ENDOGENOUS CLEAVAGE OF PHOSPHOLIPASE C-BETA-3 BY AGONIST-INDUCED ACTIVATION OF CALPAIN IN HUMAN PLATELETS

Two membrane-associated phosphoinositide-specific phospholipase Cs (mP I-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were acti vated by brain G-protein beta gamma subunits have been isolated from h uman platelets. The truncation of mPI-PLC-1 that was mediated by mu-ca lpain induced much higher activation by beta gamma subunits (Banno, Y. , Asano, T., and Nozawa, Y. (1994) FEES Lett. 340, 185-188). On the ba sis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) wa s PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI- PLC (140 kDa) was recognized by the antibody selective for internal se quences of PLC-beta 3 but not by the antibody raised against its carbo xyl terminus, indicating that it may be related to PLC-beta 3. Treatme nt of human platelets with A23187 and dibucaine, activators of calpain , caused cleavage of actin-binding protein and talin in a time-depende nt manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were obser ved on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombi n and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) an d an increase of 100 kDa PLC-beta 3 in a time- and dose dependent mann er. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modifica tion may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists . The cleavage of PLC-beta 3 evoked by thrombin and collagen but not A DP was correlated with irreversible aggregation, suggesting that the P LC-beta 3 modification may play a role in secondary irreversible aggre gation in agonist-stimulated human platelets.