U.S. patent number 5,523,238 [Application Number 08/273,549] was granted by the patent office on 1996-06-04 for method and apparatus for determining platelet function in primary hemostasis.
This patent grant is currently assigned to Ramot University Authority for Applied Research and Industrial. Invention is credited to Naphtali Savion, David Varon.
United States Patent |
5,523,238 |
Varon , et al. |
June 4, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Method and apparatus for determining platelet function in primary
hemostasis
Abstract
A method for determining a platelet function in a primary
hemostasis is provided in which a blood sample or a platelet
containing fraction thereof is introduced into a vessel having a
flat bottom which has an inner surface covered with a substrate
capable of inducing platelet adhesion thereto and aggregation such
as ECM (extracellular matrix). Preparation is then rotated inside
the vessel, and consequently shear forces develop on the surface
which bring to adhesion an aggregation of the blood platelets to
the surface. Morphological parameters of blood aggregates are then
determined.
Inventors: |
Varon; David (Kfar Bilu A',
IL), Savion; Naphtali (Givat Shmuel, IL) |
Assignee: |
Ramot University Authority for
Applied Research and Industrial (Tel Aviv, IL)
|
Family
ID: |
11065042 |
Appl.
No.: |
08/273,549 |
Filed: |
July 11, 1994 |
Foreign Application Priority Data
Current U.S.
Class: |
436/69; 436/63;
422/73; 435/2; 435/287.9; 435/288.1; 422/555 |
Current CPC
Class: |
G01N
33/4905 (20130101) |
Current International
Class: |
G01N
33/49 (20060101); G01N 33/483 (20060101); G01N
033/86 () |
Field of
Search: |
;436/63,69,164,177
;422/73,99,101,102 ;435/2,291,296,312,316 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Riess et al., American Journal of Clinical Pathology, vol. 85, No.
1, pp. 50-56, Jan. 1986. .
Tippe et al. Thrombosis Research, vol. 67, No. 4, pp. 407-418,
1992. .
Born, Nature, vol. 194, pp. 927-929, Jun. 9, 1962. .
Fukuyama et al., Thrombosis Research, vol. 54, No. 3, pp. 253-260,
1989. .
Ikeda et al., Journal of Clinical Investigation, vol. 87, pp.
1234-1240, Apr. 1991. .
Lavee et al., Journal of Thoracic+Cardiovascular Surgery, vol. 97,
No. 2, pp. 204-212, Feb. 1989..
|
Primary Examiner: Warden; Robert J.
Assistant Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Gerstenzang; William C. Sprung Horn
Kramer & Woods
Claims
We claim:
1. A method for determining platelet function in primary hemostasis
comprising:
(a) obtaining a sample being whole-blood or a platelet-containing
fraction thereof;
(b) introducing the sample obtained in (a) into a vessel having a
flat bottom, an inner surface of which is covered with a substrate
capable of inducing platelet adhesion thereto and aggregation;
(c) rotating said sample inside the vessel, whereby shear forces
develop at said inner surface; thereby causing adherence and
aggregation of blood platelets onto said inner surface;
(d) determining parameters of the adhered and aggregated blood
platelets, the parameters being selected from the group consisting
of amount of adhered platelets, aggregate size, aggregates'
morphology, total area covered by the aggregates and distribution
of adhered platelets or aggregates.
2. A method according to claim 1, wherein said substrate is
Extracellular matrix or an active component thereof capable of
inducing platelet adhesion thereto and aggregation.
3. A method according to claim 2, wherein said substrate is said
active component of said extracellular matrix and said active
component is selected from the group consisting of: basement
membrane matrix, fibrillar and non-fibrillar collagen of various
types, von Willebrand factor and fibronectin.
4. A method according to claim 1, wherein the vessel has a
cylindrical rotating element located therein.
5. A method according to claim 1, wherein the vessel has a rotating
element located therein which has a bottom portion having the shape
of an inverted cone.
6. A method according to claim 1, wherein the sample is mixed with
an anti-coagulant in an amount sufficient to inhibit blood
coagulation prior to introduction into the vessel.
7. An apparatus for determining platelet function in primary
hemostasis, comprising a flat-bottomed vessel capable of receiving
liquids and a rotating element placed within said flat-bottomed
vessel, wherein the inner face of the bottom of said vessel is
lined with a substrate capable of inducing platelet adhesion
thereto and aggregation.
8. An apparatus according to claim 7, wherein the rotating element
has an essentially conical shape.
9. An apparatus according to claim 8, in which the angles of the
conical rotating element are adjusted so that during rotational
movements thereof the shear force produced through the bottom inner
face of the vessel is essentially equal.
10. An apparatus according to claim 7 wherein said substrate is
extracellular matrix or an active component thereof capable of
inducing platelet adhesion thereto and aggregation.
11. An apparatus according to claim 10, wherein the active
component of the extracellular matrix is selected from the group
consisting of basement membrane matrix, fibrilar and non-fibrilar
collagen of various types, von Willebrand factor and fibronectin.
Description
FIELD OF THE INVENTION
The present invention concerns a method and an apparatus for
determining platelet function in primary hemostasis.
BACKGROUND OF THE INVENTION
Hemostasis, i.e. the arrest of hemorrhage, is a mechanism which
comprises essentially two consecutively functioning mechanisms:
"primary hemostasis" is responsible for the immediate arrest of
hemorrhage and is caused by the localization and aggregation of
circulating platelets on damaged vascular surfaces or exposed
tissues and subsequent formation of a thrombus; "secondary
hemostasis" is responsible for the long-term arrest of hemorrhage
and is caused by a chain of enzymatic reactions resulting
eventually in the formation of fibrin.
Abnormalities of primary hemostasis are clinical conditions
associated with bleeding tendencies which can become
life-threatening in traumatic situations such as operation,
delivery, invasive diagnostic and therapeutic procedures as well as
traumatic injury. The evaluation of the degree of primary
hemostasis is thus of a high clinical importance.
Several procedures have been hitherto employed in order to evaluate
primary hemostasis. One procedure termed "the bleeding time test",
which is the most common clinical test for determining primary
hemostasis, is carried out by inducing a controlled cut in the arm
of the tested subject and determining the duration before bleeding
is arrested. This procedure has relatively low clinical relevance
as it is able to identify only severe abnormalities in primary
hemostasis and is thus unable to distinguish between normal
subjects and those whose primary hemostasis is slightly defective.
Furthermore, this method is virtually impossible to standardize
since the duration of bleeding depends strongly on the precise size
and location of the cut as well as on the venus and arterial blood
pressures.
Platelet aggregation studies are usually performed in platelet-rich
plasma (PRP) using a turbidometric device according to Born (Born
G. V. R., Nature, 194, 927-929 (1962)). One drawback of
turbidometric aggregometry is that the use of PRP necessitates
centrifugation and separation of platelets from other blood cells
which manipulation may alter platelets' properties and behavior.
Another drawback of the turbidometric method is in that it is time
consuming and laborious. Finally, the evaluation of such a test is
limited by the optical quality of PRP which is affected by the
levels of lipids in the plasma.
Platelets' aggregation was also tested in whole blood (WB) (Riess
H., Am. J. Clin. Pathol, 85, 50-56, (1986)). According to this
technique, platelet aggregation was measured by an increase in
impedence across two electrodes placed in the blood sample.
However, in this technique as well as the turbidometric technique
described above, the platelets' aggregation was induced by
artificial reagents and consequently the conditions in which the
aggregation was measured were clearly non-physiological.
Several other procedures involved subjecting PRP to shear forces
and determining platelet aggregation under these conditions. The
shear forces in such tests were induced either by a cone-plate
apparatus in which a rotating member was rotated within a
cylindrical vessel, or by various sophisticated means adapted to
produce a continuous flow of fluids (Tippe A. et al., Thrombosis
Res., 67:407-418 (1992); Ikeda et al., J. Clin. Invest., 87,
1234-1240 (1991)); Fukiyama M. et al., Thrombosis Res. 64:253-260
(1989)). In such tests, which were generally conducted with PRP,
the shear induced adhesions took place on the surfaces of the test
vessel. The physiological significance of adhesion of platelets to
artificial matrix under such conditions is questionable and this
procedure indeed very often fails to provide clinically significant
results. In addition, the means to produce the shear force in some
of the procedures disclosed in the above publications were often
complicated and expensive rendering the procedure unsuitable as a
routine clinical procedure.
Another hitherto disclosed procedure involved determining adhesion
of platelets, in a platelet rich plasma (PRP) (Vlodavksy, I. et
al., Thrombosis Research, 18, 179-191, (1983)) and in whole blood
(Lavee J. et a., The Journal of Thoracic and Cardiovascular
Surgery, 97, (2), 204-212, (1989)) to an extracellular matrix
(ECM).
It is the object of the invention lo provide a novel method and
apparatus for evaluating platelets' function in primary
hemostasis.
GENERAL DESCRIPTION OF THE INVENTION
The present invention provides a method for determining platelet
function in primary hemostasis comprising:
(a) obtaining a sample being whole-blood or a platelet-containing
fraction thereof; and optionally mixing it with an anti-coagulant
in an amount sufficient to inhibit blood coagulation;
(b) introducing the sample obtained in (a) into a vessel having a
fiat bottom the inner surface of which is covered with a substrate
capable of inducing platelet adhesion thereto and aggregation;
(c) rotating said preparation inside the vessel, whereby shear
forces develop at said surface; and
(d) determining parameters of the adhered blood platelets, the
parameters being selected from the group consisting of amount of
adhered platelets aggregate size, aggregates' morphology, total
area covered by the aggregates and distribution of adhered
platelets or aggregates and or said substance.
Said substrate may be extracellular matrix (ECM), an active
component thereof or any other natural or artificially produced
substrates capable of inducing platelet adhesion thereto and
aggregation.
ECM is a matrix produced by endothelial cells such as corneal or
vascular endothelial cells usually obtained from bovine or human
sources. The ECM may be produced by culturing the cells inside said
the vessel and then removing the cells after production of the ECM
(Gospodarowicz D., et al., Endocr. Rev., 1, 201-207 (1980)). In
addition, rather than using ECM, the surface may also be covered by
various components thereof or artificially produced analogs which
are capable of inducing the primary hemostasis, such as basement
membrane matrix (e.g. MATRIGEL.TM., Flow Laboratories Inc., U.S.A.;
Kleinman H., et al., Biochemistry, 25, 312-318 (1986)), fibrilar
anti non-fibrilar collagen of various types, von Willebrand factor,
fibronectin, etc.
In the following description the invention will be described at
times with the reference to the use of ECM, it being understood
that this is done for the sake of convenience only and other
matrices, e.g. such consisting of said active component or analogs
may equally be used.
The tested blood sample may be whole blood, i.e. blood including
all its cellular components, or may be a platelet rich plasma
(PRP). The sample is optionally mixed with an anti-coagulant which
is added in order to neutralize the effects of the secondary
hemostasis mechanism. The anti-coagulant can be any anti-coagulants
known in the art, such as trisodium citrate, hirudin, heparin,
etc.
The rotation of the fluid inside the vessel, and hence the shear
forces, can be induced by a number of means. In accordance with one
embodiment of the invention, the shear forces are produced by
rotation of the vessel. In accordance with another embodiment,
which is preferred in accordance with the present invention, the
vessel is stationary and the rotation of the fluid is produced by a
rotating element inside the vessel. The vessel in accordance with
this embodiment is preferably cylindrical.
The rotating clement according to the second embodiment of the
invention is preferably cylindrical. In such a case, the shear
forces acting on the platelets gradually increase from the center
of the vessel towards the periphery. This embodiment is useful, for
example, when it is desired to evaluate, in a single test, the
primary hemostasis under varying shear force conditions. The
difference between platelet adhesion and aggregation at the center
of the ECM covered surface, where shear forces are in such a case
relatively low, and the periphery where shear forces are relatively
higher, can provide an indication of differences in platelet
function in primary hemostasis in various blood vessels in which
the platelets are subjected to different shear forces.
The rotating element has preferably a bottom portion having the
shape of an inverted cone. e.g. a "cone-plate" device (Ikeda et
al., J. Clin. Invest., 87, 1234-1240 (1991)). In such a device the
bottom, sloped faces of the cone are made at an angle, e.g. up to
about 3.degree. from horizontal, which is calculated to essentially
neutralize the effect of the difference in velocity of fluid
rotated by the element between its center and the periphery, and
thus the shear forces produced on the ECM covered bottom surface of
the vessel are essentially equal throughout the entire surface
(with the exception of the very periphery due to borderline
conditions which exist there). Thus, according to this embodiment,
the adhesion and aggregation of platelets to the ECM covered
surface are a result of an essentially uniform shear force acting
on the platelets regardless of their position.
After the blood sample is placed within the vessel, the blood is
rotated for a duration of 10 seconds to 10 mins., e.g. about 2
min., at a shear force of, which may for example be, in the range
of 50-3000 sec.sup.-1, preferably in the range of 100-2000
sec.sup.-1.
The adhesion and aggregation of platelets onto the ECM is
determined by any of a number of means known per se such as optical
inspection using a light microscope after appropriate staining, by
the use of a scanning electron microscope, determining changes in
light absorbance or transmission, etc. The determination may also
involve image analysis using various image analysis systems
(IAS).
The present invention also provides an apparatus for carrying out
the method in accordance with the above preferred embodiment of the
invention. The apparatus comprises a fiat bottomed vessel, and a
rotating element within the vessel. The inner surface of the bottom
of the vessel is covered with said substrate.
In accordance with a preferred embodiment, the rotating element has
a bottom portion having the shape of an inverted cone as described
above.
The rotating element in the apparatus may be driven by a number of
means, e.g. by direct mechanical coupling to a rotating motor or by
means of magnetic coupling to an external magnetic driving means
("magnetic stirrer").
The present invention thus provides means for evaluating primary
hemostasis in a manner which will yield results which have a
physiological significance, as the condition in which the adhesion
and aggregation of the platelets is induced, closely resemble
physiological conditions prevailing in the blood vessel.
Furthermore, the invention may be carried out by the use of
relatively simple and inexpensive instrumentation.
The present invention is suitable for determining both clinical
situations of low function of the primary hemostasis, giving rise
to risks of hemorrhages, as well as cases of pathologically high
rate of primary hemostasis, which increase the risk of
thrombosis.
The invention will now be described by some non-limiting
embodiments with occasional reference to the annexed drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of an apparatus of the
invention;
FIGS. 2A-B show a scanning electron microscope (SEM) picture
depicting the formation of aggregate adhered to ECM in control
blood (magnification: .times.745) under low (I) and high (h) shear
force conditions;
FIGS. 3A-B show an SEM picture depicting the formation of aggregate
adhered to ECM in blood of patients suffering from Glanzmann's
Thrombasthenia (magnification: .times.745) under low (I) and high
(h) shear force conditions;
FIGS. 4A-B show an SEM picture depicting the formation of aggregate
adhered to ECM in blood of patients suffering from von Willebrand's
disease (magnification: .times.1000) under low (I) and high (h)
shear force conditions;
FIGS. 5A-B show an SEM picture depicting the formation of aggregate
adhered to ECM in blood of patients suffering from Afibrinogenemia
(magnification: I-.times.1000; h-.times.1700) under low (I) and
high (h) shear force conditions;
FIGS. 6A-B show an SEM picture depicting the formation of aggregate
adhered to ECM in tested blood samples supplied with (A) von
Willebrand's blockers (VWFf) and (B) integrin receptor blockers
(RGDS) (magnification: vWF.sub.f, -.times.745;
RGDS-.times.1500);
FIGS. 7A-C show a particle size histogram of representative
results, obtained by image analysis, of size of platelet aggregates
adhered to ECM of blood from normal subjects (A), subjects
suffering from von Villebrand's disease (B) and from subjects
having Glanzmann's Thrombasthenia (C);
FIG. 8 shows the surface coverage and the mean aggregate size of
platelet particles (either single or aggregated) adhered onto the
ECM surface as a function of incubation time under either low (100
sec.sup.-1) or high (1000 sec.sup.-1) shear conditions;
FIG. 9 shows the surface coverage and the mean aggregate size of
platelet particles (either single or aggregated) adhered onto the
ECM surface as a function of the shear rate; and
FIGS. 10A-C show particle size histograms of platelet particles
adhered onto the ECM surface from blood from patients before
coronary angioplastic (A), blood from normal subjects (B) and blood
from patients under asperin therapy (C).
DESCRIPTION OF SPECIFIC EMBODIMENTS
Reference is first made to FIG. 1 which is a schematic
representation of an apparatus according to one embodiment of the
invention. The apparatus in accordance with this embodiment
comprises a cone-plate device 10 comprising a cylindrical vessel 11
and a rotating element 12 a bottom portion 13 having the shape of
an inverted cone. The rotating element 12 holds inside a magnetic
bar 14. The upper portion 15 of the clement 12 is held inside
stopper 16, whereby element 12 is suspended from the stopper inside
vessel 11 and can rotate freely. The bottom surface 17 of vessel 11
is covered by ECM 18. The device 10 is placed on a magnetic stirrer
apparatus 19.
In operation, a blood sample, optionally mixed with an
anti-coagulant, is introduced into the vessel 11, and then the
stopper 16 with the suspended element 12 is placed on the vessel.
The device is then placed on the magnetic stirring apparatus 19,
the operation of which creates a rotating magnetic field which
cause the rotation of element 12. Consequently, shear forces are
produced on the ECM covered surface 18, which is essentially
constant throughout the entire surface.
EXAMPLE I
Qualitative Evaluation by Scanning Electron Microscope (SEM)
Whole blood was collected from normal volunteers or patients with
an hemostatic disorder, including Glanzman's thrombasthenia (GT),
von Willebrand disease (vWD) Afibrinogenemia (AF) and other
non-defined abnormalities. Samples were collected also from
patients before coronary bypass with apparently increased primary
hemostasis who are predisposed to thrombosis.
To each sample, 0.38% of sodium citrate which acts as an
anti-coagulant, was added.
200 .mu.l-500 .mu.l of the citrated blood was added to the
apparatus described above with or without pre-incubation with
blockers or their controls.
The sample was subjected to a low or high shear force for 2 min, by
selecting the corresponding speed of the rotating disk: for low
shear force the sample was rotated at a speed of 100-200 rpm, and
for high shear force, the sample was rotated at a speed of
1000-2000 rpm. In the specific setting the rotating speed (rpm)
corresponded exactly to the shear force which was applied (in units
of sec.sup.-1).
The sample was then washed by phosphate buffered saline and the
evaluation of the level of adherence and aggregation on the ECM was
carried out by examination under a scanning electron microscope
(SEM).
FIG. 2 shows SEM pictures of normal blood samples tested under low
(l) shear force conditions (100 sec.sup.-1) and under high (h)
shear force conditions (1000 sec.sup.-1). As can be seen, normal
blood showed a reproducible mode of adherence/aggregation in both
of the shear forces employed.
When blood from different patients with hemostatic abnormalities
was tested in the hemostatometer a clear decrease in both the
adhesion and the aggregation rate was observed. The following are
some examples of such testings:
Glanzmann Thrombasthenia (GT) (FIG. 3)--There was a total lack of
aggregate formation in both low (l) and high (h) shear force and
the single adhered platelets were only partially spread, exhibiting
some pseudopodia formation.
von Willebrand Disease (vWD) (FIG. 4)--There was no aggregate
formation in both high (h) and low (l) shear force conditions and
the adhered platelets are exhibiting partial spreading (comparable
to the spreading of normal platelets on a plastic surface).
The pathologic aggregation of vWd platelets could partially be
corrected by either in vitro or in vivo treatment with vWF (data
not shown).
Afibrinogenemia (AF) (FIG. 5)--Afibrinogenemic platelets present a
pathologic interaction with the ECM with some pseudopodia formation
of the single adhered platelets. While absolute no aggregate
formation was found in the low (l) shear conditions experiment,
there was a definite aggregation when high (h) shear force was
applied. These data were in accordance with the concept that at
high (h) shear conditions (which correspond to arterial conditions)
vWF is the major interaction ligand that mediate platelets with ECM
and platelet-plalelet interaction.
Specific Receptor Blockers (FIG. 6)--The application of both a vWF
fragment which serves as a vWF blocker (VWFf) and the Integrin
receptor blocker Arginine-Glycine-Aspwotic-Serin tetra peptide
(RGDS) at concentrations of 4 .mu.M and 15 .mu.M, respectively,
under high shear force conditions in the apparatus of the invention
created pathologic interaction of platelets with ECM. The effect of
the specific receptor blockers was comparable to the corresponded
diseases vWD and GT, respectively.
EXAMPLE II
Quantitative Evaluation by an Image Analysis System (IAS)
Normal platelets in citrated whole blood were circulated over ECM,
using the device represented in FIG. 1 at varying degrees of shear
rates as indicated below. The shearing action was applied for
various periods of time ranging from 15 seconds to 5 mins.
The ECM surfaces were then examined by an IAS. The parameters that
could be evaluated in the system included:
(i) percentage of total area covered by adhered platelet
aggregation (which is designated in graphs shown below as percent
surface coverage);
(ii) mean size of observed objects (indicated in the graph below as
"mean particle size");
(iii) percentage of single adhered/spread platelets (mean area of
5-6 .mu.m.sup.2);
(iv) percentage of platelet aggregates (aggregates having a mean
area larger than 14 .mu.m.sup.2).
FIG. 7 shows representative results from three cases: normal
subjects (A), subjects suffering from von Willebrand's Disease (B)
and subjects suffering from Glanzmann's Thrombasthenia (C). As can
be seen in diseased subjects, there was a dramatic reduction in all
the above four parameters, except the percentage of the single
adhered platelets (the first column in the histogram).
The results obtained from normal subjects in patients of various
diseases are summarized in the following Table 1:
TABLE I
__________________________________________________________________________
Surface Coverage Mean % Single % Aggregates (% total area) size
(mm.sup.2) platelets >40 .mu.m.sup.2
__________________________________________________________________________
Control 100s.sup.-1 9.5 .+-. 1.7 24.1 .+-. 2.3 51.7 .+-. 8.7 12.8
.+-. 3.3 Control 1500s.sup.-1 20.1 .+-. 6.7 42.3 .+-. 11.8 37.7
.+-. 9.3 37.0 .+-. 6.5 vWD 100s.sup.-1 5.3 .+-. 1.7 13.8 .+-. 1.3
87.0 .+-. 2.0 0 vWD 1500s.sup.-1 3.7 .+-. 0.1 12.5 .+-. 0.1 92.0
.+-. 1.3 0 GT 100s.sup.-1 3.9 .+-. 1.8 10.5 .+-. 1.2 92.0 .+-. 3.0
0 GT 1500s.sup.-1 1.8 .+-. 0.5 12.6 .+-. 0.2 92.0 .+-. 1.0 0 AF
100s.sup.-1 4.4 14.2 83 0 AF 1500s.sup.-1 8.5 24.9 64 10
__________________________________________________________________________
As can be seen from the above table, the difference between normal
and control is apparent in all tested parameters and the observed
changes are apparent following applications of both low
(100s.sup.-1) or high (1500s.sup.-1) shear rates.
The time course of platelet deposition at both low and high shear
rate is shown in FIG. 8. As can be seen, the deposition progresses
with time, as evidenced both by the increase in surface coverage
and in the mean particle size. Furthermore, as can be seen, at both
shear rates, there was a time dependent increase in the percentage
of surface coverage as well as in the mean size of ECM-bound
particles (mean particle size). Maximum values of surface coverage
in mean aggregate size were obtained after about 2 mins. of blood
circulation at both low and high shear rate conditions.
The rate of platelet deposition on ECM (% surface coverage and mean
particle size) as a function of shear rate (shear force applied for
1 min.) is shown in FIG. 9. As can be seen, there is no significant
variation in the percentage of surface coverage or in the mean
aggregate size between shear rate of 100 to 1000 sec.sup.-1.
However, at 1500 sec.sup.-1, a significant increase in both
parameters can be observed.
On the basis of the time course and shear rate dependence
experiments, optimal conditions can be chosen: circulation period
of about 2 mins, which was found to be sufficient to achieve
maximal surface coverage and maximal mean aggregate size at both
high and low shear rates; a choice of 100 sec.sup.-1 as a low shear
rate and a 1500 sec.sup.-1 as a high shear rate.
Normal platelets in the citrated whole blood were circulated over
ECM for 2 mins. at either high or low shear rate. At both shear
rates, platelets adhered and aggregated onto this ECM. The extent
of aggregation, however, was significantly higher at the high shear
rate. Incubation of whole blood with ECM at high shear rate yielded
aggregates which were much larger than those formed under low shear
conditions (see FIG. 9 and Table I). The percentage of large
aggregates (>40 .mu.m.sup.2) formed at high shear rate was
37.0%.+-.6.54%, as compared to 12.83%.+-.3.25% at the low shear
rate. The higher extent of platelet aggregation at the high shear
rate was also reflected by the larger mean size of ECM-bound
particles (42.26.+-.11.78 .mu.m.sup.2 at high shear rate, as
compared to 24.06.+-.2.34 .mu.m.sup.2 at low shear rate), as well
as by a higher percentage of surface coverage (see Table I).
Incubation on ECM of citrated whole blood taken from either type
III vWD patient or a GT patient resulted in limited platelet
adhesion without aggregation at both high and low shear rates
(FIGS. 3 and 4, as well as Table I). At both low and high shear
rates, surface coverage, as well as the mean size of ECM-bound
particles were greatly reduced in both vWD and GT samples, when
compared to the corresponding normal control values. Both vWD and
GT samples have demonstrated a more pronounced decrease in surface
coverage when high shear rate conditions were applied.
Under low shear conditions, platelets in fibrinogen deficient blood
exhibited adhesion to ECM without aggregate formation (see FIG. 5
and Table I). In contrast, at high shear rate, both adhesion and
aggregation could be observed. The parameters of platelet
deposition on ECM obtained from Afibrinogenemic blood under high
shear rate conditions were very similar to those observed in
control samples at low shear rate (see Table I). These findings are
in agreement with previous reports showing that platelet
aggregation can occur in the absence of fibrinogen at high, but not
at low shear conditions.
The potential of the method of the invention to identify
hyperactive hemostasis (and prethrombotic state) is demonstrated in
FIG. 10. This figure shows an IAS evaluation of normal subjects
(middle panel), patients under aspirin therapy (lower panel) and
patients before coronary angioplasty (upper panel). As can be seen,
there is a clear difference in all parameters between the different
panels, suggesting a higher adhesion and aggregation rate in the
coronary heart disease patient and a reduced adhesion in the
aspirin treated patients.
* * * * *