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.     

Disparate effects of the calcium-channel blockers, nifedipine and verapamil, on alpha 2-adrenergic receptors and thromboxane A2-induced aggregation of human platelets

Calcium-channel blockers inhibit human platelet aggregation in vitro and ex vivo. To further evaluate the mechanism(s) responsible for the inhibition induced by this structurally heterogeneous group of compounds, we studied the effect of nifedipine and verapamil on human platelet aggregation in vitro. Neither 10 microM nifedipine nor 10 microM verapamil consistently inhibited the aggregation response of platelet-rich plasma to threshold concentrations of ADP, sodium arachidonate, epinephrine, or collagen. However, both 10 microM nifedipine and 10 microM verapamil epinephrine-potentiated, thromboxane A2 (TXA2)-induced aggregation of aspirin-incubated, gel-filtered platelets. Aggregation of similarly prepared platelets induced by TXA2 alone was abolished by 10 microM nifedipine but not by 10 microM verapamil. Even 100 microM verapamil gave only partial and inconsistent inhibition of aggregation. Both drugs had essentially the same effects on platelet aggregation induced by the stable endoperoxide and TXA2 mimic, U46619, with or without epinephrine. Neither 10 microM nifedipine nor 10 microM verapamil elevated platelet cyclic AMP. Verapamil (10 microM) inhibited binding of [3H]-yohimbine (an alpha 2-adrenergic receptor antagonist) to intact human platelets (KD 10.5 nM vs 2.4 nM for control platelets) without altering the number of binding sites. In contrast, 10 microM nifedipine had no effect on KD or number of binding sites. These results indicate that nifedipine and verapamil inhibit epinephrine-potentiated, TXA2-induced human platelet aggregation by different mechanisms. Verapamil inhibits the epinephrine contribution to the aggregation response by blocking alpha 2-adrenergic receptor binding. Nifedipine blocks the platelet response to TXA2 without affecting alpha-adrenergic receptor binding. These observations have potential clinical implications with regard to the mechanisms by which calcium-channel blockers inhibit vascular spasm and myocardial ischemia.     

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.     

Thrombospondin promotes platelet aggregation

Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

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.     

Von Willebrand factor can support platelet aggregation via interaction with activated GPIIb-IIIa and GPIb

In this study we investigated mechanisms of platelet interaction with von Willebrand factor (vWF) induced by activating anti-glycoprotein (GP)IIb-IIIa antibody CRC54 directed against LIBS (ligand-induced binding site epitope) in GPIIIa. It was demonstrated that aggregation of washed platelets (measured in Born aggregometer) could be stimulated by CRC54 not only in the presence of fibrinogen but vWF as well. The level of aggregation induced in the presence of saturating concentrations of vWF (approximately 80 microg/ml) was even higher than that in the presence of 1 mg/ml of fibrinogen. Aggregation supported by vWF unlike fibrinogen supported aggregation was almost completely inhibited not only by GPIIb-IIIa antagonists (F(ab')2 fragment of blocking anti-GPIIb-IIIa antibody CRC64 and peptidomimetic aggrastat) but also by anti-GPIb blocking antibody AK2. Aggregation response induced by CRC54 in the presence of vWF was much lower when normal platelets were substituted with GPIb-deficient platelets and this residual aggregation was not affected by anti-GPIb antibody AK2 but still inhibited by anti-GPIIb-IIIa blocking antibody fragment. CRC54-induced aggregation supported by vWF (as well as by fibrinogen) was only partially inhibited by prostaglandin E1, indicating that at least its initiation does not require activation of platelets. CRC54, both in the presence of vWF and fibrinogen, failed to stimulate serotonin secretion at physiological Ca2+ concentration of 1 mM, although substantial release reaction was detected when Ca2+ concentration was decreased to 0.1 mM. CRC54 could also stimulate platelet interaction with immobilized vWF and fibrinogen. However, unlike platelet aggregation in suspension mediated by flow phase vWF, platelet adhesion to adsorbed vWF (in a same way as to fibrinogen) was inhibited only by GPIIb-IIIa but not GPIb antagonists. The data obtained indicated that vWF support platelet aggregation induced by activating anti-GPIIb-IIIa via interaction with two receptors - activated GPIIb-IIIa and GPIb.     

A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.     

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6,200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10(-5) M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 microM) and inhibited U46619-27, or collagen-induced platelet aggregation, with ED50-values of 27 microM and 4.7 microM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as defined in functional tests, coincides with inhibition of specific ligand binding to the receptors.     

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.     

Identification of FcgammaRIIa as the ITAM-bearing receptor mediating alphaIIbbeta3 outside-in integrin signaling in human platelets

Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin alphaIIbbeta3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding-dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, FcgammaRIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase Cgamma2 (PLCgamma2). Addition of Fab fragments of an FcgammaRIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of FcgammaRIIa, Syk, and PLCgamma2, and platelets from a patient whose platelets express reduced levels of FcgammaRIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding-induced FcgammaRIIa phosphorylation did not occur in human platelets expressing a truncated beta3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet alphaIIbbeta3 induces integrin cytoplasmic domain-dependent phosphorylation of FcgammaRIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.     

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.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

Differential inhibition of adenosine diphosphate- versus thrombin receptor-activating peptide-stimulated platelet fibrinogen binding by abciximab due to different glycoprotein IIb/IIIa activation kinetics

The exposure of internal glycoprotein (GP) IIb/IIIa receptors has been proposed to explain the incomplete inhibition of aggregation of thrombin receptor-activating peptide (TRAP)-stimulated platelets by abciximab. However, a marked and rapid externalization of GPIIb/IIIa was also observed upon stimulation with 30 microM adenosine diphosphate (ADP). ADP-induced fibrinogen binding was completely inhibited by 10 microg/mL abciximab, 30 nM tirofiban, or 3 microg/mL eptifibatide, while fibrinogen binding induced by 100 microM TRAP was inhibited only by 50%. Interestingly, striking differences in fibrinogen binding kinetics in ADP- versus TRAP-stimulated platelets were observed. ADP-induced fibrinogen binding was much slower than that of abciximab. These differences in the fibrinogen binding rate were due to differential GPIIb/IIIa activation kinetics because the actual fibrinogen binding rate (measured by adding fibrinogen after platelet activation) was similar in ADP- and TRAP-stimulated platelets. Thus, the TRAP-induced GPIIb/IIIa activation rate would allow significant amounts of fibrinogen to occupy externalized GPIIb/IIIa receptors even in the presence of the inhibitor.     

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.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

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.     

A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100-solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde-fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard-Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.     

The role of fibronectin in platelet aggregation

A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.     

FbsA, a fibrinogen-binding protein from Streptococcus agalactiae, mediates platelet aggregation

The bacterium Streptococcus agalactiae is an etiologic agent in the pathogenesis of endocarditis in humans. FbsA, a fibrinogen-binding protein produced by this pathogen, is considered an important virulence factor. In the present study we provide evidence that S agalactiae clinical isolates bearing FbsA attach to fibrinogen and elicit a fibrinogen-dependent aggregation of platelets. Mutants of S agalactiae lacking the fbsA gene lost the ability to attach to fibrinogen and to aggregate platelets. Plasmid-mediated expression of fbsA restored the capability for fibrinogen binding and platelet aggregation in S agalactiae fbsA mutants, and allowed Lactococcus lactis to interact with fibrinogen and to aggregate human platelets. Moreover, a monoclonal anti-FbsA antibody inhibited bacterial adherence to fibrinogen and S agalactiae-induced platelet aggregation. Platelet aggregation was inhibited by aspirin, prostaglandin E(1,) the peptide RGDS, and the antibody abciximab, demonstrating the specificity of platelet aggregation by S agalactiae and indicating an involvement of integrin glycoprotein IIb/IIIa in the induction of platelet aggregation. Aggregation was also dependent on anti-FbsA IgG and could be inhibited by an antibody against the platelet FcgammaRIIA receptor. These findings indicate that FbsA is a crucial factor in S agalactiae-induced platelet aggregation and may therefore play an important role in S agalactiae-induced endocarditis.