Human PAR1 expressed on mouse platelets contributes to hemostasis and arterial occlusion

Figures

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  Figure 1.. Different effects of FIIa binding to GPIbα on Ca2+ signaling in human and mouse platelets. Washed platelets were prepared from human healthy donors (A) or mice expressing on the platelet surface hGPIbα-WT or hGPIbα-D277N defective in FIIa binding (B). These platelets were loaded with 5 μM Fura 2-AM to measure changes in intracytoplasmic Ca2+ concentration after stimulation with different concentrations of FIIa in the absence or presence of MoAb LJ-Ib10, which binds to human GPIbα interfering with FIIa-dependent platelet activation. WEDE, blocking PAR1 function, was used only with human platelets. (C) Schematic representation of the targeting construct used for creating a mouse strain expressing hPAR1.

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  Figure 2.. Expression of hPAR1 on mouse platelets. (A) Identification by flow cytometry of mouse platelets expressing EGFP and hPAR1Tg or hGPIbαTg generated using the same targeting vector. (B) Aggregation of hPAR1Tg mouse and human washed platelets induced by 10 μM P1-AP. (C) Expression of hPAR1 in P3−/−hP1Tg compared to human platelets analyzed by flow cytometry using the anti-hPAR1 antibody ATAP2. (D) Expression of hPAR1 on platelets from human controls and P3−/−hP1Tg mice measured as mean fluorescence intensity (MFI) of bound antibody divided by forward side scatter (FSC) to correct for the difference in platelet size. (E) Western blotting of platelet lysates after reaction with the anti-hPAR1 antibody ATAP2; red arrows point to the hPAR1 signal in human and P3−/−hP1Tg platelets. (F) BM cells were prepared from P3−/− and P3−/−hP1Tg mice (n = 3 in each group) to analyze by flow cytometry surface expression of integrin αIIb (CD41) and hPAR1 on MKs of different ploidy. Results are shown as scatter dot plots with mean ± SD.

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  Figure 3.. Functional analyses of hPAR1 in P3−/−hP1Tg mouse platelets stimulated by PAR1 activation peptide (P1-AP). Aggregation induced by different concentrations of P1-AP was evaluated in washed platelet suspensions prepared from P3−/−hP1Tg mice (A) or healthy human donors (B). The inhibitory effect of the PAR1 antagonist drug, vorapaxar, was examined using the minimum P1-AP concentration enabling full aggregation, corresponding to (C) 7.5 μM for P3−/−hP1Tg platelets and (D) 2.5 μM (for human platelets. (E) Washed platelets prepared from P3−/−hP1Tg mice or human healthy donors were stimulated with different concentrations of P1-AP and platelet lysates were analyzed by Western blotting. HSC70 was used as a loading control. (F) The signal intensity of protein bands as in (B) was quantified with the Licor ImageStudio instrument. These experiments were repeated two times and the quantified results (mean ± SD) are shown as scatter plots with mean value.

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  Figure 4.. Aggregation of P3−/−hP1Tg as compared to P3−/− mouse platelets stimulated by α-thrombin (FIIa). Washed platelets from P3−/− (A) and P3−/−hP1Tg (B) mice were induced to aggregate by various FIIa concentrations. Mean ± S.E.M. of amplitude from six independent biological experiments at the indicated FIIa concentration (C). (D, E) Aggregation of P3−/− and P3−/−hP1Tg mouse platelets were induced by FIIa in the presence of the PAR4 inhibitor, BMS-986120 (BMS, 80 nM). Note the complete aggregation inhibition by BMS of P3−/− platelet, but only partial of P3−/−hP1Tg platelets at the two higher FIIa concentrations. (F) The residual aggregation of P3−/−hP1Tg compared to P3−/− platelets induced by 1.6 nM FIIa in the presence of BMS was abolished by vorapaxar (VOR; 400 nM).

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  Figure 5.. Effect of PAR1 and PAR4 inhibition on FIIa-induced aggregation of human and P3−/−hP1Tg mouse platelets. Washed platelets prepared from healthy donors (A) and P3−/−hP1Tg mice (B) were stimulated with 1.6 nM FIIa in the presence of different concentrations of BMS-986120 (BMS), vorapaxar (VOR) or both and evaluated for aggregation.

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  Figure 6.. Hemostasis after tail clipping in P3−/−hP1Tg and control mice. (A) The arrest of bleeding after tail clipping was evaluated in C57BL/6J wild type (B6 WT), P3−/− and P3−/−hP1Tg mice; in B6 WT and P3−/−hP1Tg mice the test was also performed 5 minutes after intravenous administration of vorapaxar (3.6 mg/kg). N = 10 in each group. The percentage of tails bleeding within 600 seconds after injury is shown by Kaplan-Meier analysis, with differences between groups evaluated by the log-rank (Mantel-Cox) test with Bonferroni correction for pairwise comparison. *P<0.01. P = 0.012 for the other bracketed comparison.

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  Figure 7.. FeCl3-induced carotid artery occlusion model. Time to first occlusion (A) and time to stable occlusion (B) were measured following ferric chloride (0.22 M) induced injury to the left carotid artery. Flow index (C) was calculated as described in Materials and Methods. Indicated groups were treated with vehicle or vorapaxar (VOR) (3.6 mg/kg). Censored values (1980 s) indicate absence of occlusion within the recording window (33’). Statistical differences were determined using Kruskal-Wallis test with Dunn’s multiple comparisons. *P < 0.05, **P < 0.01.