title: Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets.
abstract: Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction.
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors: Amal
authors: A
authors: Arachiche
authors: María
authors: M
authors: de la Fuente
authors: Marvin T
authors: MT
authors: Nieman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D018797:Cell Cycle Proteins; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D018345:Mice, Knockout; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000378:metabolism*; D055503:Protein Multimerization; D058925:Receptors, Purinergic P2Y12 / Q000737:chemistry / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; C548577:P2ry12 protein, mouse; C115084:Pard3 protein, mouse; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein; C112908:protease-activated receptor 4; D002118:Calcium