1998). Activation also increases the amount of
.alpha..sub.IIb.beta..sub.3-integrin and its avidity for fibrinogen
(Shattil, 1999), but cooling does not have these effects (Table 1).
The normal survival of thrombin-activated platelets is consistent
with our findings.
[0202] We have shown that CR3 on liver macrophages is primarily
responsible for the recognition and clearance of cooled platelets.
The predominant role of CR3 bearing macrophages in the liver in
clearance of chilled platelets despite abundant CR3-expressing
macrophages in the spleen is consistent with the principally
hepatic clearance of IgM-coated erythrocytes (Yan et al., 2000) and
may reflect blood filtration properties of the liver that favor
binding and ingestion by macrophage CR3. The extracellular domain
of GP1b.alpha. binds avidly to CR3, and under shear stress in vitro
supports the rolling and firm adhesion of THP-1 cells (Simon et
al., 2000). Cleavage of the extracellular domain of murine
GP1b.alpha. results in normal survival of chilled platelets
transfused into mice. GP1b.alpha. depletion of human chilled
platelets greatly reduces phagocytosis of the treated platelets by
macrophage-like cells in vitro. We propose, therefore, that
GP1b.alpha. is the co-receptor for liver macrophage CR3 on chilled
platelets leading to platelet clearance by phagocytosis.
[0203] The normal clearance of cold platelets lacking the
N-terminal portion of GP1b.alpha. rules out the many other
CR3-binding partners, including molecules expressed on platelet
surfaces as candidates for mediating chilled platelet clearance.
These ligand candidates include ICAM-2, fibrinogen bound to the
platelet integrin .alpha..sub.IIb.beta..sub.3, iC3b, P-selectin,
glucosaminoglycans, and high molecular weight kininogen. We
excluded deposition of the opsonic C3b fragment iC3b as a mechanism
for chilled platelet clearance using mice deficient in complement
factor 3, and the expression level of .alpha..sub.IIb.beta..sub.3
and fibrinogen binding are also unchanged after chilling of
platelets.
Binding to Activated vWf and Cold-Induced Binding to CR3 Appear to
be Separate Functions of GP1b.alpha.
[0204] GP1b.alpha. on the surface of the resting discoid platelet
exists in linear arrays (FIG. 5A-5C) in a complex with GP1b.alpha.,
GP1X and V, attached to the submembrane actin cytoskeleton by
filamin-A and Filamin B (Stossel et al., 2001). Its role in
hemostasis is to bind the activated form of vWf at sites of
vascular injury. GP1b.alpha. binding to activated vWf is
constitutive and requires no active contribution from the platelet,
since activated vWf binds equally well to GP1b.alpha. on resting or
on stimulated platelets. Stimulation of platelets in suspension by
thrombin and other agonists causes GP1b.alpha. to redistribute in
part from the platelet surface into an internal membrane network,
the open canalicular system, but does not lead to platelet
clearance in vivo (Berger et al., 1998; Michelson et al., 1996) or
to phagocytosis in vitro (unpublished observations). Cooling of
platelets however, causes GP1b.alpha. clustering rather than
internalization. This clustering is independent of barbed end actin
assembly, because it occurs in the presence of cytochalasin B.
[0205] Despite cold's promoting recognition of platelet GP1b.alpha.
by CR3, it has no effect on interaction between GP1b.alpha. and
activated vWf in vitro, and chilled platelets transfused into
vWf-deficient mice disappear as rapidly as in wild-type mice. The
separability of GP1b.alpha.'s interaction with vWf and CR3 suggests
that selective modification of GP1b.alpha. might inhibit
cold-induced platelet clearance without impairment of GP1
b.alpha.'s hemostatically important reactivity with vWf. Since all
tests of platelet function of cooled platelets in vitro and after
infusion into CR3-deficient mice yielded normal results, suitably
modified platelets would predictably be hemostatically
effective.
Physiological Importance of Cold-Induced Platelet Clearance.
[0206] Although gross platelet shape changes become obvious only at
temperatures below 15.degree. C., accurate biochemical analyses
show that cytoskeletal alterations and increased responsiveness to
thrombin are detectable as the temperature falls below 37.degree.
C. (Faraday and Rosenfeld, 1998; Hoffmeister et al., 2001; Tablin
et al., 1996). We refer to those changes as "priming" because of
the many functional differences that remain between cold-exposed
and thrombin- or ADP-stimulated platelets. Since platelet
activation is potentially lethal in coronary and cerebral blood
vessels subjected to core body temperatures, we have proposed that
platelets are thermosensors, designed to be relatively inactive at
the core body temperature of the central circulation but to become
primed for activation at the lower temperatures of external body
surfaces, sites most susceptible to bleeding throughout
evolutionary history (Hoffmeister et al., 2001). The findings
reported here suggest that irreversible changes in GP1 b.alpha. are
the reason for the clearance of cooled platelets. Rather than
allowing chilled platelets to circulate, the organism clears low
temperature-primed platelets by phagocytosis.
[0207] A system involving at least two clearance pathways, one for
removal of locally activated platelets and another for taking out
excessively primed platelets (FIG. 7), can possibly explain why
chilled platelets circulate and function normally in CR3-deficient
mice and have a slightly prolonged circulation following removal of
GP1b.alpha.. We propose that some primed platelets enter
microvascular clots on a stochastic basis. Others are susceptible
to repeated exposure to body surface temperature, and this
repetitive priming eventually renders these platelets recognizable
by CR3-bearing liver macrophages. Platelets primed by chilling are
capable of normal hemostatic function in CR3-deficient mice, and
coagulation contributes to their clearance. However, the slightly
shorter survival time of autologous platelets in CR3-deficient mice
examined is probably not ascribable to increased clearance of
normally primed platelets in microvascular clots, because the
clearance rate of refrigerated platelets was indistinguishable from
that of platelets kept at room temperature.
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Mocarhagin, a novel cobra venom metalloproteinase, cleaves the
platelet von Willebrandt factor receptor glycoprotein Ib.alpha..
Identification of the sulfated tyrosine/anionic sequence
Tyr-276-Glu-282 of glycoprotein Ib.alpha. as a binding site for von
Willebrandt factor and .alpha.-thrombin. Biochemistry. 28,
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Generation and rescue of a murine model of platelet dysfunction:
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Example 2
Implication of the .alpha..sub.M.beta..sub.2 (CR3) Lectin Domain in
Chilled Platelet Phagocytosis
[0252] .alpha..sub.M|.sub.2 (CR3) has a cation-independent
sugar-binding lectin site, located "C-T" to its I-domain (Thornton
et al, J. Immonol. 156, 1235-1246, 1996), which binds to mannans,
glucans and N-Acetyl-D-glucosamine (GlcNAc). Since
CD16b/.alpha..sub.M.beta..sub.2 membrane complexes are disrupted by
.beta.-glucan, N-Acetyl-D-galactosamine (GalNAc), and
methyl-.alpha.-mannoside, but not by other sugars, it is believed
that this interaction occurs at the lectin site of the
.alpha..sub.M.beta..sub.2 integrin (CR3) (Petty et al, J. Leukoc.
Biol. 54, 492-494, 1993; Sehgal et al, J. Immunol. 150, 4571-4580,
1993).
[0253] The lectin site of .alpha..sub.M.beta..sub.2 integrin has a
broad sugar specificity (Ross, R. Critical Reviews in Immunology
20, 197-222, 2000). Although sugar binding to lectins is usually of
low affinity, clustering can cause a more robust interaction by
increasing avidity. The clustering of GP1b.alpha. following
cooling, as shown by electron microscopy, suggests such a
mechanism. The most common hexosamines of animal cells are
D-glucosamine and D-galactosamine, mostly occurring in structural
carbohydrates as GlcNAc and GalNAc, suggesting that the
.alpha..sub.M.beta..sub.2 integrin lectin domain might also bind to
mammalian glycoproteins containing carbohydrates that are not
covered by sialic acid. The soluble form of GP1b.alpha.,
glycocalicin, has a carbohydrate content of 60% comprising N- as
well as O-glycosidically linked carbohydrate chains (Tsuji et al,
J. Biol. Chem. 258, 6335-6339, 1983). Glycocalicin contains 4
potential N-glycosylation sites (Lopez, et al, Proc. Natl. Acad.
Sci., USA 84, 5615-5619, 1987). The 45 kDa region contains two
sites that are N-glycosylated (Titani et al, Proc Natl Acad Sci 16,
5610-5614, 1987). In normal mammalian cells, four common core
structures of O-glycan can be synthesized. All of them may be
elongated, sialylated, fucosylated and sulfated to form functional
carbohydrate structures. The N-linked carbohydrate chains of
GP1b.alpha. are of the complex-type and di-, tri- and
tetra-antennary structures (Tsuji et al, J. Biol. Chem. 258,
6335-6339, 1983). They are sialylated GalNAc type structures with
an .alpha.(1-6)-linked fucose residue at the Asn-bound GlcNAc unit.
There is a structural similarity of Asn-linked sugar chains with
the Ser/Thr-linked: i.e., their position is of a common Gal-GlcNAc
sequence. Results suggested that removal of sialic acid and
galactose has no influence on the binding of vWf to glycocalicin,
but partial removal of GlcNac resulted in the inhibition of vWf
binding (Korrel et al, FEBS Lett 15, 321-326, 1988). A more recent
study proposed that the carbohydrate patterns are involved in
maintaining an appropriate functional conformation of the receptor,
without participating directly in the binding of vWf (Moshfegh et
al, Biochem. Biophys. Res. Communic. 249, 903-909, 1998).
[0254] A role of sugars in the interaction between chilled
platelets and macrophages has the important consequence that
covalent modification, removal or masking of oligosaccharide
residues could prevent this interaction. We hypothesized that if
such prevention does not impair normal platelet function, we may be
able to modify platelets and enable cold platelet storage. Here, we
show evidence that favor this hypothesis: 1) Saccharides inhibited
phagocytosis of chilled platelets by macrophages in vitro, and the
specific sugars that are effective implicated .beta.-glucans as the
relevant targets. Low concentrations of .beta.-GlcNAc were
surprisingly effective inhibitors, consistent with the idea that
interference with a relatively small number of clustered sugars may
be sufficient to inhibit phagocytosis. Addition of sugars at
concentrations that maximally inhibited phagocytosis of chilled
platelets has no effect on normal GP1b.alpha. function
(vWf-binding); 2) A .beta.-GlcNAc-specific lectin, but not other
lectins, bound avidly to chilled platelets; 3) Removal of
GP1b.alpha. or .beta.-GlcNAc residues from platelet surfaces
prevented this binding (since .beta.-GlcNAc removal exposed mannose
residues, it did not prevent phagocytosis by macrophages which have
mannose receptors); 4) Blocking of exposed .beta.-Glucans on
chilled platelets by enzymatic addition of galactose markedly
inhibited phagocytosis of chilled platelets by macrophages in vitro
and extended the circulation times of chilled platelets in normal
animals.
Effect of Monosaccharides on Phagocytosis of Chilled Platelets.
[0255] To analyze the effects of monosaccharides on platelet
phagocytosis, phagocytes (differentiated monocytic cell line THP-1)
were incubated in monosaccharide solutions at various
concentrations, and the chilled or room temperature platelets were
added. Values in the Figures are means.+-.SD of 3-5 experiments
comparing percentages of orange-positive monocytes containing
ingested platelets incubated with RT or chilled platelets). While
100 mM D-glucose inhibited chilled platelet phagocytosis by 65.5%
(P<0.01), 100 mM D-galactose did not significantly inhibit
chilled platelet phagocytosis (n=3) (FIG. 8A). The D-glucose
.alpha.-anomer (.alpha.-glucoside) did not have an inhibitory
effect on chilled platelet phagocytosis, although 100 mM inhibited
by 90.2% (FIG. 8B) In contrast, .beta.-glucoside inhibited
phagocytosis in a dose-dependent manner (FIG. 8B). Incubation of
the phagocytes with 100 mM .beta.-glucoside inhibited phagocytosis
by 80% (p<0.05) and 200 mM by 97% (P<0.05), therefore we
concluded that the .beta.-anomer is preferred. D-mannose and its
.alpha.- and .beta.-anomers (methyl-.alpha.-D-mannopyranoside (FIG.
8C) and methyl-.beta.-D-mannopyranoside (FIG. 8C) had no inhibitory
effect on chilled or RT platelet phagocytosis. Incubation of
phagocytes using 25 to 200 mM GlcNAc (N-acetyl-D-glucosamine)
significantly inhibited chilled platelet phagocytosis. Incubation
with 25 mM GlcNac was sufficient to inhibit the phagocytosis of
chilled platelets by 86% (P<0.05) (FIG. 8D), whereas 10 .mu.M of
the .beta.-anomer of GlcNAc inhibited the phagocytosis of chilled
platelets by 80% (p<0.01) (FIG. 8D). None of the monosaccharides
had an inhibitory effect on RT platelet phagocytosis. Table 2
summarizes the inhibitory effects of monosaccharides at the
indicated concentrations on chilled platelet phagocytosis
(**P<0.01, *P<0.05). None of the monosaccharides inhibited
thrombin or ristocetin induced human platelet aggregation or
induced .alpha.-granule secretion as measured by P-selectin
exposure.
TABLE-US-00002 TABLE 2 Inhibitory effects of monosaccharides on
chilled platelet phagocytosis Monosaccharides % inhibition
phagocytosis mM D-(+)-glucose 65.5 100 D-(+)-galactose -- 100
Methyl-.alpha.-D- 90.2* 100 glucopyranoside Methyl-.beta.-D- 80.2*
100 gludopyranoside 97.1* 200 D-(+)-mannose -- 100
Methyl-.alpha.-D- -- 100 mannopyranoside Methyl-.beta.-D- -- 100
mannopyranoside .beta.-GlcNac 80.9* 0.01 GlcNac 86.3* 25 83.9* 100
83.1* 200
Binding of Various Lectins to Room Temperature Platelets or Chilled
Platelets.
[0256] .beta.-GlcNAc strongly inhibited chilled human platelet
phagocytosis in vitro at .mu.M concentrations, indicating that
GlcNac is exposed after incubation of platelets in the cold. We
then investigated whether wheat germ agglutinin (WGA), a lectin
with specificity towards the terminal sugar (GlcNAc), binds more
effectively to chilled platelets than to room temperature
platelets. Washed, chilled or room temperature platelets were
incubated with 2 .mu.g/ml of FITC coupled WGA or FITC coupled
succinyl-WGA for 30 min at room temperature and analyzed by flow
cytometry. FIGS. 9A and 9B show the dot plots after incubation with
FITC-WGA of room temperature (RT) or chilled (Cold) human
platelets. WGA induces platelet aggregation and release of
serotonin or ADP at concentrations between 25-50 g/ml WGA
(Greenberg and Jamieson, Biochem. Biophys. Acta 345, 231-242,
1974). Incubation with 2 .mu.g/ml WGA induced no significant
aggregation of RT-platelets (FIG. 9A, RT w/WGA), but incubation of
chilled platelets with 2 .mu.g/ml WGA induced massive aggregation
(FIG. 9B, Cold/w WGA). FIG. 9C shows the analysis of FITC-WGA
fluorescence binding to chilled or room temperature platelets. To
verify that the increase of fluorescence binding is not aggregation
related, we used succinyl-WGA (S-WGA), a dimeric derivate of the
lectin that does not induce platelet aggregation (Rendu and Lebret,
Thromb Res 36, 447-456, 1984). FIGS. 9D and 9E show that
succinyl-WGA (S-WGA) did not induce aggregation of room temperature
or chilled platelets, but resulted the same increase in WGA binding
to chilled platelets versus room temperature platelets (FIG. 9F).
The enhanced binding of S-WGA after chilling of platelets cannot be
reversed by warming of chilled platelets to 37.degree. C.
[0257] Exposed .beta.-GlcNAc residues serve as substrate for a
.beta.1,4glactosyltransferase enzyme that catalyses the linkage
Gal.beta.-1GlcNAc.beta.1-R. In support of this prediction, masking
of .beta.-GlcNAc residues by enzymatic galactosylation inhibited
S-WGA binding to cold platelets, phagocytosis of chilled platelets
by THP-1 cells, and the rapid clearance of chilled platelets after
transfusion into mice. The enzymatic galactosylation, achieved with
bovine .beta.1,4galactosyltransferase and its donor substrate
UDP-Gal, decreased S-WGA binding to chilled human platelets to
levels equivalent to room temperature platelets. Conversely, the
binding of the galactose-specific RCA I lectin increased by
.about.2 fold after galactosylation. UDP-Glucose and UDP alone had
no effect on S-WGA or RCA I binding to chilled or room temperature
human platelets.
[0258] We found that the enzymatic galactosylation of human and
mouse platelets is efficient without addition of exogenous
.beta.1,4galactosyltransferase. The addition alone of the donor
substrate UDP-Gal reduces S-WGA binding and increases RCA I binding
to chilled platelets, inhibits phagocytosis of chilled platelets by
THP1 cells in vitro, and prolongs the circulation of chilled
platelets in mice. An explanation for this unexpected finding is
that platelets reportedly slowly release endogenous
galactosyltransferase activity. A least one form of
.beta.1,4galactosyltransferases, .beta.4Gal T1, is present in human
plasma, on washed human platelets and in the supernatant fluids of
washed platelets. Galactosyltransferases may associate specifically
with the platelet surface. Alternatively, the activity may be
plasma-derived and leak out of the platelet's open canalicular
system. In either case, modification of platelet glycans
responsible for cold-mediated platelet clearance is possible by
simple addition of the sugar-nucleotide donor substrate,
UDP-Gal.
[0259] Importantly, both chilled and non-chilled platelets show the
same increase in RCA I binding after galactosylation, implying that
.beta.-GlcNAc residues are exposed on the platelet surface
independent of temperature. However chilling is a requirement for
recognition of .beta.-GlcNAc residues by S-WGA and by the
.alpha..sub.M.beta..sub.2 integrin. We have demonstrated that
chilling of platelets induces an irreversible clustering of GP1b.
Generally lectin binding is of low affinity and multivalent
interactions with high density of carbohydrate ligands increases
binding avidity. Possibly the local densities of exposed
.beta.-GlcNAc on the surface of non-chilled platelets are too low
for recognition, but cold-induced clustering of GP1b.alpha.
provides the necessary density for binding to S-WGA or the
.alpha..sub.M.beta..sub.2 integrin lectin domain. We confirmed by
S-WGA and RCA-I binding flow cytometry that UDP-Gal transfers
galactose onto murine platelets in the presence or absence of added
galactosyl transferase and documented that chilled, galactosylated
murine platelets circulate and initially survive significantly
better than untreated room temperature platelets.
[0260] Although the earliest recoveries (<2 min) did not differ
between transfused RT, chilled and chilled, galactosylated
platelets, galactosylation abolished an initial platelet loss of
about 20% consistently observed with RT platelets.
[0261] Galactosylation of murine and human platelets did not impair
their functionality in vitro as measured by aggregation and
P-selectin exposure induced by collagen related peptide (CRP) or
thrombin at concentrations ranging from maximally effective to
three orders of magnitude lower. Importantly, the aggregation
responses of unmodified and galactosylated chilled human platelets
to a range of ristocetin concentrations, a test of the interaction
between GP1b and activated VWF, were indistinguishable or slightly
better. The attachment points for N-linked glycans on GP1b.alpha.
are outside of the binding pocket for VWF. Moreover, mutant
GP1b.alpha. molecules lacking N-linked glycans bind VFW
tightly.
[0262] Using FITC labeled lectins with specificities towards
.beta.-galactose (R. communis lectin/RCA), 2-3 sialic acid
(Maackida amurensis lectin/MAA) or 2-6 sialic acid (Sambucus Nigra
bark lectin/SNA), we could not detect increased binding after
chilling of platelets by flow cytometry (FIG. 10), showing that
exposure after chilling of platelets is restricted to GlcNAc.
[0263] We localized the exposed .beta.-GlcNAc residues mediating
.alpha..sub.M.beta..sub.2 lectin domain recognition of GP1b.alpha.
N-glycans. The extracellular domain of GP1b.alpha. contains 60% of
total platelet carbohydrate content in the form of N- and
O-glycosidically linked carbohydrate chain. Accordingly, binding of
peroxidase-labeled WGA to GP1b.alpha. is easily detectable in
displays of total platelet proteins resolved by SDS-PAGE,
demonstrating that GP1b.alpha. contains the bulk of the
.beta.-GlcNAc-residues on platelets, and binding of WGA to
GP1b.alpha. is observable in GP1b.alpha. immunoprecipitates.
UDP-Gal with or without added galactosyltransferase diminishes
S-WGA binding to GP1b.alpha., whereas RCA I binding to GP1b.alpha.
increases. These findings indicate that galactosylation
specifically covers exposed .beta.-GlcNAc residues on GP1b.alpha..
Removal of the N-terminal 282 residues of GP1b.alpha. from human
platelet surfaces using the snake venom protease mocarhagin, which
inhibited phagocytosis of human platelets by THP-1 cells in vitro,
reduces S-WGA binding to chilled platelets nearly equivalent to
S-WGA room temperature binding levels. WGA binds predominantly to
the N-terminus of GP1b.alpha. released by mocarhagin into
.quadrature.platelet supernatant fluids as a polypeptide band of 45
kDa recognizable by the monoclonal antibody SZ2 specific for that
domain. The glycans of this domain are N-linked. A small portion of
GP1b.alpha. remains intact after mocarhagin treatment, possibly
because the open canalicular system of the platelet sequesters it.
Peroxidase-conjugated WGA weakly recognizes the residual platelet
associated GP1b.alpha. C-terminus after mocarhagin cleavage,
identifiable with monoclonal antibody WM23.
[0264] The cold-induced increase in binding of human platelets to
.alpha..sub.M.beta..sub.2 integrin and to S-WGA occurs rapidly
(within minutes). The enhanced binding of S-WGA to chilled
platelets remained stable for up to 12 days of refrigerated storage
in autologous plasma. RCA I binding remained equivalent to room
temperature levels under the same conditions. Galactosylation
doubled the binding of RCA I lectin to platelets and reduced S-WGA
binding to baseline RT levels. The increase in RCA I and decrease
in S-WGA binding were identical whether galactosylation proceeded
or followed storage of the platelets in autologous plasma for up to
12 days. These findings indicate that galactosylation of platelets
to inhibit lectin binding is possible before or after refrigeration
and that the glycan modification is stable during storage for up to
12 days. Platelets stored at room temperature rapidly lose
responsiveness to aggregating agents; this loss does not occur with
refrigeration. Accordingly, refrigerated platelets with or without
galactosylation, before or after storage, retained aggregation
responsiveness to thrombin for up to 12 days of cold storage.
Effects of .beta.-hexosaminidase (.beta.-Hex) and Mocarhagin (MOC)
on FITC-WGA Lectin Binding to Chilled Versus Room Temperature
Stored Platelets.
[0265] The enzyme .beta.-hexosaminidase catalyzes the hydrolysis of
terminal .beta.-D-N-acetylglucosamine (GlcNAc) and galactosamine
(GalNAc) residues from oligosaccharides. To analyze whether removal
of GlcNAc residues reduces the binding of WGA to the platelet
surface, chilled and room temperature washed human platelets were
treated with 100 U/ml .beta.-Hex for 30 min at 37.degree. C. FIG.
11A shows the summary of FITC-WGA binding to the surface of room
temperature or chilled platelets obtained by flow cytometry before
and after .beta.-hexosaminidase treatment. FITC-WGA binding to
chilled platelets was reduced by 85% after removal of GlcNac (n=3).
We also checked whether, as expected, removal of GP1b.alpha. from
the platelet surface leads to reduced WGA-binding after platelet
chilling. GP1b.alpha. was removed from the platelet surface using
the snake venom mocarhagin (MOC), as described previously (Ward et
al, Biochemistry 28, 8326-8336, 1996). FIG. 11B shows that
GP1b.alpha. removal from the platelet surface reduced FITC-WGA
binding to chilled platelets by 75% and had little influence on
WGA-binding to GP1b.alpha.-depleted room temperature platelets
(n=3). These results indicate that WGA binds mostly to
oligosaccharides on GP1b.alpha. after chilling of human platelets,
and it is very tempting to speculate that the Mac-1 lectin site
also recognizes these exposed sugars on GP1b.alpha. leading to
phagocytosis.
Masking of Human Platelet GlcNAc Residues by Galactose-Transfer
Greatly Reduces their Phagocytosis after Chilling In Vitro and
Dramatically Increases their Survival in Mice.
[0266] To achieve galactose transfer onto platelets, isolated human
platelets were incubated with 200 .mu.M UDP-galactose and 15 mU/ml
galactose transferase for 30 min at 37.degree. C., followed by
chilling or maintenance at room temperature for 2 h.
Galactosylation reduced FITC-WGA binding almost to resting room
temperature levels. Platelets were fed to the monocytes and
platelet phagocytosis was analyzed as described above. FIG. 12
shows that galactose transfer onto platelet oligosaccharides
reduces greatly chilled platelet (Cold) phagocytosis, but does not
affect the phagocytosis of room temperature (RT) platelets (n=3).
These results show that in vitro the phagocytosis of chilled
platelets can be reduced through coverage of exposed GlcNAc
residues. We tested whether this approach could be extended to
animals and used to increase the circulation time of chilled
platelets. Murine platelets were isolated and stained with CMFDA.
Using the same approach of galactose transfer described for human
platelets above, wild type murine platelets were galactosylated and
chilled, or not, for 2 hours. 10.sup.8 Platelets were transfused
into wild type mice and their survival determined. FIG. 13 shows
the survival of these chilled, galactosylated murine platelets
relative to untreated platelets. Both platelets kept at room
temperature (RT) and the galactosylated chilled platelets
(Cold+GalT) had almost identical survival times, whereas chilled
untreated platelets (Cold) were cleared rapidly as expected. We
believe galactosylated chilled platelets will circulate in
humans.
[0267] We noted that our control reaction, in which galactose
transferase was heat-inactivated also resulted in glycan
modification of platelets as occurred in the experimental reaction
with active galactose transferase, as judged by WGA binding (FIG.
14A), in vitro phagocytosis results in human platelets (FIG. 14B),
and survival of murine platelets (FIG. 14C). Therefore, we conclude
that platelets contain galactose transferase activity on their
surface, which is capable of directing to glycan modification using
only UDP-galactose without the addition of any exogenous galactose
transferase. Thus, glycan modification of platelets can be achieved
simply by incubation with UDP-galactose.
UDP-Galactose Incorporate into Human Platelets in a Time Dependent
Matter.
[0268] In another set of experiments we have shown that
.sup.14C-labeled UDP-galactose incorporates into human platelets in
a time dependent manner in the presence or absence of the enzyme
galactosyl transferase. FIG. 15 shows the time course of
.sup.14C-labeled UDP-galactose incorporation into washed human
platelets. Human platelets were incubated with .sup.14C-labeled
UDP-galactose for different time intervals in the absence of
galactosyl transferase. The platelets were then washed and the
.sup.14C radioactivity associated with platelets measured.
Example 3
Enzymatic Modification of Platelet .beta.-Glycans Inhibit
Phagocytosis of Cooled Platelets by Macrophages In Vitro and
Accommodate Normal Circulation In Vivo
[0269] Our preliminary experiments have demonstrated the enzymatic
covering of GlcNAc residues on GP1b.alpha. using galactose-transfer
(glycan modification) onto chilled human platelet surfaces greatly
reduced their in vitro phagocytosis. One interpretation of these
findings is that GP1b.alpha. structure is altered on the surface of
chilled human and murine platelets. This causes the exposure or
clustering of GlcNAc, which is recognized by the lectin binding
domain of .alpha.M.beta.2 leading to platelet removal .beta.-GlcNAc
exposure can be measured by WGA binding and possibly by binding of
recombinant .alpha.M.beta.2 lectin domain peptides. Resting human
platelets bind WGA, which increases greatly after chilling. We
propose that galactose transfer (glycan modification) will prevent
GP1b.alpha.'s interaction with .alpha.M.beta.2-lectin but not with
vWf. This modification (galactose transfer onto platelet surface)
leads to normal survival of chilled platelets in WT mice as shown
by our preliminary experiments.
Example 4
[0270] This example shows that the .alpha.M.beta.2 lectin site
mimics WGA and sugar modifications prevent the engagement of the
recombinant lectin site with chilled platelets. Dr. T. Springer
(Corbi, et al., J. Biol Chem. 263, 12403-12411, 1988) provided the
human .alpha.M cDNA and several anti-.alpha.M antibodies. The
smallest r-hu.alpha.M construct exhibiting lectin activity that has
been reported includes its C-T and a portion of its divalent cation
binding region (residues 400-1098) (Xia et al, J Immunol 162,
7285-7293, 1999). The construct is 6.times.His-tagged for ease of
purification. We first determined if the recombinant lectin domain
can be used as a competitive inhibitor of chilled platelet
ingestion in the phagocytic assay. Competition proved that the
.alpha.M lectin site mediates binding to the platelet surface and
initiates phagocytosis. As controls, a construct lacking the
lectin-binding region of .alpha.M was used and the recombinant
protein was denatured. Lectin binding domain functions as a
specific inhibitor of chilled platelet ingestion. We made a
.alpha.M construct that include GFP and express and labeled the
.alpha.M-lectin binding site with FITC and used it to label the
surface of chilled platelets by flow cytometry. Platelets were
labeled with CMFDA. We found that chilled platelets bind more
efficiently to the .alpha.M lectin side of .alpha.M.beta.2 integrin
compared to room temperature platelets. The lectin side and whole
.alpha.M-construct (Mac-1) was expressed in Sf9 insect cells.
[0271] The platelet sugar chains are modified to inhibit the
platelet-oligosaccharide interaction with the r-hu.alpha.M-lectin
site. The efficiency of sugar modifications is also monitored by
inhibition of the binding of fluorescent-lectin domain binding to
platelets by flow cytometry.
[0272] The recovery and circulation times of room temperature,
chilled and chilled-modified platelets are compared to establish
that galactose transfer onto chilled murine platelets results in
longer circulating platelets. Room temperature, chilled and
chilled-modified platelets are stained with CMFDA, and 10.sup.8
platelets transfused into wild type mice as described above. The
mice are bled immediately (<2 min.), 30 min, 1 h, 2, 24, 48 and
72 hours after transfusion. The blood obtained is analyzed using
flow cytometry. The percentage of fluorescent labeled platelets
within the gated platelet population measured immediately after
injection is set as 100%. The recovery of fluorescently labeled
platelets obtained at the various time points is calculated
accordingly.
Example 5
[0273] This example demonstrates that chilled, unmodified and
chilled, galactosylated (modified) platelets have hemostatic
function in vitro and in vivo. Chilled platelets are not
"activated" in the sense of agonist-stimulated platelets. Patients
undergoing surgery under hypothermic conditions may develop
thrombocytopenia or show severe hemostatic post-operative
impairments. It is believed that under these hypothermic
conditions, platelets might lose their functionality. However, when
patients undergo hypothermic surgery, the whole organism is exposed
to hypothermia leading therefore to changes in multiple tissues.
Adhesion of non-chilled platelets to hepatic sinusoidal endothelial
cells is a major mechanism of cold preservation injury (Takeda, et
al. Transplantation 27, 820-828, 1999). Therefore, it is likely
that it is the interaction between cold hepatic endothelium and
platelets, not platelet chilling per se, that leads to deleterious
consequences under hypothermic conditions of surgery or
trans-plantation of cold preserved organs (Upadhya et al,
Transplantation 73, 1764-1770, 2002). Two approaches showed that
chilled platelets have hemostatic function. In one approach, the
circulation of chilled platelets in .alpha.M.beta.2-deficient mice
facilitates studies of platelet function after cooling. In the
other approach, the function of modified chilled and (presumably)
circulating platelets was tested.
[0274] Human and murine unmodified and modified (galactosylated)
chilled platelets were tested for functionality, including in vitro
aggregation to agonists, P-selectin exposure and fibrinogen
binding.
[0275] .alpha.M.beta.2 deficient or WT mice are transfused with
murine chilled/RT platelets modified or not, and allowed to
circulate for 30 min., 2 and 24 hours. We determine if chilled
platelets contribute to clotting reactions caused by tail vein
bleeding and if these platelets bind agents such as fibrinogen
after activation. We also determine how chilled platelets, modified
or not, contribute to clotting on ferric chloride injured and
exteriorized mouse mesenteries, an in vivo thrombus-formation model
that we developed. This method detects the number of platelets
adherent to injured vessels and has documented impaired platelet
vessel wall interactions of platelets lacking glycoprotein V or
.beta.3-integrin function (Ni et al., Blood 98, 368-373 2001;
Andre, et al. Nat Med 8, 247-252, 2002). Last, we determine the
storage parameters of the modified platelets.
[0276] In vitro platelet function is compared using aggregation
with thrombin and ADP and botrocetin induced vWf-binding to murine
platelets. Murine and human chilled platelets modified
(galactosylated) or unmodified platelets are normalized to a
platelet concentration of 0.3.times.10.sup.9/mm.sup.3, and
aggregation induced using the various agonists according to
standard protocols (Bergmeier, et al. 2001 276, 25121-25126, 2001).
To study vWf-binding we activate murine vWf using botrocetin and
analyze the binding of fluorescently labeled vWf to chilled
platelets modified or not in PRP (Bergmeier, et al. 2001 276,
25121-25126, 2001). To evaluate whether degranulation of platelets
occurs during modification, we also measure P-selectin exposure of
chilled murine and human platelets modified or not using
fluorescent labeled anti-P-selectin antibodies by flow cytometry
(Michelson et al., Proc. Natl. Acad. Sci., USA 93, 11877-11882,
1996).
[0277] 10.sup.9 CMFDA-labeled platelets are transfused into mice,
first verifying that these platelets are functional in vitro. We
determine whether chilled platelets contribute to aggregation by
transfusing chilled or room temperature CMFDA-labeled platelets
into .alpha.M.beta.2 deficient mice. At 30 min., 2 hours and
twenty-four hours after the infusion of platelets, a standard tail
vein bleeding test is performed (Denis, et al. Proc Natl Acad Sci
USA 95, 9524-9529, 1998). The emerging blood is fixed immediately
in 1% formaldehyde and platelet aggregation is determined by whole
blood flow cytometry. Platelet aggregates appear as bigger sized
particles in the dot plot analysis. To verify that the transfused
platelets do not aggregate in the normal circulation we also bleed
the mice through the retroorbital eye plexus into an anticoagulant.
Platelets do not form aggregates under these bleeding conditions.
The emerging blood is fixed immediately and platelets are analyzed
by flow cytometry in whole blood as described above. Platelets are
identified through binding of a phycoerythrin-conjugated
.alpha..sub.IIb.beta..sub.3 specific monoclonal antibody. The
infused platelets in the blood sample are identified by their
CMFDA-fluorescence. Non-infused platelets are identified by their
lack of CMFDA fluorescence (Michelson, et al, Proc. Natl. Acad.
Sci., U.S.A. 93, 11877-11882, 1996). The same set of tests is
performed with CMFDA modified (galactosylated) chilled platelets
transfusing these platelets into .alpha.M.beta.2 and WT. This
experiment tests aggregation of chilled platelets modified or not
in shed blood.
[0278] 10.sup.9 CM-orange labeled unmodified chilled or room
temperature platelets are transfused into .alpha.M.beta.2 deficient
mice to verify that these platelets are functional in vitro. At 30
min., 2 h and twenty-four hours after the infusion of CM-orange
labeled platelets, PRP is isolated as described and analyzed by
flow cytometry. P-selectin exposure is measured using an anti
FITC-conjugated anti P-selectin antibody (Berger, et al, Blood 92,
4446-4452, 1998). Non-infused platelets are identified by their
lack of CM-orange fluorescence. The infused platelets in the blood
sample are identified by their CM-orange fluorescence. CM-orange
and P-selectin positive platelets appear as double positive
fluorescently (CM-orange/FITC) stained platelets. To verify that
chilled platelets still expose P-selectin after thrombin
activation, PRP is activated through the addition of thrombin (1
U/ml, 2 min at 37.degree. C.) and P-selectin exposure is measured
as described. To analyze the binding of fibrinogen to
.alpha..sub.IIb.beta..sub.3, isolated platelets are activated
through the addition of thrombin (1 U/ml, 2 min, 37.degree. C.) and
Oregon-green coupled fibrinogen (20 .mu.g/ml) added for 20 min at
37.degree. C. (Heilmann, et al, Cytometry 17, 287-293, 1994). The
samples are analyzed immediately by flow cytometry. The infused
platelets in the PRP sample are identified by their CM-orange
fluorescence. CM-orange and Oregon-green positive platelets appear
as double positive fluorescently stained (CM-orange/Oregon green)
platelets. The same sets of experiments are performed with
CM-orange labeled modified (galactosylated) chilled platelets
transfused into .alpha.M.beta.2 deficient and WT mice.
Example 6
In Vivo Thrombosis Model
[0279] First, we show the delivery of RT and unmodified chilled
platelets to injured endothelium of .alpha.M.beta.2 deficient mice
using double fluorescently labeled platelets. The resting blood
vessel is monitored for 4 min., then ferric chloride (30 .mu.l of a
250-mM solution) (Sigma, St Louis, Mo.) is applied on top of the
arteriole by superfusion, and video recording resumed for another
10 min. Centerline erythrocyte velocity (Vrbc) is measured before
filming and 10 min after ferric chloride injury. The shear rate is
calculated on the basis of Poiseuille's law for a Newtonian fluid
(Denis, et al, Proc Natl Acad Sci USA 95, 9524-9529, 1998). These
experiments show if chilled platelets have normal hemostatic
function. We repeat these experiments in WT mice comparing RT and
galactosylated chilled platelets using two different, fluorescently
labeled platelet populations injected into the same mouse and
analyze the thrombus formation and incorporation of both platelet
populations.
[0280] We then compare in vitro platelet functions and survival and
in vivo hemostatic activity of chilled and modified chilled murine
platelets stored for 1, 5, 7 and 14 days under refrigeration as
described above. We compare the recovery and circulation times of
these stored chilled and modified chilled platelets and prove that:
1) the modification through galactose transfer onto chilled murine
platelets is stable after the long term refrigeration; and 2) that
these platelets function normally. Survival experiments are
performed as described above. We use WGA binding, to verify that
GlcNAc residues remain covered by galactose after the longer
storage time points. As an ultimate test that these modified,
stored platelets are functionally intact and contribute to
hemostasis, we transfuse them into total-body-irradiated mice
(Hoyer, et al, Oncology 49, 166-172, 1992). To obtain the
sufficient numbers of platelets, we inject mice with commercially
available murine thrombopoietin for seven days to increase their
platelet count (Lok, et al Nature 369, 565-558, 1994). Isolated
platelets are modified using the optimized galactose transfer
protocol, stored under refrigeration, transfused, and tail vein
bleeding times measured. Since unmodified chilled platelets do not
persist in the circulation, a comparison of modified cooled
platelets with room temperature stored platelets is not necessary
at this point. The murine platelets are stored under refrigeration
in standard test tubes. If a comparison with room temperature
stored murine platelets is necessary we switch to primate
platelets. Rather than engineer special down-scale, gas-permeable
storage containers to accommodate mouse platelets, such comparisons
are more appropriate for primates (including humans) for which room
temperature storage bags have been designed.
Example 7
Galactosylation of Platelets in a Platelet Concentrate
[0281] Four different platelet concentrates were treated with
increasing concentrations of UDP galactose: 400 .mu.M, 600 .mu.M,
and 800 .mu.M. Future experiments will use between 10 .mu.M and
5000 .mu.M UDP galactose. RCA binding ratio measurements showed a
dose dependent increase in galactosylation in the four samples
tested. (FIG. 16). Our results provide evidence that
galactosylation is possible in platelet concentrates.
[0282] It should be understood that the preceding is merely a
detailed description of certain preferred embodiments. It therefore
should be apparent to those skilled in the art that various
modifications and equivalents can be made without departing from
the spirit and scope of the invention. It is intended to encompass
all such modifications within the scope of the appended claims. All
references, patents and patent publications that are recited in
this application are hereby incorporated by reference herein in
their entirety.
Example 8
Evaluation of the In Vivo Survival of UDP-Galactose Treated
Platelets Stored in the Cold
[0283] The technology for galactosylating human platelets with the
use of the activated carbohydrate substrate UDP-galactose may allow
large-scale human platelet storage under refrigeration (4.degree.
C.). Untreated platelets stored at 4.degree. C. are rapidly cleared
from the circulation. In contrast, untreated platelets stored at
room temperature survive for .about.5-7 days following transfusion.
The present study is intended to demonstrate that the
galactosylated modified human platelets circulate in vivo when
infused autologously into individuals.
[0284] The reason for the removal of chilled platelets from the
circulation has recently been defined. Cooling of platelets causes
clustering of the platelet GPIb/V/IX complex on the platelet
surface. The .alpha.M.beta.2 integrin receptor (CR3, Mac-1) present
on hepatic macrophages recognizes clustered GPIb.beta. molecules,
and platelets are ingested by the macrophages. The .alpha.M.beta.2
integrin receptor contains a carbohydrate binding domain (lectin
domain) that is critical for the recognition of exposed
.beta.-N-acetylglucosamine (.beta.GlcNAc) residues on the platelet
surface by macrophages. Covering of exposed .beta.GlcNAc residues
by enzymatic galactosylation prevents recognition and phagocytosis
of chilled platelets. This has been extensively demonstrated in a
mouse model, where chilled and galactosylated murine platelets have
survival superior to that of room temperature stored platelets. In
vitro studies using human platelets indicate that galactosylated
platelets stored at 4.degree. C. are likely also to circulate when
transfused into humans.
[0285] To determine and demonstrate that galactosylated modified
human platelets survive and circulate in vivo when infused
autologously into individuals. This will be determined by comparing
the survival rates of radiolabeled refrigerated
(2.degree.-8.degree. C.) platelets with or without galactosylation
to radiolabeled non-galactosylated platelets stored at room
temperature (22.degree..+-.2.degree. C.) and in the cold (Stored
for 36 to 48 hrs).
[0286] The following describes a Phase I study in which in vivo
recovery and half-life of autologously-infused galactosylated
platelets in normal, healthy volunteer group subjects is
determined.
[0287] Six (6) healthy donors will donate a unit of apheresis
platelets. The collected apheresis product will be divided into two
bags. One bag will have the platelets treated with UDP-galactose
and stored under refrigeration for 36-48 hours. The other platelet
bag will either be stored under refrigeration or as per current FDA
guidelines at room temperature for 36-48 hours. The two bags of
platelets will each be radiolabeled with a different radioactive
isotope, .sup.51Chromium or .sup.111Indium and 5-10 mL of labeled
platelets will be injected in the healthy volunteers. Blood samples
will be drawn before and at 2 hours after the transfusion and then
on days 1, 2, 3, 5, 7 and 10 after reinfusion, and the
post-transfusion recovery and survival of the platelets will be
determined.
[0288] The experimental material injected in the healthy volunteers
will be 5-10 mL aliquots of platelets that have been taken from the
study subjects, with or without modification by galactosylation and
either stored at room temperature (22.+-.2.degree. C.) or stored in
the cold (4.+-.2.degree. C.).
[0289] Upon confirmation of eligibility and enrollment in the
study, healthy donors will be recruited to donate a unit of
platelets on the Haemonetics MCS+ apheresis machine. This machine
draws whole blood from a donor's arm, centrifuges the blood to
separate the platelets from the plasma and the red cells, collects
the platelets with a small amount of plasma and returns most of the
plasma and the red cells back to the donor. The collected platelets
and plasma will be divided into two bags. Each bag will be weighed
and the platelet count determined on the day of collection, day 1
and day of infusion. After collection the platelets will be rested
for 1 hour. After the resting period one platelet bag will be
treated with a naturally occurring sugar substance, UDP-galactose.
This bag will be incubated for 1 hour at 37.degree. C. and stored
under refrigeration. The other platelet bag will likewise be
incubated for 1 hour at 37.degree. C. and stored under
refrigeration or as per current FDA guidelines at room temperature.
On Day 1 following collection a sample from each bag will be sent
to a microbiology lab for culture.
[0290] The platelet culture results will be recorded along with the
results of a gram stain sample that will be sent to the lab on the
day of reinfusion. If either report is positive the platelet units
will not be reinfused. The two bags of platelets will each be
radiolabeled with a different radioactive isotope, .sup.51Chromium
or .sup.111Indium. Blood samples will be drawn before and at 2
hours after and then on days 1, 2, 3, 5, 7 and 10 after the
reinfusion. The blood samples will be analyzed for radioactivity to
determine the post-transfusion recovery and survival of the
platelets. Since the two units of platelets have been tagged with
different radioactive isotopes, we will be able to distinguish
between the platelets that were subjected to the UDP Galactose and
those that are untreated.
[0291] UDP-galactose (Uridine-5'-diphosphogalactose) is a natural
sugar compound found in the human body. It is used in this study as
a donor for the addition of galactose to the surface of the human
platelets to be transfused. The UDP-galactose was manufactured by
Roche Diagnostics GmbH and is over 97% pure. It contains trace
quantities of by-products of the manufacturing process. It was
formulated and filled into syringes by a licensed filling facility,
and tested for sterility and pyrogenicity.
[0292] Blood samples taken from each study subject will be tested
for platelet count and anti-platelet antibodies before and at two
weeks and three months after the platelet infusion.
[0293] Between 5 and 10 mL of platelets radiolabeled with the two
different radioactive isotope, .sup.51Chromium or .sup.111Indium,
will be injected at day 0. Blood samples will be drawn before and
at 2 hours and on days 1, 2, 3, 5, 7 and 10 after reinfusion.
[0294] During each reinfusion, the subject will be carefully
monitored for adverse reactions, most usually fever, chills,
dyspnea, urticaria or pain (infusion site, chest pain or other), or
significant changes in vital signs. In addition, each subject will
be queried during the follow up period visits up to three months
after the infusion to obtain information on any occurrence of
adverse events during that time. Non-modified and modified
platelets will be characterized by a number of in vitro analyses
including but not limited to: pH, pO2, pCO2, bicarbonate, hypotonic
shock response, morphology, extent of shape change, ATP levels,
glucose, O2 consumption, p-Selectin, and Annexin V binding.
REFERENCES
Incorporated Herein in their Entirety
[0295] 1. Becker, Tucecelli et al. G. Transfusion 13, 61 (1973).
[0296] 2. Hoffmeister, Felbinger et al. Cell 10, 87 (2003). [0297]
3. Valeri, Ragno et al. Transfusion 44(6):865-70 (2004). [0298] 4.
Murphy S, Oski F A et al N Engl J Med. 1969 16; 281(16):857-62
[0299] 5. Dumont, VandenBroeke et al. Transfus Med Rev. 13(1):31-42
(1999). [0300] 6. Michelson, Adelman et al. J Clin Invest.
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66(6):619-27 (1992). [0302] 8. Jaremo, Rubach-Dahlberg et al.
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et al. Science September 12; 301(5639):1531-4 (2003). [0304] 10. J
Pediatr Gastroent Nutr 13:26 0-266 (1991). [0305] 11. J Pediatr
Gastroent Nutr 19:100-108 (1994). [0306] 12. Mizoguchi, Ono et al.,
Eur J Pediatr 159: 851-853 (2000). [0307] 13. Lancet 346:1073-1074
(1995). [0308] 14. Acta Medica Scandinav Suppl 177:1-125 (1947).
[0309] 15. Lazarowski, Shea et al. Mol Pharmacol 63: 1190-1197
(2003). [0310] 16. Josefsson et al J Biol Chem. 2005 Mar. 1; [Epub
ahead of print] [0311] 17. Puget Sound Blood Center SOP,
"Radiolabeling Fresh Platelets with .sup.111Indium Oxine or
.sup.51Chromium", Rev. Jan. 12, 2005 [0312] 18. Puget Sound Blood
Center SOP, "Radiolabeling Stored Apheresis Platelets with
.sup.51Chromium", Rev. Jan. 12, 2005 [0313] 19. Puget Sound Blood
Center SOP, "Radiolabeling Stored Apheresis Platelets with
.sup.111Indium Oxine", Rev. Jan. 12, 2005
* * * * *