Mechanisms of action of proteinase-activated receptor agonists on human platelets

Abstract

1. We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)-activating peptides (PAR-APs) [SFLLRNPNDKYEPF-amide (TRAP), TFLLR-amide (PAR1AP) and AYPGKF-amide (PAR4AP)]. 2. PAR agonist-induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence. 3. Aggregation inhibitors, including prostacyclin (PGI(2)), nitric oxide-releasing agent (S-nitroso-glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist-induced aggregation to pharmacological inhibition. 4. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. 5. The aggregatory potencies of PAR-APs were not modified by the aminopeptidase inhibitor, amastatin. 6. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. 7. Both PGI(2) and GSNO reduced PAR agonist-induced aggregation and diminished GPIIb/IIIa up-regulation. 8. PAR agonist-induced aggregation was aspirin-insensitive indicating a minor role for TXA(2). 9. In contrast, phenanthroline and apyrase significantly enhanced the anti-aggregatory effects of aspirin against thrombin-, PAR1AP- and TRAP-induced aggregation suggesting the involvement of ADP- and MMP-2-dependent pathways. 10. PAR4AP-induced aggregation (but not PAR1AP-induced aggregation) was entirely ADP-dependent (abolished by apyrase) and resistant to phenanthroline (MMP-2-independent). 11. Thus, the mechanisms of PAR1 and 4-induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors.

Figure 1

PAR agonist-induced platelet aggregation. Panel a shows concentration-response curves to PAR agonists. Data points are mean±s.e.mean, n=5. Superimposed aggregatory tracings (panel b) demonstrate amplification of platelet aggregation by PAR1AP (3 μM) and PAR4AP (30 μM). Tracings are representative of six similar experiments.

Figure 2

Flow cytometry analysis of GPIb, GPIIb/IIIa and activated GPIIb/IIIa on platelets stimulated with PAR agonists. PAR agonists result in a significant reduction of GPIb (panel a) and up-regulation of GPIIb/IIIa and activated GPIIb/IIIa (panel b). PAR agonists were used at concentrations that resulted in submaximal (95%) aggregatory response (shown in Table 1). Control: unstimulated platelets. Bars are mean±s.e.mean, n=3 – 9. ^*P<0.05 treatments versus control.

Figure 3

Inhibition of PAR agonist-induced aggregation by PGI₂. Aggregation was induced by submaximal concentrations of PAR agonists. Numbers beside the tracings show the concentrations of PGI₂ in nM. Traces are representative of 3 – 7 independent experiments.

Figure 4

Flow cytometry analysis of GPIIb/IIIa (panel a) and activated GPIIb/IIIa (panel b) in platelets stimulated with PAR agonists in the presence of PGI₂ (3 nM) or GSNO (5 μM). Data are mean±s.e.mean, n=3 – 9. ^*P<0.05 treatments versus control (PAR agonists in the absence of inhibitors).

Figure 5

Effects of aspirin (ASA, 300 μM), phenanthroline (Phe, 100 μM) and apyrase (300 μg ml⁻¹) on PAR agonist-induced aggregation. Results are expressed as a per cent of aggregation induced by the corresponding agonist without inhibitors. Data are mean±s.e.mean, n=3 – 9.

Figure 6

Role of ADP in PAR4AP-induced aggregation. Superimposed aggregatory and the corresponding chemiluminescence tracings showing inhibition of PAR4AP-stimulated aggregation with apyrase (panel a and b). Panel c shows correlation between the concentrations of ATP and the aggregatory responses during inhibition of aggregation induced by apyrase (square: 0, triangle: 10, cross: 20, diamond: 35 and circle: 300 μg ml⁻¹). Panel d demonstrates inhibition of PAR4AP-induced GPIIb/IIIa activation by apyrase (50 μg ml⁻¹). Data are mean±s.e.mean, n=3. ^*P<0.05 treatments versus control.