U.S. patent application number 10/807614 was filed with the patent office on 2004-12-30 for therapeutic platelets and method.
Invention is credited to Crowe, John H., Tablin, Fern, Tsvetkova, Nelly M..
Application Number | 20040265293 10/807614 |
Document ID | / |
Family ID | 23994957 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265293 |
Kind Code |
A1 |
Crowe, John H. ; et
al. |
December 30, 2004 |
Therapeutic platelets and method
Abstract
A dehydrated composition is provided that includes freeze-dried
platelets. The platelets are loaded with trehalose which preserves
biological properties during freeze-drying and rehydration. The
trehalose loading is conducted at a temperature of from greater
than about 25.degree. C. to less than about 40.degree. C., most
preferably at 37.degree. C., with the loading solution having
trehalose in an amount from about 10 mM to about 50 mM. These
freeze-dried platelets are substantially shelf-stable and are
rehydratable so as to have a normal response to an agonist, for
example, thrombin, with virtually all of the platelets
participating in clot formation within about three minutes at
37.degree. C.
Inventors: |
Crowe, John H.; (Davis,
CA) ; Tablin, Fern; (Davis, CA) ; Tsvetkova,
Nelly M.; (Davis, CA) |
Correspondence
Address: |
CARPENTER & KULAS, LLP
1900 EMBARCADERO ROAD
SUITE 109
PALO ALTO
CA
94303
US
|
Family ID: |
23994957 |
Appl. No.: |
10/807614 |
Filed: |
March 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10807614 |
Mar 24, 2004 |
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09828627 |
Apr 5, 2001 |
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6723497 |
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09828627 |
Apr 5, 2001 |
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09501773 |
Feb 10, 2000 |
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Current U.S.
Class: |
424/93.72 ;
435/2 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 35/19 20130101; A01N 1/0231 20130101; A61K 2300/00 20130101;
A01N 1/0221 20130101; A61K 47/6901 20170801; A01N 1/0284 20130101;
A61K 35/19 20130101; Y10T 436/108331 20150115 |
Class at
Publication: |
424/093.72 ;
435/002 |
International
Class: |
A01N 001/02; A61K
045/00 |
Goverment Interests
[0001] This invention was made with Government support under Grant
No. HL67810-03, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1-32. (canceled)
33. A process for increasing the loading efficiency of trehalose
into platelets comprising: providing platelets having a first phase
transition temperature range and a second phase transition
temperature range which is greater than the first phase transition
temperature; disposing the platelets into a trehalose solution to
commence loading trehalose into the platelets by uptaking trehalose
from the trehalose solution by fluid phase endocytosis; and heating
the trehalose solution to the second phase transition temperature
range to increase the loading efficiency of trehalose into the
platelets.
34. The process of claim 33 wherein said platelets comprise human
platelets.
35. The process of claim 33 wherein said second phase transition
temperature range is greater than about 25.degree. C.
36. The process of claim 34 wherein said second phase transition
temperature range is greater than about 25.degree. C.
37. The process of claim 35 wherein said second phase transition
temperature ranges from a temperature greater than about 25.degree.
C. to a temperature less than about 40.degree. C.
38. The process of claim 34 wherein said second phase transition
temperature ranges from a temperature greater than about 25.degree.
C. to a temperature less than about 40.degree. C.
39. The process of claim 37 wherein said temperature ranges from
about 30.degree. C. to less than about 40.degree. C.
40. The process of claim 38 wherein said temperature ranges from
about 30.degree. C. to less than about 40.degree. C.
41. A process of preparing loaded platelets comprising: providing
platelets selected from a mammalian species; and loading an
oligosaccharide by fluid phase endocytosis into the platelets at a
temperature greater than about 25.degree. C. to produce loaded
platelets.
42. The process of claim 41 wherein said loading comprises loading
with an oligosaccharide solution.
43. The process of claim 42 wherein said fluid phase endocytosis
comprises uptaking external oligosaccharide from the
oligosaccharide solution at the temperature greater than about
25.degree. C.
44. The process of claim 42 wherein said loading comprises
incubating the platelets at the temperature greater than about
25.degree. C. with the oligosaccharide solution.
45. The process of claim 41 wherein said loading is without a
fixative.
46. The process of claim 41 wherein said oligosaccharide comprises
trehalose.
47. The process of claim 41 wherein said loading of the
oligosaccharide into the platelets is at a temperature ranging from
greater than about 25.degree. C. to less than about 40.degree.
C.
48. The process of claim 47 wherein said temperature ranges from
about 30.degree. C. to less than about 40.degree. C.
49. The process of claim 47 wherein said temperature is about
37.degree. C.
50. The process of claim 41 wherein said platelets comprise human
platelets.
51. Loaded platelets produced in accordance with the process of
claim 41.
52. Loaded platelets produced in accordance with the process of
claim 33.
Description
FIELD OF THE INVENTION
[0002] The present invention generally relates to the therapeutic
uses of blood platelets, and more particularly to manipulations or
modifications of platelets, such as in preparing freeze-dried
compositions that can be rehydrated at the time of application and
which when rehydrated have a normal response to thrombin and other
agonists with respect to that of fresh platelets. The inventive
compositions are useful in applications such as transfusion
therapy, as hemostasis aids and for drug delivery.
BACKGROUND OF THE INVENTION
[0003] Blood transfusion centers are under considerable pressure to
produce platelet concentrates for transfusion. The enormous quest
for platelets necessitates storage of this blood component, since
platelets are important contributors to hemostasis. Platelets are
generally oval to spherical in shape and have a diameter of 2-4
.mu.m. Today platelet rich plasma concentrates are stored in
bloodbags at 22.degree. C.; however, the shelf life under these
conditions is limited to five days. The rapid loss of platelet
function during storage and risk of bacterial contamination
complicates distribution and availability of platelet concentrates.
Platelets tend to become activated at low temperatures. When
activated they are substantially useless for an application such as
transfusion therapy. Therefore the development of preservation
methods that will increase platelet lifespan is desirable.
[0004] Several techniques for preservation of platelets have been
developed over the past few decades. Cryopreservation of platelets
using various agents, such as glycerol (Valeri et al., Blood, 43,
131-136, 1974) or dimethyl sulfoxide, "DMSO" (Bock et al.,
Transfusion, 35, 921-924, 1995), as the cryoprotectant have been
done with some success. The best results have been obtained with
DMSO. However, a considerable fraction of these cells are partly
lysed after thawing and have the shape of a balloon. These balloon
cells are not responsive to various agonists, so that overall
responsiveness of frozen thawed platelets to various agonists is
reduced to less than 35% compared with fresh platelets. The shelf
life of cryopreserved DMSO platelets at -80.degree. C. is reported
to be one year, but requires extensive washing and processing to
remove cryoprotective agents, and even then the final product has a
severe reduction in ability to form a clot.
[0005] Attempts to dry platelets by lyophilization have been
described with paraformaldehyde fixed platelets (Read et al., Proc.
Natl. Acad. Sci. USA, 92, 397-401, 1995). U.S. Pat. No. 5,902,608,
issued May 11, 1999, inventors Read et al. describe and claim a
surgical aid comprising a substrate on which fixed, dried blood
platelets are carried. These dried blood platelets are fixed by
contacting the platelets to a fixative such as formaldehyde,
paraformaldehyde, gutaraldehyde, or permanganate. Proper
functioning of lyophilized platelets that have been fixed by such
fixative agents in hemostasis is questionable.
[0006] Spargo et al., U.S. Pat. No. 5,736,313, issued Apr. 7, 1998,
have described a method in which platelets are loaded overnight
with an agent, preferably glucose, and subsequently lyophilized.
The platelets are preincubated in a preincubation buffer and then
are loaded with carbohydrate, preferably glucose, having a
concentration in the range of about 100 mM to about 1.5 M. The
incubation is taught to be conducted at about 10.degree. C. to
about 37.degree. C., most preferably about 25.degree. C.
[0007] U.S. Pat. No. 5,827,741, Beattie et al., issued Oct. 27,
1998, discloses cryoprotectants for human platelets, such as
dimethylsulfoxide and trehalose. The platelets may be suspended,
for example, in a solution containing a cryoprotectant at a
temperature of about 22.degree. C. and then cooled to below
15.degree. C. This incorporates some cryoprotectant into the
cells.
[0008] Trehalose is a disaccharide found at high concentrations in
a wide variety of organisms that are capable of surviving almost
complete dehydration (Crowe et al., Anhydrobiosis. Annu. Rev.
Physiol., 54, 579-599, 1992). Trehalose has been shown to stabilize
certain cells during freezing and drying (Leslie et al., Biochim.
Biophys. Acta, 1192, 7-13, 1994; Beattie et al., Diabetes, 46,
519-523, 1997).
[0009] Other workers have sought to load platelets with trehalose
through use of electroporation before drying under vacuum. However,
electroporation is very damaging to the cell membranes and is
believed to activate the platelets. Activated platelets have
dubious clinical value.
[0010] Platelets have also been suggested for drug delivery
applications in the treatment of various diseases, as is discussed
by U.S. Pat. No. 5,759,542, issued Jun. 2, 1998, inventor Gurewich.
This patent discloses the preparation of a complex formed from a
fusion drug including an A-chain of a urokinase-type plasminogen
activator that is bound to an outer membrane of a platelet.
[0011] Accordingly, a need exists for the effective and efficient
preservation of platelets such that they maintain, or preserve,
their biological properties, particularly their response to
platelet agonists such as thrombin, and which can be practiced on a
large, commercially feasible scale. Further, it would also be
useful to expand the types of present vehicles that are useful for
encapsulating drugs and used for drug delivery to targeted
sites.
SUMMARY OF THE INVENTION
[0012] In one aspect of the present invention, a dehydrated
composition is provided comprising freeze-dried platelets that are
effectively loaded with trehalose to preserve biological properties
during freeze-drying and rehydration. These platelets are
rehydratable so as to have a normal response to at least one
agonist, such as thrombin. For example, substantially all
freeze-dried platelets of the invention when rehydrated and mixed
with thrombin (1 U/ml) form a clot within three minutes at
37.degree. C. The dehydrated composition can include one or more
other agents, such as antibiotics, antifungals, growth factors, or
the like, depending upon the desired therapeutic application.
[0013] In another aspect of the invention, a hemostasis aid is
provided where the above-described freeze-dried platelets are
carried on or by a biocompatible surface. A further component of
the hemostasis aid may be a therapeutic agent, such as an
antibiotic, an antifungal, or a growth factor. The biocompatible
surface may be a bandage or a thrombic surface, such as
freeze-dried collagen. Such a hemostasis aid can be rehydrated just
before the time of application, such as by hydrating the surface on
or by which the platelets are carried, or, in case of an emergency,
the dry hemostasis treatment aid could be applied directly to the
wound or burn and hydrated in situ.
[0014] Methods of making and using inventive embodiments are also
described. One such method is a process of preparing a dehydrated
composition comprising providing a source of platelets, effectively
loading the platelets with trehalose to preserve biological
properties, cooling the trehalose loaded platelets to below their
freezing point, and lyophilizing the cooled platelets. The
trehalose loading includes incubating the platelets at a
temperature from greater than about 25.degree. C. to less than
about 40.degree. C. with a trehalose solution having up to about 50
mM trehalose therein. The process of using such a dehydrated
composition further may comprise rehydrating the platelets. The
rehydration preferably includes a prehydration step wherein the
freeze-dried platelets are exposed to warm, moisture saturated air
for a time sufficient to bring the water content of the
freeze-dried platelets to between about 35 weight percent to about
50 weight percent.
[0015] In yet another aspect of the invention, a drug delivery
composition is provided comprising platelets having a homogeneously
distributed concentration of a therapeutic agent therein. The drug
delivery composition is particularly useful for targeting the
encapsulated drug to platelet-mediated sites.
[0016] Practice of the invention permits the manipulation or
modification of platelets while maintaining, or preserving,
biological properties, such as a response to thrombin. Further, use
of the method to preserve platelets can be practiced on a large,
commercially feasible scale, and avoids platelet activation. The
inventive freeze-dried platelets, and hemostasis aids including the
freeze-dried platelets, are substantially shelf stable at ambient
temperatures when packaged in moisture barrier materials.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In the drawings:
[0018] FIG. 1 graphically illustrates the loading efficiency of
trehalose plotted versus incubation temperature of human
platelets;
[0019] FIG. 2 graphically illustrates the percentage of
trehalose-loaded human platelets following incubation as a function
of incubation time;
[0020] FIG. 3 graphically illustrates the internal trehalose
concentration of human platelets versus external trehalose
concentration as a function of time at a constant temperature of
37.degree. C.;
[0021] FIG. 4 graphically illustrates the loading efficiency of
trehalose into human platelets as a function of external trehalose
concentration;
[0022] FIG. 5 graphically illustrates the recovery of platelet
embodiments after lyophilization and direct rehydration with
various concentrations of trehalose in the drying buffer, and in a
combination of 30 mM trehalose and one percent HSA in the drying
buffer;
[0023] FIG. 6 graphically illustrates the uptake of FITC dextran
versus the external concentration compared with that of the marker,
LYCH (with an incubation time of four hours);
[0024] FIG. 7 graphically illustrates the effect of prehydration on
optical density of platelets;
[0025] FIG. 8 illustrates the response of 500 .mu.l platelets
solution (with a platelet concentration of 0.5.times.10.sup.8
cells/ml) that was transferred to aggregation vials, thrombin added
(1U/ml) to each sample, and the samples stirred for three minutes
at 37.degree. C., where panel (A) are the prior art platelets and
panel (B) are the inventive platelets; and,
[0026] FIG. 9 graphically illustrates clot formation where the
absorbance falls sharply upon addition of thrombin (1U/ml) and the
platelet concentration drops from 250.times.10.sup.6 platelets/ml
to below 2.times.10.sup.6 platelets/ml after three minutes for the
inventive platelets.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] Compositions and embodiments of the invention include
platelets that have been manipulated (e.g. by freeze-drying) or
modified (e.g. loaded with drugs), and that are useful for
therapeutic applications, particularly for platelet transfusion
therapy, as surgical or hemostasis aids, such as wound dressings,
bandages, and as sutures, and as drug-delivery vehicles. As has
been known, human platelets have a phase transition between
12.degree. C. and 20.degree. C. We have found that platelets have a
second phase transition between 30.degree. C. and 37.degree. C. Our
discovery of this second phase transition temperature range
suggests the possible use of platelets as vehicles for drug
delivery because we can load platelets with various useful
therapeutic agents without causing abnormalities that interfere
with normal platelet responses due to changes, such as in the
platelet outer membranes.
[0028] For example, platelets may be loaded with anti-thrombic
drugs, such as tissue plasminogen activator (TPA) so that the
platelets will collect at the site of a thrombus, as in an heart
attack, and release the "clot busting" drug or drugs that are
encapsulated and have been targeted by the platelets. Antibiotics
can also be encapsulated by the platelets, since
lipopolysaccharides produced by bacteria attract platelets.
Antibiotic loaded platelets will bring the selected antibiotics to
the site of inflammation. Other drugs that can be loaded include
anti-mitotic agents and anti-angiogenic agents. Since platelets
circulate in newly formed vessels associated with tumors, they
could deliver anti-mitotic drugs in a localized fashion, and likely
platelets circulating in the neovasculature of tumors can deposit
anti-angiogenic drugs so as to block the blood supply to tumors.
Thus, platelets loaded with a selected drug in accordance with this
invention can be prepared and used for therapeutic applications.
The drug-loaded platelets are particularly contemplated for
blood-borne drug delivery, such as where the selected drug is
targeted to a site of platelet-mediated forming thrombi or vascular
injury. The so-loaded platelets have a normal response to at least
one agonist, particularly to thrombin. Such platelets can be loaded
additionally with trehalose, if preservation by freeze-drying is
intended.
[0029] The key component for compositions and apparatus of the
invention, when preservation will be by freeze-drying, is an
oligosaccharide, preferably trehalose, because we have found that
platelets which are effectively loaded with trehalose preserve
biological properties during freeze-drying (and rehydration). This
preservation of biological properties, such as the normal clotting
response in combination with thrombin, is necessary so that the
platelets following preservation can be successfully used in a
variety of therapeutic applications.
[0030] Normal hemostasis is a sequence of interactions in which
blood platelets contribute, beginning with adhesion of platelets to
an injured vessel wall. The platelets form an aggregate that
accelerates coagulation. A complex, termed the glycoprotein (GP)
1b-IX-V complex, is involved in platelet activation by providing a
binding site on the platelet surface for the potent agonist,
.alpha.-thrombin. .alpha.-thrombin is a serine protease that is
released from damaged tissue. Thus, it is important that the
manipulations and modifications in accordance with this invention
do not activate the platelets. Further, it is normally preferred
that the platelets be in a resting state. Otherwise, the platelets
will activate.
[0031] Although for most contemplated therapeutic applications the
clotting response to thrombin is key, the inventive freeze-dried
platelets after rehydration will also respond to other agonists
besides thrombin. These include collagen, ristocetin, and ADP
(adenosine diphosphate), all of which are normal platelet agonists.
These other agonists typically pertain to specific receptors on the
platelet's surface.
[0032] Broadly, the preparation of preserved platelets in
accordance with the invention comprises the steps of providing a
source of platelets, loading the platelets with a protective
oligosaccharide at a temperature above about 25.degree. C. and less
than about 40.degree. C., cooling the loaded platelets to below
-32.degree. C., and lyophilizing the platelets.
[0033] In order to provide a source of platelets suitable for the
inventive preservation process, the platelets are preferably
isolated from whole blood. Thus, platelets used in this invention
preferably have had other blood components (erythrocytes and
leukocytes) removed prior to freeze-drying. The removal of other
blood components may be by procedures well known to the art, which
typically involve a centrifuge step.
[0034] The amount of the preferred trehalose loaded inside the
inventive platelets is from about 10 mM to about 50 mM, and is
achieved by incubating the platelets to preserve biological
properties during freeze-drying with a trehalose solution that has
up to about 50 mM trehalose therein. Higher concentrations of
trehalose during incubation are not preferred, as will be more
fully explained later. The effective loading of trehalose is also
accomplished by means of using an elevated temperature of from
greater than about 25.degree. C. to less than about 40.degree. C.,
more preferably from about 30.degree. C. to less than about
40.degree. C., most preferably about 37.degree. C. This is due to
the discovery of the second phase transition for platelets. As can
be seen by FIG. 1, the trehalose loading efficiency begins a steep
slope increase at incubation temperatures above about 25.degree. C.
up to about 40.degree. C. The trehalose concentration in the
exterior solution (that is, the loading buffer) and the temperature
during incubation together lead to a trehalose uptake that seems to
occur primarily through fluid phase endocytosis (that is,
pinocytosis). Pinocytosed vesicles lyse over time, which results in
a homogeneous distribution of trehalose in the platelets, does not
activate the platelets, and can be applied for large scale
production. FIG. 2 illustrates the trehalose loading efficiency as
a function of incubation time.
[0035] As may be gathered from various of the figures, in preparing
particularly preferred embodiments, platelets may be loaded with
trehalose by incubation at 37.degree. C. for about four hours. The
trehalose concentration in the loading buffer is preferably 35 mM,
which results in an intracellular trehalose concentration of around
20 mM, but in any event is most preferably not greater than about
50 mM trehalose. At trehalose concentrations below about 50 mM,
platelets have a normal morphological appearance.
[0036] Human platelets have a phase transition between 12.degree.
C. and 20.degree. C. We found relatively poor loading when the
platelets were chilled through the phase 1.5 transition. Thus, in
practicing the method described by U.S. Pat. No. 5,827,741, of
which some of us are conventors, only a relatively modest amount of
trehalose may be loaded into platelets.
[0037] In this application, we have further investigated the phase
transition in platelets and have found a second phase transition
between 30.degree. C. and 37.degree. C. We believe that the
excellent loading we obtain at about 37.degree. C. is in some way
related to this second phase transition. Without being limited by
theory, we also believe that pinocytosis is involved, but it may be
that the second phase transition itself stimulates the pinocytosis
at high temperatures. It may be that other oligosaccharides when
loaded in this second phase transition in amounts analogous to
trehalose could have similar effects.
[0038] In any case, it is fortuitous that the loading can be done
at elevated temperatures in view of the fact that chilling
platelets slowly--a requirement for using the first, or lower,
phase transition between 20.degree. C. and 12.degree. C. to
introduce trehalose--is well known to activate them (Tablin et al.,
J. Cell. Physiol., 168, 305-313, 1996). Our relatively high
temperature loading, regardless of the mechanism, is thus
unexpectedly advantageous both by providing increased loading as
well as surprisingly, obviating the activation problem.
[0039] Turning to FIG. 6, one sees that we have loaded other,
larger molecules into the platelets. In FIG. 6 an illustrative
large molecule (FITC dextran) was loaded into the platelets. This
illustrates that a wide variety of water-soluble, therapeutic
agents can be loaded into the platelets by utilizing the second
phase transition, as we have shown may be done with trehalose and
with FITC dextran, while still maintaining characteristic platelet
surface receptors and avoiding platelet activation.
[0040] We have achieved loading efficiencies by practicing the
invention with values as high as 61% after four hours incubation.
The plateau is not yet reached after four hours. The high loading
efficiency of trehalose is a strong indication that the trehalose
is homogeneously distributed rather than located in pinocytosed
vesicles, and we expect similar results for loading other
therapeutic agents. A loading efficiency of 61% in an external
concentration of 25 mM corresponds to a cytosolic concentration of
15 mM. If trehalose was only located in endosomes of 0.1
micrometer, the vesiculation number would be more than 1000. It is
unlikely that such a high number of vesicles would be present in
platelets next to the other platelet organelles. We therefore
believe that the pinocytosed vesicles lyse in the cytoplasm. This
results in a homogeneous distribution of trehalose rather than
punctuated loading in small vesicles. It is also possible that the
trehalose is crossing the membrane due to the phase transition
between 30.degree. C. and 37.degree. C.
[0041] We have found that the endocytotic uptake route is blocked
at sugar concentrations above 0.1 M. Consequently, we prefer not to
use sugar concentrations higher than about 50 mM in the loading
buffer, because at some point above this value we have found
swelling and morphological changes of the platelets. Thus, we have
found that platelets become swollen after four hours incubation at
37.degree. C. in 75 mM trehalose. Further, concentrations higher
than 50 mM the internal trehalose concentration begins to decrease.
By contrast to the present invention, the platelet method taught by
Spargo et al., U.S. Pat. No. 5,736,313, loads with carbohydrate in
the range beginning at about 100 mM and going up to 1.5 M. As
noted, we find a high concentration of loading buffer, at least
with trehalose, to lead to swelling and morphological changes.
[0042] The effective loading of platelets with trehalose is
preferably conducted by incubating for at least about two hours,
preferably for at least about four hours. After this loading, then
the platelets are cooled to below their freezing point and
lyophilized.
[0043] Before freezing, the platelets should be placed into a
resting state. If not in the resting state, platelets would likely
activate. In order to place the platelets in a resting state, a
variety of suitable agents, such as calcium channel blockers, may
be used. For example, solutions of adenine, adenosine or iloprost
are suitable for this purpose. Another suitable agent is PGE1. It
is important that the platelets are hot swollen and are completely
in the resting state prior to drying. The more they are activated,
the more they will be damaged during freeze-drying.
[0044] After the platelets have been effectively loaded with
trehalose and are in a resting state, then the loading buffer is
removed and the platelets are contacted with a drying buffer.
Drying of platelets after trehalose loading may be carried out by
suspending the platelets in a solution containing a suitable water
replacing molecule (or drying buffer), such as albumin. If albumin
is used, it should be from the same species as the platelets. The
drying buffer should also include trehalose, preferably in amounts
up to about 100 mM. The trehalose in the drying buffer assists in
spatially separating the platelet as well as stabilizing the
platelet membranes on the exterior. The drying buffer preferably
also includes a bulking agent (to further separate the platelets).
As already mentioned, albumin may serve as a bulking agent, but
other polymers may be used with the same effect. Suitable other
polymers, for example, are water-soluble polymers such as HES and
dextran.
[0045] The trehalose loaded platelets in drying buffer are then
cooled to a temperature below about -32.degree. C. A cooling, that
is, freezing, rate is preferably between -30.degree. C. and
-1.degree. C./min. and more preferably between about -2.degree.
C./min to -5.degree. C./min.
[0046] The lyophilization step is preferably conducted at a
temperature below about -32.degree. C., for example conducted at
about -40.degree. C., and drying may be continued until about 95
weight percent of water has been removed from the platelets. During
the initial stages of lyophilization, the pressure is preferably at
about 1.times.10.sup.-6 torr. As the samples dry, the temperature
can be raised to be warmer than -32.degree. C. Based upon the bulk
of the sample, the temperature and the pressure it can be
emperically determined what the most efficient temperature values
should be in order to maximize the evaporative water loss.
Freeze-dried compositions of the invention preferably have less
than about 5 weight percent water.
[0047] The freeze-dried platelets may be used by themselves,
dissolved in a physiologically acceptable solution, or may be a
component of a biologically compatible (biocompatible) structure or
matrix, which provides a surface on or by which the freeze-dried
platelets are carried. The freeze-dried platelets can be, for
example, applied as a coating to or impregnated in a wide variety
of known and useful materials suitable as biocompatible structures
for therapeutic applications. The earlier mentioned U.S. Pat. No.
5,902,608, for example, discusses a number of materials useful for
surgical aid, wound dressings, bandages, sutures, prosthetic
devices, and the like. Sutures, for example, can be monofilament or
braided, can be biodegradable or nonbiodegradabie, and can be made
of materials such as nylon, silk, polyester, cotton, catgut,
homopolymers, and copolymers of glycolide and lactide, etc.
Polymeric materials can also be cast as a thin film, sterilized,
and packaged for use as a wound dressing. Bandages may be made of
any suitable substrate material, such as woven or nonwoven cotton
or other fabric suitable for application to or over a wound, may
optionally include a backing material, and may optionally include
one or more adhesive regions on the face surface thereof for
securing the bandage over the wound.
[0048] The freeze-dried platelets, whether by themselves, as a
component of a vial-compatible structure or matrix, and optionally
including other dry or freeze-dried components, may be packaged so
as to prevent rehydration until desired. The packaging may be any
of the various suitable packagings for therapeutic purposes, such
as made from foil, metallized plastic materials, and moisture
barrier plastics (e.g. high-density polyethylene or plastic films
that have been created with materials such as SiOx), cooling the
trehalose loaded platelets to below their freezing point, and
lyophilizing the cooled platelets. The trehalose loading includes
incubating the platelets at a temperature from greater than about
25.degree. C. to less than about 40.degree. C. with a trehalose
solution having up to about 50 mM trehalose therein. The process of
using such a dehydrated composition comprises rehydrating the
platelets. The rehydration preferably includes a prehydration step
sufficient to bring the water content of the freeze-dried platelets
to between 35 weight percent to about 50 weight percent.
[0049] When reconstitution is desired, prehydration of the
freeze-dried platelets in moisture saturated air followed by
rehydration is preferred. Use of prehydration yields cells with a
much more dense appearance and with no balloon cells being present.
Prehydrated, previously lyophilized platelets of the invention
resemble fresh platelets. This is illustrated, for example, by FIG.
7. As can be seen, the previously freeze-dried platelets can be
restored to a condition that looks like fresh platelets.
[0050] Before the prehydration step, it is desirable to have
diluted the platelets in the drying buffer to prevent aggregation
during the prehydration and rehydration. At concentrations below
about 3.times.10.sup.8 cells/ml, the ultimate recovery is about 70%
with no visible aggregates. Prehydration is preferably conducted in
moisture saturated air, most preferably is conducted at about
37.degree. C. for about one hour to about three hours. The
preferred prehydration step brings the water content of the
freeze-dried platelets to between about 35 weight percent to about
50 weight percent.
[0051] The prehydrated platelets may then be fully rehydrated.
Rehydration may be with any aqueous based solutions, depending upon
the intended application. In one preferred rehydration, we have
used plasma, which has resulted in about 90% recovery.
[0052] Since it is frequently desirable to dilute the platelets to
prevent aggregation when the freeze-dried platelets are once again
hydrated, it may then be desired or necessary for particular
clinical applications to concentrate the platelets. Concentration
can be by any conventional means, such as by centrifugation. In
general, a rehydrated platelet composition will preferably have
10.sup.6 to 10.sup.11 platelets per ml, more preferably 10.sup.8 to
10.sup.10 platelets per ml.
[0053] By contrast with the previous attempts at freeze drying
platelets, we show here that with a very simple loading,
freeze-drying and rehydration protocol one obtains platelets that
are morphologically intact after rehydration, and have an identical
response to thrombin as do fresh platelets. Moreover, the
concentration of thrombin to give this response is a physiological
concentration--1 U/ml.
[0054] For example, FIG. 8, panel (A), illustrates the clot
formation for fresh platelets and in panel (B) for platelets that
have been preserved and then rehydrated in accordance with this
invention. The cell counts that were determined after three minutes
exposure to thrombin were zero for both the fresh platelets and the
previously freeze-dried platelets of the invention.
[0055] FIG. 9 graphically illustrates clotting as measured with an
aggregometer. With this instrument one can measure the change in
transmittance when a clot is formed. The initial platelet
concentration was 250.times.10.sup.6 platelets/ml, and then
thrombin (1 U/ml) was added and the clot formation was monitored
with the aggregometer. The absorbance fell sharply and the cell
count dropped to below 2.times.10.sup.6 platelets/ml after three
minutes, which was comparable to the results when the test was run
with fresh platelets as a control.
[0056] Although platelets for use in this invention preferably have
had other blood components removed before freeze-drying,
compositions and apparatuses of the invention may also include a
variety of additional therapeutic agents. For example, particularly
for embodiments contemplated in hemostasis applications, antifungal
and antibacterial agents are usefully included with the platelets,
such as being admixed with the platelets. Embodiments can also
include admixtures or compositions including freeze-dried collagen,
which provides a thrombogenic surface for the platelets. Other
components that can provide a freeze-dried extracellular matrix can
be used, for example, components composed of proteoglycan. Yet
other therapeutic agents that may be included in inventive
embodiments are growth factors. When the embodiments include such
other components, or admixtures, they are preferably in dry form,
and most preferably are also freeze-dried. We also contemplate
therapeutic uses of the composition where additional therapeutic
agents may be incorporated into or admixed with the platelets in
hydrated form. The platelets, as earlier mentioned, can also be
prepared as to encapsulate drugs in drug delivery applications. If
trehalose is also loaded into the platelet interiors, then such
drug-encapsulated platelets may be freeze-dried as has been earlier
described.
[0057] The platelets should be selected of the mammalian species
for which treatment is intended (e.g. human, equine, canine,
feline, or endangered species), most preferably human.
[0058] The injuries to be treated by applying hemostasis aids with
the platelets include abrasions, incisions, bums, and may be wounds
occurring during surgery of organs or of skin tissue. The platelets
of the invention may be applied or delivered to the location of
such injury or wound by any suitable means. For example,
application of inventive embodiments to bums can encourage the
development of scabs, the formation of chemotactic gradients, of
matrices for inducing blood vessel growth, and eventually for skin
cells to move across and fill in the burn.
[0059] For transfusion therapy, inventive compositions may be
reconstituted (rehydrated) as pharmaceutical formulations and
administered to human patients by intravenous injection. Such
pharmaceutical formulations may include any aqueous carrier
suitable for rehydrating the platelets (e.g., sterile,
physiological saline solution, including buffers and other
therapeutically active agents that may be included in the
reconstituted formulation). For drug delivery, the inventive
compositions will typically be administered into the blood stream,
such as by i.v.
[0060] Aspects of the invention will now be illustrated by the
following examples, which are not intended to limit the invention.
Abbreviations used in the examples, and elsewhere, are as
follows.
[0061] DMSO dimethylsulfoxide
[0062] ADP=adenosine diphosphate
[0063] PGE1=prostaglandin E1
[0064] HES=hydroxy ethyl starch
[0065] EGTA=ethylene glycol-bis(2-aminoethyl ether)N,N,N',N',
tetra-acetic acid
[0066] TES=N-tris (hydroxymethyl) methyl-2-aminoethane-sulfonic
acid
[0067] HEPES=N-(2-hydroxylethyl) piperarine-N'-(2-ethanesulfonic
acid)
[0068] PBS=phosphate buffered saline
[0069] HSA=human serum albumin
EXPERIMENTAL
Example 1
[0070] Washing of Platelets. Platelet concentrations were obtained
from the Sacramento blood center or from volunteers in our
laboratory. Platelet rich plasma was centrifuged for 8 minutes at
320.times.g to remove erythrocytes and leukocytes. The supernatant
was pelleted and washed two times (480.times.g for 22 minutes,
480.times.g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10
mM EGTA, 10 mM imidazole, pH 6.8). Platelet counts were obtained on
a Coulter counter T890 (Coulter, Inc., Miami, Fla.).
[0071] Loading of Lucifer Yellow CH into Platelets. A fluorescent
dye, lucifer yellow CH (LYCH), was used as a marker for penetration
of the membrane by a solute. Washed platelets in a concentration of
1-2.times.10.sup.9 platelets/ml were incubated at various
temperatures in the presence of 1-20 mg/ml LYCH. Incubation
temperatures and incubation times were chosen as indicated. After
incubation the platelets suspensions were spun down for 20.times.
at 14,000 RPM (table centrifuge), resuspended in buffer A, spun
down for 20 s in buffer A and resuspended. Platelet counts were
obtained on a Coulter counter and the samples were pelleted
(centrifugation for 45 s at 14,000 RPM, table centrifuge). The
pellet was lysed in 0.1% Triton buffer (10 mM TES, 50 mM KCl, pH
6.8). The fluorescence of the lysate was measured on a Perkin-Elmer
LS5 spectrofluorimeter with excitation at 428 nm (SW 10 nm) and
emission at 530 nm (SW 10 nm). Uptake was calculated for each
sample as nanograms of LYCH per cell using a standard curve of LYCH
in lysate buffer. Standard curves of LYCH, were found to be linear
up to 2000 nm ml.sup.-1.
[0072] Visualization of cell-associated lucifer yellow. LYCH loaded
platelets were viewed on a fluorescence microscope (Zeiss)
employing a fluorescein filter set for fluorescence microscopy.
Platelets were studied either directly after incubation or after
fixation with 1% paraformaldehyde in buffer. Fixed cells were
settled on poly-L-lysine coated cover slides and mounted in
glycerol.
[0073] Loading of Platelets with Trehalose. Washed platelets in a
concentration of 1-2 10.sup.9 platelets/ml were incubated at
various temperatures in the presence of 1-20 mg/ml trehalose.
Incubation temperatures were chosen from 4.degree. C. to 37.degree.
C. Incubation times were varied from 0.5 to 4 hours. After
incubation the platelet solutions were washed in buffer A two times
(by centrifugation at 14,000 RPM for 20 s in a table centrifuge).
Platelet counts were obtained on a coulter counter. Platelets were
pelleted (45 S at 14,000 RPM) and sugars were extracted from the
pellet using 80% methanol. The samples were heated for 30 minutes
at 80.degree. C. The methanol was evaporated with nitrogen, and the
samples were kept dry and redissolved in H.sub.2O prior to
analysis. The amount of trehalose in the platelets was quantified
using the anthrone reaction (Umbreit et al., Mamometric and
Biochemical Techniques, 5.sup.th Edition, 1972). Samples were
redissolved in 3 ml H.sub.2O and 6 ml anthrone reagents (2 g
anthrone dissolved in 1 l sulfuric acid). After vortex mixing, the
samples were placed in a boiling water bath for 3 minutes. Then the
samples were cooled on ice and the absorbance was measured at 620
nm on a Perkin Elmer spectrophotometer. The amount of platelet
associated trehalose was determined using a standard curve of
trehalose. Standard curves of trehalose were found to be linear
from 6 to 300 .mu.g trehalose per test tube.
[0074] Quantification of Trehalose and LYCH Concentration. Uptake
was calculated for each sample as micrograms of trehalose or LYCH
per platelet. The internal trehalose concentration was calculated
assuming a platelet radius of 1.2 .mu.m and by assuming that 50% of
the platelet volume is taken up by the cytosol (rest is membranes).
The loading efficiency was determined from the cytosolic trehalose
or LYCH concentration and the concentration in the loading
buffer.
[0075] FIG. 1 shows the effect of temperature on the loading
efficiency of trehalose into human platelets after a 4 hour
incubation period with 50 mM external trehalose. The effect of the
temperature on the trehalose uptake showed a similar trend as the
LYCH uptake. The trehalose uptake is relatively low at temperatures
of 22.degree. C. and below (below 5%), but at 37.degree. C. the
loading efficiency of trehalose is 35% after 4 hours.
[0076] When the time course of trehalose uptake is studied at
37.degree. C., a biphasic curve can be seen (FIG. 2). The trehalose
uptake is initially slow (2.8.times.10.sup.-11 mol/m.sup.2 s from 0
to 2 hours), but after 2 hours a rapid linear uptake of
3.3.times.10.sup.-10 mol/m.sup.2 s can be observed. The loading
efficiency increases up to 61% after an incubation period of 4
hours. This high loading efficiency is a strong indication that the
trehalose is homogeneously distributed in the platelets rather than
located in pinocytosed vesicles.
[0077] The uptake of trehalose as a function of the external
trehalose concentration is shown in FIG. 3. The uptake of trehalose
is linear in the range from 0 to 30 mM external trehalose. The
highest internal trehalose concentration is obtained with 50 mM
external trehalose. At higher concentrations than 50 mM the
internal trehalose concentration decreases again. Even when the
loading buffer at these high trehalose concentrations is corrected
for isotonicity by adjusting the salt concentration, the loading
efficiency remains low. Platelets become swollen after 4 hours
incubation in 75 mM trehalose.
[0078] The stability of the platelets during a 4 hours incubation
period was studied using microscopy and flow cytometric analysis.
No morphological changes were observed after 4 hours incubation of
platelets at 37.degree. C. in the presence of 25 mM external
trehalose. Flow cytometrit analysis of the platelets showed that
the platelet population is very stable during 4 hours incubation.
No signs of microvesicle formation could be observed after 4 hours
incubation, as can be judged by the stable relative proportion of
microvesicle gated cells (less than 3%). The formation of
microvesicles is usually considered as the first sign of platelet
activation (Owners et al., Trans. Med. Rev., 8, 2744, 1994).
Characteristic antigens of platelet activation include:
glycoprotein 53 (GP53, a lysosomal membrane marker), PECAM-1
(platelet-endothelial cell adhesion molecule-1, an alpha granule
constituent), and P-selectin (an alpha granule membrane
protein).
Example 2
[0079] Washing Platelets. Platelets were obtained from volunteers
in our laboratory. Platelet rich plasma was centrifuged for 8
minutes at 320.times.g to remove erythrocytes and leukocytes. The
supernatant was pelleted and washed two times (480.times.g for 22
minutes, 480.times.g for 15 minutes) in buffer A (100 mM NaCl, 10
mM KCl, 10 mM EGTA, 10 mM imidazole, 10 .mu.g/ml PGE1, pH 6.8).
Platelet counts were obtained on a Coulter counter T890 (Coulter,
Inc., Miami, Fla.).
[0080] Loading Platelets with Trehalose. Platelets were loaded with
trehalose as described in Example 1. Washed platelets in a
concentration of 1-2.times.10.sup.9 platelets/ml were incubated at
37.degree. C. in buffer A with 35 mM trehalose added. Incubation
times were typically 4 hours. The samples were gently stirred for 1
minute every hour. After incubation the platelet solutions were
pelleted (25 sec in a microfuge) and resuspended in drying buffer
(9.5 mM HEPES, 142.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl.sub.2, 30 mM
Trehalose, 1% Human Serum Albumin, 10 .mu.g/ml PGE1). In the
aggregation studies no PGE1 was added in the drying buffer.
Trehalose was obtained from Pfahnstiehl. A 30% human serum albumin
was obtained from Sigma.
[0081] Freezing and Drying. Typically 0.5 ml platelet suspensions
were transferred in 2 ml Nunc cryogenic vials and frozen in a
Cryomed controlled freezing device. Vials were frozen from
22.degree. C. to -40.degree. C. with freezing rates between -30 and
-1.degree. C./min and more often between -5 and -2.degree. C./min.
The frozen solutions were transferred to a -80.degree. C. freezer
and kept there for at least half an hour. Subsequently the frozen
platelet suspensions were transferred in vacuum flasks that were
attached to a Virtis lyophilizer. Immediately after the flasks were
hooked up to the lyophilizer, they were placed in liquid nitrogen
to keep the samples frozen until the vacuum returned to
20.times.10.sup.-6 Torr, after which the samples were allowed to
warm to the sublimation temperature. The condenser temperature was
-45.degree. C. Under these conditions, sample temperature during
primary drying is about -40.degree. C., as measured with a
thermocouple in the sample. It is important to maintain the sample
below T.sub.g for the excipient during primary drying (-32.degree.
C. for trehalose).
[0082] Rehydration. Vials with originally 0.5 ml platelet
suspension were rehydrated in 1 ml PBS buffer/water (1/1). PBS
buffer was composed of 9.4 mM Na.sub.2HPO.sub.4, 0.6 mM
KH.sub.2PO.sub.4, 100 mM NaCl). In a few experiments PGE1 was added
to the rehydration buffer in a condition of 10 .mu.g/ml or
rehydration was performed in plasma/water (1/1).
[0083] Prehydration. Platelet lyophilisates were prehydrated in a
closed box with moisture saturated air at 37.degree. C.
Prehydration times were between 0 and 3 hours.
[0084] Recovery. The numerical recovery of lypophilized and
(p)rehydrated platelets was determined by comparing the cell count
with a Coulter count T890 (Coulter, Inc., Miami, Fla.) before
drying and after rehydration. The morphology of the rehydrated
platelets was studied using a light microscope. For this purpose
platelets were fixed in 2% paraformaldehyde or gutaraldehyde and
allowed to settle on poly-L-lysine coated coverslides for at least
45 minutes. After this the coverslides were mounted and inspected
under the microscope. The Optical density of freeze-dried and
rehydrated platelets was determined by measuring the absorbance of
a platelet suspension of 1.0.times.10.sup.8 cells/ml at 550 nm on a
Perkin Elmer absorbance spectrophotometer.
[0085] Aggregation studies. Dried platelets were rehydrated (after
2 hour prehydration) with 2 aliquots of platelet free plasma
(plasma was centrifuged for 5 minutes at 3800.times.g) diluted with
water in 1/1 ratio. Half ml aliquots of this platelet suspension
were transferred to aggregation cuvettes with a magnetic stirrer.
The response of the platelets to thrombin was tested by adding
thrombin (1 U/ml) to the platelet suspension at 37.degree. C. under
stirring conditions. After 3 minutes thrombin treated platelet
suspensions were inspected for clots and cell counts were done on a
Coulter Counter T890.
[0086] Direct rehydration tends toward cell lysis and prehydration
leads to aggregation when the cell concentration is 10.sup.9
cells/ml in the drying buffer. We found also that recovery of
prehydrated and rehydrated platelets depends on the cell
concentration in the drying buffer. The recovery drops to very low
values if the cell concentration is higher than 3.times.10.sup.8
cells/ml. At concentrations below 3.times.10.sup.8 cells/ml, the
recovery is around 70%, and no aggregates were visible.
Prehydration resulted in denser cells and the absence of balloon
cells.
[0087] Longer prehydration times than 90 minutes did not further
improve the cellular density, but slightly activated the platelets.
The water content of the pellet increases with increasing
prehydration time, and preferably is between about 35% and 50% at
the moment of rehydration. At higher water contents than 50% water
droplets become visible in the lyophilisate (which means that the
platelets are in a very hypertonic solution).
[0088] As described by Example 1, platelets were loaded with
trehalose by incubation at 37.degree. C. for 4 hours in buffer A
with 35 mM trehalose, which yielded platelets with intracellular
trehalose concentration of 15-25 mM. After incubation, the
platelets were transferred to drying buffer with 30 mM trehalose
and 1% HSA as the main excipients.
[0089] The directly rehydrated platelets had a high numerical
recovery of 85%, but a considerable fraction (25-50%) of the cells
was partly lysed and had the shape of a balloon. Directly
rehydrated platelets were overall less dense when compared with
fresh platelets.
[0090] The numerical recovery of platelets that were prehydrated in
moisture saturated air was only 25% when the platelet concentration
was 1.times.10.sup.9 cells/ml in the drying buffer. This low
recovery was due to aggregates that were formed during the
prehydration period. But the cells that were not aggregated were
more dense than the directly rehydrated platelets and resembled
that of fresh platelets.
[0091] Since it appears desirable to dilute the platelets to
prevent aggregation during the prehydration step, it may be
necessary for clinical applications to concentrate the platelets
following rehydration. We therefore also tested the stability of
the rehydrated platelets with respect to centrifugation and found
that the directly rehydrated platelets had 50% recovery after
centrifugation, while the prehydrated ones had 75% recovery
following centrifugation. Thus, we conclude that the inventive
platelets can be concentrated without ill effect.
Example 3
[0092] We view trehalose as the main lyoprotectant in the drying
buffer. However, other components in the drying buffer, such as
albumin, can improve the recovery. In the absence of external
trehalose in drying buffer, the numerical recovery is only 35%.
With 30 mM trehalose in the drying buffer the recovery is around
65%. A combination of 30 mM trehalose and 1% albumin gave a
numerical recovery of 85%.
Example 4
[0093] Typically 0.5 ml platelet suspensions were transferred in 2
ml Nunc cryogenic vials and frozen in a Cryomed controlled freezing
device. Vialswere frozen from 22.degree. C. to -40.degree. C. with
freezing rates between -30.degree. C./min and -1.degree. C./min and
more often between -5.degree. C. and -2.degree. C./min. The frozen
solutions were transferred to a -80.degree. C. freezer and kept
there for at least half an hour. Subsequently the frozen platelet
suspensions were transferred in vacuum flasks that were attached to
a Virtus lyophilizer Immediately after the flasks were hooked up to
the lyophilizer, they were placed in liquid nitrogen to keep the
samples frozen until the vacuum returned to 20.times.10.sup.-6
Torr, after which the samples were allowed to warm to the
sublimation temperature. The condenser temperature was 45.degree.
C. Under these conditions, sample temperature during primary drying
is about -40.degree. C., as measured with a thermocouple in the
sample. In is important to maintain the sample below T.sub.g, for
the excipient during primary drying (-32.degree. C. for trehalose).
Only minor differences in recovery were found as a function of the
freezing rate. The optimal freezing rate was found to be between
2.degree. C. and 5.degree. C./minute.
Example 5
[0094] Response of freeze-dried platelets to thrombin (1 U/ml) was
compared with that of fresh platelets. The platelet concentration
was 0.5.times.10.sup.8 cells/ml in both samples. 500 .mu.l
platelets solution was transferred into aggregation vials. Thrombin
was added to the samples and the samples were stirred for 3 minutes
at 37.degree. C. The cell counts that were determined after 3
minutes were 0 for both the fresh and the freeze-dried platelets.
The response to thrombin was determined by a cleavage in
glycoprotein 1b-(GP1b). This was detected by using monoclonal
antibodies and flow cytometry. Thus, the pattern seen after
addition of thrombin was a reduced amount of GP1b on the platelet
surface.
[0095] The response of lyophilized, prehydrated, and rehydrated
platelets (Examples 1 and 2) to thrombin (1 U/ml) was found to be
identical compared with that of fresh platelets. In both fresh and
rehydrated platelets a clot was formed within 3 minutes at
37.degree. C. These clots are illustrated by FIG. 8, panels (A) and
(B). When cell counts were done with the Coulter counter, we found
no cells present, indicating that all platelets participated in
forming the clot illustrated in panel (B).
Example 6
[0096] Reactions with other agonists were studied. Platelet
suspensions of the inventive platelets were prepared with
50.times.10.sup.6 platelets/ml. Different agonists were then added
and subsequently counted with a Coulter counter to determine the
percentage of platelets involved in the visually observable clot
formation. The cell count was between 0 and 2.times.10.sup.6
platelets/ml:
[0097] after 5 minutes with 2 mg/ml collagen
[0098] after 5 minutes with 20 .mu.M ADP
[0099] after 5 minutes with 1.5 mg/ml ristocetin
[0100] This means that the percentage of platelets that are
involved in clot formation is between 95-100% for all the agonists
tested. The agonist concentrations that were used are all
physiological. In all cases the percentage of clotted platelets was
the same as fresh control platelets.
[0101] It is to be understood that while the invention has been
described above in conjunction with preferred specific embodiments,
the description and examples are intended to illustrate and not
limit the scope of the invention, which is defined by the scope of
the appended claims.
* * * * *