Protease-Activated Receptors 1 and 4 Mediate the Synergistic Effect of Thrombin on Collagen-Stimulated Phosphatidylserine Expression, That Occurs on the Smallest Platelets.
Authors: Rand Margaret L. , Wang Hong , Bang K.W. Annie , Freedman John 
Published: April 2020

Abstract
Activated platelets promote hemostasis and thrombosis by expressing a procoagulant surface that accelerates thrombin generation. The procoagulant nature of this surface is largely attributable to exposure of the anionic aminophospholipid phosphatidylserine (PS) that is normally confined to the inner membrane leaflet of unstimulated platelets. Thrombin synergizes with collagen to expose more PS on the platelet surface than occurs with either agonist alone. The roles of the protease-activated receptors PAR1 and PAR4 in PS expression involving thrombin are not yet settled. In this study, we examined whether activation of PAR1 and/or PAR4 with collagen stimulation of platelets results in synergistic PS expression; since PS expression was restricted to a subpopulation of platelets, we also examined PS expression on different sizes of platelets. Citrated-whole blood was mixed gently with 2.5 mM GPRP (to inhibit platelet aggregation and fibrin polymerization), 2 mM Ca2+, and the following concentrations of agonists (previously determined to give maximal aggregation responses, and, with the exception of collagen, maximal granule content release): 20 μg/mL collagen (C), 1 U/mL thrombin (T), 50 μM of the PAR1-activating peptide (AP) SFLLRN, 250 μM of the PAR4-AP AYPGKF, and combinations of C with T and the PAR APs (Table 1). Platelet PS expression was measured by flow cytometry as the mean channel fluorescence (MCF) of annexin A5-FITC bound to CD41-positive events. T synergized with C to give increased PS expression, as did PAR1-AP, PAR4-AP and their combination, and PS expression was limited to 14–21% of platelets (Table 1). On the subpopulations of PS-expressing platelets, the annexin A5 MCF was similar (~300 arbitrary units), indicating the same level of PS exposure. To further characterize the subpopulations of PS-expressing platelets, the total platelet population was divided into quartiles based on size (forward scatter) and the % of annexin A5-positive platelets in each quartile was examined separately. The % of PS-expressing platelets was highest in the first size quartile, ie, the smallest platelets, and progressively decreased in quartiles of increasing size. For example, with platelets stimulated with the combinations of agonists, the proportion of PS-expressing platelets (31–43%) was significantly higher in the first quartile than in the second quartile (13–23%), the third quartile (6–11%), and the fourth quartile (4–7%) (n=5; P&lt;0.05–0.001, 1-way ANOVA). In contrast, the proportions of platelets expressing another activation marker, P-selectin, were distributed equally among the size quartiles. Taken together, our results indicate that PAR1 and PAR4 can both mediate the synergistic effect of thrombin on collagen-stimulated PS expression, and that since this PS expression occurs on a subpopulation of platelets that are the smallest platelets, these are the ones most capable of contributing to procoagulant activity.
Table 1. Platelet PS Expression Treatment Annexin A5 MCF % Annexin A5-positivity Values are mean±SEM, n=5. Significantly different from control: *P&lt;0.01; **P&lt;0.001 (1-way ANOVA). control 16.7±8.5 1.3±0.4 C 34.0±3.6 7.0±1.5 T 10.8±1.6 1.3±0.4 PAR1-AP 13.4±0.7 1.5±0.3 PAR4-AP 16.9±2.1 2.2±0.5 C+T 61.4±10.7 13.5±1.7* C+PAR1-AP 87.9±18.8* 16.4±3.1** C+PAR4-AP 96.6±20.5* 18.4±3.7** C+PAR1-AP+PAR4-AP 106.1±19.0** 20.6±3.4**