Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

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.     

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.     

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.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Exposure of platelet fibrinogen receptors by a monoclonal antibody to the GPIIb-IIIa complex, PMA4.

We found that a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, PMA4, induces fibrinogen binding to platelets, and we examined the mechanism involved. Affinity chromatography and crossed immunoelectrophoresis showed that PMA4 recognized an epitope on the GPIIb-IIIa complex-specific domain. The binding of 125I-fibrinogen to platelets was induced by PMA4 in a concentration-dependent manner and was blocked by EDTA, RGDS peptides and an anti-GPIIb-IIIa monoclonal antibody, PMA1. Binding of the divalent antibody to the GPIIb-IIIa complex was necessary to induce fibrinogen binding and subsequent platelet aggregation, since Fab fragments, unlike PMA4 IgG and F(ab')2 fragments, did not induce fibrinogen binding or aggregation. The PMA4 IgG induced fibrinogen binding, serotonin secretion, and Ca2+ mobilization, whereas F(ab')2 induced fibrinogen binding only. In addition, F(ab')2-induced fibrinogen binding was not abolished in the presence of aspirin, H-7, a protein kinase C inhibitor, PGE1 or dibutyryl cyclic AMP. These results demonstrate that the binding of PMA4 divalent molecules to the GPIIb-IIIa complex can expose platelet fibrinogen receptors in the absence of the stimulatory effects of intracellular mediators on platelets. Thus, we conclude that the fibrinogen receptors on the GPIIb-IIIa complex can be exposed by direct action of the antibody on the complex molecules.     

Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.     

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.     

Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta-thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.     

The effect of dietary saturated fat and cholesterol on platelet function, platelet survival and response to continuous aortic injury in rats

Induction of hypercholesterolemia in rats by diets containing milk fat, cholesterol and taurocholate caused increased sensitivity of platelets to thrombin-induced aggregation and release, but not to ADP- or collagen-induced aggregation or release. This hypersensitivity to thrombin persisted in the presence of CP/CPK to convert released ADP to ATP, and aspirin to block formation of thromboxane A2. The increased sensitivity of platelets to thrombin in hypercholesterolemic animals was associated with an increase in 18:1 omega 9, 18:2 omega 6 and 20:3 omega 6 and a decrease in 20:4 omega 6 and 22:4 omega 6 in their phospholipids. Hypercholesterolemic animals also had a shortened platelet survival that did not appear to be due to an alteration in the lipid composition of the platelets. The diet-induced changes in platelet function were not associated with enhanced thrombosis in animals with indwelling aortic catheters, but were associated with increased platelet accumulation on the exposed subendothelium.     

A monoclonal anti-human platelet antibody: a new platelet aggregating substance

A monoclonal anti-human platelet antibody, TP82, is described, which caused irreversible aggregation of platelets in association with the release of adenosine triphosphate or [14C] serotonin, and which inhibited ristocetin-induced agglutination. Immunofluorescence assay showed that the antibody binds to platelets, megakaryocytes, and common acute lymphoblastic leukemia cells. The antibody (IgG1) immunoprecipitated a polypeptide of 23,000 daltons with an isoelectric point of about 7.0. The aggregation induced by the purified antibody and/or F(ab')2 fragments occurred in platelet-rich plasma and with washed platelets, but not with formalin-fixed washed platelets. TP82- induced aggregation was completely inhibited by disodium ethylendiaminotetraacetate, diltiazem, W-7, PGE1, and several metabolic inhibitors. At a concentration of apyrase or CP/CPK, which inhibited adenosine 5-diphosphate-induced aggregation. TP82-induced aggregation was only partially affected. Thrombin was not required for the antibody- mediated effects, since two thrombin inhibitors failed to block the reaction. The antibody, at least at a high concentration, induced platelet aggregation by a mechanism almost independent of thromboxane A2 formation, since cyclooxygenase inhibitors had little inhibitory effect on aggregation. TP82 monoclonal antibody is a new platelet- aggregating substance that interacts with a low-molecular-weight binding site on the platelet membrane.     

Potentiation by adrenaline of agonist-induced responses in normal human platelets in vitro

Adrenaline is not a true platelet agonist, but enhances aggregation, dense granule secretion, and phospholipase C induced by other agonists. In the present work we investigated the effect of adrenaline on other platelet responses. It strongly potentiated ADP-induced shape change in platelet-rich plasma, particularly when aggregation was prevented by EDTA. The degree of potentiation increased with increasing concentrations of ADP. Thrombin-induced α-granule secretion, measured by the release of fibrinogen in gel-filtered platelets, was also potentiated by adrenaline at thrombin concentrations above 0.05 U/ml. In contrast, adrenaline had little effect on thrombin-induced secretion of β-acetyl-hexosaminidase and potentiated very little liberation of arachidonate at high thrombin concentrations. When autocrine stimulation was inhibited by the removal of secreted ADP by creatine phosphate/creatine phosphate kinase and specific blocking of the thromboxane A(2) and fibrinogen receptors, the potentiation of thrombin-induced ADP?+?ATP secretion by adrenaline was reduced and this reduction was mostly due to the blocking of the thromboxane A(2) receptor. Protein tyrosine phosphorylation by both thrombin and collagen was reduced by adrenaline, and inhibitors of autocrine stimulation counteracted this reduction.     

Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.     

Anti-fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb-IIIa

Summary The binding of fibrinogen to platelets requires the agonist activation of platelet membrane glycoprotein IIb/IIIa. We have now found an anti-fibrinogen polyclonal antibody (YCU-R3) that increases the fibrinogen affinity of GPIIb/IIIa-binding function (activation) and subsequent platelet aggregation. The addition of intact IgG, F(ab)₂ fragments or Fab fragments induced platelet aggregation. The antibody-mediated fibrinogen binding was specific and saturable. This binding was inhibited by native fibrinogen, the RGDS peptide, the peptide of the C-terminus γ chain of fibrinogen (γ397–411), and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The antibody-dependent fibrinogen binding was similar to that induced by ADP. Moreover, after pretreatment with the anti-fibrinogen antibody and fibrinogen, formalin-fixed platelets bound to fibrinogen saturably. These results suggest that this anti-fibrinogen antibody may function as partial agonist.     

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.     

Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.     

Mechanism and characteristics of platelet activation by haematin

The mechanism as well as some characteristics of haematin-induced human platelet aggregation were investigated. Haematin-induced platelet aggregation required the presence of devalent cations; Mg2+, and to a lesser extent, Co2+, were just as effective as Ca2+ in supporting the aggregation. Mono- and trivalent cations were ineffective. Verapamil inhibited the aggregation. The aggregation was accompanied by thromboxane formation which could be abolished by aspirin. The release of adenine nucleotides was only slightly inhibited by aspirin. The rate of aggregation and the ultrastructure of the aggregated platelets were comparable between control and aspirin-treated samples. It is concluded therefore that haematin-induced aggregation is not dependent on platelet prosta-glandin synthesis. Haematin induced binding of fibrinogen to platelets, and failed to aggregate thrombasthenic platelets. These findings indicate that haematin may induce platelet aggregation by promoting influx of divalent cations in association with increased fibrinogen binding and release of adenine nucleotides.     

Effect of platelet-activating factor (PAF) on human platelets

The effect of pure synthetic PAF (1-0-alkyl-2-acetyl-sn-glycero-3- phosphorylcholine) was studied in human platelets. PAF (0.2--2.0 micrograms/ml) produced a dose-dependent aggregation in human platelet- rich plasma (PRP) or platelet suspension obtained by gel-filtration (GFP). In addition, PAF (0.8 microgram/ml) induced secretion of 14C- serotonin (45% +/- 10%; mean +/- SD, n = 9) and platelet factor 4 (PF4) (12.89 +/- 3.81 micrograms/10(9) platelets; n = 9) in PRP. Similar results were obtained in GFP. Aggregation and release of 14C-serotonin and PF4 were inhibited by the metabolic inhibitors 2-deoxyglucose (16.7 mM) and antimycin-A (8.3 micrograms/ml), by the membrane-active drugs mepacrine (10 microM) and chlorpromazine (0.025 mM), by PGI2 (5.34 nM), which elevates intracellular c-AMP, by indomethacin (10 microM) or aspirin (100 microM). The ADP scavengers, creatine phosphate and creatine phosphokinase (CP/CPK), inhibited the second wave of aggregation but not secretion. These data suggest that the major effect of PAF on human platelets is mediated through the cyclo-oxygenase pathway and not through a third pathway.