Genetic Variant in Human PAR (Protease-Activated Receptor) 4 Enhances Thrombus Formation Resulting in Resistance to Antiplatelet Therapeutics

Abstract

Objective: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased G_q signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention.

Approach and results: Membranes expressing human PAR4-120 variants were reconstituted with either G_q or G₁₃ to determine the kinetics of G protein activation. The kinetics of G_q and G₁₃ activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y₁₂ receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy.

Conclusions: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both G_q and G₁₃ activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y₁₂ receptor.

Keywords: blood platelets; humans; pharmacogenetics; signal transduction; thrombin.

Figure 1.

The PAR4 (protease-activated receptor 4)-Thr120 variant enhances G_q and G₁₃ activation compared with the PAR4-Ala120 variant. A, Left, The relative abundance of recombinant human PAR4-Ala120 and PAR4-Thr120 in prepared membranes was visualized by Western blot analysis in comparison with control noninfected membranes. Right, Intact cells expressing the recombinant human PAR4 variants were subjected to cell surface biotinylation. Biotinylated cell surface proteins were isolated, and relative PAR4 levels were compared by Western blot analysis. B–E, Prepared membranes of PAR4-Ala120 (blue) or PAR4-Thr120 (red) were preincubated with PAR4-AP (PAR4-activating peptide; closed symbols) or buffer control (open symbols) before reconstitution with the purified G protein heterotrimers: (B) G₁₃, (C) G_q, (D) Gi₁, or (E) Gs_short. The kinetics of PAR4-stimulated [³⁵S]-GTPγS binding to G proteins (activation) are shown. B and C, (inset) Initial linear [³⁵S]-GTPγS binding rates of (B) G₁₃ or (C) G_q stimulated by PAR4-AP–activated PAR4-Ala120 and PAR4-Thr120. An unpaired t test, 2-tailed, was performed. Data represent mean±SEM. Ala indicates alanine; and Thr, threonine. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2.

After PAR4 (protease-activated receptor 4) stimulation, RhoA activation and shape change are heightened in platelets from individuals expressing PAR4-T120. A, Active RhoA was selectively precipitated from the lysates of PAR4-stimulated (PAR4-AP [PAR4-activating peptide]; 25 µmol/L) platelets and normalized to the amount of total RhoA in each sample as determined by Western blot. RhoA activation data, reported as fold change relative to the unstimulated control, were analyzed by PAR4-120 variants (alanine and threonine). B, Shape change was measured in a lumi-aggregometer after PAR4 stimulation of EGTA-treated platelets, and data were analyzed by PAR4-120 variant. Two-way statistical ANOVA was performed. Data represent mean±SEM. AA indicates arachidonic acid.

Figure 3.

No difference in platelet spreading and clot retraction by PAR4 (protease-activated receptor 4) Ala120/Thr120 dimorphism. A, Platelets treated with indomethacin (20 μM) and apyrase (50 U/mL) were allowed to spread on fibrinogen-coated (50 μg/mL) glass cover slips in the presence or absence of PAR4-AP (PAR4-activating peptide; 25 μM). The surface area of 5 spread platelets from 5 donors was measured every 30 s for 300 s. B, Platelet-rich plasma was incubated with thrombin (2.5 or 5 nmol/L), and clot size was quantified by subtracting the volume of plasma expelled from the contracting clot from the starting/original volume (500 µL) at the indicated time. Dashed line depicts the total volume of platelet-rich plasma in samples that were not treated with thrombin after 60 min. Two-way statistical ANOVA was performed. Data represent mean±SEM. AA indicates arachidonic acid.

Figure 4.

Thrombin-dependent platelet accumulation is enhanced under arterial shear ex vivo with whole blood from individuals who express PAR4 (protease-activated receptor 4)-Thr120 compared with those who are homozygous for the PAR4-Ala120 variant. A, Recalcified citrated or (B) heparinized blood was stained with 3,3′-dihexyloxacarbocyanine iodide and perfused through collagen-coated perfusion chamber at arterial shear (1500 s⁻¹) for 4 min. Platelet accumulation as quantified by mean fluorescence intensity using an inverted fluorescent microscope (20X objective) was analyzed by PAR4-120 variants (alanine and threonine). C, Recalcified citrated blood was treated with RWJ-56110 (10 µmol/L) for 15 min before being perfused through a collagen-coated chamber. D, Recalcified citrated blood was perfused through a chamber coated with collagen and tissue factor at arterial shear (1500 s¹).Two-way statistical ANOVA was performed. Data represent mean±SEM. AA indicates arachidonic acid; and MFI, mean fluorescence intensity.

Figure 5.

The difference in PAR4 (protease-activated receptor 4)-mediated platelet activation by PAR4 Ala120/Thr120 dimorphism persists in individuals treated with either aspirin or Plavix. Representative aggregation tracings of platelets from individuals who are homozygous for the PAR4-Ala120 variant or individuals who express at least 1 copy of the PAR4-Thr120 variant on aspirin (A) or Plavix (B) in response to 50 µmol/L PAR4-AP (PAR4-activating peptide). Quantification of the maximum aggregation by PAR4 variant of platelets isolated from individuals on aspirin (A) or Plavix (B) in response to increasing concentrations of PAR4-AP. Platelets isolated from healthy individuals on aspirin (81 mg) or Plavix (75 mg) were stimulated in an aggregometer with (C) arachidonic acid (AA) or (D) ADP, respectively. Donors on single-antiplatelet therapy who had an aggregation response ≥20% (dashed line) to either AA or ADP, respectively, were excluded from the study. Recalcified citrated whole blood was treated with dual-antiplatelet therapy (DAPT), aspirin (100 µmol/L), and 2-methylthioadenosine 5′-monophosphate triethylammonium (50 µmol/L), before being perfused through a collagen-coated chamber at arterial shear (1500 s⁻¹) in the (E) presence or (F) absence of TF (tissue factor). Two-way statistical ANOVA was performed. Data represent mean±SEM. MFI indicates mean fluorescence intensity.