Calpains are involved in phosphatidylinositol 3′,4′‐bisphosphate synthesis dependent on the α_IIbβ₃ integrin engagement in thrombin‐stimulated platelets

Abstract

In thrombin-stimulated platelets (XiibP3 integrin engagement triggers both phosphatidylinositol 3',4'-6wphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dosedependent inhibition of phosphatidylinositol 3,4-6wphosphate synthesis by calpeptin from 50 jig/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated an b p 3 integrin, did not induce additive effects on phosphatidylinositol 3,4-/>/vphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptintreated platelets. These data indicate that calpains are involved in the regulation of a 1Ib p 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.

Biphasic Activation of PKBα/Akt In Platelets

“…As in Fig. 1 tion of PtdIns4P 3-K (which utilized either commercial PtdIns4P or synthetic diC 16 PtdIns4P with similar results) was inhibited about 50% by ␤ARK-PH, but not by calpeptin treatment of platelets. The sensitivity to ␤ARK-PH also decreased with time, but this was not improved by calpeptin treatment.…”

“…EGTA (20 mM) and calpeptin were included in Triton buffer for lysis. Washed CSK (50 g) was incubated with 1 mg/ml mixtures (23) of diC 16 PtdIns3P, PtdIns, PtdIns4P, diC 16 PtdIns4P, PtdIns(4,5)P 2 , or diC 16 PtdIns(3,4)P 2 , and phosphatidylserine, for assays of lipid kinase activity (13,18). In some cases, ␤ARK-PH (5 M; Ref.…”

“…An antibody to PIP5KII␣ (25)(26)(27)(28) was used for immunoprecipitations from cytosol. After immunoprecipitation or after "mock" immunoprecipitation (resulting from incubation with antibody buffer and protein G-Sepharose), supernatants were assayed as above for PtdInsP kinase activities, and in some cases, synthetic diC 16 PtdIns5P or diC 16 PtdIns4P was used as a substrate. For separations of glycerophospho-Ins(3,5)P 2 (derived from PtdIns(3,5)P 2 ) and glycerophospho-Ins(3,4)P 2 (derived from PtdIns(3,4)P 2 ), HPLC fractions were collected every 10 s and monitored by scintillation spectrophotometry, rather than by Flo-One Beta detection (23).…”

A Novel Integrin-activated Pathway Forms PKB/Akt- stimulatory Phosphatidylinositol 3,4-Bisphosphate via Phosphatidylinositol 3-Phosphate in Platelets

“…Late (post-aggregation) accumulations of PtdIns(3,4)P 2 , but not the levels of PtdIns(3,4,5)P 3 (5), have been found to be regulated by extracellular Ca 2ϩ and binding of FIB to ␣ IIb ␤ 3 (5,6). Other work has shown that THR-R-dependent accumulation of PtdIns(3,4)P 2 can be impaired by calpeptin, an inhibitor of the Ca 2ϩ -dependent protease calpain, which is activated under these conditions (7)(8)(9). Norris et al (9) have suggested that calpain hydrolytically inactivates PtdIns(3,4)P 2 4-phosphatase, thereby elevating PtdIns(3,4)P 2 .…”

Vascular Smooth Muscle Cell Spreading onto Fibrinogen Is Regulated by Calpains and Phospholipase C

“…Two key enzymes of phosphoinositide metabolism, phosphoinositide 3-kinase (PI 3-kinase) and phospholipase C (PLC), have been involved in integrin-dependent cell spreading (13,14). In platelets, different groups including ours, have demonstrated that calpains were involved in the regulation of ␣IIb␤3 integrin-dependent PI 3-kinase signaling pathway (15)(16)(17). By regulating PI 3-kinase, PI-3-P 4-kinase, and PI-3,4-P2 4-phosphatase activities, calpains induced the accumulation of PtdIns-3,4-P2 (16,17), whose production was correlated with platelet spreading (13).…”