Double Knockouts Reveal That Protein Tyrosine Phosphatase 1B Is a Physiological Substrate of Calpain-1 in Platelets.

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b₃ subunit of α_Iibβ₃ integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b₃ subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.