© 2001 American Heart Association, Inc.
Thrombosis |
Procoagulant Activity on Platelets Adhered to Collagen or Plasma Clot
From the Wihuri Research Institute and Helsinki University Central Hospital (R.L.), Helsinki, Finland.
Correspondence to Riitta Lassila, Wihuri Research Institute, Kalliolinnantie 4, FIN-00140, Helsinki, Finland. E-mail riitta.lassila{at}wri.fimnet.fi
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Abstract—In a new 2-stage assay of platelet procoagulant activity (PCA), we first subjected gel-filtered platelets to adhesion on collagen (as a model of primary hemostasis) or plasma clots (as a model of preformed thrombus) for 30 minutes, and then the adherent platelets were supplemented with pooled, reptilase-treated, diluted plasma. Defibrinated plasma provided coagulation factors for assembly on platelet membranes without uncontrolled binding of thrombin to fibrin(ogen). Platelet adhesion to both surfaces showed modest individual variation, which increased at platelet densities that allowed aggregation. However, adhesion-induced PCA varied individually and surface-independently >3-fold, suggesting a uniform platelet procoagulant mechanism. Permanently adhered platelets showed markedly enhanced PCA when compared with the platelet pool in suspension, even after strong activation. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than on collagen-adherent platelets during the initial phase of coagulation, whereas collagen-induced PCA proceeded slowly, strongly promoted by tissue thromboplastin. Therefore at 10 minutes, after adjustment for adhered platelets, collagen supported soluble thrombin formation as much as 5 times that of the thrombin-retaining clots. Activation of platelets by their firm adhesion was accompanied by formation of microparticles, representing about one third of the total soluble PCA. Collagen-adhered platelets provide soluble thrombin and microparticles, whereas the preformed clot serves to localize and accelerate hemostasis at the injury site, with the contribution of retained thrombin and microparticles.
Key Words: platelet adhesion • procoagulant activity • arterial thrombosis • collagen
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Introduction |
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During hemostasis and thrombosis, primary platelet activation and aggregation and secondary clotting are recognized to interact, thereby amplifying each other.1 2 3 4 Activated platelets accelerate coagulation by exposing a procoagulant phospholipid surface5 and factor V,6 whereas thrombin, the enzymatic end product of coagulation, is considered the most potent activator of platelets.7 This interplay establishes a powerful resonance loop, which localizes itself at the site of vessel injury.
The platelet surface also localizes the negative-feedback reactions of coagulation, such as inactivation of factors Va and VIIIa by activated protein C8 and inactivation of activated protein C by protein C inhibitor.9 Despite the important functional role of platelets in directing the coagulation system and its regulation at the site of injury, relatively little is known about the procoagulant activity (PCA) of adherent platelets on thrombogenic surfaces.10 11 12 13 These few kinetic studies have essentially used purified components, factors Va and Xa, instead of plasma or have been performed with plasma and platelets in suspension or in whole blood. In fact, the reports currently available do not directly concern PCA triggered in situ by firmly adhered platelets when supplemented with plasma. These considerations are validated by the fact that platelet adhesion and subsequent activation may significantly vary, depending on the adhesive receptors, their signaling abilities, and procoagulant capacity. Also, thrombin generation must overcome the natural anticoagulants to support hemostasis or thrombosis on the very same platelet phospholipid surfaces.3 In addition to activated platelets, platelet-derived microvesicles provide the mandatory procoagulant phospholipids and lower the threshold at which activated clotting factors will induce explosive thrombin generation.14 15 16 17
The aim of this study therefore was to compare the PCA of platelets activated by permanent adhesion with that of platelets activated in suspension. Thus, we studied adhesion-induced PCA by using (1) collagen as an initial platelet-adhesive surface and (2) a plasma fibrin clot as a preformed thrombus promoting sustained interaction between platelets and coagulation. Moreover, we assessed individual differences in adhesion-induced PCA, the role of the extrinsic pathway, and the contribution of adhesion-induced microparticles (MPs).
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Methods |
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Blood Collection
The present study was approved by the institutional review board. Blood was obtained from healthy, nonsmoking volunteers who had not taken any medication during the previous 14 days. Free-flowing blood was collected by venipuncture of a cubital vein with use of a polytetrafluoroethylene cannula (BOC Ohmeda AB). The first 3 mL was collected in EDTA for blood cell counts by a Coulter T-540 Thrombocounter (Coulter Electronics, Inc). For gel-filtered platelets (GFPs), 6 volumes of blood were collected in 1 volume of 131 mmol/L acidic citrated dextrose (pH 4.5), and for plasma separation, 9 volumes of blood were collected into 1 volume of 110 mmol/L sodium citrate.
Pooled and Defibrinated Plasma
Pooled, citrated, platelet-poor plasma (PPP) was
prepared by repeated centrifugation
(1500g, 10 minutes, 22°C) and
used as an adhesive substrate. Fibrinogen content was assessed by the
functional method of Clauss (bovine 
-thrombin; Dade, Baxter
Healthcare Corp). Prothrombin fragment 1+2 (F1+2),
thrombin–antithrombin III complex (TAT), and D-dimer levels were
analyzed with the corresponding ELISAs (Enzygnost F1+2 micro
and TAT micro, Behring; and Asserachrom D-Di, Diagnostica
Stago, respectively). Their values in pooled plasma were as follows:
fibrinogen 3.1 mg/mL, F1+2 0.9 nmol/L, TAT 3.8 ng/mL, and D-dimer 157
ng/mL. Also, activated partial thromboplastin time, thrombin
time, and prothrombin time were within the normal ranges.
Pooled, defibrinated plasma was prepared essentially by the method of Hemker and coauthors18 and used as the source of procoagulant and anticoagulant factors to avoid clot formation and uncontrolled binding of thrombin during the assay.19 20 One volume of reptilase (STA-Reptilase, 20 BU/mL, Diagnostica Stago) was added to 49 volumes of the plasma and incubated (5 minutes, 37°C, 10 minutes on ice). The clot was centrifuged (2100g, 4°C, 10 minutes) and plasma was separated. In this reptilase-treated plasma, F1+2 was 1.1 nmol/L, TAT 4.3 ng/mL, and D-dimer <500 ng/mL (NycoCard D-Dimer, Nycomed Pharma AS). Ristocetin (1 mg/mL, Sigma Chemical Co) -induced aggregation of GFPs in defibrinated plasma indicated the presence of functional von Willebrand factor.
GFPs and Labeling With
[3H]Serotonin
GFPs were prepared from platelet-rich plasma
(PRP) as previously
reported.21 22 23
In brief, platelets suspended in HEPES buffer without cations were
eluted through a Sepharose CL-2B column (Pharmacia LKB Biotechnology
Inc). To control platelet activation during filtration, small doses
of apyrase (1 U/mL, Sigma) and prostaglandin
E1 (25 ng/mL, Sigma) were added to the PRP.
Isolated platelets were diluted in HEPES buffer to different
densities (10 to 300x106/mL), and 2
mmol/L CaCl2 and 1 mmol/L
MgCl2 were added just before assay.
For adhesion studies, GFPs were incubated (15 minutes, 37°C) with [3H]serotonin [5-hydroxy(G-3H)tryptamine creatinine sulfate, Amersham International plc]. The final concentration of added [3H]serotonin in GFPs (300x106 platelets per mL) was 10 nmol/L. Labeling and quantification of deposited 3H-positive platelets after their activation were performed as previously reported.22 Serotonin release during platelet adhesion to collagen was 3% (range, 1% to 4%) and to clot, 30% (range, 23% to 39%).
Preparation of Collagen-Coated Coverslips and
Plasma Clots as Adhesive Substrates for Platelets
Before clots were induced, pooled, citrated plasma
was first diluted 1:2 in PBS (Life Technologies). Plasma (100 µL per
coverslip) clots were generated with calcium (5 mmol/L
CaCl2) and thrombin (0.1 U per coverslip) on
round, plastic Thermanox coverslips (area 1.77
cm2; Nunc), incubated for 30 minutes at
37°C in a humid chamber and for 30 minutes at 22°C, and washed 6
times in PBS. The final concentration of ionized calcium under the
conditions of the clot was physiological: 1.1
mmol/L (Microlyte 6, Kone Instruments; reference values, 1.1 to
1.3 mmol/L). Collagen-coated coverslips were prepared from Horm
collagen reagent (Nycomed Arzneimittel; 25 µg/mL in isotonic glucose
solution), incubated (1 hour, 35°C) in a humid chamber, and washed 3
times in PBS. Bovine serum albumin (2% BSA, Sigma) in PBS was
used as a control substrate and a blocking agent.
Two-Stage PCA Assay
Phase 1: Platelet Deposition on Adhesive
Substrates
GFPs (500 µL) in HEPES buffer supplemented with
2 mmol/L CaCl2 and 1 mmol/L
MgCl2 were first allowed to adhere to the
substrate-coated coverslips in 24-well, flat-bottomed multidish plates
(Nunc) blocked with BSA. The samples were incubated (37°C, 30
minutes) under slow rotation (70 rpm) and washed 3 times in PBS. Buffer
without platelets served as a control. Adhesive surfaces, prepared
as previously described,23
were studied by scanning electron microscopy (JEOL model 820). F1+2 was
measured to estimate adhesion-induced soluble thrombin generation. The
samples (9 volumes) were collected in 1 volume of 35 mmol/L citric
acid, 75 mmol/L sodium citrate, 0.14 mol/L dextrose, 6 mmol/L
EDTA, 5 mmol/L adenosine, 25 U/mL hirudin, and 25 U/mL
heparin (1 volume)24 and
centrifuged (11 000g,
5 minutes, 4°C). F1+2 was measured from the
supernatant.
Phase 2: Generation of Soluble Thrombin on
Adherent Platelets
PCA was initiated on adherent platelets by
incubating (37°C, under slow rotation) pooled, defibrinated plasma
(1:20 dilution in PBS with Ca2+ [0.9
mmol/L] and Mg2+ [0.76 mmol/L]; 500
µL) with or without tissue thromboplastin (1:5000 final concentration
of Thromborel S, Behringwerke AG). The time dependence of
platelet-induced thrombin generation after adhesion to collagen or
clot was estimated during incubation periods ranging between 2 and 30
minutes in the procoagulation assay.
Thrombin generation was stopped by diluting the supernatant
to an equal volume with a stopping buffer (125 mmol/L NaCl,
50 mmol/L Tris-HCl, and 2 mmol/L EDTA; pH
7.9).13 Twenty microliters
of chromogenic substrate S-2238 (4.7 mmol/L,
Chromogenix AB) was added to 180 µL of the diluted sample and
incubated for 6 minutes; the reaction was terminated by addition of
20% acetic acid (50 µL), and absorbance was measured at 405 nm by a
Labsystems Multiscan MCC (Labsystems). Duplicate samples were assayed,
and the amount (in units per milliliter) of thrombin was estimated with
a standard curve of -thrombin
(Dade).
PCA of Permanently Adhered Platelets in
Comparison With PCA of Platelets Activated in
Suspension
The PCA of adherent platelets was compared with
that of GFPs in suspension at the same platelet amount that had
adhered, after activation with 10 µmol/L SFLLRN (Bachem, Bubendorf)
and 10 µg/mL collagen, or with 10 µmol/L calcium ionophore A23187
(Sigma). Platelets were first activated either by a
collagen surface or by the soluble agonists under slow rotation (30
minutes, 37°C). Four hundred sixty-five microliters of this
platelet suspension was incubated with 25 µL of defibrinated
plasma and 10 µL of 1:100-diluted Thromborel S for 15 minutes.
Samples were diluted in stopping buffer and centrifuged
(11 000g, 5 minutes, 22°C)
to discharge the platelets, and thrombin generation was
measured.
One-Stage PCA Assay: Generation of Soluble
Thrombin on PRP Clots
The procoagulant effect of platelets was also
studied in a 1-stage assay with PRP clots. PRP was separated after
centrifugation
(180g, 12 minutes, 22°C), and
PPP was centrifuged twice from PRP
(1500g, 10 minutes). PRP was
adjusted to a density of 200x106
platelets per milliliter with PPP and diluted 1:2 in PBS. PRP clots
(100 µL, corresponding to a final number of
10x106 platelets) were prepared on a
coverslip by recalcification (15 mmol/l
CaCl2) and addition of 0.1 U -thrombin. The
PRP clots and their PPP controls were incubated at 37°C for 15
minutes and washed 3 times in PBS. The coverslips were incubated for 15
minutes under slow rotation with 1:20-diluted, defibrinated plasma and
traces of thromboplastin (37°C, 10 minutes). The clots were also
incubated with PBS buffer to estimate the amount of clot-derived
soluble thrombin. These absorbance values were subtracted from the
results obtained with plasma.
Generation and Contribution of
Platelet-Derived MPs
MP generation during adhesion-induced platelet
activation was studied in GFP supernatants after the first phase of the
assay. GFPs (100x106/mL in HEPES buffer) or
buffer control first adhered onto BSA, collagen, or clot for 30 minutes
in the presence of 2 mmol/L CaCl2 and
1 mmol/L MgCl2. Nonadherent platelets
were centrifuged in the presence of
platelet-inhibitory
anticoagulant,24 and tissue
factor–triggered PCA of the supernatant (465 µL) was studied 10
minutes after the addition of defibrinated plasma (25 µL) and
1:100-diluted Thromborel S (10 µL). To study the role of soluble
platelet-derived MPs after adhesion-induced platelet
activation, the PCA of the supernatant was measured both in the
presence of MP and after their filtration (0.1-µm filter, Millipore).
Furthermore, the PCA of the MP supernatant was adjusted with the
amount of originally adhered platelets (on collagen or clot) from
which the MPs were generated.
Statistical Analysis
N refers to number of donors. The experiments were
performed in duplicate or triplicate. Data were expressed as mean±SD
unless indicated otherwise. Student’s
t test was used for comparison
of 2 groups of data.
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Results |
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Determination of Thrombin Generation in Plasma
Generation of soluble thrombin during the PCA assay was estimated by adding known amounts of
-thrombin to undiluted or
1:20-diluted, defibrinated plasma or to PBS buffer
(Figure 1
). Absorbance values due to exogenous thrombin in
1:20-diluted, defibrinated plasma were equal to those in the buffer
control. However, in undiluted, defibrinated plasma, absorbance values
were markedly lower, reflecting the role of natural anticoagulants in
plasma. Loss of thrombin to the forming clot during the procoagulation
assay was studied by comparing 1:20-diluted, citrated plasma with the
same 1:20-diluted, plasma after its defibrination. Absorbances due to
soluble thrombin activity at 10 minutes were, on average, 28% (range,
14% to 53%) lower in citrated plasma. To avoid uncontrolled binding
of thrombin to forming fibrin during the PCA assay, 1:20-diluted,
defibrinated plasma was chosen as the source of coagulation
factors.
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Plasma PCA on Platelets Adherent to
Collagen or Plasma Clot
Next, we determined the time scale of thrombin
generation, ranging between 2 and 30 minutes, in 1:20-diluted,
defibrinated plasma on the adhered platelets
(Figures 2A and 2B). The rate at which thrombin was generated
by platelets adherent to the clot was significantly greater than
that generated by platelets bound to collagen. After initial
activation over the clots, thromboplastin did not improve the maximal
rate or extent of thrombin generation. In contrast, collagen-induced
thrombin generation progressed slowly, reaching a maximum after 20
minutes, but thromboplastin significantly accelerated it.
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At 10 minutes, adhered platelets increased the generation of soluble thrombin >2-fold on the 2 thrombogenic surfaces. The collagen-bound platelets alone did not generate significant amounts of thrombin, whereas in the presence of thromboplastin, collagen enhanced PCA almost 4-fold (1.1±0.5 vs 0.3±0.1 U/mL, n=3, P<0.05). In contrast to collagen, PCA on clot-bound platelets did not benefit further from thromboplastin at 10 minutes (1.8±0.5 vs 1.5±0.3 U/mL, n=3).
In addition to adhered platelets on a clot surface, we studied the PCA of PRP clots in a 1-stage procoagulation assay. As in the 2-stage assay of PCA on clot-adherent platelets (3.6±0.8x106 platelets on the clot surface), PCA induced by PRP clots with 10x106 platelets (0.4±0.1 U) increased thrombin generation by 2.3-fold when compared with PPP clots (P=0.03).
Direct Coagulation Activity of the Substrates
Without and With Adherent Platelets
To further assess the extent of soluble thrombin
generation, we measured F1+2 in a suspension of GFPs
(100x106/mL) or HEPES buffer after 30
minutes of adhesion
(the
Table). Soluble F1+2 in the buffer or on GFPs
incubated over collagen did not differ from that obtained over BSA.
After incubation of the buffer with thrombin-induced clot, F1+2
reflected minor thrombin generation on the clot surface or release of
soluble F1+2, although the clots had been carefully washed. We detected
a 15-fold increase in the amount of F1+2 in the supernatant of GFPs.
This difference demonstrates thrombin generation induced by the
clot-adherent platelets: 26.7 nmol/L F1+2 was supported by
3.6x106 adherent platelets (vide
infra).
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Effect of Platelet Density on Deposition to
Collagen or Plasma Clot and on Subsequent PCA
The specific aim here was to dissociate the effects of
platelet adhesion vs their aggregation. To study the effect of
platelet density on the extent of their deposition on the
thrombogenic surfaces, GFPs (300x106/mL in
HEPES buffer) were first labeled with
[3H]serotonin (10 nmol/L) and
thereafter adjusted to suspensions having different platelet counts
(10 to 300x106/mL). At platelet
densities 
100x106/mL, mainly single
adherent platelets were visualized on the thrombogenic
surfaces, whereas the higher platelet densities supported
aggregation on the initial adherent platelets (see Figures 3 and 4).
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Individual deposition of platelets from 6 healthy donors
is illustrated in
Figures 3A
and 3B. At a density of
100x106/mL, platelet deposition on the
plasma clot was 3 times that on collagen. However, with
platelet densities up to 100x106/mL,
only minor interindividual differences in deposition occurred on both
substrates, but higher densities had increased variability. This
individual variation in platelet deposition was triggered mainly by
varying aggregation, especially with clots. The platelet counts of
the supernatants after deposition to collagen or clot
(Figures 3C and 3D, respectively) were diminished owing to
platelet aggregation. The effect of platelet density on
deposition to collagen or clot is also demonstrated in scanning
electron photomicrographs
(Figures 4A through 4D). GFPs at
100x106/mL formed an adhesive layer on
collagen, showing a fully spread "fried egg" form
(Figure 4A), whereas at a higher density
(300x106/mL), extensive platelet
aggregation was triggered on the initial adherent layer
(Figure 4B). Also, on clots, single platelets adhered
with pseudopod extensions at a lower platelet density, but they
strongly aggregated at a higher density
(Figures 4C and 4D).
The individual PCAs initiated on these same adherent
platelets (without the
[3H]serotonin label) by
1:20-diluted, pooled, defibrinated plasma with thromboplastin are
presented in
Figures 5A and 5B. Although platelet deposition on
collagen and clots did not markedly vary with platelet densities up
to 100x106/mL
(Figures 3A and 3B), thrombin generation induced by these
adhered platelets (1.2±0.3x106 on
collagen and 3.6±0.8x106 on clot) did vary
>3-fold.
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These adhesion and procoagulation data were used to adjust
platelet-induced thrombin generation to the number of
surface-adhered platelets
(Figure 5C). The platelet adhesion–quantified PCA
(measured as soluble thrombin) was 5-fold greater on collagen than
on clots (P<0.05), and it
became attenuated with increasing platelet densities, suggesting
the importance of the initial adherent layer.
PCA of Adhesion-Activated
Platelets in Comparison With That of Platelets
Activated in Suspension
The effect of permanent surface adhesion on
platelet PCA was then compared with that of activated GFPs
in suspension at the same platelet number that had adhered to
collagen
(Figure 6). When adjusted to platelet number,
collagen-adherent platelets supported 30% more thrombin generation
than did the same number of strongly activated platelets in
suspension. Thus, even when GFPs in suspension were maximally
activated with SFLLRN and collagen or with calcium ionophore,
their PCAs at 15 minutes still remained markedly lower than that
induced by an equal amount of adhesion-activated platelets
on collagen.
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Effect of Soluble MPs on Coagulation Activation
After Adhesion-Induced Platelet Activation
Formation of MPs during platelet adhesion and the
subsequent MP-induced PCA were studied in GFP supernatants after
adhesion to collagen or clot, with BSA as a control
(Figure 7). Nonadherent platelets were removed from the
supernatant by centrifugation, and the tissue
factor–triggered PCA was measured in the presence and after removal of
soluble MPs by filtration. On collagen, the thromboplastin-induced
thrombin generation after MP filtration decreased by 32±6% (n=3).
After clot-induced platelet activation, MP filtration reduced the
PCA of the supernatant by 43±14%. When the PCA of the soluble MPs was
adjusted to the amount of initially adhered platelets on collagen
or clot, MP-induced thrombin generation per
106 adhered platelets on collagen was
>3-fold that on clot
(P=0.01).
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Discussion |
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It is now well established that platelets and clotting factors have several interactive roles in hemostasis and thrombosis. Previous procoagulant studies mainly used soluble platelets in PRP or whole blood.25 26 27 In earlier adhesion-type studies, isolated platelets were first allowed to interact with collagen and then resuspended to react with purified coagulation factors.13 Our study design provided activation of platelets by their firm adhesion, with subsequent in situ procoagulant membrane alteration. We observed a >3-fold individual variability in adhesion-induced PCA at a given level of platelet adhesion, independent of the adhesive surface. Importantly, locally adhered and activated platelets had markedly enhanced procoagulant capacity in comparison with platelet suspensions, even on strong activation. This could well be due to enhanced calcium signaling by adhesion-induced activation of the spread platelets and their larger membrane accessibility for coagulation factors, as we have reported earlier.13
Platelet deposition during phase 1 of the assay was studied both with a radiolabel and by scanning electron microscopy. At 100x106/mL, platelet deposition on plasma clot was 3-fold greater compared with that on collagen, but the individual variation in deposition on both surfaces was relatively modest in the presence of cations. At higher platelet densities, however, platelet deposition on collagen or clot markedly (2- to 4-fold) varied between donors. Aggregation of GFPs during the first step of the assay reflected activation, because in the absence of plasma, it was mediated by platelet-derived ligands: fibrinogen, von Willebrand factor, or thrombospondin.
Instead of using purified clotting factors, we chose to use
defibrinated plasma as a source of procoagulants to (1) eliminate
uncontrolled loss of the forming thrombin on fibrin and (2) overcome
the natural anticoagulants. In the presence of fibrin(ogen) under
identical conditions, measurable soluble thrombin was, on average, 28%
lower owing to clot formation. The report by Kumar et
al20 also indicated that
30% of all thrombin formed during coagulation activation is
adsorbed onto fibrin. Thrombin generation during PCA was quantified
with a thrombin standard. In undiluted plasma, anticoagulants fully
blocked the chromogenic action of added thrombin. Plasma
dilution of 1:20 seemed to provide enough coagulation factors to
overcome natural anticoagulant activity in the PCA assay. Compatible
with the idea that the diluted coagulation factors did not become rate
limiting during the assay, we observed that collagen-adherent
platelets at prolonged incubation and clot-bound platelets at
higher densities continued to increase their PCA in this 1:20 plasma
dilution. In addition to the use of defibrinated and diluted plasma,
the limitations of our study include the absence of flow conditions and
other blood cells.
The importance of permanent adhesion on platelet PCA became evident when it was compared with that of platelets in suspension. Because the PCA of adherent platelets clearly exceeded that of the strongly activated platelets in suspension, it is evident that adhesion- and activation-related platelet receptors have a significant impact on PCA. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than that on collagen-adherent platelets during the initial phase of coagulation activation, already reaching a plateau at 10 minutes, and probably due to clot-bound thrombin and MPs. As previously reported,13 on collagen-adherent platelets, PCA proceeded slowly in the absence of thromboplastin, compatible with the idea that although tissue factor is not absolutely necessary, it induces a local, rapid thrombin "burst" at the site of vascular injury. In experiments with isolated platelets and purified coagulation factors, thrombin generation was prolonged for 15 minutes in the absence of tissue factor.28 Endothelial injury simultaneously exposes collagen and tissue factor, an integral membrane protein of subendothelial cells, and tissue factor is also carried by monocytes.29 When the clot has already formed, it contains thrombin and additional procoagulant factors, so that the role of tissue factor is less crucial. However, when PCA at 10 minutes was adjusted with adhesion, the collagen-adherent platelets in the presence of thromboplastin generated up to 5 times as much soluble thrombin as did the clot-adherent platelets. This surface-associated discrepancy may be related to the retention of forming thrombin on the clot. In addition, this relative platelet-adjusted PCA decreased at increased platelet densities, supporting the importance of the PCA of the first adhesive layer of platelets, with restriction on excessive platelet accumulation.30 31 32 33
Activation of platelets by their firm adhesion to collagen or plasma clot was accompanied by formation of soluble procoagulant MPs. When MP-derived thrombin generation was adjusted to the number of adherent platelets from which the MPs originated, soluble PCA induced by collagen exceeded that of the clot. This finding is explained by the quantity and/or quality of the MPs produced by the collagen-adherent platelets. Also, as we have shown earlier, platelet-derived MPs bind to fibrin23 ; the retained MPs may contribute to the rapid initial burst of PCA on the clot while fewer MPs are detected in the supernatant. These findings demonstrate the major differences between collagen and clot as adhesive and thrombogenic surfaces. First, the initial activation of platelets by adhesion to subendothelial collagen leads (in the presence of tissue factor) to a marked burst of thrombin and platelet-derived MPs into the circulation. Second, the subsequently forming clot, though contributing to explosive platelet adhesion and PCA as well, seems to retain significant amounts of thrombin and MPs, thus localizing the process at the site of vascular injury.
Although platelet deposition on collagen or plasma clot did not markedly vary with platelet densities up to 100x106/mL, thrombin generation induced by these adhered platelets varied surface-independently, reflecting donor-dependent differences in the procoagulant capacity of platelets. Similar evidence of individual variation in platelet activity has previously been reported,25 34 35 although the precise pathophysiological mechanisms are still unknown. There is already good evidence that inherited alterations in platelet membrane adhesive glycoproteins are partly responsible for the functional heterogeneity36 and that these differences even modulate the risk for arterial thrombosis.37 38 39
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Acknowledgments |
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Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. This study was financially supported by the Aarne Koskelo Foundation and the Finnish Heart Research Foundation. The excellent technical assistance of Tuula Järvenpää and Marja Lemponen is warmly acknowledged.
Received September 26, 2000; accepted January 10, 2001.
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