PAR1-Induced Human Platelet Shape Change, Aggregation and Secretion Requires Gα13 Switch Region I Signaling.

While it is known that platelets possess multiple G protein signaling pathways that contribute to the different platelet functional responses, the relative participation of these individual pathways in platelet shape change, aggregation and secretion is not well characterized. To a large extent this is due to the lack of suitable reagents which selectively interfere with specific G protein signaling events, and which can be applied to the study of intact human platelets. With the exception of pepducins, which modulate receptor-G protein coupling (Kuliopulos, A. and Covic, L. Life Sciences 74, 255–262, 2003), the field has for the most part been limited to agents which interfere with different downstream kinases or other downstream effectors. However, the G protein pathways share many of these downstream targets, and consequently, it has been difficult to assign a specific platelet function to a certain G protein. In order to address this issue, it was reasoned that more direct information about specific G protein involvement in human platelet activation might be obtained by interfering with the initial G protein signal transduction events, rather than by interfering with the secondary downstream consequences of this transduction process. Based on this consideration, the present study used a specific Gα13 switch region I (SRI) peptide to investigate the involvement of Gα13 signaling in protease-activated receptor 1 (PAR1)-mediated human platelet function. Specifically, a myristoylated peptide representing the Gα13 SRI (Myr-G13SRIpep) was synthesized and evaluated for its effects on PAR1 activation. Initial studies using dot blot and mass spectrum analysis demonstrated that Myr-G13SRIpep, and its random sequence control (Myr-G13SRIRandom-pep), were equally taken up by intact human platelets. Radioligand binding experiments revealed that Myr-G13SRIpep did not interfere with PAR1-ligand interaction. Subsequent experiments demonstrated that G13SRIpep specifically bound to platelet p115Rho guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF, and indicate a possible mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies revealed that Myr-G13SRIpep inhibited PAR1-stimulated platelet shape change, aggregation and dense granule secretion in a dose-dependent manner. On the other hand, Myr-G13SRIpep did not inhibit platelet activation induced by ADP, A23187 or PAR4 activating peptide (AYPGKF). Taken together, these findings demonstrate that the inhibitory effects of Myr-G13SRIpep are limited to PAR1 signaling mechanisms and are not due to nonspecific effects on platelet function. These results also suggest a significant role for Gα13 SRI signaling in the process of PAR1-mediated human platelet activation. In additional studies it was found that Myr-G13SRIpep also inhibited low-dose thrombin-induced aggregation and PAR1-induced intraplatelet calcium mobilization. Collectively, these results provide evidence that: 1. interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2. signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes; 3. Gα13 SRI signaling is involved in low-dose thrombin-induced platelet aggregation as well as PAR1-mediated calcium mobilization; and 4. permeable peptides representing SRI of Gα-subunits should be a useful approach for studying individual G protein signaling pathways in intact cells.