Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion.
This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.
Huang
Jin-Sheng
JS
Department of Pharmacology, College of Medicine University of Illinois at Chicago, Chicago, Illinois 60612.
Dong
Lanlan
L
Kozasa
Tohru
T
Le Breton
Guy C
GC
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D048430:Cell Shape / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism* / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D044463:Receptor, PAR-1; D064067:Rho Guanine Nucleotide Exchange Factors; D013917:Thrombin / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D006490:Hemostatics; D007476:Ionophores; D010455:Peptides; D044463:Receptor, PAR-1; D064067:Rho Guanine Nucleotide Exchange Factors; D000001:Calcimycin; D000244:Adenosine Diphosphate; D013917:Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein