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We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

Although calpain (the Ca2(+)-dependent protease) is widely distributed, its function is poorly understood. One cell in which it becomes activated as a consequence of activation of the cell is the blood platelet. The aim of the present study was to determine whether activation of calpain was responsible for any of the responses of platelets to stimulation. Platelets were incubated with calpeptin, a membrane-penetrating inhibitor of calpain, before being exposed to an agonist. Concentrations of calpeptin that totally inhibited the agonist- induced hydrolysis of actin-binding protein (ABP) by calpain had no effect on many other responses associated with platelet activation: phosphorylation of myosin light chain, phosphorylation of P47, platelet shape change, aggregation of platelets, secretion of granule contents, or retraction of fibrin clots. However, these concentrations of inhibitor decreased the agonist-induced generation of procoagulant activity (assayed as the ability of platelets to catalyze the conversion of prothrombin to thrombin in the presence of factor Va and factor Xa). When thrombin was the agonist, the amount of ABP that was hydrolyzed was small; only a small component of the total agonist- induced procoagulant activity was inhibited by calpeptin. When collagen was the agonist, more ABP was hydrolyzed and the amount of procoagulant activity generated was greater; calpeptin decreased the collagen- induced procoagulant activity to levels comparable with those induced by thrombin in the presence of the inhibitor. We suggest that there are at least two mechanisms by which procoagulant activity is generated on activated platelets and that the agonist-induced activation of calpain mediates one of these mechanisms. These results show that activation of calpain is a component of the stimulus-response pathway in platelets.