title: Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in human platelets.
abstract: Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was 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 sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent 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 observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification 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 ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.
authors.affiliation: Department of Biochemistry, Gifu University School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Banno
authors.forename: S
authors.initials: S
authors.lastname: Nakashima
authors.forename: T
authors.initials: T
authors.lastname: Hachiya
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
mesh_terms: D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000819:agonists / Q000378:metabolism*; D008969:Molecular Sequence Data; D054799:Phospholipase C beta; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000819:agonists / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D003094:Collagen; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D013917:Thrombin; D002154:Calpain; D019204:GTP-Binding Proteins