Binding of Thrombin to Human Platelets and its Possible Significance

SUMMARY Thrombin binds tightly to human platelets. The binding reaction is dependent on the thrombin concentration used. At a physiologically significant thrombin concentration, there are about 500 binding sites per platelet with an apparent dissociation constant of 0.02 u/ml. Autoradiography studies of platelets treated with labelled thrombin showed that the thrombin was located on the platelet surface. Separation of the subcellular fractions of platelets treated with labelled thrombin by density gradient centrifugation revealed that the membrane area contained over 80% of the radioactivity initially applied. Furthermore, isolated platelet membranes bind thrombin similar to intact platelets. These data suggest that the receptors for thrombin are located on the platelet membrane. Cytochalasin A, cytochalasin B or prostaglandin E₁ did not have any effect on thrombin binding although these agents inhibited platelet aggregation. Thus, binding of thrombin is not sufficient for aggregation of platelets and other steps are involved. These agents do not affect the induction of stimulation but interfere at a later step in the thrombin-platelet interaction. On the other hand, hirudin completely inhibited binding of thrombin to platelets. It appears that the platelet receptor recognizes that part of the thrombin molecule on its surface which is blocked by hirudin. Binding studies with serotonin loaded platelets showed a close correlation between thrombin binding and the release reaction.     

Platelet procoagulant complex assembly in a tissue factor-initiated system

Summary. The aim of this study was to examine the assembly of the factor IXa/VIIIa (Xase) and factor Xa/Va (IIase) complexes on the platelet surface in a system designed to mimic tissue factor-initiated coagulation. The experimental system contained tissue factor-bearing monocytes, unactivated platelets, and plasma concentrations of factors V, VIII, IX, X, prothrombin, tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and small amounts of factor VIIa. The time courses of platelet activation, coagulation factor binding and thrombin generation were compared. In this system, thrombin generation by the combination of monocytes and platelets was synergistic compared to each cell type alone. Platelet activation and thrombin generation were minimal in the absence of prothrombin or factor X. After a lag period, platelet activation began, followed by progressive binding of factors Va and VIIIa. This was followed by factor IXa and Xa binding and the onset of thrombin generation. Unexpectedly, a transient early increase in platelet-associated factor IX and X was also seen, that was due to release from platelets. The amount of factor IX bound to isolated activated platelets was increased by addition of factor VIIIa, or by activation of factor IX to IXa. In contrast, factor VIIIa binding was not altered by the presence of factor IX or IXa. We conclude that in a tissue factor-initiated system, assembly of the procoagulant complexes on the platelet surface begins after platelet activation occurs. Platelet activation requires thrombin generation in the vicinity of the tissue factor bearing cells. The cofactors Va and VIIIa bind to the platelets and facilitate subsequent binding of factors IXa and Xa to form functional procoagulant complexes.     

Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder.

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.     

Binding of thrombin-activated human factor VIII to platelets

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.     

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.     

Histidine-rich glycoprotein binding to activated human platelets

Specific binding of purified histidine rich glycoprotein (HRGP) to human platelets stimulated with either bisdiazoniumbenzidine-crosslinked immunoglobulin G (BDB-IgG), with thrombin or with collagen was dose- and divalent cation dependent. A 5-10-fold increase of platelet bound 125I-HRGP was obtained when 0.5-0.8 x 10(9) platelets/ml were activated with 100 micrograms BDB-IgG/ml, 0.1 U thrombin/ml or 15 micrograms collagen/ml. At maximal binding tested 16,000 molecules of HRGP became bound per platelet, but saturation was not achieved. Such platelet inhibitors as acetylsalicylic acid, prostaglandin E1 and cytochalasin B reduced the capacity of platelets to bind ligand, and by kinetic experiments involving enzymatic digestion of radiolabelled bound HRGP the ligand revealed to remain surface bound rather than being taken up to inner parts of the cell.     

Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa.

Factor Xa binds to platelets provided that factor Va is present on the platelet surface, an interaction that results in a striking acceleration of the conversion of prothrombin to thrombin. Thrombin then initiates fibrin formation, induces platelet aggregation, and stimulates the intraplatelet synthesis of thromboxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured as thromboxane B2, from less than 0.5 pmol per 10(8) platelets to (mean +/- SEM) 100 +/- 22 and 250 +/- 10 pmol per 10(8) platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations of factor Xa (1-12 nM) prior to the addition of thrombin progressively inhibited the production of TXA2. Thrombin (9.6 nM), which produced 93% of the maximal formation of TXA2, was inhibited 70% by factor Xa (10 nM). To identify which of these steps in thromboxane synthesis was inhibited by factor Xa, platelets labeled with [14C]arachidonic acid were exposed to thrombin and products of prostaglandin synthesis were separated by thin-layer chromatography. In contrast to the inhibition of TXA2 synthesis, prostaglandin E2 and prostaglandin F2 alpha synthesis were not inhibited suggesting that neither phospholipase(s) nor cycloxygenase was involved. The inhibition of TXA2 formation by factor Xa could be reversed by increasing the molar ratio of thrombin to factor Xa to 5.5. Incubation of platelets with an IgG fraction of a human monoclonal antifactor V antibody, previously shown to inhibit factor Xa binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence of the active site serine of factor Xa and is not specific for TXA2 formation induced by thrombin because it is also demonstrable when the agonist is ADP. Further, factor Xa does not require additional plasma components for its action because its inhibitory effects are detected in gel-filtered platelets. The effect of factor Xa was evident at physiological (1.3 mM) calcium concentrations. These results indicate that factor Xa binding to platelets through factor Va not only stimulates thrombin formation but also has a countervailing effect by inhibiting TXA2 formation.     

Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va.

The blood coagulation protein factor Va forms the receptor for the serine protease factor Xa on the platelet surface. This membrane-bound complex of factor Va and factor Xa plus Ca2+ comprises the prothrombinase complex, the enzyme that catalyzes the proteolytic conversion of prothrombin to the clotting enzyme thrombin. Factor Va is a two-subunit protein composed of component D (Mr = 94,000) and component E (Mr = 74,000); subunit interaction is Ca2+ dependent. Factor Va bound to platelets consists of three peptides: component D, component E, and component D'(Mr = 90,000) which appears as the result of a platelet-associated protease cleavage of component D. The present studies were undertaken to determine which peptide(s) mediates the binding of factor Va to the platelet membrane surface and which peptide(s) serves as the binding site for factor Xa. These interactions were assessed by direct measurements of radiolabeled factor Va and factor Xa binding to platelets as well as autoradiographic visualization of the factor Va peptides associated with the platelet. Experiments were performed to determine the interaction of components D and E with platelets under reaction conditions in which components D and E were present as either the intact, functional two-subunit protein or as nonfunctional discrete peptides dissociated by the addition of Na2EDTA. The results suggest that component E mediates the binding of factor Va to the platelet and also serves as the binding site for the interaction of factor Xa with platelet-bound factor Va.     

Comparison of thrombin and ristocetin in the interaction between von Willebrand factor and platelets

It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that low concentrations of thrombin also cause platelet membrane receptors to become available for FVIII/vWF. As a consequence, the suspicion has been raised that thrombin provides similar or equivalent activity in vivo to that observed for ristocetin under in vitro conditions. In this study, we quantitated the extent to which thrombin promotes the binding of FVII/vWF to platelets and determined whether or not this interaction initiates or complements platelet aggregation. With ristocetin present, the amount of 125I-FVIII/vWF that became platelet-bound correlated closely with the onset, rate, and extent of platelet aggregation. In contrast, at every thrombin concentration tested, the amount of 125I- FVIII/vWF that specifically bound to platelets was about 6% of that observed with ristocetin. Significantly, FVIII/vWF did not augment the rate of aggregation of platelets in response to thrombin or initiate platelet aggregation when subaggregating doses of thrombin were used. These observations indicate that the minimal association that occurs between FVIII/vWF and the platelet membrane in the presence of thrombin does not correlate with platelet aggregation and therefore is not analogous to the effects of ristocetin. Whether the low level of binding relates to another process, such as platelet-endothelial interactions, remains unknown.     

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).     

Heterogeneity of fibrinogen receptor expression on platelets activated in normal plasma with ADP: analysis by flow cytometry

Fibrinogen receptor expression of platelets activated in normal plasma by ADP was measured by flow cytometry after labelling bound fibrinogen with fluorescein-conjugated antifibrinogen antibody. The platelet response to ADP was heterogeneous both with respect to number of platelets binding fibrinogen and the amount of fibrinogen bound per platelet. The proportion of platelets showing positive antifibrinogen antibody binding increased with increasing ADP concentration; however, even at 10(-3) M ADP, usually about one-fifth of the platelets failed to demonstrate bound fibrinogen. The non-responsive platelets tended to be the smaller ones. The relative fluorescence intensity of individual platelets also increased as ADP concentration was increased, indicating that the average number of fibrinogen molecules bound was also related to agonist concentration. The amount of fibrinogen bound following platelet activation directly correlated with the quantity of surface glycoprotein IIb detected by Tab antibody and with platelet size. This study demonstrates that platelet response to ADP in native plasma is heterogeneous in both the proportion of platelets activated and in the number of available fibrinogen receptors per platelet. This heterogeneity is related to platelet size and glycoprotein IIb-IIIa content. These observations indicate that models of ligand interaction with membrane receptors on intact cells requiring an exposure step must take into account the heterogeneity of response within a cell population. In addition to providing new insights into the response of individual platelets to activation, these results suggest that study of platelet bound fibrinogen by flow cytometry may be useful for the detection of platelet activation in vivo.     

Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.

Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated with a time course and concentration dependence consistent with a role for these lipids in platelet activation. We now report that when human platelets are stimulated with thrombin, a complex forms between phosphatidylinositol 3-kinase, a protein-serine/threonine kinase, and an uncharacterized platelet membrane protein. The complex is immunoprecipitated from detergent lysates of thrombinstimulated platelets by a rabbit antiserum prepared against a peptide from the cytoplasmic domain of the mouse platelet-derived growth factor (PDGF) receptor. The antigen is not the PDGF receptor, since complex formation is not stimulated by PDGF and thrombin-induced complexes are not precipitated by another rabbit antiserum against the same peptide or by monoclonal anti-human PDGF receptor antibodies. Formation of the complex is rapid (within 30 sec) and occurs at thrombin concentrations that stimulate platelet aggregation and secretion (50% of maximal complex formation at 0.03 unit of thrombin per ml). We propose that the complex initiates formation of 3-phosphate-containing phosphatidylinositols that may function in platelet activation.     

Platelet Antithrombins: Role of Thrombin Binding and the Release of Platelet Fibrinogen

The nature of platelet antithrombin was elucidated by comparison of thrombin binding and antithrombin activities of intact platelets and by purification of antithrombin from platelet lysates using glycerol osmotic lysis, ethanol precipitation and Sephadex gel filtration techniques. The major portion of the antithrombin and thrombin binding activity of intact platelets is lost after brief sonication. The antithrombin activity in destroyed platelets is found to be due to platelet fibrinogen. Treatment of platelets with PGE₁ (100 μg/ml) markedly inhibits (>80%) the release of platelet fibrinogen induced by thrombin. However, the PGE₁ treatment produced slight (<30%) but significant decrease of antithrombin activity of intact platelets, whereas the binding of thrombin to platelets was not affected by PGE₁ treatment. The amounts of thrombin bound to and inactivated by PGE₁-treated platelets at the same cell concentration are identical. The above results suggest that platelets contain at least two antithrombin activities. One, which accounts for the major portion of platelet antithrombin is mediated by thrombin binding to platelets. The other, which attributes to a lesser extent to platelet antithrombin activity, is due to the release of platelet fibrinogen. Also, antithrombin is readily demonstrated in a plasma medium indicating physiological significance of platelet antithrombin.     

Fibronectin-mediated inhibition of human platelet activation by crosslinked immunoglobulin G

The effect of purified human plasma fibronectin on stimulation of human platelets with crosslinked immunoglobulin G (IgG) was tested by means of aggregometry, 14C-serotonin release and fibronectin-to-platelet binding experiments. Incremental additions of fibronectin to gel-filtered platelets (8 X 10(8)/ml) followed by 25 micrograms/ml bisdiazoniumbenzidine (BDB)-crosslinked IgG produced a dose-related inhibition of platelet aggregation and suppression of 14C-serotonin release from the platelets. Evidence was obtained that upon stimulation of the platelets with BDB-IgG, fibronectin becomes bound to its receptor and that further platelet activation is inhibited when a sufficient number of receptors is occupied.     

Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.     

Streptokinase-induced platelet aggregation. Prevalence and mechanism

BACKGROUND. Streptokinase (SK) is a bacteria-derived protein and one of the plasminogen activators that is currently available for therapeutic use. Exposure to SK induces synthesis of specific antibodies that may initiate platelet aggregation and paradoxical clot propagation during treatment. METHODS AND RESULTS. Using platelet-rich plasma (PRP), we found that SK (5,000 units/ml) but not urokinase (2,500 units/ml) or recombinant tissue-type plasminogen activator (2,500 units/ml) caused platelet aggregation in PRP from 14 of 100 normal volunteers. In 13 consecutive patients treated with SK for acute myocardial infarction, SK-mediated platelet aggregation was induced in five patients within 1 week after treatment. SK-mediated platelet aggregation was associated with significantly increased titers of both anti-SK antibodies and SK-neutralizing activity in plasma; it was partially inhibited by aspirin (1 mM) and by aprotinin (500 kallikrein inhibitor units/ml) and completely inhibited by tranexamic acid (1 mM) and by prostaglandin E1 (9 microM). Addition of SK (1,000 or 5,000 units/ml) induce a statistically significant dose-dependent thromboxane B2 release in mixtures of PRP with plasma from subjects with SK-induced aggregation but not in samples of PRP mixed with plasma from nonresponders; addition of recombinant tissue-type plasminogen activator (1 or 50 micrograms/ml) did not induce thromboxane B2 release. Mixing experiments with PRP and immunoglobulin G from reactive and nonreactive donors revealed that SK-induced aggregation requires the presence of anti-SK antibodies. When 125I-SK (50 nM) was used, platelets preincubated with plasminogen (0.5 microM) bound 9,500 +/- 600 (mean +/- SEM, n = 6) molecules SK/platelet, which increased to 25,000 +/- 3,100 molecules/platelet after thrombin stimulation. Tranexamic acid (1 mM) blocked specific binding of SK to resting platelets. CONCLUSIONS. These data demonstrate that SK-induced platelet aggregation is initiated by the binding of anti-SK antibodies to the SK-plasminogen complex located on the platelet surface. SK-induced platelet activation may limit the therapeutic effectiveness of the drug, and in view of the high prevalence of aggregation in a normal population, prospective evaluation of the effects of platelet aggregation during treatment with SK is warranted.     

Platelet Aggregation and Release by ADP and Thrombin: Evidence for Two Separate Effects of ADP on Platelets, Involvement of Fibrinogen in Release, and Mechanism of Inhibitory Action of Acetylsalicylic Acid

S ummary . The previously demonstrated roles of thrombin and fibrinogen in platelet interaction (Ardlie & Han, 1974) suggested a possible relationship between platelet aggregation and the release reaction. The present results show that a product of the action of thrombin on fibrinogen induced the release of platelet constituents. This activity was not inhibited by heparin. The thrombin-fibrinogen reaction which occurs when platelets come into contact could be the cause of the subsequent release reaction. ADP was shown to be essential for the thrombin-induced aggregation, but not the alteration in platelet morphology. Evidence of involvement of ADP in cell contact was obtained and it is possible that its role may be to facilitate aggregation through an effect on platelet surface charge. Thus, ADP induces aggregation through two distinct actions: (1) alteration of platelet morphology; and (2) reduction in net negative surface charge of platelets. Incubation of thrombin with aspirin inhibited its action on platelets and it is suggested that acetylation of thrombin (or prothrombin) by aspirin may account for the action of this compound on platelets. The significance of these observations and a possible mechanism for the release reaction is discussed.     

The interaction of thrombin with blood platelets

Thrombin is a potent agonist of platelets. In the current article, the research on the interaction of thrombin with blood platelets is reviewed starting with the first studies demonstrating the direct action of thrombin on platelets and ending with an analysis of the importance of the protease-activated receptors (PARs) and the GpIb complex. The antithrombin activity of platelets is discussed in terms of the binding of thrombin to receptor(s) on the platelet surface. Evaluation of the PAR receptors and the GpIb supports a model where thrombin binds to the GpIb receptor prior to the proteolysis of the PAR receptor(s). Thus, the maximal hemostatic response requires both PAR receptors and the GpIb receptors.     

Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400-411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.     

Interaction of a Monoclonal Antibody to Glycoprotein IV (CD36) with Human Platelets and its Effect on Platelet Function

FA6-152, a monoclonal antibody to platelet membrane glycoprotein IV (CP IV), was used to quantify the expression of this glycoprotein on platelets, as well as to evaluate its role in platelet aggregation. On resting platelets, 19 400 ± 7700 molecules of the (125)I-labelled IgC could bind per platelet (n = 20). Binding was not modified following stimulation of the platelets with ADP (10 μmol/l) or thrombin (0.1 U/ml). Fab fragments prepared from the antibody by papain digestion also bound to the platelet surface in a saturable manner. Both the intact IgC and its Fab fragments were found to inhibit platelet aggregation and secretion induced by ADP or collagen in platelet-rich plasma and by thrombin in platelet suspensions. Under nonstirred conditions, whereby the release reaction was only minimally affected, the antibody markedly inhibited thrombin-induced surface expression of α-granule thrombospondin (TSP), whereas it did not alter the concomitant expression of α-granule fibrinogen. In addition, electron microscopy revealed a predominant distribution of TSP and T;P IV on pseudopodia and between adherent cells on thrombin-stimulated platelets. These findings thus support the hypothesis that the interaction of TSP with GP IV on the platelet surface is required for an optimal platelet aggregation/secretion process to occur.