Inhibition of Protein Kinase a Induces Platelet Apoptosis

Background:

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis.

Methods and Results:

Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm.

Conclusions:

These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.