Regulation of Platelet Survival by Protein Phosphatase 1 Gamma

Abstract 2026

Platelets are key players in hemostasis and their senescence is intrinsically associated with the activation of apoptotic pathways that shows similarities to the apoptosis of nucleated cells. Anti-apoptotic protein Bcl-xl restrains the pro-apoptotic Bak activity and maintains platelet survival in circulation. In nucleated cells, serine phosphorylation of a pro-apoptotic protein Bad can also promote cell survival. Serine phosphorylation of Bad is regulated by the action of serine/threonine (Ser/Thr) protein kinases and Ser/Thr protein phosphatases. Although alterations in the activities of either enzyme can change the rate of apoptosis, whether Ser/Thr phosphatases participate in regulating platelet apoptosis is unknown. In this study, we report that the mice lacking the catalytic subunit of protein phosphatase 1 gamma (PP1cγ) exhibit a moderate but significant increase in the number of circulating platelets [596 × 10³/μL ± 17.9 in WT compared with 694 × 10³/μL ± 19.8 in PP1cγ^–/– mice; P = .0003]. Examination of the bone marrow from the PP1cγ^–/– mice revealed a non-significant increase in the number of morphologically matured megakaryocytes. A trend towards decreased plasma thrombopoietin levels were also noticed in PP1cγ^–/– mice. These observations suggest that an increased megakaryopoiesis/thrombopoiesis may not fully account for the increased platelet numbers in PP1cγ^–/– mice. In vivo platelet survival studies revealed that the loss of PP1cγ modestly increased platelet half life in circulation (t1/2 ∼69 hours in WT compared to ∼78 hours in PP1cγ^–/– mice). PP1cγ^–/– platelets had decreased mean platelet volume, suggesting the PP1cγ^–/– mice may harbor a greater proportion of older circulating platelets. These studies are consistent with the delayed clearance of platelets from PP1cγ^–/– mice. Pro-apoptotic Bad possess PP1c binding motif and mechanistically, PP1cγ interacts with Bad protein in platelets. Phosphorylation of Bad Ser112, which promotes cell survival, was enhanced ∼50% in PP1cγ^–/– platelets. Consistent with the increased BAD phosphorylation, co-immunoprecipitation studies revealed increased BAD-14-3-3 protein complexes from the PP1cγ^–/– platelets. It is reported that in the phosphorylated state, BAD can interact with 14-3-3 and is sequestered in the cytoplasm, thereby preventing the binding of Bad with the mitochondrial anti-apoptotic Bcl-xl. Interaction of Bad with Bcl-xl has the potential to displace the pro-apoptotic Bak from the Bcl-xl-Bak protein complex to trigger apoptosis. Finally, immunoblotting with anti-caspase 9 antibody revealed decreased caspase 9 cleavage product (∼37 kDa) in the PP1cγ^–/– platelets, suggesting decreased activation of caspase 9 dependent apoptotic pathway. These data indicate that the increased platelet counts in PP1cγ^–/– mice could be in part due to delayed platelet apoptosis. Loss of PP1cγ leads to the hyperphosphorylation of BAD, which via an interaction with 14-3-3, delays caspase mediated apoptosis to prolong the life span of platelets.