Fibrinogen-coated albumin microcapsules reduce bleeding in severely thrombocytopenic
rabbits
Marcel Levi1, Philip W. Friederich1, Sarah Middleton2, Philip G. de Groot3, Ya Ping Wu3, Roy Harris2, Bart J. Biemond1, Harry F.G. Heijnen3, Jack Levin4
& Jan Wouter ten Cate1
1 Center for Hemostasis, Thrombosis, Atherosclerosis
and Inflammation Research, Academic Medical Center, University of Amsterdam
, Meibergdreef 9, 1105 AZ
Amsterdam, the Netherlands
3 Department of Hematology, University of Utrecht,
P.O. Box 85500, 3508 GA Utrecht,
the Netherlands
4 Department of Laboratory Medicine, University of California
School of Medicine, and VA Medical Center, 4150 Clement Street
, San Francisco, California 94121,
USA
Severe thrombocytopenia frequently occurs in patients receiving chemotherapy
and in patients with autoimmune disorders. Thrombocytopenia is associated
with bleeding, which may be serious and life threatening1,
2,
3.
Current treatment strategies for thrombocytopenia may require transfusion
of allogeneic platelets, which is associated with serious drawbacks4.
These include the occurrence of anti-platelet antibodies, which may result
in refractoriness to further platelet transfusions, and the potential risk
of transfer of blood-borne diseases5,
6. Therefore, we have
recently developed a platelet substitute product (Synthocytes), which is composed
of human albumin microcapsules with fibrinogen immobilized on their surface.
Here we show that the intravenous administration of these microcapsules not
only corrects the prolonged bleeding time in rabbits rendered thrombocytopenic
either by anti-platelet antibodies or by chemotherapy, but also reduces bleeding
from surgical wounds inflicted in the abdominal skin and musculature. No potential
systemic prothrombotic effect of the microcapsules was observed in a model
of rabbit venous thrombosis. As for the mechanism of action, experiments with
normal and thrombocytopenic human blood in an endothelial cell matrix-coated
perfusion chamber demonstrated an interaction between the fibrinogen-coated
albumin microcapsules and native platelets. It was shown that the fibrinogen-coated
albumin microcapsules could facilitate platelet adhesion to endothelial cell
matrix and correct the impaired formation of platelet aggregates in relatively
platelet-poor blood. This study indicates that fibrinogen-coated albumin microcapsules
can act to improve primary hemostasis under thrombocytopenic conditions and
may eventually be a promising agent for prophylaxis and treatment of bleeding
in patients with severe thrombocytopenia.
To determine the effect of the fibrinogen-coated albumin microcapsules
on thrombocytopenic bleeding, we used models of immune thrombocytopenia and
of chemotherapy-induced thrombocytopenia in rabbits. In rabbits that had received
an intravenous bolus injection of goat anti-rabbit platelet antibodies, there
was a rapid and substantial decline of the platelet count from 452 (87)
109 per liter to 30 (13) 10
9 per liter (Fig. 1a). The thrombocytopenia
was associated with a substantial increase in the ear template bleeding time
from 1.7 (0.4) minutes to 21.7 (4.4) minutes (
Fig. 1b). A subsequent intravenous bolus injection of fibrinogen-coated
microcapsules at a dose of 1.5 109 microcapsules/kg
or 0.75 109 microcapsules/kg significantly shortened
the prolonged bleeding time to 5.2 (1.7) minutes (P < 0.001)
and 6.5 (1.7) minutes (P < 0.001), respectively, at 15 minutes after
the injection. At 60 minutes, the correction of the bleeding time was still
apparent, although the bleeding time in the rabbits that were treated with
the lower dose of Synthocytes was longer than in the rabbits that had received
the higher dose of Synthocytes (8.8 (2.1) minutes in the low-dose
group compared with 4.3 (0.8) minutes in the high-dose group;
P < 0.05). Two control groups of rabbits that had received saline or
non-coated albumin microcapsules did not show any change in the prolonged
bleeding time throughout the period of observation (Fig. 1
b).
Figure 1. Effect of the bolus administration of goat antibody against rabbit
platelet and the subsequent administration of fibrinogen-coated albumin microcapsules
(Synthocytes) or control agents (n = 6 per group).
a, Platelet count before the administration of the anti-platelet
antibody (−30 minutes) and throughout the experiment. , Synthocytes
1.5 109/kg; , Synthocytes 0.75 10
9/kg; , non-coated albumin microcapsules 1.5 10
9/kg; and , saline. Differences in platelet levels between the
groups were not statistically significant. b, Template ear bleeding
time measurements at the start of the experiment (−30 minutes), 30 minutes
after the administration of the anti-platelet antibody but before the administration
of the study agents (0 minutes), and at 15 and 60 minutes after the administration
of Synthocytes, 1.5 109/kg (first bar at each time
point); Synthocytes, 0.75 109/kg (second bar); non-coated
albumin microcapsules; 1.5 109/kg (third bar); and
saline (fourth bar). The administration of the fibrinogen-coated albumin microcapsules
resulted in a statistically significant shortening of the prolonged bleeding
time,* P < 0.001,compared with saline or non-coated albumin microcapsules).
At 60 minutes after the administration of the relatively lower dose of Synthocytes,
the bleeding time was less reduced compared with the bleeding time after administration
of the higher dose, but remained significantly shorter than in the controls
(#, P < 0.05).
In a model of chemotherapy-induced thrombocytopenia produced by repeated
busulfan administration, a similar degree of thrombocytopenia was observed,
without reduction of the hemoglobin or white blood cell levels (
Table). Busulfan-treated rabbits with a mean platelet count of 15
(5.2) 109 per liter had a prolonged ear template
bleeding time of 18.6 (2.1) minutes and a blood loss from a standardized
abdominal surgical incision of 2354 (351) mg per 5 minutes (Fig. 2). The administration of the fibrinogen-coated albumin
microcapsules (1.5 109 microcapsules/kg) resulted in
a shortening of the ear bleeding time to 5.3 (2.7) minutes, 28% of
the bleeding time before microcapsule administration, and a reduction of the
blood loss from the surgical incision to 276 (102) mg per 5 minutes,
12% of the pre-administration blood loss. Each effect lasted for at least
3 hours. In rabbits treated with a lower dose of Synthocytes (0.75
109 microcapsules/kg), a similar effect was observed initially.
Although at 2 and 3 hours after the bolus administration of the lower dose
the shortening of the bleeding time gradually lessened, both measures of hemostasis
remained significantly improved. At 3 hours after the administration of the
lower dose, the ear bleeding time was 12.5 (2.5) minutes and the blood
loss from the surgical incision 923 (121) mg per 5 minutes, compared
with 7.9 (1.9) minutes and 400 (61.9) mg per 5 minutes in
the rabbits that had received 1.5 109 microcapsules/kg
(P < 0.05). At 8 and 24 hours after administration of the fibrinogen-coated
albumin microcapsules, however, no substantial reduction in ear bleeding time
or surgical wound bleeding remained detectable (Fig. 2).
Figure 2. Effect of the administration of fibrinogen-coated microcapsules
(Synthocytes: , 1.5 109/kg; , 0.75
109/kg), non-coated albumin microcapsules (,1.5
109/kg), and , saline on the ear bleeding time (
a) and the 5-minute blood loss from a standardized abdominal surgical
incision (b) in busulfan-treated thrombocytopenic rabbits (n
= 6 per group). Statistical significance compared with saline
or non-coated albumin microcapsules is indicated: *, P < 0.001;
, P < 0.01. At 120 and 180 minutes after the administration of Synthocytes,
the difference in effect between the two doses of Synthocytes is statistically
significant (#, P < 0.05).
To exclude the possibility of a systemic prothrombotic effect of the fibrinogen-coated
microcapsules, we investigated their effect on thrombus accretion in a rabbit
jugular vein thrombosis model. Thrombus growth in control animals 2 hours
after treatment with saline or non-coated albumin microcapsules was 46.6 (4.5)%
and 49.6 (3.7)%, respectively, of initial thrombus volume. The administration
of fibrinogen-coated microcapsules (1.5 109 microcapsules/kg)
did not significantly affect thrombus accretion (50.3 (5.0)%;
P, not significant compared with controls). Similarly, at 4 hours after
administration of the fibrinogen-coated microcapsules or saline, there was
no difference in thrombus growth between the groups (64.4 (7.1)% after
fibrinogen-coated microcapsules and 69.8 (6.6%) after saline administration;
P, not significant).
Single-dose toxicity studies in rats and cynomolgus monkeys, using histological
analysis of the organs up to 15 days after dosing, did not demonstrate adverse
effects of the administration of fibrinogen-coated albumin microcapsules at
doses up to 7.5 109 microcapsules/kg. In addition,
studies of cardiorespiratory function and pulmonary resistance and compliance
in rabbits and dogs did not show substantial effects of the administration
of fibrinogen-coated albumin microcapsules (at doses up to 7.5 10
9 microcapsules/kg) on hemodynamics, heart rate and rhythm, blood
gases or other pulmonary parameters.
Studies of the tissue distribution and clearance from the blood of
125I-labelled fibrinogen-coated albumin microcapsules showed that
the amount of microcapsules in the circulation initially rapidly declined
to about 15% of the total injected at 15 minutes after administration and
disappeared more gradually thereafter. A rapid tissue distribution of the
albumin microcapsules was observed, with peak levels of radioactivity in lungs
and liver (28.8% and 41.5% of the injected dose, respectively) occurring between
5 minutes and 1 hour. Thereafter, levels of radioactivity gradually declined
and were no longer detectable at 24 to 72 hours after administration of the
radiolabelled fibrinogen-coated albumin microcapsules.
To determine the mechanism of action of the fibrinogen-coated albumin microcapsules
and to investigate whether they could also function in human blood, we did
experiments in which the microcapsules (40 103 per l)
were added to whole human blood and perfused over a PMA-stimulated endothelial
cell matrix for 5 minutes at a shear rate of 300 per second. When the fibrinogen-coated
albumin microcapsules were added to relatively platelet-poor blood (40
109 platelets/l) the surface covered with aggregates increased
from 4.6 (0.9)% to 8.6 (0.7)% (P < 0.01). The addition
of the fibrinogen-coated albumin microcapsules to normal blood (containing
160 109 platelets/l) resulted in an enhancement of the area
covered with aggregates from 12.0 (0.8)% to 16.4 (1.6)% (
P < 0.01). Addition of non-coated albumin microcapsules (control) did
not influence total aggregate formation in either normal or thrombocytopenic
blood. Incubation of blood with the potent and specific platelet inhibitor
dRGDW resulted in a complete prevention of fibrinogen-coated albumin microcapsule-induced
aggregate formation, whereas incubation with the thrombin inhibitor hirudin
inhibited aggregate formation from 16.2 (0.2)% to 10.4 (1.9)%
(P < 0.01). These results may indicate a direct interaction between
platelets and microcapsules, and a partial role for thrombin and fibrin strand
formation in the action of the microcapsules. When the coverslips were examined
with scanning electron microscopy to visualize the interaction between the
fibrinogen-coated albumin microcapsules and platelets, combined aggregates
of platelets and microcapsules, with connecting fibrin fibers, were seen (Fig. 3). Moreover, in biopsies taken from bleeding-time wounds
in the rabbit experiments, the presence of the fibrinogen-coated albumin microcapsules
surrounded by platelets and fibrin was confirmed (Fig. 4).
Figure 3. Scanning electron microscope photograph showing the interaction of
the fibrinogen-coated albumin microcapsules and platelet aggregates with connecting
fibrin fibers.
Figure 4. Micrograph of a biopsy from a bleeding-time wound of a rabbit 15 minutes
after the administration of fibrinogen-coated albumin microcapsules (1.5
109 microcapsules/kg), showing the presence of the microspheres
at the site of the hemostatic plug, surrounded by platelets and fibrin.
Thus, the administration of fibrinogen-coated albumin microcapsules results
in a significant reduction of enhanced bleeding in rabbits made thrombocytopenic
with either platelet antiserum or with chemotherapy. These results are in
agreement with preliminary observations using another preparation of cross-linked
human serum albumin spheres7. The mechanism of the Synthocytes-induced
improvement of primary hemostasis seems to rely on the facilitation of adhesion
of the remaining platelets to the endothelium. A limitation of our observations
may be that measurement of the bleeding time may represent a function of the
primary hemostatic system in vivo, but may poorly predict the occurrence
of bleeding in clinical situations8. Therefore, studies in patients
with severe thrombocytopenia are required to establish a beneficial effect
of this agent in the treatment or prevention of thrombocytopenic bleeding.
However, our data here indicate that fibrinogen-coated albumin microcapsules
may be an effective new replacement therapy for platelet transfusion in the
management of thrombocytopenia.
Methods Production and characterization of fibrinogen-coated microcapsules.
Human albumin and human fibrinogen were recovered from large
'pools' of human plasma by low-temperature controlled fractionation using
the Cohn process. Albumin microcapsules were formed by spray-drying a 10%
weight/volume solution of human albumin. The albumin was atomized with compressed
air and the microcapsules were subsequently formed by rapid drying of the
droplets. Control of the pressure of the atomization air and the feed rate
limited the size and size distribution of the microcapsules to a median diameter
of 3.5−4.5 m. Less than 2% of the microcapsules were greater than
6 m in diameter, to facilitate transit across the pulmonary capillary
bed. Fibrinogen was immobilized onto the surface of sterile albumin microcapsules
by incubation for up to 4 hours at a controlled ionic strength at a pH range
of 6.0−6.5. Tangential flow microfiltration was used to remove unbound
fibrinogen, and the washed fibrinogen-coated microcapsules were suspended
in 25 mM phosphate buffered isotonic mannitol, to achieve a final concentration
of 1.5 109 microcapsules/ml (total protein 20 mg/ml).
Fibrinogen bound to the microcapsules was determined by a sandwich ELISA using
a polyclonal antibody to human fibrinogen to capture the immobilized fibrinogen.
The fibrinogen concentration represents less than 2% of the total protein
of the microcapsules. Activity of the immobilized fibrinogen was confirmed
by measuring the rate of aggregation of the fibrinogen-coated microcapsules
after the addition of thrombin.
Immune and busulfan-induced thrombocytopenia models. The studies were approved by the Institutional Animal Experiment Committee
and were done according to the Dutch Law on Animal Experiments and according
to the guidelines of the American Physiological Society. New Zealand white
rabbits (approximately 2.5 kg) were anesthetized with an intramuscular injection
of a 'cocktail' of 9 mg (1.5 mL) Ketamin (Aescoket, Boxtel, the Netherlands)
and 0.5 ml Rompun 2% (Bayer, Leverkusen, Germany) in 3 compared with 1 volume
units. To maintain adequate anesthesia, intramuscular injections of Ketamin
were repeated when appropriate.
To produce acute immune thrombocytopenia, the rabbits received a bolus
infusion for 5 minutes of goat polyclonal antibody against rabbit platelet
(0.8 ml/rabbit), derived from goats that had been repeatedly immunized with
washed rabbit platelets. The antiserum had been adsorbed with washed rabbit
red blood cells to remove anti-erythrocyte antibodies9,
10.
A busulfan-induced thrombocytopenia model was used as described11.
On day −12 and day −9 (day 0 = day of bleeding time experiments0,
rabbits received a subcutaneous injection of busulfan (1,4 butanediol dimethanesulfonate;
Sigma) in polyethylene glycol (average MW 400, Sigma) at a dose of 15 mg/kg
on day -12 and 10 mg/kg on day −9. These injections invariably resulted
in a reduction of the platelet count on day 0 to 10 109−20
109 per liter.
Ear bleeding time and 5-minute blood loss. Operators
who were not aware of the treatment allocation of the rabbits measured bleeding
time and blood loss in this controlled experiment. The template ear bleeding
time was measured using a Surgicutt device (International Technidyne, Edison,
New Jersey). The dorsal surface of the rabbit ear was shaved and a standardized
incision was made at a site where no visible vessel was seen. Blood from the
incision was carefully removed at exactly 15-second intervals with a filter
paper, until bleeding completely stopped10,
12.
The 5-minute blood loss from a standardized surgical incision was measured
as reported13. Using a template, a standard surgical incision
5 cm long and 0.5 cm deep was made in the anterior abdominal wall, incising
the first layer of the anterior abdominal wall muscles. A pre-weighed gauze
pad (5 5 cm) was placed in the incision and left in place for 5 minutes.
Then, the gauze pad was removed and its increase in weight was determined
as an objective measure of blood loss (normal value 150−300 mg per 5
minutes).
To examine the presence of the fibrinogen-coated albumin microcapsules
at the site of the hemostatic plug, biopsies were taken from bleeding-time
wounds that were made 15 minutes after injection of the fibrinogen-coated
albumin microcapsules. The biopsies were immediately immersed in 2% glutaraldehyde,
3% paraformaldehyde in 0.1 M cacodylate buffer, postfixed for 24 hours, dehydrated
in methanol, and stained with May-Grünwald-Giemsa.
Thrombus growth model. New Zealand white rabbits (approximately
2.5 kg) were anesthetized as described above. The jugular veins were exposed
by a median incision in the neck and were cleared at both sites for a distance
of 2 cm; all side branches were ligated. The venous segments were isolated
by application of vessel clamps proximally and distally. To assess the extent
of thrombus growth, non-radiolabeled thrombi were formed in both isolated
jugular vein segments by injection of 150 l homologous rabbit blood, which
had been previously aspirated into a 1 ml syringe containing 25 l human
thrombin (Human Thrombin T7009, 150 U/ml; Sigma;) and 45 l 0.25 MCaCl
2, into the isolated venous segments14. After 30 minutes
of 'aging', blood flow was restored by removing the vessel clamps, and 100 l
of 125I-radiolabeled human fibrinogen (approximately 2 Ci/ml;
Amersham) was injected intravenously, followed immediately by the intravenous
administration of the study compounds. Thrombi were removed 2 hours or 4 hours
after restoration of the blood flow and administration of the study medication.
Thrombus growth was assessed by measuring the accretion of 125I-radiolabeled
human fibrinogen onto the pre-formed non-radioactive thrombi, and was expressed
as percentage of the initial thrombus volume (200 l).
125I-labelled fibrinogen-coated albumin microcapsules.
Fibrinogen-coated albumin microcapsules were radiolabelled with
125I, using the Iodogen method. Stability of the label was confirmed
by incubation in vitro in plasma for 72 hours. Rats were injected with
the radiolabelled microcapsules and tissue distribution of radioactivity was
measured in organs, tissues and body fluids at 5, 15 and 30 minutes and 1,
6, 24 and 72 hours after the administration of the compound (n = 4
per time point).
Perfusion experiments. Perfusion experiments were done
in a parallel perfusion chamber15. Whole blood, obtained by
venipuncture from healthy subjects taking no medication, was anticoagulated
with 1/10 (volume/volume) low-molecular-weight heparin (dalteparin, 50 U/ml;
Pharmacia). To vary the platelet count of the blood, platelets and red cells
were separated from the blood and washed, and the blood was subsequently reconstituted
to obtain a hematocrit of 40% (volume/volume) and the desired platelet count16. The fibrinogen-coated albumin microcapsules or control non-coated
albumin microcapsules (40 103/l) were added to
the blood and the mixture was prewarmed at 37 °C for 10 minutes. The perfusate
was then recirculated though the perfusion chamber for 5 min at a wall shear
rate of 300 per second, over an endothelial cell matrix of endothelial cells
stimulated with PMA (4—-phorbol-12-myristate 13-acetate, 20 ng/ml for
6 hours; Sigma)(17). After perfusion, the
system was rinsed with HEPES-buffered saline (10 mM HEPES, 150 mM NaCl, pH
7.4). The coverslips were removed, fixed in 0.5% glutardialdehyde/PBS, dehydrated
in methanol, and stained with May-Grünwald-Giemsa. The extent of platelet
deposition was determined by light microscopy at a magnification of 1000
using an image analyzer that was interfaced with the microscope. The results
were expressed as the percentage surface covered with platelets. Similar experiments
were done with platelets from blood that was pre-incubated with either the
potent platelet inhibitor dRGDW (a synthetic RGD-containing peptide; Rhône-Poulenc
Rorer, Centre de Recherches de Vitry, France)(18)
or the thrombin inhibitor hirudin (30 U/ml; Ciba Geigy, Horsham, United Kingdom).
Platelet−microcapsule interactions were also examined using scanning
electron microscopy. Coverslips from the perfusion chamber were fixed in 2%
glutardialdehyde for 1 hour, postfixed in 1% osmium tetroxide (Sigma) for
1 hour, and then dehydrated through a graded ethanol series (50−100%).
Samples were 'sputtered' with a thin layer of platinum, and examined with
a scanning electron microscope (Philips XL30; Eindhoven, the Netherlands).
Statistical analysis. Statistical analysis was done
by ANOVA and the Newman-Keuls test. A P value < 0.05 was considered
statistically significant. All values are presented as mean s.d.
Roy, A.J., Jaffe, N. & Djerassi, I. Prophylactic platelet transfusions in children with acute leukemia. A dose response study. Transfusion13, 283-290 (1973). | PubMed | ISI |
Rebulla, P. et al. The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. N. Engl. J. Med.337, 1870-1875 (1997). | PubMed | ISI |
National Institute of Health Consensus Conference. Platelet transfusion therapy.Transfus. Med. Rev.1, 195-200 (1987). | PubMed |
Slichter, S.J. inHematology: Basic Principles and Practice 2nd edn. (eds. Hoffman, R. et al. ) 419-421 (Churchill Livingstone, New York, 1995).
The Trial to Reduce Alloimmunization to Platelets Study Group. Leucocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N. Engl. J. Med.337, 1861-1869 (1997). | PubMed |
Dodd, R.Y. The risk of transfusion-transmitted infection. N. Engl. J. Med.327, 419-421 (1992). | PubMed | ISI |
Yen, R.C.K., & Ho, T.W.C. Blajchman M.A. A novel approach to correcting the bleeding time in thrombocytopenic rabbits. Transfusion35, 41S (1995).
Rodgers, R.P. & Levin, J. A critical reappraisal of the bleeding time. Semin. Thromb. Hemost.16, 1-20 (1990). | PubMed | ISI |
Evatt, B.L., Levin, J. & Algazy, K.M. Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits. Blood54, 377-388 (1979). | PubMed | ISI |
Blajchman, M.A. & Lee, D.H. The thrombocytopenic rabbit bleeding time model to evaluate the in vivo hemostatic efficacy of platelets and platelet substitutes. Transf. Med. Rev.11, 99-105 (1997).
Kuter, D.J. & Rosenberg, R.D. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood85, 2710-2730 (1995).
Blajchman, M.A. et al. Shortening of the bleeding time in rabbits by hydrocortisone caused inhibition of prostacyclin generation by the vessel wall. J. Clin. Invest.63, 1026-1035 (1979). | PubMed | ISI |
Benedict, C.R. et al. New variant of human tissue plasminogen activator (TPA) with enhanced efficacy and lower incidence of bleeding compared with recombinant human TPA. Circulation92, 3032-3040 (1995). | PubMed | ISI |
Levi, M., Biemond, B.J., van Zonneveld, A.J., ten Cate, J.W. & Pannekoek, H.: Inhibition of plasminogen activator inhibitor 1 (PAI-1) activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation85, 305-312 (1992). | PubMed | ISI |
Sakariassen, K.S., Aarts, P.A., de Groot, P.G., Houdijk, W.P. & Sixma, J.J. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J. Lab. Clin. Med.102, 522-535 (1983). | PubMed | ISI |
Zwaginga, J.J. et al. Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts. Arteriosclerosis10, 437-448 (1990). | PubMed | ISI |
van Breugel, H.F., de Groot, P.G., Heethaar, R.M. & Sixma, J.J. Role of plasma viscosity in platelet adhesion. Blood80, 953-959 (1992). | PubMed | ISI |
Saelman, E.U.M. et al. Aggregate formation is more strongly inhibited at high shear rates by dRGDW, a synthetic RGD-containing peptide. Arterioscler. Thromb.13, 1164-1170 (1993). | PubMed | ISI |
87)
109 per liter to 30 (
, Synthocytes
1.5 
, Synthocytes 0.75 
, non-coated albumin microcapsules 1.5 
, saline. Differences in platelet levels between the
groups were not statistically significant. b, Template ear bleeding
time measurements at the start of the experiment (−30 minutes), 30 minutes
after the administration of the anti-platelet antibody but before the administration
of the study agents (0 minutes), and at 15 and 60 minutes after the administration
of Synthocytes, 1.5 
l)
were added to whole human blood and perfused over a PMA-stimulated endothelial
cell matrix for 5 minutes at a shear rate of 300 per second. When the fibrinogen-coated
albumin microcapsules were added to relatively platelet-poor blood (40 
