Protein Phosphatase1 gamma (PP1cγ) Is Required for Thrombin-Induced Integrin α_IIbβ₃ Activation.

Integrin α_IIbβ₃ activation is critical for platelet physiology and is controlled by agonists- induced signal transduction through kinases and phosphatases. Compared to kinases, a role of phosphatases in platelet integrin α_IIbβ₃ signaling and activation is less understood. We have previously demonstrated that the catalytic subunit of protein phosphatase 1 (PP1c) associates constitutively with the integrin α_IIbβ₃ and regulates myosin light chain phosphorylation. Platelets express three isoforms of PP1c namely PP1cα, PP1cβ/δ and PP1cγ1, however their role in platelet physiology is unclear. In this study, we explored a role for the PP1cγ in integrin α_IIbβ₃ activation using the PP1cγ null mice that lacks both PP1cγ1 and PP1cγ2 splice variants. Deletion of PP1cγ did not alter the expression of PP1cα and PP1cβ/δ in platelets. Compared to the wild type mice, PP1cγ null mice had significantly (P=0.03) more platelets but not other blood cells and increased megakaryocytes in an in vitro bone marrow culture system, suggesting that PP1cγ may regulate platelet production. Compared to the wild type platelets, PP1cγ null platelets exhibited a ∼50% decreased activation (P=0.007) of integrin α_IIbβ₃ in response to low doses of thrombin as measured by the binding of soluble Alexa-flour 488-conjugated fibrinogen or Jon/A, an antibody to high affinity integrin α_IIbβ₃. Consistent with this observation, PP1cγ null platelets showed decreased aggregation in response to thrombin or protease activated receptor activating peptide. Soluble fibrinogen binding and aggregation to ADP, collagen and cross linked collagen -related peptide was not different between the PP1cγ null and wild type platelets. Surface expression of integrin α_IIbβ₃ was comparable in the PP1cγ null platelets indicating that the decreased activation of α_IIbβ₃ in PP1cγ platelets was not due to decreased integrin expression. These studies suggest that PP1cγ is required for thrombin-induced α_IIbβ₃ inside-out signaling process. In contrast, α_IIbβ₃ outside-in signaling process was not altered in PP1cγ null mice as revealed by normal adhesion to immobilized fibrinogen. Intravital microscopy revealed that the thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly (P=0.03) delayed in PP1cγ null mice compared to wild type mice. These studies illustrates that PP1cγ is an essential component of thrombin signaling that is downstream of protease activated receptors and promotes thrombus formation in part by supporting soluble fibrinogen binding and platelet aggregation.