The roles of protein kinase G in platelet activation

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DESCRIPTION (provided by applicant): The platelet adhesion and aggregation play critical roles in the development of thrombotic diseases such as heart attack and stroke. In normal circulation, platelets are in a "resting" state. At sites of vascular injury or atherosclerotic plagues, exposure of platelets to soluble platelet agonists or sub endothelial adhesive proteins triggers platelet activation. A common consequence and characteristic of platelet activation is the activation of the platelet integrin aIIb beta3, which mediates platelet adhesion, spreading and aggregation. Over the last 20 years, it has been accepted that platelet activation is inhibited by the cGMP-dependent protein kinase (protein kinase G, PKG). However, there have been recently reports that a cGMP-enhancing drug, sildenafil, was associated with heart attack and thrombosis in some patients. In our study, we have found that expression of recombinant human PKG in a reconstituted integrin activation model promotes GPIb-IX-mediated integrin alphaIIbbeta3 activation. In addition, integrin dependent platelet aggregation induced by vWF or low dose thrombin was inhibited by various PKG inhibitors and enhanced by PKG activators. Furthermore, we found that sildenafil promoted vWF- or thrombin-induced platelet aggregation. Thus, cGMP-PKG may play a stimulatory role in platelet activation. Interestingly, we found that cGMP is stimulatory when elevated immediately following agonist simulation, but is inhibitory after a prolonged preincubation with platelets. Thus, we hypothesize that, when elevated by the platelet agonists during hemostasis, cGMP induces biphasic platelet responses: an initial phase stimulatory response leading to platelet activation and thrombus formation, and a secondary phase of inhibitory responses that desensitizes platelets and prevent overgrowth of thrombus. To test this hypothesis, we propose (1) to investigate the stimulatory roles of cGMP-PKG pathway in platelet adhesion and activation; (2) to investigate the mechanisms of the second phase platelet inhibitory response to cGMP; (3) to identify the roles and mechanisms of guanyl cyclase regulation during platelet activation; (4) to investigate the structure-function relationship and topographic regulatory mechanisms of PKG; and (5) to initiate preliminary studies on the downstream pathways of PKG-mediated integrin activation. Understanding the biphasic roles of cGMP-PKG pathway in platelets should provide new insight into molecular mechanisms of platelet activation and the mechanisms of thrombosis associated with popularly used cGMP enhancing drugs such as sildenafil.