The system of calpain (Ca2+-activated protease) and its inhibitor calpastatin in platelets have been well characterized in our laboratory but the role of the system has not been fully elucidated yet, though various endogenous substrates have been identified. Employing SH inhibitors as calpain antagonists, We have proposed a possible role of platelet calpain in myosin light chain (20K) phosphorylation (Biochem.Int. 6,767,1986) but more specific calpain antagonists were required to draw conclusion. Recentry, series of peptide calpain antagonists(PCAs) were syn-thetized such as Ac-Leu-Leu-Nle.al(PCA-I), Ac-Leu-Leu-Met.al(PCA-II) and Leu-Leu-Phe.CH2Cl(PCA-III)(J.Biochem.99,173,1986) and attempts were made to apply them to platelet reaction. ID50 of PCAs against platelet calpain I was as follows; PCA-I:0.04uM,PCA-II:0.luM and PCA-III:0.4uM. Thus, these antagonists were 1000 times more potent than N-ethylmaleimide(NEM), and they did not inhibit Mg2+-ATPase activity of platelet myosin B in contrast to NEM. When PCAs were applied to intact platelets, no effect was obtained against stimulus-linked proteolysis of ABP(aotin binding protein) and P235, indicating poor permeability of the antagonists across the plasma membrane. Thereby PCA was applied to lysed platelets or to permeabilized platelets. Lysed platelets suspension with 2mM EGTA, 2mM EDTA was incubated at 37 C with 32P -ATP,2mM MgCl2, 3mM CaCl2 in the presence or absence of PCA-II and proteolysis of. ABP,P235 and phosphorylation of 20K,47K were studied. The proteolysis and phosphorylation were inhibited by PCA-II in a dose-related manner. Then, PCA-II was applied to permeabilized platelet prepared by a high voltage electric charge to which acriflavine was loaded. PCA-II inhibited Ca2+ stimulated proteolysis and phosphsrylation of the permeabilized platelets likewise but no effect was obtained on Ca2+ stimulated acriflavine secretion from dense granules. These observations indicated that the proteolysis and protein phosphorylation are mediated by calpain but that these phenomena may not be related to secretory process.
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