Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets

Abstract

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.

Fig. 1.

Specificity and potency of PAR tool compounds. (A) Chemical structures of PAR1 (SCH602539) and PAR4 (VU0652925) antagonists. (B) The effect of PAR1 and PAR4 antagonists on PAR1-AP–induced aggregation. Washed human platelets were preincubated for 20 minutes with 1 μM SCH602539 or 316 nM VU0652925 before initiating aggregation with 20 μM PAR1-AP. Representative tracings of three independent experiments are shown. (C) The effect of PAR1 and PAR4 antagonists on PAR4-AP–induced aggregation. Platelets were pretreated with antagonist as in (B). Aggregation was initiated with 200 μM PAR4-AP. Representative tracings of three independent experiments are shown. (D) The effect of SCH602539 on PAR1-AP– and PAR4-AP–induced platelet activation. GPIIbIIIa inside-out activation (PAC1) and P-selectin expression (P-Sel) were measured by flow cytometry with PAC1 and CD62p antibodies. Platelets were treated with increasing concentrations of SCH602539 for 20 minutes prior to activation with 20 μM PAR1-AP or 200 μM PAR4-AP. Data are normalized to vehicle control. Means ± S.E.M. are shown (n = 3). (E) The effect of VU0652925 on PAR1-AP– and PAR4-AP–induced platelet activation. Platelets were treated as in (D). Data are normalized to vehicle control. Means ± S.E.M. are shown (n = 3). DMSO, dimethylsulfoxide; P-Sel, P-selectin.

Fig. 2.

Inhibition of PAR1 and PAR4 abolish thrombin-mediated signaling on human platelets. (A) The effect of PAR1 and PAR4 antagonists individually on thrombin-mediated platelet activation. Platelets were treated with increasing doses of SCH602539 or VU0652925 for 20 minutes prior to activation with 2 or 10 nM α-thrombin. GPIIbIIIa activation was monitored by PAC1 binding. Means ± S.E.M. are shown (n = 4). (B) The effect of PAR1 and PAR4 antagonists, combined, on platelet activation by thrombin. Platelets were treated for 20 minutes with 3.16 μM SCH62539 or 1 μM VU652965 prior to activation with 2 or 10 nM α-thrombin. Means ± S.E.M. are shown (n = 3). Two-sided t-test was used. Software used was GraphPad Prism (GraphPad Software, La Jolla, CA). *, P < 0.05; ***, P < 0.005. (C) The effect of PAR1 and PAR4 antagonists on platelet aggregation induced by thrombin. Platelets were pretreated for 20 minutes with antagonist prior to activation with 10 nM α-thrombin. Representative tracings of three independent experiments are shown. DMSO, dimethylsulfoxide.

Fig. 3.

Relative contributions of PAR1 and PAR4 to thrombin-mediated platelet signaling. (A) The effect of increasing doses of SCH602539 on α-thrombin–induced GPIIbIIIa activation (PAC1) CRCs. CRCs were constructed with PAC1 binding data measured by flow cytometry. (B) The effect of increasing doses of VU0652925 on α-thrombin PAC1 CRCs. (C) The effect of increasing doses of SCH602539 on PAR1 isolated thrombin-mediated platelet activation. Platelets were pretreated with a fixed concentration of 316 nM VU0652925 and the indicated concentrations of SCH602539 before activation with increasing concentrations of α-thrombin. (D) The effect of increasing concentrations of VU0652925 on PAR4 isolated thrombin-mediated platelet activation. Platelets were pretreated with a fixed concentration of 1 μM SCH602539 and the indicated concentrations of VU0652925 before activation with increasing concentrations of α-thrombin. Means are shown (n = 3). DMSO, dimethylsulfoxide.

Fig. 4.

Structures of VU0652925 analogs.

Fig. 5.

Schild analysis and identification of competitive PAR4 antagonists. Progressive fold-shift experiments and accompanying Schild analysis with VU0652925 fragments. A) VU0652925, B) VU0661247, C) VU0661245, D) VU0807074, E) VU0807081, F) VU0806526. Platelet activation was monitored by PAC1 binding. Platelets were pretreated with increasing concentrations of each antagonist for 20 minutes prior to activation with increasing concentrations of PAR4-AP. Each curve was constructed from at least three independent experiments. DRs were calculated from the EC₅₀s of each individual experiment (vehicle_EC50/VU#_EC50) and plotted against the administered concentration of antagonist. A) Shown on the right are the means ± S.E.M. of log DR-1 (n = 3). In the graph insert, m is the slope from linear regression. DMSO, dimethylsulfoxide.

Fig. 6.

Novel PAR4 antagonists are effective against the TL and completely reversible. Progressive fold-shift experiments with VU0652925 (A) and novel PAR4 antagonist VU0661224 (B). Platelet activation was monitored by PAC1 binding. Platelets were pretreated with increasing concentrations of each antagonist for 20 minutes prior to activation with increasing concentrations of PAR4-AP. Each curve was constructed from at least three independent experiments. DMSO, dimethylsulfoxide.