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Endothelium-dependent phosphorylation of vasodilator-stimulated protein in platelets during coronary passage
Insights
Endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) and prostaglandin I2 (PGI2) regulate vasodilator-stimulated protein (VASP) phosphorylation in human platelets. Both signaling molecules contribute to VASP phosphorylation, impacting platelet function.
Area of Science:
- Cardiovascular Biology
- Platelet Physiology
- Molecular Signaling
Background:
- Platelet activation involves signaling pathways regulating adhesion and aggregation.
- Vasodilator-stimulated protein (VASP) phosphorylation is linked to cyclic nucleotide signaling.
- Endogenous compounds like nitric oxide and prostaglandins influence platelet function.
Purpose of the Study:
- To investigate the role of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) and prostaglandin I2 (PGI2) in VASP phosphorylation.
- To determine how acetylcholine stimulation affects VASP phosphorylation in human platelets.
- To elucidate the contribution of EDRF/NO and PGI2 to platelet signaling.
Main Methods:
- Human platelets were perfused through rabbit hearts.
- Platelets were collected after coronary passage and analyzed for VASP phosphorylation.
- cGMP, cAMP, and PGI2 levels were measured in coronary effluent and platelets.
- Inhibition of EDRF/NO synthesis (NG-nitro-L-arginine) and PGI2 synthesis (Indomethacin) was employed.
Main Results:
- Acetylcholine (ACh) significantly increased platelet cGMP, cAMP, and VASP phosphorylation.
- EDRF/NO inhibition attenuated ACh-induced cAMP elevation and decreased VASP phosphorylation.
- PGI2 inhibition abolished ACh-induced cAMP and VASP phosphorylation increases.
- Both EDRF/NO and PGI2 were found to regulate VASP phosphorylation.
Conclusions:
- EDRF/NO and PGI2 synergistically regulate VASP phosphorylation in human platelets.
- VASP phosphorylation is a common downstream target for NO and PGI2 signaling pathways.
- These findings suggest a shared mechanism for the anti-aggregatory effects of NO and PGI2 on platelets.
Abstract:
Compounds that elevate intraplatelet guanosine 3',5'-cyclic monophosphate (cGMP) or adenosine 3',5'-cyclic monophosphate (cAMP) stimulate the phosphorylation of a 46- to 56-kDa thrombocyte protein designated "vasodilator-stimulated protein" (VASP), which is most likely involved in the regulation of adhesion/aggregation. We investigated whether endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) and prostaglandin I2 (PGI2) affected VASP phosphorylation in washed human platelets that were injected into the coronaries of saline-perfused rabbit hearts (n = 22) and collected immediately after passage. The endothelial stimulator acetylcholine (ACh; 1 microM) significantly increased the concentration of cGMP (indicating release of EDRF) and PGI2 in the coronary venous effluent, as well as the concentration of cGMP and cAMP in platelets. Phosphorylation state of VASP increased from 32.1 +/- 2.9 to 64.8 +/- 2.7%. Inhibition of EDRF/NO synthesis by NG-nitro-L-arginine (30 microM) completely abolished the ACh-induced cGMP increase, attenuated the cAMP-elevation without affecting PGI2, and caused a 20.5 +/- 5.8% decrease of the phosphorylation state of VASP. Indomethacin (30 microM) virtually abolished ACh-induced increases of PGI2, cAMP (but not cGMP), and phosphorylated VASP. These results indicate that both EDRF/NO and PGI2 contribute to the regulation of VASP phosphorylation in platelets collected after a single coronary passage. Their synergistic inhibitory effects on platelet function may thus be mediated by a common effect on target proteins like VASP as well as by a secondary increase in cAMP in response to cGMP-elevating compounds such as EDRF.