Objective-Protein Kinase C delta (PKC␦) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC␦ in platelets. Methods and Results-We evaluated the role of PKC␦ in platelets using two approaches-pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (␦ V1-1)TAT, and in the murine platelets lacking PKC␦, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A 2 (TXA 2 ). Furthermore, TXA 2 generation was differentially regulated by PKC␦. However, PKC␦ had a small effect on platelet P-selectin expression. Calcium-and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKC␦ null mouse platelets and in human platelets pretreated with (␦ V1-1)TAT, was inhibited.

Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.

Protein kinase C-␦ (PKC-␦) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-␦ positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-␦ in platelets. SH2 domain-containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-␦ on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-␦-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. IntroductionPlatelet-collagen interactions are thought to have the greatest significance at medium or high shear rates found in arteries and diseased vessels. 1 Glycoprotein VI (GPVI) is the major signaling receptor for collagen on the platelet surface. 1 The critical role played by platelets in hemostasis, thrombosis and vascular remodeling, and healing is related to their function as exocytotic cells that secrete important effector molecules at the site of vascular injury. Platelets normally contain at least 3 types of large intracellular granules, such as ␣, dense, and lysosomal granules. There are 3 to 8 dense granules per platelet, and the contents from these dense granules are important for recruiting more platelets to the site of injury. 2,3 Protein kinase C (PKC) has been implicated in platelet secretion. 4 Protein kinase Cs are members of the extended AGC (protein kinases A, G, and C) family of differentially expressed serine/ threonine kinases implicated in a diverse array of cellular functions. After activation, these kinases migrate to different subcellular locations, including the plasma membrane and cytoskeletal elements where they regulate different physiologic functions. 5 PKC isoforms are subdivided into 3 groups based on their lipid and cofactor requirements: the diacylglycerol and calcium-sensitive conventional isoforms (␣, ␤I, ␤II and ␥), the diacylglycerolsensitive and calcium-insensitive novel isoforms (␦, , , and ⑀), and the phosphatidylinositide trisphosphate-sensitive atypical isoforms (, , , and ). 6 PKC-␦ plays a key role in growth regulation and tissue remodeling in other cells 7 and differentially regulates dense granule secretion in platelets. It positively regulates proteaseactivated receptor (PAR)-mediated dense granule release and negatively regulates GPVI-mediated dense granule release. [8][9][10] Intrinsic function of PKCs is regulated by 3 mechanisms: (1) binding of the cofactor that allosterically activates the enzyme, (2) phosphorylation on the activation loop r...

Phosphorylation of activation loop threonine (Thr(505)) and regulatory domain tyrosine (Tyr(311)) residues are key regulators of PKC (protein kinase C) delta function in platelets. In the present study, we show that G(q) and G(12/13) pathways regulate the Thr(505) and Tyr(311) phosphorylation on PKCdelta in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr(505), but not Tyr(311), phosphorylation on PKCdelta, whereas selective activation of G(12/13) pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr(505) and Tyr(311) phosphorylation on PKCdelta. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G(12/13)-mediated Tyr(311) phosphorylation of PKCdelta. These results were confirmed using G(q)-deficient mouse platelets. Finally, we investigated whether Thr(505) phosphorylation is required for Tyr(311) phosphorylation. A T505A PKCdelta mutant failed to be phosphorylated at Tyr(311), even upon stimulation of both G(q) and G(12/13) pathways. We conclude that (i) PKCdelta binding to DAG, downstream of G(q) pathways, and its translocation results in Thr(505) phosphorylation, (ii) G(12/13) pathways activate SFKs required for the phosphorylation of Tyr(311) on Thr(505)-phosphorylated PKCdelta, and (iii) Thr(505) phosphorylation is a prerequisite for Tyr(311) phosphorylation on PKCdelta.

Cardiovascular diseases (CVDs) such as atherosclerosis, hypertension and diabetes, are major global health problems and one of the leading causes of death. Thrombosis associated with multiple CVDs such as atherosclerosis and diabetes further increase morbidity by causing myocardial infarction or stroke. The members of Protein Kinase C (PKC) family are serine threonine kinases, abundantly expressed in cells that maintain cardiovascular health. Studies done using pharmacological tools that block wide range of PKCs or specific PKC isoforms and PKC gene knockout animals revealed that these enzymes regulate critical functional responses in cardiovascular cells. Interestingly, PKC isotype activity is context specific and PKC isotypes may have opposing functional roles depending on cell type and cellular environment (eg., cardiomyocytes, platelets). Furthermore, precise structural differences that occur amongst these isoforms have lead to development of compounds that inhibit or activate specific PKC isoforms. Thus, it is feasible to enhance the protective effects of a PKC isoform, while minimizing the damage caused by other members of PKC family. In this review, we summarize the role of each of these PKC isoforms in various cardiovascular diseases. In addition, we detail the specific PKC isoform modulators, their mechanism of action and ability to treat cardiovascular diseases, as evaluated in animal models or human subjects.