G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca 2؉ mobilization. Present studies demonstrated that platelet phospholipase A 2 (cytosolic and membrane) activity in the patient was normal. Receptormediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca 2؉ release induced by guanosine 5-[␥-thio]triphosphate (GTP[␥S]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of ␣-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A 2 analog U46619. Binding of 35 S-labeled GTP[␥S] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I 2 analog) -induced rise in cAMP (mediated by G␣ s ) and its inhibition (mediated by G␣ i ) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of G␣ subunits in the patient's platelet membranes showed a decrease in G␣ q (<50%) but not G␣ i , G␣ z , G␣ 12 , and G␣ 13 . These studies provide evidence for a hitherto undescribed defect in human platelet G-protein ␣-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of G␣ q in thrombin-induced responses.

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.

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SummaryEchicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin (<0.2 U/ml), by binding to platelet glycoprotein lb (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin (>0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.