title: Protein kinase Ctheta negatively regulates store-independent Ca2+ entry and phosphatidylserine exposure downstream of glycoprotein VI in platelets.
abstract: 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, PKCtheta, 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 PKCtheta(-/-) platelets. PKCtheta acts at multiple distinct sites. PKCtheta limits secretion, reducing autocrine ADP signaling that enhances Ca(2+) release from intracellular Ca(2+) stores. PKCtheta thereby indirectly regulates activation of store-operated Ca(2+) entry. However, PKCtheta 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 PKCtheta(-/-) 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 PKCtheta(-/-) platelets. We propose that PKCtheta 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.
authors.affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD Bristol, United Kingdom.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Harper
authors.forename: Alastair W
authors.initials: AW
authors.lastname: Poole
mesh_terms: D000081:Acetamides / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology; D005434:Flow Cytometry; D017136:Ion Transport / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010718:Phosphatidylserines / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000081:Acetamides; D004075:Diglycerides; D007527:Isoenzymes; D007546:Isoquinolines; D010718:Phosphatidylserines; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; C081356:LOE 908; D000244:Adenosine Diphosphate; C038237:1-oleoyl-2-acetylglycerol; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D002118:Calcium
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