Download Proteolysis of the platelet surface: dissociation of shape change from

Proteolysis of the platelet surface:
dissociation of shape change from aggregation
ELIZABETH
KORNECKI,
AND ROBERT
H. LENOX
YIGAL
H. EHRLICH,
DAVID
H. HARDWICK,
Neuroscience Research Unit, Departments
of Psychiatry and Biochemistry,
University of Vermont College of Medicine, Burlington, Vermont 05405
Stimulation of intact platelets by ADP resultsin a shapechange
followed by aggregationin the presenceof fibrinogen. ADP was
found to induce a shapechangein chymotrypsin-treated platelets that was similar in extent and initial velocity to that of
intact (untreated) platelets. Scanning-electron microscopyverified an ADP-induced shape change in chymotrypsin-treated
platelets. This shapechange could be completely blocked by
stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation
of chymotrypsin-treated platelets by fibrinogen was not dependent on the presenceof ADP and could not be blocked by
forskolin, prostaglandin E1, or prostacyclin, even though the
levels of cyclic AMP (CAMP) formed in chymotrypsin-treated
platelets were comparable to levels that completely inhibited
the ADP-induced aggregation of intact platelets. This lack of
inhibition of platelet aggregationwasnot due to degradation of
the adenylate cyclase or prostaglandin receptors, sincechymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin,
prostaglandin E1, and prostacyclin and inhibited by ADP and
epinephrine, similar to that of intact platelets. These results
provide direct evidence that CAMP does not interact with
fibrinogen binding sites once they have becomepermanently
exposedon the surface of platelets. Pretreatment of platelets
with chymotrypsin therefore appearsto be a useful tool that
allowsfor the dissociationof platelet shapechangefrom aggregation, without inhibiting either response.
chymotrypsin treatment of human platelets; cyclic AMP; shape
change;fibrinogen-induced aggregation
MEMBRANES
contain fibrinogen receptors consisting of glycoproteins IIb and IIIa that are responsible
for platelet aggregation. Platelets that are not activated
do not bind radiolabeled fibrinogen; such fibrinogen receptors remain latent, allowing platelets to circulate as
single disks. However, when platelets are stimulated by
ADP (1, 12, 16, 25), thrombin
(9, 26), epinephrine
(1,
27), or prostaglandin
endoperoxides (2, 20), the platelets
change shape from disk to sphere, extend long pseudopods, and expose specific fibrinogen binding sites on the
cell surface. The exposure of these sites on adjoining
platelets results in fibrinogen binding and platelet aggrePLATELET
H550
0363-6135/86
gation. The aggregation and shape change of platelets
can be inhibited
by prostaglandin
E1, forskolin, and
prostacyclin I2 (8), agents that raise levels of intracellular
cyclic AMP (CAMP) by stimulating the adenylate cyclase
of platelets (7, 8, 10, 18, 19). Hawiger et al. (9) and
Graber and Hawiger (5) have shown that the binding of
radiolabeled fibrinogen to ADP or thrombin-activated
platelets can be inhibited by these agents.
Several laboratories have demonstrated that fibrinogen binding sites become exposed on the platelet surface
by pretreating platelets with proteolytic enzymes such as
chymotrypsin or pronase (6,13,23). These platelets have
been shown to specifically bind fibrinogen and to aggregate spontaneously
on the addition of fibrinogen. In
contrast to intact (untreated) platelets, proteolytically
treated platelets do not require the presence of ADP or
any other platelet agonist for fibrinogen binding and
aggregation. The treatment of platelets with chymotrypsin has been used as a tool for the identification
of
fibrinogen binding domains of the platelet fibrinogen
receptor (13, 17). Furthermore,
proteolysis by chymotrypsin provides a platelet model for the investigation of
pathological conditions in which chymotrypsin and other
enzymes are found in high concentrations in the circulation. The effects of pancreatic enzymes on platelet
activation have been implicated in the development of
systematic complications of pancreatitis (28). Proteolysis
of platelet surface glycoproteins in vivo has been associated with liver cirrhosis (24). Egbring et al. (3) measured
high concentrations of granulocyte elastase in the plasma
of patients with septicemia or acute leukemia, which may
be associated with thrombocytopenia.
Damage of fibrinogen receptors resulting in thrombocytopenia
and platelet dysfunction, concurrent with increased concentrations of neutrophil elastase, were found to occur during
simulated extracorporeal circulation (2 1, 35).
The purpose of the present study was to determine
whether treatment of the platelet surface with chymotrypsin has differential effects on platelet shape change,
aggregation, the platelet adenylate cyclase system, and
on the function of several receptors coupled to the system. The results demonstrate that treatment of the platelet surface with an extracellular protease provides a most
useful tool for investigating the mechanisms of receptormediated platelet functions.
$1.50 Copyright 0 1986 the American
Physiological Society
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KORNECKI, ELIZABETH,
YIGAL H. EHRLICH, DAVID H.
HARDWICK, AND ROBERT H. LENOX. Proteolysis of the platelet
surface: dissociation of shape change from aggregation. Am. J.
Physiol. 250 (Heart Circ. Physiol. 19): H550-H557, 1986.-
ACTIVATION
MATERIALS
AND
OF
PLATELETS
METHODS
SURFACE
PROTEOLYSIS
H551
Suspensions containing
10’ platelets/ml
were preincubated with chymotrypsin
(500 or 1,000 pg) for 1 h at
37OC. Control (untreated) platelet suspensions were incubated for 1 h at 37°C in the absence of chymotrypsin.
Both types of platelet suspensions were washed by centrifugation
for 10 min at 1,600 g and resuspended in
Tyrode-albumin
containing buffer at a concentration of
2 x log/ml. Four hundred fifty microliters of each suspension were aliquoted into an aggregometer cuvette
under stirring conditions at 37OC. Aliquots of ADP (50
~1; 50 PM, final concn) or distilled H20 were added to
both intact and chymotrypsin-treated
platelets. After 1
min, the platelets were fixed with 1.5% glutaraldehyde
(final concn) buffered with 0.1 M phosphate buffer, pH
7.4. After fixation, coverslips coated with platelets were
dehydrated in alcohol, critical point dried from Freon
mounted on carbon stubs, vacuum coated with goldpalladium, and examined in a JEOL JSM-35C scanning
electron microscope.
RESULTS
Human platelets that have been pretreated with various concentrations
of chymotrypsin
were aggregated
directly upon addition of fibrinogen (Fig. 1). In contrast
to intact (untreated) platelets, ADP was not necessary
for aggregation to occur. Moreover, the fibrinogen-induced aggregation of chymotrypsin-treated
platelets was
not accompanied by a shape change. The fibrinogeninduced aggregation was dependent on the concentration
of chymotrypsin
used during the preincubation
period.
Maximal aggregation to fibrinogen was observed with
platelets preincubated with 1,000 pg of chymotrypsin,
whereas minimal
fibrinogen-induced
aggregation occurred with platelets preincubated with less than 500 pg
of chymotrypsin.
To determine the changes in surface
proteins resulting from the preincubation
of platelets
with chymotrypsin,
the platelets were surface radiolabeled with 1251using the iodogen method (34). The results
of the surface labeling of intact and chymotrypsintreated platelets were identical to those reported in our
previous studies (13) using these concentrations of chymotrypsin. The main change was in the appearance of a
66-K dalton derivative of glycoprotein IIIa on the surface
of chymotrypsin-treated
platelets (13). As shown recently, this 66-K dalton protein could be immunoprecipitated from detergent-solubilized,
chymotrypsin-treated
platelets by human anti-PIA1 antibody (11).
The fibrinogen-induced
aggregation of chymotrypsintreated platelets was not inhibited by prostaglandin El,
forskolin, or prostacyclin. These agents, however, inhibited the ADP-induced,
fibrinogen-dependent
aggregation of intact (untreated) platelets (Fig. 2). We tested
whether the lack of inhibition
of fibrinogen-induced
aggregation of chymotrypsin-treated
platelets by these
agents could be due to the absence of prostaglandin
receptors on the platelet surface or due to degradation of
the adenylate cyclase complex by chymotrypsin
treatment. To test these possibilities,
we measured CAMP
accumulation
in both intact and chymotrypsin-treated
platelets. In response to prostaglandin
El, forskolin, or
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CoZZection of blood. Blood was obtained from healthy
individuals with the approval of the Institutional
Human
Experimentation
Committee
at the University of Vermont, Burlington,
VT.
Reagents. ADP, epinephrine, prostaglandin
El, forskolin, prostacyclin,
albumin,
chymotrypsin
(grade IS),
apyrase, and heparin were obtained from Sigma Chemical, St. Louis, MO. Kabi fibrinogen was purchased from
Helena Laboratories, Beaumont, TX.
Human washed platelets. Platelets from blood freshly
collected in the anticoagulant
acid-citrate dextrose were
washed in the presence of apyrase and heparin by the
method of Mustard et al. (22). Platelets were suspended
in Tyrode solution, pH 7.35, containing 0.35% albumin,
2 mM CaC12, 1 mM MgC12, 0.36 mM NaH2P04, 135 mM
NaCl, 2.68 mM KCl, 11.9 mM NaHC03, and 5.55 mM
glucose.
Platelet count. Platelets were counted by using a hemacytometer
and an Olympus phase-contrast
microscope.
Treatment of platelets with chymotrypsin. Treatment
of washed human platelets by chymotrypsin was carried
out as previously described (13). Suspensions of washed
platelets (1 x 10’ platelets/ml)
were incubated with
chymotrypsin
for 1 h at 37OC. Untreated (intact) platelets were incubated for 1 h at 37°C without the addition
of chymotrypsin.
The platelets were then washed three
times by centrifugation,
and the platelet pellets were
resuspended in Tyrode’s albumin buffer.
Surface labeling of platelet proteins. Surface labeling of
platelet proteins of intact and chymotrypsin-treated
platelets with 12Y and sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out as previously
described (11, 13, 34).
CAMP accumulation. Washed, intact (untreated) platelets and chymotrypsin-treated
platelets suspended in
Tyrode buffer, pH 7.4, were incubated with C3H]adenine
(10 &i/log platelets) for 1 h at 37OC. The platelets were
then washed and resuspended in Tyrode buffer. The
enzyme reaction (conversion of [3H]ATP to [3H]cAMP)
was initiated by the addition of 10 PM prostaglandin E1,
1 PM prostacyclin, or 50 PM forskolin and terminated
after 2 min at 37°C by the addition of 0.5 ml of 0.75 n&I
CAMP and heating to 100°C for 10 min. The reaction
volume was 100 ~1, and the final platelet concentration
was 108/ml. ADP (50 PM) and epinephrine (10 PM) were
also added at the time of initiation.
The C3H]cAMP
formed was isolated using a modification
of the method
of Salomon et al. (30) as described by Lenox et al. (14).
Reaction products were centrifuged at 2,500 g for 20 min,
the supernatants were applied on cation exchange columns (Bio-Rad AG50W-X8),
and the [3H] precursor
nucleotides were eluted with water. After the contents
were eluted into alumina columns, the [3H]cAMP
was
eluted with 0.1 M imidazole, pH 7.4. Aliquots of the
alumina eluates and [3H] -nucleotide precursor fractions
were counted with 5 ml scintillation
cocktail.
Morphological studies. Platelets were washed twice in
the presence of heparin and apyrase as described above.
BY
ACTIVATION
OF PLATELETS
BY SURFACE
INTACT
PROTEOLYSIS
PLATELETS
A
1 OOONg
PQE,
ADP
CHYMOTRYPSIN
ADP
TREATED
PGI,
ADP
f-i
PLATELETS
z
of chymotrypsin on sponFIG. 1. Effect of various concentrations
taneous aggregation of platelets by fibrinogen. Intact platelets (10’
platelets/ml) were divided into 5 equal aliquots and treated for 1 h with
the concentrations of chymotrypsin shown above at 37°C. After the
incubation period, platelets were washed in presence of phenylmethanesulfonyl fluoride and soybean trypsin inhibitor (23) and resuspended in Tyrode buffer containing albumin, pH 7.35 (450 ~1). Aliquots
(2 x 108/ml) of platelet suspensions were tested for their ability to be
aggregated by fibrinogen. Preincubation of platelets with chymotrypsin
(in pg. 10’ platelets-’ *ml-l): A, 1,000; B, 750; C, 500; D, 300; and E, no
chymotrypsin added. Twenty-five microliters of fibrinogen (200 pg/ml,
final concn) was added to initiate platelet aggregation.
prostacyclin, we found that chymotrypsin-treated
platelets increased CAMP to a similar extent as did intact
platelets (Table l), and no significant differences were
observed between preparations (P > 0.2). The uptake of
[3H] adenine by intact or chymotrypsin-treated
platelets
was approximately
90%, and we observed no significant
differences in [3H]adenine uptake between intact and
chymotrypsin-treated
platelets.
We also studied the ability of ADP or epinephrine to
inhibit the stimulation of CAMP generation in intact and
chvmotrypsin-treated
platelets. Table 1 shows that the
response of the adenylate cyclase system of chymotrypsin-treated platelets to epinephrine and ADP was similar
to the response found with intact platelets. The treatment of platelets with 500 or 1,000 pg of chymotrypsin
had no significant effect on the ability of ADP or epinephrine to inhibit stimulation
of platelet adenylate
cyclase by prostaglandin
E1, prostacyclin, or forskolin.
Since ADP acts through specific receptors to induce
shape change, we have investigated whether platelet
FIG. 2. Comparison of effects of stimulators of adenylate cyclase on
aggregation of intact and chymotrypsin-treated platelets. Upper panel:
aliquots (450 ~1) of intact platelets (3 x lO’/ml) were tested for their
aggregation at 37°C by ADP and fibrinogen (200 pg/ml) under the
following conditions: A, no inhibitors added; B, prostaglandin E1
(PGE1), 10 PM; C, forskolin (FSK), 50 PM; and D, prostacyclin (PGI&,
1 PM. ADP (50 PM) was added to initiate platelet aggregation. Bottom
panel: aliquots (450 ~1) of chymotrypsin-treated
platelets (1,000 pg 10’
platelets-‘-ml-l)
at a concentration of 3 x 108/ml were aggregated by
adding fibrinogen under the following conditions: A, no agents added;
B, PGE1, 10 PM; C, FSK, 50 PM; and D, PG12, 1 PM. Fibrinogen (200
pg/ml) was added to initiate platelet aggregation.
l
ADP receptors are sensitive to proteolysis. We found
that ADP was able to induce a similar shape change in
both intact and chymotrypsin-treated
platelets as shown
by the scanning-electron
micrographs in Fig. 3, A-D.
These observations could be quantitated
by measuring
the decrease in light transmission
that occurs when
platelets change shape. Table 2 shows that neither the
extent nor the initial velocity of ADP-induced
shape
change was altered by pretreatment
of platelets with 500
or 1,000 pg of chymotrypsin, when compared with intact
(untreated) platelets.
DISCUSSION
This study demonstrates that ADP is able to induce a
shape change in chymotrypsin-treated
platelets, a phenomenon previously reported only with intact platelets.
This shape change is similar in its extent and initial
velocity to that seen with intact platelets. The shape
change could be blocked by stimulators of the platelet
adenylate cyclase such as prostaglandin
El. Therefore,
the ADP receptor responsible for ADP-induced
shape
change appears to be present on the surface of chymotrypsin-treated
platelets. However, in contrast to intact
(untreated) platelets, which require ADP for platelet
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uI8
FSK
-+-I-
+I---
D
C
B
Chymotrypsin
ACTIVATION
OF
PLATELETS
BY
SURFACE
H553
PROTEOLYSIS
TABLE 1. Inhibition by epinephrine and ADP of CAMP generation in intact
and chymotrypsin-treated platelets
Platelet
Treatment
PGE,
0.57t0.11
of [‘H]ATP
% Inhibition
to [3H]Cyclic
Forskolin
AMP:Inducing
%Inhibition
Agent
PG12
%Inhibition
(7)
(3)
(7)
72
2.36t0.73 (6)
0.49t0.04 (3)
60
7.15tl.43
1.94kO.60
2.37t0.93
66
0.26t0.02
(6)
80
89
(6)
(3)
(6)
53
57
5.71tl.49
2.8OkO.70
1.59t0.38
(5)
(3)
(5)
50
72
2.4OkO.80
0.51-3-0.11
0.23t0.07
(3)
(3)
(3)
78
90
(3)
(3)
(3)
42
53
6.7lt2.10
3.20t0.39
2.3620.82
(3)
(3)
(3)
52
64
2.11t0.40
O.BOt0.16
0.2lt0.01
(3)
(3)
(3)
62
90
(6)
0.30t0.03 (3)
48
0.23rtO.03
(6)
0.55t0.08
0.2620.03
0.24t0.02
0.47t0.04
0.27t0.04
0.22kO.04
Values represent
means t SEM.
Intact
platelets
and chymotrypsin-treated
platelets
(CTP,
500 or 1,000 gg.10’
platelets-loml-‘)
were
suspended
in Tyrode
buffer.
Platelet
suspensions
(1 x log/ml)
were first incubated
with [3H]adenine
for 1 h and then washed. Conversion
of
[3H]precursor
to [3H]cyclic
AMP (CAMP)
by the addition
of prostaglandin
E1 (PGE1, 10 PM), forskolin
(50 PM), or prostacyclin
Iz (PG12, 1 pM)
was measured
in presence of 10 PM epinephrine
or 50 PM ADP. Each experiment
was performed
in duplicate,
and number
of experiments
is
indicated
in parentheses.
%Inhibition
of conversion
of [3H]ATP
to [3H]cAMP
by ADP or epinephrine
is also indicated.
Basal 1eveIs of [3H]
CAMP accumulation
for intact and CTP (500 and 1,000 pg) were 0.06 * 0.01 (7), 0.09 t 0.01 (6), and 0.07 t 0.005 (3), respectively.
Counts in
fraction
1 of the Dowex column, representing
predominantly
(>80%)
labeled ATP, were found to be about the same in intact and CTP (500 and
1,000 pg) (0.94 t 0.19, 1.16 t, 0.20, and 0.92 t 0.15 X lo6 cpm/108 platelets,
respectively).
aggregation, prior stimulation
of the ADP receptor of
chymotrypsin-treated
platelets is not required for fibrinogen binding and aggregation. This ADP receptor appears to be coupled to the adenylate cyclase of chymotrypsin-treated
platelets as shown by the ability of ADP
to inhibit the stimulation
of adenylate cyclase by forskolin, prostaglandin El, or prostacyclin. Whether the ADPinduced shape change and the inhibition
of adenylate
cyclase by ADP are the result of the stimulation
of one
or more types of ADP receptor(s) on chymotrypsintreated platelets presently is not known.
Our findings indicate that chymotrypsin-treated
platelets possess adenylate cyclase activity similar to that of
intact platelets. In chymotrypsin-treated
platelets, stimulators such as forskolin, which acts directly on the
adenylate cyclase complex (32), or prostaglandin
El and
prostacyclin, were able to raise the levels of CAMP to an
extent similar to that seen with intact platelets. We
conclude therefore that membrane receptors for prostaglandin or prostacyclin that are coupled to the adenylate
cyclase are functional in chymotrypsin-treated
platelets
and similar in their responsivity to those found, in intact
platelets. These conclusions are based on our findings
that similar levels of [3H]ATP were found in intact and
chymotrypsin-treated
platelets (see legend to Table 1)
and that the amount of [3H]adenine taken up by the
three platelet preparations was also the same. Since these
are all relative measurements, however, we cannot exclude the possibility that small differences in adenylate
cyclase activity do exist between intact platelets and
chymotrypsin-treated
platelets.
The effect of chymotrypsin
on isolated platelet membranes has been previously studied and shown to result
in the activation of basal adenylate cyclase activity and
loss of the inhibitory influence of epinephrine and GTP
(4, 33). These results are clearly different from those
that we have observed after chymotrypsin
treatment of
the surface of intact pl .atelets. We h ave found th .at both
AD P and epinephrine were able to inhibit the prostaglandin El, for&Olin, or prostacyclin-stimulated
adenylate cyclase activity of chymotrypsin-treated
platelets.
These differences are not surprising, since the guanine
nucleotide regulatory proteins required for inhibition
of
adenylate cyclase are found on the inner surface of the
plasma membrane and hence n .ot exposed to proteolysis
under the conditions used in the present study. We also
found that epinephrine receptors on the surface of platelets coupled to adenylate cyclase are resistant to treatment with extracellular chymotrypsin.
Our findings provide direct evidence that once fibrinogen binding sites become permanently exposed on the
platelet surface by iimited surface proteolysis, potent
stimulators of platelet adenylate cyclase such as prostacyclin, forskolin, or prostaglandin El are unable to inhibit
fibrinogen-induced
aggregation.
ADP-induced
shape
change of chymotrypsin-treated
platelets, on the other
hand, can be inhibited by these: agents. Our results
suggest therefore that inhibition
of the ADP-induced
aggregation of intact platelets by increased levels of
CAMP must involve interaction with events that precede
the exposure of fibrinogen binding sites on the platelet
surface. We provide direct evidence that CAMP does not
interact with the fibrinogen binding sites themselves, as
evidenced by the lack of effect of adenylate cyclase
activators on the aggregation of chymotrypsin-treated
platelets. Previous studies have shown that chymotrypsin-treated platelets can aggregate spontaneously in the
presence of fibrinogen (6, 13, 23). The present study
confirms these findings and shows that the degree of
fibrinogen-induced
aggregation is dependent on the concentration of chymotrypsin
used during the preincubation period.
Pretreatment
of platelets with chymotrypsin appears
to be a useful tool that allows for the dissociation of
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Intact
No agonist added
+ ADP
+ Epinephrine
CTP, 500 pg
No agonist added
+ ADP
+ Epinephrine
CTP, 1,000 pg
No agonist added
+ ADP
+ Epinephrine
% Conversion
Hi554
ACTIVATION
OF
PLATELETS
BY
SURFACE
PROTEOLYSIS
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FIG. 3. Scanning-electron
micrographs
showing
ADP-induced
shape change of intact and chymotrypsin-treated
D: chymotrypsin-treated
platelets. A: intact platelets.
B: intact platelets
plus ADP. C: chymotrypsin-treated
platelets.
platelets
plus ADP. Platelets
were washed and treated
with chymotrypsin
as described
in METHODS.
ADP (50 PM)
was added to initiate
shape change in the presence
of 5 mM EGTA.
One minute
after addition
of ADP, 1.5%
glutaraldehyde
(final concn) in 0.2 M phosphate
buffer, pH 7.4, was added, and samples were prepared
for scanningelectron microscope.
ACTIVATION
OF
PLATELETS
BY
SURFACE
PROTEOLYSIS
H555
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L
FIG.
3 . I 3 and D. See legend
on facing
page.
H556
ACTIVATION
OF PLATELETS
2. Effect of chymotrypsin on ADP-induced
shape change
TABLE
Shape Change
Treatment
Extent,
LTU
Initial Velocity,
LTU/min
platelet shape change from the aggregation process, without blocking these responses to ADP or fibrinogen, respectively. The mechanisms underlying this dissociation
may involve proteolysis of proteins that mask the fibrinogen binding site in intact, resting platelets. Proteolysis
of surface proteins by extracellular proteases may prove
to be useful also in studies of other receptors in a variety
of cells. Several lines of investigation have demonstrated
a physiological role for proteolysis as a mechanism of
activation of processes ranging from blood coagulation
to cell receptor activation (29). Proteolysis by extracellularly applied chymotrypsin
also has been shown to
have profound biological effects resulting in the differentiation of murine erythroleukemia
cells (31). Several
studies have implicated
proteolysis in various pathophysiological states (3, 24, 28). The present study demonstrates that surface proteolysis has selective effects on
specific platelet functions. It can be speculated that
under certain conditions, such proteolysis may occur in
vivo. Further investigations
should determine whether
proteolysis of platelet surface proteins plays a role in
hemostatic regulation under certain physiological and/
or pathological conditions.
The authors thank Dr. Dan Hendley, Dee Van Riper, Daniel De
Mars, and Ann Wood for excellent technical assistance and Pat Smith
for the preparation of the manuscript.
E. Kornecki is a recipient of a New Investigator Research Award
HL-32594 from the National Heart, Lung, and Blood Institute.
A preliminary report of this work was presented at the Xth International Congress on Thrombosis and Haemostasis, San Diego, CA,
July 15-19, and the abstract has been published (Thromb.
Haemostas.
54: 186, 1985).
Received 15 May 1985; accepted in final form 1 November 1985.
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