An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.     

Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats

Fibrinogen is a cofactor in the aggregation of human platelets, and is required for ADP-induced aggregation of washed platelets; however, exogenous fibrinogen is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Because with human platelets the cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), inhibits aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets, its effects on rabbit and rat platelets were studied to investigate the differences in the fibrinogen requirements of platelets from the three species. RGDS (50 mumol/L) caused greater than 80% inhibition of thrombin- induced or (ADP + fibrinogen)-induced aggregation of human platelets, but only 3% to 9% inhibition of the aggregation of rabbit or rat platelets, regardless of whether fibrinogen was added. RGDS inhibited the binding of 125I-fibrinogen to ADP-stimulated human platelets by 80% to 90%, but by only 15% to 27% in the case of rabbit or rat platelets. The differences were due to the species of platelets, since human and rabbit fibrinogens gave similar results. In addition, RGDS failed to displace fibrinogen from the surface of rabbit platelets that had been stimulated with ADP. Thus, there are species differences in the ability of the cell adhesion peptide, RGDS, to block the platelet fibrinogen receptor, even within the mammalian species.     

Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.     

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.     

Exposure of fibrinogen receptors on fresh and stored platelets by ADP and epinephrine as single agents and as a pair

Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10-15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.     

Recognition of platelet-associated fibrinogen by polyclonal antibodies: correlation with platelet aggregation.

Progressive decreases in platelet-bound fibrinogen accessibility to antibody and enzymes were recently reported to occur after adenosine diphosphate (ADP)-induced fibrinogen binding. Because previous studies also indicated that platelets that are activated but not aggregated by ADP in the presence of fibrinogen lose their ability to aggregate in a time-dependent manner despite negligible changes in fibrinogen binding, the present study examined the relationship between platelet aggregation and accessibility of platelet-bound fibrinogen to specific polyclonal antibody F(ab')2 fragments over a 60-minute time course. Although 125I-fibrinogen binding remained virtually unchanged, comparison of antifibrinogen antibody F(ab')2 binding and platelet aggregation 5 minutes and 60 minutes after platelet stimulation with ADP or thrombin showed decreases in F(ab')2 binding of 62% +/- 13% and 73% +/- 7% (mean +/- SD, n = 5), respectively, and decreases of 65% +/- 16% and 60% +/- 10% in platelet aggregation. In contrast, platelets stimulated with A23187 or chymotrypsin retained 87% +/- 16% and 76% +/- 12% of their ability to aggregate over the same time course, and lost only 39% +/- 14% and 36% +/- 12% of their ability to bind antifibrinogen antibody F(ab')2 fragments, respectively. Pretreatment of ADP-stimulated platelets with chymotrypsin largely prevented the progressive loss of platelet aggregability and the accompanying decreased recognition of bound fibrinogen by antifibrinogen F(ab')2 fragments. Preincubation of platelets with cytochalasin D (30 micrograms/mL) also inhibited the decrease in platelet aggregation after exposure of ADP-treated platelets to fibrinogen over a 60-minute time course. This was accompanied by only a 25% +/- 18% decrease in antifibrinogen antibody F(ab')2 binding. Present data support the hypothesis that qualitative changes in platelet-bound fibrinogen correlate with loss of the ability of platelets to aggregate, and implicate both the platelet cytoskeleton and chymotrypsin-sensitive surface membrane structures in modulating qualitative changes in bound fibrinogen on the platelet surface.     

Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot-reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti-glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non-stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.     

ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is not dependent on ADP. Human fibrinogen inhibited in a competitive manner the ADP-induced binding of 125I-labeled vWF (9 micrograms/ml) with an IC50 of 25 micrograms/ml. Conversely, unlabeled vWF inhibited ADP-induced binding of 125I-labeled fibrinogen (60 micrograms/ml) with an IC50 of 16 micrograms/ml. A synthetic dodecapeptide (Mr, 1188), analogous with the specific platelet receptor recognition site of human fibrinogen gamma chain (gamma 400-411), inhibited binding of both 125I-labeled vWF and 125I-labeled fibrinogen to ADP-treated platelets, whereas it was without effect on binding of 125I-labeled vWF to ristocetin-treated platelets. These data indicate that vWF and fibrinogen have a common receptor mechanism for their interaction with human platelets that is dependent on ADP occupancy of its binding sites and is recognized by the sequence of 12 amino acid residues at the carboxyl terminus of the human fibrinogen gamma chain.     

gamma and alpha chains of human fibrinogen possess sites reactive with human platelet receptors.

Fibrinogen, a clottable plasma protein, agglutinates both prokaryotic cells (e.g., staphylococci) and eukaryotic cell fragments (e.g., platelets) through interaction with specific receptors. To identify the region of the fibrinogen molecule responsible for its interaction with human platelets, we prepared polypeptide chain subunits (alpha, beta, and gamma) of human fibrinogen by reduction and carboxymethylation. A mixture of the chains induced aggregation (clumping) of human platelets separated from plasma proteins and treated with ADP. When individual chains of fibrinogen were tested, gamma-chain multimers caused platelet aggregation at a molar concentration comparable with that of intact human fibrinogen. The beta chain remained inactive, and the alpha chain was 1/4th to 1/5th as reactive as the gamma chain. Monospecific antibody fragments against the gamma chain inhibited binding of 125I-labeled fibrinogen to the human platelet receptor and blocked aggregation of platelets induced by ADP in the presence of fibrinogen or gamma-chain multimers. These results indicate that the gamma chain of human fibrinogen bears the main site for interaction with the platelet receptor.     

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.     

Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin

Thrombopoietin (TPO) is a recently identified growth factor that regulates megakaryocytopoiesis. Its receptor, c-Mpl, is expressed in megakaryocyte progenitors, mature megakaryocytes, and human blood platelets. We have observed that TPO treatment of human platelets resulted in tyrosine phosphorylation of several cellular proteins, including the c-Mpl receptor and the 85-kD subunit of phosphatidylinositol 3-kinase (PI3-K). TPO stimulated this tyrosine phosphorylation in a time-dependent manner, reaching a maximum in 5 minutes. The tyrosine phosphorylation of PI 3-K was dependent on the concentration of TPO and reached a maximum at concentrations between 50 and 100 ng/mL. This phosphorylation was independent of extracellular fibrinogen and ligation of the alpha IIb beta 3 integrin. In contrast, TPO, in the presence of exogenous fibrinogen, induced concentration- dependent platelet aggregation, which was blocked by the soluble c-Mpl receptor. Increasing TPO concentrations modulated the degree of the primary wave of aggregation and the lag phase, but not the slope or maximum of the secondary wave of aggregation. This secondary aggregation was controlled by the addition of apyrase, suggesting an adenosine diphosphate (ADP)-dependent mechanism. Treatment of platelets with TPO resulted in augmented binding of 125I-fibrinogen to intact platelets, with a 50% effect (EC50) occurring between 5 and 10 ng/mL. TPO-induced binding of fibrinogen to platelets was comparable in degree with that observed by stimulation with 10 mumol/L ADP. In an immobilized collagen-platelet adhesion assay, a significant increase in the attachment of TPO-stimulated platelets was observed. This effect was dependent on the concentration of TPO. At 50 ng/mL of TPO, platelet attachment to collagen increased threefold compared with the buffer control. Furthermore, the presence of fibrinogen did not significantly alter TPO augmentation of the platelet-collagen interaction. This interaction was mediated by the Arg-Gly-Asp (RGD) adhesion recognition sequence, as it was completely abolished by 100 mumol/L of the RGDS peptide. A fraction of the TPO-dependent platelet attachment to a collagen-coated surface was insensitive to treatment with prostaglandin E1. Furthermore, antibody to alpha IIb integrin partially inhibited platelet attachment to collagen, suggesting that the integrin alpha IIb beta 3 participates in this association. These data indicate that TPO might function not only as a cytokine in megakaryocyte growth and differentiation, but may also participate in direct platelet activation and modulate platelet-extracellular matrix interactions.     

Comparison of fibrinogen association with normal and thrombasthenic platelets on exposure to ADP or chymotrypsin.

Although 125I-fibrinogen becomes associated with washed platelets from normal human subjects during ADP-induced shape change and aggregation, 125I-fibrinogen did not become associated with washed plateletes from a thrombasthenic subject during ADP-induced shape change and the platelets did not aggregate. Platelets from control and thrombasthenic subjects were treated with chymotrypsin, which is known to degrade platelet membrane glycoproteins. More 125I-fibrinogen became associated with chymotrypsin-pretreated platelets from normal subejcts than with untreated platelets, and fibrinogen caused the enzyme-treated platelets to aggregate. 125I-fibrinogen did not become associated with chymotrypsin-pretreated thromobasthenic platelets, and fibrinogen did not aggregate them. Thus, there appears to be a defect in thrombasthenic platelets that prevents the association of fibrinogen with them.     

Platelet GPIIb/IIIa antagonist, XV459, in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a serious, immune-related complication of heparin therapy. One of the most severe manifestations of HIT is the development of thromboembolic events, which is based on platelet activation and aggregation caused by HIT-associated antibodies. Therapeutic options for patients with HIT are limited despite advancement toward the development of alternative (nonheparin) anticoagulants, such as direct thrombin inhibitors and indirect anti-factor Xa agents. Platelet GPIIb/IIIa receptor antagonists have been shown to be the final common pathway for platelet aggregation regardless of the use of activator or anticoagulant. In this study, the ability of a novel platelet GPIIb/IIIa antagonist, a free acid form of roxifiban (XV459), to block platelet activation/aggregation in response to highly characterized heparin-PF4 antibody-positive plasma/heparin was examined using light transmittance aggregometry, serotonin release, and (125)I-fibrinogen binding assays to human platelets. XV459 at 20 nM maximally inhibited (P < 0.001) the platelet-activation/aggregation responses as mediated by the HIT antibody-positive plasma (in the presence of therapeutic heparin concentrations). Compared with controls, both HIT antibodies/heparin and TEAC (a mixture of thrombin [0.1 IU/ml], epinephrine [1 microg/ml], arachidonate [0.1 mM], and collagen [10 microml]) resulted in significantly higher levels of fibrinogen binding to human platelets (5-7-fold increase; P < 0.001). Concentration-dependent profiles of XV459 on the mean percent inhibition of (125)I-fibrinogen binding in the presence of HIT antibodies and TEAC were achieved ( approximately 50% inhibition at 10 nM XV459). The platelet GPIIb/IIIa receptor antagonist (XV459) might be of potential benefit in the management of thrombotic thrombocytopenia produced by heparin and/or related glycosaminoglycans.     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Platelet activation by sustained exposure to low-dose plasmin.

Plasmin has been reported to activate and inhibit platelet function depending on dose and exposure temperature. The present study examines the induction of fibrinogen-dependent platelet aggregation following prolonged (60 min) platelet exposure to very low doses of plasmin (0.05 CU/ml) at either 22 or 37 degrees C. Maximum aggregation [mean +/- SD, 60 +/- 19 light transmission units (LTU); n = 43] occurred following platelet exposure to plasmin at 22 degrees C, but significant platelet aggregation (28 +/- 4 LTU, n = 3) also occurred following plasmin treatment at 37 degrees C. Plasmin-induced platelet aggregates appeared microscopically larger than aggregates of adenosine diphosphate (ADP)-activated platelets, and were less reversible. Aggregated plasmin-treated platelets also expressed more procoagulant activity than platelets aggregated with ADP, as reflected by shortening of the plasma kaolin recalcification time. Aggregation of platelets exposed to very low doses of plasmin was not accompanied by dense or alpha-granule secretion, and was unaffected by ADP antagonists or aspirin. Partial inhibition of platelet aggregation, however, was achieved with metabolic inhibitors, PGE1, and inhibitors of phosphoinositide 3-kinase or protein kinase C. Although fibrinogen was required for plasmin-treated platelet aggregation, [125I]-fibrinogen binding comprised only 58 +/- 3% (n = 3) of fibrinogen binding associated with ADP aggregated platelets. This was consistent with observed decreases in reptilase-induced fibrin clot retraction. Taken together, these data suggest that sustained exposure of platelets to very low plasmin doses leads to platelet activation and thus may contribute to thrombotic complications in vivo.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.     

Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.     

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.     

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.