Protein Kinase Cθ Negatively Regulates Store-independent Ca(2+) Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca(2+) signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca(2+) signals in PKCθ(−/−) platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca(2+) release from intracellular Ca(2+) stores. PKCθ thereby indirectly regulates activation of store-operated Ca(2+) entry. However, PKCθ also directly and negatively regulates store-independent Ca(2+) entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ(−/−) platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry but not store-operated Ca(2+) entry, blocked the enhanced GPVI-dependent Ca(2+) signaling and PS exposure seen in PKCθ(−/−) platelets. We propose that PKCθ normally acts to restrict store-independent Ca(2+) entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.