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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Klinik und Poliklinik für
Anästhesiologie und operative Intensivmedizin, Experimental and
Clinical Haemostasis, University of Münster, Germany; Theodor
Kocher Institute, University of Berne, Switzerland.
The role of the platelet glycoprotein (GP) Ib-V-IX receptor in
thrombin activation of platelets has remained controversial although
good evidence suggests that blocking this receptor affects platelet
responses to this agonist. The mechanism of expression of procoagulant
activity in response to platelet agonists is also still obscure. Here,
the binding site for thrombin on GPIb is shown to have a key role in
the exposure of negatively charged phospholipids on the platelet
surface and thrombin generation, in response to thrombin, which also
requires protease-activated receptor-1, GPIIb-IIIa, and
platelet-platelet contact. Von Willebrand factor binding to GPIb is not
essential to initiate development of platelet procoagulant activity.
Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block
platelet procoagulant activity. Both heparin and low molecular weight
heparin block thrombin-induced platelet procoagulant activity,
which may account for part of their clinical efficacy. This study
demonstrates a new, critical role for platelet GPIb in hemostasis,
showing that platelet activation and coagulation are tightly
interwoven, which may have implications for alternative therapies for
thrombotic diseases.
(Blood. 2000;96:2469-2478) The platelet glycoprotein (GP) Ib complex is
important in primary hemostasis as demonstrated by the bleeding
problems in patients with Bernard-Soulier syndrome where this receptor
is either absent or defective.1 GPIb is the receptor for
von Willebrand factor (vWf) on resting platelets and slows down
platelets by a transient interaction so that they adhere to damaged
vessel wall subendothelium in the first stage of a complex repair
process.2,3 The primary structure of the components of the
complex is known4-7 and models of the tertiary structure
have been proposed.8 These indicate that the binding site
for vWf is held out from the platelet surface by a rodlike, highly
O-glycosylated domain. Binding of vWf to GPIb induced by
ristocetin or botrocetin causes signal transduction and activation of
GPIIb-IIIa.9-13
Although much evidence has accumulated for GPIb as a thrombin
receptor,14-18 particularly at low doses of thrombin, this
has sometimes been controversial with several authors claiming that the
protease-activated receptor-1 (PAR-1) is sufficient to explain all
aspects of platelet activation by thrombin.19,20 In most cases this conflict was due to inadequate attention being paid to the
GPIb effect being apparent only at low doses so that amounts were used
such that its role might have been masked. As well as these problems,
studies on the platelet thrombin receptors have been complicated by the
inability to explain the high-affinity receptor
(KD~10 The lack of GPIb in Bernard-Soulier syndrome affects not only the
platelet responses to vWf and thrombin, which show defects in
activation similar to those when GPIb receptor sites are blocked, but
in addition there is a defect in procoagulant activity. Basal levels of
procoagulant activity are higher than controls,29 and yet
the expression of procoagulant activity in response to agonists is
reduced.30 The raised basal levels could be due to a
reduced stabilization of the membrane by fewer links with the
membrane-associated cytoskeleton, but the reduced response on
activation has never been satisfactorily explained. Development of
procoagulant activity on platelets is a complicated process including
the formation of prothrombinase complex. After platelet activation,
negatively charged aminophospholipids (phosphatidylserine, -ethanolamine, and -inositol) are exposed in the outer leaflet of the
plasma membrane and bind factors Va and Xa in the presence of
Ca++. Prothrombin can then bind to this complex and is
rapidly cleaved to thrombin. Measurement of thrombin generation is a
classic method of assessing this activity.31 Because
exposure of negatively charged aminophospholipids at the platelet
surface is essential for procoagulant activity, its measurement by
binding of suitably labeled annexin V is a good method for determining
the first steps of this process.32,33 We found that
measurement of thrombin generation or of annexin V binding led to
comparable estimates of procoagulant activity. The development of
procoagulant activity and formation of microvesicles, by shedding of
membrane, are linked processes.34,35
Some controversy still exists over the binding between GPIb and
thrombin and the sites that are involved on each of these molecules.
The anionic exosite II on thrombin36 or the anonic exosite
I37 may be the binding site for GPIb involving the highly negatively charged 268 to 287 sequence on GPIb Several recent papers have addressed the mechanism of activation of
platelets involving GPIb. Béguin and coworkers40
observed a requirement for GPIb in procoagulant enhancement by fibrin
in tissue factor-initiated thrombin generation. In this case vWf was
also necessary, which could be explained by vWf binding to GPIb
providing additional linking and signaling interactions between platelets. The vWf-fibrin-GPIb interactions might play a role in
flip-flop events. Hayes and Tracy41 tried to identify the platelet high-affinity thrombin receptor by preparing polyclonal antibodies against a hirudin-like sequence similar to the thrombin anionic exosite I binding sequences present in hirudin, GPIb In this paper we present results supporting a unique role for GPIb in
thrombin-platelet interactions in the development of platelet
procoagulant activity in response to thrombin activation.
Materials
Preparation of platelets
Platelet aggregation Platelet aggregation studies were performed in an aggregometer (Chrono-Log, Haverton, PA) within 3 hours of venipuncture using gel-filtered platelets at 1 × 108 platelets/mL.Thrombin generation on gel-filtered platelets Gel-filtered platelets were diluted to 2.5 × 107 platelets/mL with TBS (50 mmol/L Tris, 175 mmol/L NaCl, 6 mmol/L CaCl2, pH 7.5). Platelets (200 µL) were activated with thrombin (0.5 U/mL) or TRAP (50 µmol/L) for 25 minutes (room temperature, stirring). Factor Xa (0.4 U/mL) was added to activated or control platelets and thrombin generation by the prothrombinase complex was started by adding prothrombin (10 µg/mL). Aliquots (10 µL) were removed at set times and added to microtiter-plate wells containing 140 µL TBS, 50 mmol/L Tris,175 mmol/L NaCl, 20 mmol/L EDTA, pH 7.9, which stops further prothrombin cleavage. The diluted samples were then assayed for thrombin activity after adding the p-nitroanilide thrombin substrate (2 mmol/L). Absorbance at 405 nm was measured at 30-second intervals, and the data were calculated as the maximum slope (mOD/min) in each well. Thrombin concentrations in each sample were obtained by comparison to a reference thrombin preparation.Flow cytometry Samples were analyzed using a Becton Dickinson FACScan flow cytometer (Heidelberg, Germany) with excitation by an argon laser at 488 nm. The FACScan was used in a standard configuration with a 530-nm bandpass filter. Standard beads containing specific amounts of "mean equivalent soluble fluorescein molecules" were used for calibration. Standard beads or platelets were gated and data were obtained from fluorescence channels in a logarithmic mode. A total of 5000 events were analyzed. Specific binding of MoAbs was calculated by subtracting nonspecific binding as determined with a FITC-labeled mouse isotype-specific IgG.Annexin V-FITC binding Gel-filtered platelets were diluted to 2.5 × 107 platelets/mL with solution II (127 mmol/L NaCl, 2.7 mmol/L KCl, 0.42 mmol/L NaH2PO4, 12 mmol/L NaHCO3, 1 mmol/L MgCl2, 4 mmol/L CaCl2, 5.5 mmol/L glucose, 3.5% BSA, pH 7.35). Platelets (200 µL) were activated with thrombin (0.05-2 U/mL) or TRAP (1-200 µmol/L) for 25 minutes (room temperature, with gentle shaking) in a total volume of 250 µL in solution II. Thrombin- or TRAP-treated or control platelets were incubated with annexin V-FITC (1.5 µg/mL) in the dark (room temperature, shaken gently). After 15 minutes, 0.5 mL of solution II was added and after another 15 minutes samples were analyzed by flow cytometry. Inhibitors (antibodies, peptides, heparin, or low molecular weight heparin) were added directly to the platelets after gel filtration. After 5 minutes, platelets were activated with thrombin and treated as described above. Specific binding was calculated by subtracting nonspecific binding as determined in the presence of 20 mmol/L EDTA.Analysis of ristocetin-induced vWf binding to platelets The PRP was diluted to 2.5 × 107 platelets/mL with solution II. Platelets (200 µL) were treated with ristocetin (0.1-1.0 mg/mL) for 3 minutes and fixed with formaldehyde. After washing platelets and resuspending in solution II, they were incubated with FITC-conjugated anti-vWf MoAb (4F9, 10 µg/mL). After 1 hour the platelets were washed and then analyzed by flow cytometry.Analysis of CD62 expression on platelets The PRP was diluted to 2.5 × 107 platelets/mL with solution II. Platelets (200 µL) were activated with -thrombin
(0.5-20 nmol/L) or  -thrombin (5-200 nmol/L) in the presence of GPRP
(1.25 mmol/L) for 3 minutes and fixed with formaldehyde. After washing
platelets and resuspending in solution II, they were incubated with
anti-CD62P-FITC MoAb (10 µg/mL). After 1 hour platelets were again
washed and analyzed by flow cytometry.
Fibrinogen-FITC binding In the presence of GPRP (1.25 mmol/L), gel-filtered platelets (200 µL, 2.5 × 107 platelets/mL in solution II) were incubated with fibrinogen-FITC (100 µg/mL), which was preincubated with an antifibrinogen Ab (100 µg-1 mg/mL, control 0 µg/mL, 30 minutes), for 10 minutes. Platelets were activated with 1 U/mL thrombin or 50 µmol/L TRAP for 3 minutes and fixed with formaldehyde. After washing platelets and resuspending them in solution II, they were analyzed by flow cytometry. Fab fragments of MoAb IV.3 (20 µg/mL) against FcRII were first added to prevent possible platelet
activation by antibody binding to FcRII.
Measurement of annexin V-FITC binding sites/platelet The bead standard containing beads with 5 different levels of fluorescence was analyzed on the flow cytometer. The beads were treated as the platelets. All samples using microbeads were analyzed in triplicate. A linear regression curve plotted the mean fluorescence intensity channel for each bead peak against the known mean equivalent soluble fluorescein molecules level for each bead population. To determine the mean number of annexin V-FITC molecules bound per platelet, the mean equivalent soluble fluorescein molecules per cell was first determined by comparison of the cellular mean fluorescence intensity with the standard curve, and then dividing the difference in the number of FITC molecules for each cell and negative control by the F/P molar ratio for the marker. Specific binding was calculated by subtracting nonspecific binding as determined in the presence of 20 mmol/L EDTA.
Thrombin induced procoagulant activity on platelets The exposure of negatively charged phospholipids on gel-filtered thrombin-stimulated platelets (0.05-2 U/mL) detected by annexin V-FITC is shown in Figure 1A. The expression of negatively charged aminophospholipids depended on the thrombin concentration. Maximal expression of negatively charged phospholipids was reached at approximately 0.2 to 0.4 U/mL thrombin, whereas these platelets aggregated to 0.01 U/mL thrombin (Figure 1A, insert). In all experiments 60% to 80% of the platelets measured are procoagulant after thrombin activation (data not shown). The activation of prothrombin by the prothrombinase complex in the presence of 0.4 U/mL factor Xa and 6 mmol/L CaCl2 plus resting or thrombin-activated gel-filtered platelets (0.5 U/mL) was measured after 30 seconds and 1, 2, 5, 10, and 15 minutes (Figure 1B). Under these conditions activated platelets provide the required cofactors, factor Va and the negatively charged phospholipid membrane. On thrombin-activated platelets the added prothrombin was completely converted to thrombin after 15 minutes. In the absence of prior activation of the platelets, thrombin generation shows a characteristic lag phase, indicating a requirement for feedback activation of the platelets during the incubation, by low amounts of thrombin generated from prothrombin cleavage by factor Xa.
Thrombin but not TRAP induced procoagulant activity on platelets There was a slight increase in annexin V-FITC binding, that is, exposure of aminophospholipids, after activation of gel-filtered platelets with TRAP at concentrations up to 10 µmol/L, which was maintained as a plateau up to 200 µmol/L (Figure 2A). This increase was insignificant compared to that obtained with thrombin (Figure 2B). However, aggregation was induced at a concentration of 25 µmol/L TRAP (Figure 2A, insert). Thrombin failed to stimulate annexin V-FITC binding to gel-filtered platelets after pretreatment with 50 µmol/L TRAP for 10 minutes (Figure 2B). No annexin V-FITC binding could be detected even when platelets were activated with high thrombin concentrations, up to 2 U/mL. Thus, pretreatment with 50 µmol/L TRAP prevented expression of negatively charged phospholipids on platelets in response to thrombin. Pretreatment of gel-filtered platelets with 50 µmol/L TRAP for 10 minutes also reduced thrombin generation by the prothrombinase complex on thrombin-activated platelets (0.5 U/mL) as shown in Figure 2C. Thrombin generation on TRAP-activated platelets (50 µmol/L) was similar to that on resting platelets (Figure 2C).
Cell-cell contact is necessary for expression of platelet procoagulant activity Figure 3A shows thrombin-induced expression of negatively charged phospholipids on gel-filtered platelets detected by annexin V-FITC binding in samples containing 1000 to 100 000 platelets/µL. Samples were shaken gently during thrombin activation (0.05-2 U/mL). Only in platelet suspensions containing more than 12 500 platelets/µL did annexin V-FITC binding reach saturating concentrations, meaning maximum expression of procoagulant activity. Unshaken platelets (25 000/µL) stimulated with 1 U/mL thrombin did not bind annexin V-FITC (Figure 3B). In contrast, shaken thrombin-stimulated platelets (1 U/mL) bound 160 000 ± 19 000 molecules of annexin V/platelet.
Blockage of GPIIb-IIIa or its absence prevents thrombin-induced expression of platelet procoagulant activity Figure 4A shows that incubation of gel-filtered platelets with the GPIIb-IIIa inhibiting peptide GRGDSP (1 mmol/L), which prevents RGD-containing ligands of GPIIb-IIIa such as fibrinogen binding to GPIIb-IIIa, for 10 minutes before activation with thrombin (0.05-2 U/mL), strongly inhibited annexin V-FITC binding. Incubation of gel-filtered platelets with the GPIIb-IIIa blocking MoAb 7E3 (20 µg/mL) also completely prevented exposure of procoagulant activity in response to thrombin (Figure 4A). Similarly, gel-filtered platelets from a patient with Glanzmann thrombasthenia did not develop procoagulant activity in response to thrombin (Figure 4A) compared to a normal control. Procoagulant activity, measured as thrombin generation by the prothrombinase complex, was also reduced after blockage of GPIIb-IIIa with MoAb 7E3 (20 µg/mL) as shown in Figure 4B.
Preventing fibrinogen binding to platelets does not inhibit the exposure of procoagulant activity Figure 5A shows gel-filtered platelets activated with 1 U/mL thrombin or 50 µmol/L TRAP with or without antifibrinogen Ab (100 µg-1 mg/mL in the presence of 1.25 mmol/L GPRP and 100 µg/mL fibrinogen-FITC). FITC-fibrinogen and endogenous fibrinogen binding to platelets was inhibited by a large excess of antifibrinogen Ab and possible platelet aggregation by cross-linking fibrin monomers was inhibited by the peptide GPRP. Maximal inhibition of fibrinogen-FITC binding was reached at approximately 400 µg/mL Ab. The exposure of procoagulant activity detected by annexin V-FITC binding was not influenced by preventing fibrinogen binding to platelets (Figure 5B). Gel-filtered platelets were preincubated with high amounts of antifibrinogen Ab (100 µg-1 mg/mL) and were activated with 1 U/mL thrombin. Because 400 µg/mL antifibrinogen Ab totally blocked binding of 100 µg/mL of added FITC-fibrinogen, in the presence of all endogenous fibrinogen from the platelets, we were sure that all released fibrinogen was also inhibited. To prevent fibrin monomers binding to each other or cross-linking platelets, the peptide GPRP (1.25 mmol/L) was added to each sample. In all samples Fab fragments of MoAb IV.3 (20 µg/mL) against FcRII were first added to platelets to prevent platelet
activation by antibody binding to FcRII.
Platelet procoagulant activity exposure is inhibited by monoclonal
antibodies to GPIb MoAb SZ2 (Figure
6A) or anti-GPIb MoAb 2128 (data not
shown), which prevent both thrombin and vWf binding to GPIb. The
antibodies were used at 20 µg/mL and were incubated with gel-filtered
platelets for 10 minutes before thrombin activation (0.05-2 U/mL).
Incubation of gel-filtered platelets with glycocalicin (0.4 µmol/L)
led to a marked reduction in platelet procoagulant activity over the
total range of thrombin concentrations (0.05-2 U/mL) (Figure 6A).
Heparin-Na (400 IE) (data not shown) or Fragmin (40 IE) (Figure 6A)
added to the platelet suspension for 10 minutes before thrombin
activation (0.05-2 U/mL) also led to a total inhibition of platelet
procoagulant activity across the full range of thrombin concentrations.
The optimal antibody, glycocalicin, heparin, and Fragmin concentrations
were determined separately. Thrombin generation by the prothrombinase
complex was also clearly inhibited after blocking GPIb with the MoAb
SZ2 (Figure 6B).
Comparison of platelet procoagulant activity and CD62 exposure in
response to -thrombin compared to -thrombin at a 10-fold lower
dose. Whereas the -thrombin gives a normal procoagulant activity,
the -thrombin, like TRAP, barely induces a procoagulant response.
However, both forms of thrombin gave equivalent surface exposure of
CD62 at these relative doses (Figure 7B) showing that the platelets
were otherwise activated to an equivalent extent.
The thrombin- but not the vWf-binding site on GPIb and vWf. Fab fragments of MoAb IV.3 (20 µg/mL) against FcRII were first added to the platelet suspension
to prevent possible platelet activation by antibody binding to
FcRII. A MoAb to GPIb, which inhibits thrombin binding but not
vWf binding (VM16d), inhibited the procoagulant response of platelets
to low but not to high doses of thrombin. MoAbs to vWf that either
block (AVW3) or do not affect (AVW1) vWf binding to GPIb induced by
ristocetin did not affect procoagulant activity after thrombin
activation. Figure 8B shows that ristocetin-induced vWf binding to
platelets was not affected by the MoAb (VM16d) to the thrombin-binding
site on GPIb. The optimal antibody concentrations were determined separately. To test whether vWf binding to GPIIb-IIIa plays a role in
thrombin-induced platelet procoagulant activity we used a MoAb 9 (20 µg/mL) against the GPIIb-IIIa binding site of vWf. MoAb 9 did not
inhibit procoagulant activity, whereas it clearly inhibited TRAP
induced vWf binding to GPIIb-IIIa (data not shown).
Inhibition of platelet procoagulant activity by blocking PAR-1 Incubation of gel-filtered platelets with the PAR-1 partial agonist peptide YFLLRNP (100 µmol/L, 10 minutes), which induces platelet shape change but does not cause further activation or aggregation, nearly completely inhibited annexin V-FITC binding to thrombin-stimulated (0.05-2 U/mL) platelets (Figure 9). The peptide bradykinin, which acts as a total antagonist of PAR-1, gave similar results (Figure 9). Gel-filtered platelets were incubated with bradykinin at 1 mmol/L for 10 minutes. Blocking PAR-1 with MoAb IIaR-A (20 µg/mL) inhibited annexin V-FITC binding at thrombin concentrations up to 1 U/mL (Figure 9). In this experiment Fab fragments of MoAb IV.3 (20 µg/mL) against FcRII were also added to the platelet suspension to prevent possible
platelet activation by cross-linking through antibody Fc domain binding
to FcRII.
The differences in platelet responses to thrombin compared to the
thrombin receptor peptide SFLLRN (TRAP) have been the object of several
studies48-51 but have not been completely elucidated. It
seems clear that thrombin gives a more extensive platelet activation than TRAP. Recently, the discovery that platelets have more than one
protease-activated receptor suggested a possible solution to this
problem; however, the other thrombin-sensitive receptor, PAR-4,
requires higher levels of thrombin than PAR-1.26,27 The
earliest platelet thrombin receptor described was GPIb,52 but it is not cleaved by thrombin and because proteolytic activity is
essential for thrombin-induced platelet activation, following the
discovery of the PAR-153 there has been a tendency to
neglect these earlier findings. Nevertheless, considerable evidence
supports a role for GPIb in platelet activation by thrombin. In
particular, it is well established that reagents interacting with the
thrombin-binding site on GPIb One major difference between platelet activation by thrombin, which can use all the thrombin receptors, and TRAP, which only activates the PAR-1, is the exposure of procoagulant activity48 (Figures 1 and 2). Development of procoagulant activity involves flip-flop of lipids within the platelet plasma membrane so that negatively charged aminophospholipids, phosphatidylserine, ethanolamine, and inositol are exposed on the outer surface of the plasma membrane. Exposure of these phospholipids can be conveniently measured by using the property of annexin V to bind to negatively charged phospholipid surfaces but not to the neutral phospholipids of the resting platelet surface. To ensure that annexin V binding was representative of platelet procoagulant activity, basic experiments were also duplicated measuring thrombin generation by a chromogenic method. It was shown previously58 that thrombin desensitizes platelets to a subsequent challenge by TRAP and that preactivation by TRAP desensitizes to a later treatment with thrombin. In the case of procoagulant activity we show (Figure 2B,C) that activation of platelets by TRAP desensitizes them to a later dose of thrombin, even when this was high (2 U/mL). This shows that preactivation of PAR-1 also down-regulates the mechanisms leading to procoagulant expression. Examination of the procoagulant activity in different platelet concentrations treated with the same amount of thrombin clearly showed that there was a concentration-dependent response (Figure 3A). This suggested that the exposure of procoagulant surface in response to thrombin was dependent on platelet-platelet contact. This was confirmed by comparing the exposure of procoagulant surface in unshaken versus shaken platelets at the same concentration treated with the same amount of thrombin (Figure 3B). In unshaken platelets there was no exposure of procoagulant activity, whereas in the shaken platelets this was exposed normally. This role of activated platelet-platelet contact suggested that GPIIb-IIIa might also be implicated in the procoagulant activity because it is well known that linkage of GPIIb-IIIa on different platelets by fibrinogen is the basis for platelet aggregation.59 Blockage of GPIIb-IIIa by a specific peptide antagonist GRGDSP60 (Figure 4A), or by a specific MoAb 7E3 directed to GPIIb-IIIa61 (Figure 4), also inhibited exposure of procoagulant phospholipids. This involvement of GPIIb-IIIa in exposure of procoagulant activity in response to thrombin was confirmed using platelets from a patient with Glanzmann thrombasthenia where GPIIb-IIIa is missing and there was no procoagulant response to thrombin (Figure 4A). This effect of blockage or absence of GPIIb-IIIa could have been due to prevention of the best known ligand, fibrinogen, binding to GPIIb-IIIa and therefore of platelet aggregation via this mechanism. To check whether this was the case, fibrinogen binding to activated GPIIb-IIIa was blocked by a large excess of polyclonal antibodies to fibrinogen. Although, as measured by FITC-fibrinogen binding, fibrinogen binding was successfully prevented (Figure 5A), this did not affect expression of procoagulant activity (Figure 5B). This suggests that GPIIb-IIIa has an additional function in expression of procoagulant activity and also that the platelet-platelet contact, necessary for procoagulant expression might involve receptor-protein interactions other than GPIIb-IIIa/fibrinogen such as GPIb/thrombin/PAR-1 or GPIb/thrombin/prothrombin. Byzova and Plow62 showed that prothrombin binding to GPIIb-IIIa was necessary for normal procoagulant activity. It should be noted that in Bernard-Soulier syndrome where GPIIb-IIIa and fibrinogen are present normally there is still a defect in procoagulant activity.30 The role of GPIb in exposure of procoagulant activity was tested using
MoAbs known to block thrombin-binding sites on GPIb. One was SZ2 where
the epitope lies in the middle of the highly negatively charged domain
of GPIb This role of GPIb was confirmed by some alternative approaches.
Glycocalicin, the soluble extracellular domain of GPIb Heparin inhibits thrombin-induced platelet activation via
GPIb63 either by blocking the GPIb The differences in platelet responses to Because none of the above experiments can completely exclude a role for
GPIb in thrombin-induced platelet procoagulant activity via an indirect
mechanism, it was important to rule out a possible role for vWf-GPIb
interactions. MoAbs to vWf were incubated with platelets before adding
thrombin. MoAb AVW1 with an epitope on the C-terminal part of vWf does
not affect function, whereas the other (AVW3), with an epitope on the
A1 domain66 of vWf, blocks vWf binding to GPIb. Neither
affected thrombin-induced platelet procoagulant activity (Figure 8A).
Fab fragments of Fc VM16d, a MoAb against GPIb, which blocks thrombin but not vWf binding
to GPIb,67 only inhibited the procoagulant response to
thrombin at lower concentrations (< 0.5 U/mL) (Figure 8A). This
difference compared to SZ2 and 2128 may be due either to the position
of the epitope or to their relative avidity for GPIb compared to
thrombin. Both SZ254 and 2128 (unpublished) have epitopes
in or near the highly negatively charged sulfated tyrosine-containing peptide of GPIb Although cleavage of PAR-1 is essential to thrombin activation of platelets, its role in procoagulant expression was unclear. As noted above, preactivation of platelets with TRAP desensitized the procoagulant response to thrombin most likely via down-regulation of signaling via PAR-1. Two peptides block the effect of thrombin on PAR-1. YFLLRNP behaves as a partial agonist, inducing shape change but no release or aggregation.71 It may block PAR-1 or cause the same kind of desensitization as TRAP. It prevented procoagulant expression at all concentrations of thrombin (Figure 9). Bradykinin, which may block PAR-1,72 also completely inhibited thrombin-induced procoagulant expression (Figure 9). Blocking PAR-1 with MoAb IIaR-A prevented expression of procoagulant activity by up to 1 U/mL thrombin (Figure 9). These results show that thrombin binding to GPIb, activation of PAR-1,
platelet-platelet contact and GPIIb-IIIa are all essential for
procoagulant expression in response to thrombin but that vWf is not
involved and fibrinogen binding to GPIIb-IIIa may not be essential.
Although signaling via PAR-1 alone is enough for several aspects of
platelet activation, it is not adequate for expression of procoagulant
activity assessed by annexin V binding or thrombin generation. Several
studies found differences in platelet responses to thrombin and
TRAP48-51; however, others showed that TRAP could stimulate
platelets as strongly as thrombin.73 Because vWf binding to GPIb induces tyrosine phosphorylation and other signals, either by
clustering or shear-stress mechanisms,9,10,13 binding of
thrombin to GPIb might cause similar ancillary signals. Blockage of
GPIb affects platelet procoagulant activity across a wide range of
thrombin concentrations but only affected calcium or aggregation responses at low levels.16 Either the signals via GPIb
have a particular role in the development of procoagulant activity or
the thrombin-binding site on GPIb has supplementary roles in procoagulant activity in addition to signaling. Our results clearly show that GPIb
We thank Dr B. Coller, Dr A.V. Mazurov, and Dr D. Meyer for 7E3, VM16d, and MoAb9 antibodies, and the blood donors and patient A.M. with Glanzmann thrombasthenia type I for giving fresh blood.
Submitted December 28, 1999; accepted June 5, 2000.
K.J.C. was supported by grants from the Swiss National Science Foundation, no. 31-52396.97, and from Hoffmann-LaRoche Ltd., Basle.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Beate E. Kehrel, Mendelstrasse 11, D 48149, Münster, Germany; e-mail: kehrel{at}uni-muenster.de.
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Top
Abstract
Introduction
9-10
indicates 25 000
platelets/µL. The fluorescence of 5000 platelets per measuring point
was determined by flow cytometry. Insert: aggregation of gel-filtered
platelets induced by 0.01 U/mL thrombin. The platelet counts were
adjusted to 1 × 108/mL. The data shown are
representative of 3 similar experiments. (B) Thrombin generation by
prothrombinase complex was measured on resting and thrombin-activated
(0.5 U/mL) gel-filtered platelets in the presence of 0.4 U/mL factor Xa
and 6 µmol/L CaCl2 (n = 3); 
indicates 25 000
platelets/µL, 
, 25 000
platelets/µL activated with 50 µmol/L TRAP; 
, 25 000
platelets/µL pretreated with TRAP, activated with thrombin. Data are
the mean of the indicated number (n) of experiments using platelets
from different donors.
, 25 000 platelets/µL; 
, 12 500
platelets/µL; 
3-mediated TXA2 synthesis.
Thromb Haemost.
1998;79:1184-1190