Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Inhibition of platelet-surface-bound proteins during coagulation under flow I: TFPI

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Tissue factor pathway inhibitor (TFPI) is one such inhibitor, well known for its inhibitory action on the active enzyme complex comprising tissue factor (TF) and activated clotting factor VII. This complex forms when TF embedded in the blood vessel wall is exposed by injury and initiates coagulation. A different role for TFPI, independent of TF:VIIa, has recently been discovered whereby TFPI binds a partially cleaved form of clotting factor V (FV-h) and impedes thrombin generation on activated platelet surfaces. We hypothesized that this TF-independent inhibitory mechanism on platelet surfaces would be a more effective platform for TFPI than the TF-dependent one. We examined the effects of this mechanism on thrombin generation by including the relevant biochemical reactions into our previously validated mathematical model. Additionally, we included the ability of TFPI to bind directly to and inhibit platelet-bound FXa. The new model was sensitive to TFPI levels and, under some conditions, TFPI could completely shut down thrombin generation. This sensitivity was due entirely to the surface-mediated inhibitory reactions. The addition of the new TFPI reactions increased the threshold level of TF needed to elicit a strong thrombin response under flow, but the concentration of thrombin achieved, if there was a response, was unchanged. Interestingly, we found that direct binding of TFPI to platelet-bound FXa had a greater anticoagulant effect than did TFPI binding to FV-h alone, but that the greatest effects occurred if both reactions were at play. The model includes activated platelets’ release of FV species, and we explored the impact of varying the FV/FV-h composition of the releasate. We found that reducing the zymogen FV fraction of this pool, and thus increasing the fraction that is FV-h, led to acceleration of thrombin generation.     

Effects of chlorpromazine and other calmodulin antagonists on phosphatidylcholine-induced vesiculation of platelet plasma membranes

Dilauroylglycerophosphocholine (C12:0PC)-induced vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218; Kobayashi, T., Yamada, J.-I., Satoh, N., Setaka, M. and Kwan, T. (1985) Biochim. Biophys. Acta 817, 307-312) was inhibited by chlorpromazine. Preincubation of platelets with chlorpromazine was required for inhibition but incorporation of chlorpromazine into C12:0PC liposomes was not necessary for it, indicating that the observed inhibition of vesiculation was mainly due to the effect of chlorpromazine on platelets and not that on liposomes. The change in platelet membrane fluidity caused by chlorpromazine was not the cause of inhibition of vesiculation. The inhibition of vesiculation by various other calmodulin antagonists was also observed. The inhibitory activities of these calmodulin antagonists and chlorpromazine correspond very well to their abilities to bind to calmodulin. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited vesiculation but a structural analogue of it, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had no inhibitory activity. These results suggest the involvement of calmodulin in membrane vesiculation.     

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.     

Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard

The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.     

Human platelet factor 4 is a direct inhibitor of human osteoblast-like osteosarcoma cell growth.

The effect of purified human platelet factor 4, a platelet alpha-granule protein, on the growth of the human osteoblastic osteosarcoma cell lines Saos-2 and G-292 was investigated. Platelet factor 4 (20 ng/ml to 2 micrograms/ml) caused a significant, dose-dependent inhibition of human osteoblast-like osteosarcoma cell proliferation. Platelet factor 4 exerted its inhibitory effect under all growth conditions tested: serum-free, serum-stimulated and thrombin-stimulated. The platelet factor 4-induced cell inhibition was not associated with a cytotoxic effect on the cells (assessed by lactate dehydrogenase release). The inhibitory effect of platelet factor 4 was not affected by the presence of indomethacin in the cultures, indicating that the effect was prostaglandin-independent. These results suggest that platelet factor 4 has direct antitumor effects and that it may be important in pathological and physiological processes of bone.     

Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.     

Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions

We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.     

若干苄基异喹啉类化合物的HPLC保留因子与抑制钙调素活性的关系

苄基异喹啉类化合物具有较强的拮抗钙调素(Calmodulin,Cam)的活性,测定这类化合物HPLC参数LogK′作为疏水参数与抑制钙调素的活性进行相关分析,结果表明活性随化合物亲脂性的增大而升高,二者较符合抛物线关系。     

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.     

Purification of a troponin I-like factor from pig platelet

A troponin I-like factor has been purified from pig platelet by G150 Sephadex filtration of a low ionic strength extract, acidification at pH 4.2, ion exchange on DE-52 cellulose, and affinity chromatography on calmodulin-Sepharose. This protein (Mr 17000), together with pig brain calmodulin and platelet tropomyosin, is able to participate to the reconstitution in vitro of a thin filament-like complex which modulates with 55% calcium sensitivity the platelet actin-activated Mg2+-dependent ATPase activity of rabbit skeletal muscle myosin.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

Rab3B in human platelet is membrane bound and interacts with Ca(2+)/calmodulin.

The subcellular distribution of Rab3B in fresh and aged platelets was determined and majority of the protein was localized with the particulate fraction with only a minor amount detected in the cytosol. Rab3B was pulled out from platelet particulate fraction with GST-RabGDI-alpha fusion protein. Using GST-Rab3B in in vitro pull-down experiments, the binding of calmodulin from platelet cytosol to Rab3B was demonstrated. In the reverse experiment, binding of Rab3B from platelet particulate and cytosolic fractions to Sepharose-CaM beads was also observed. The interaction between Rab3B and calmodulin was Ca(2+)-dependent but independent of the guanine nucleotide status of Rab3B. These findings provide evidence that Rab3B is primarily localized with the particulate fraction and that Ca(2+)/calmodulin could regulate function of this GTPase in the platelet.     

Calmodulin antagonist W-7 inhibits aggregation of human platelets induced by platelet activating factor

Experiments were done to test the hypothesis that aggregation of human platelets induced by platelet activating factor (PAF) may be mediated by calmodulin-dependent processes. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide], a potent calmodulin antagonist, caused dose-dependent inhibition of PAF induced aggregation of human platelets in vitro. The ED50 for W-7 was 51.5 +/- 9.5 microM (mean +/- SEM). This concentration is known to be platelet calmodulin-specific. These data are consistent with the hypothesis.     

Platelet activating factor-induced aggregation of calf platelets: Apparent positive cooperativity in the kinetics and non competitive inhibition by diltiazem

Aggregation of calf platelets by platelet activating factor was characterized by a spectrophotometric method. The aggregation kinetics of both platelet-rich plasma and purified platelets showed concave up double-reciprocal plots and linear Hill plots withh > 1 (1.7 ± 02) consistent with positive cooperativity. Comparable values of maximum rates of aggregation(R) were obtained with platelet-rich plasma (0.25 ± 0.08) and purified platelets (0.28 ± 0.18) but the half-maximal saturation concentration (S_0.5) differed greatly between platelet-rich plasma (6 ± 3 nM) and purified platelets (0.28 ± 0.18 nM). An Arrhenius activation energy of 21 ±2 kcal/mol was found for aggregation of purified platelets. Diltiazem was inhibitory with half-maximal inhibitory concentration (I_0.5) of 4 M but the inhibition was not competitive. Diltiazem inhibited rates but not the extent of shape-change. The receptor-antagonist and sulphydryl reagent N-ethylmaleimide and the platelet antagonistic omega-3-fatty acid, 5,8,11,14,17-eicosa pentaenoic acid, inhibited both rates and extent of shape-change reactions and inhibited aggregation competitively (I_0.5 ∼ 5 M). Eicosa pentaenoic acid at > 25 M could abolish shape-change reactions and at 50 M served as an activator of platelets and the activation was enhanced by aspirin (1 mM). Although N-ethylmaleimide at > 20 M could also induce platelet activation it failed to induce aggregation and aspirin had no effect on the shape-change reactions induced by it.     

Characterization of a calmodulin-dependent protein phosphatase from human platelets

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.     

内毒素对人血小板聚集,释放的体外作用

大肠杆菌 O55B5内毒素在体外可抑制人血小板自发性聚集,并使血小板内 cAMP、钙调素明显增高,但对肾上腺素诱发的血小板聚集无作用,也不能引起血小板致密颗粒和α-颗粒的释放。此外还观察到内毒素组无血小板上清液中5-HT 含量减少。     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.