The predominant PAR4 variant in individuals of African ancestry worsens murine and human stroke outcomes
Frederik Denorme, … , Paul F. Bray, Robert A. Campbell
Frederik Denorme, … , Paul F. Bray, Robert A. Campbell
Published July 20, 2023
Citation Information: J Clin Invest. 2023;133(18):e169608. https://doi.org/10.1172/JCI169608.
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Research Article Hematology Neuroscience

The predominant PAR4 variant in individuals of African ancestry worsens murine and human stroke outcomes

Abstract

Protease-activated receptor 4 (PAR4) (gene F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not known whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice in which F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared with hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7,620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of nonstandard antiplatelet therapies for patients expressing PAR4 Thr120.

Authors

Frederik Denorme, Nicole D. Armstrong, Michelle L. Stoller, Irina Portier, Emilia A. Tugolukova, Rikki M. Tanner, Emilie Montenont, Seema Bhatlekar, Mark Cody, John L. Rustad, Abigail Ajanel, Neal D. Tolley, Darian C. Murray, Julie L. Boyle, Marvin T. Nieman, Steven E. McKenzie, Christian Con Yost, Leslie A. Lange, Mary Cushman, Marguerite R. Irvin, Paul F. Bray, Robert A. Campbell

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Figure 1

Characterization of hPAR4 mice.

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Characterization of hPAR4 mice. (A) Schematic showing generation of hPAR...
(A) Schematic showing generation of hPAR4 mice. PCR primers are shown for F2rl3 (mouse sense 1 [mS1] and mouse antisense [mAS]), the 5′ insertion of F2RL3 (mouse sense 2 [mS2] and human antisense [hAS]), and the 3′ insertion of F2RL3 (hS and mAS). (B) Agarose gel of PCR products amplified from genomic DNA from hPAR4 and C57BL/6 (mPAR4) mice. Image is representative of 3 independent experiments. (C) PAR4 sequence with SNP for each allele in bold and codon in red underline. Boxed sequence shows a Nrul restriction site only in the F2RL3 variant containing the A allele (hPAR4Thr/Thr). Agarose gel shows PCR-amplified genomic DNA digested with NruI. Image is representative of 3 independent experiments. (D) F2RL3 expression was assessed by quantitative real-time PCR using mRNA isolated from platelets from hPAR4Ala/Ala and hPAR4Thr/Thr mice. Normality determined by Shapiro-Wilk test, with significance determined by unpaired t tests. n = 6 per group. (E) Platelets from C57BL/6 mice (mPAR4) and global PAR4-deficient mice (mPAR4–/–) were stained for human PAR4 (upper blot) or murine PAR4 (middle blot). Human platelets (hPAR4) were used as controls. (F) Platelet PAR4 protein levels in hPAR4Ala/Ala and hPAR4Thr/Thr mice (n = 13 per group). Normality determined by Shapiro-Wilk test with significance determined by Mann-Whitney U test. (G–L) Platelets were isolated from hPAR4Ala/Ala and hPAR4Thr/Thr mice and stimulated with either AYPGKF or human thrombin. Representative tracings for AYPGKF (n = 10–11 per group; G and I) and thrombin (n = 4–8 per group; H and J). (K and L) Maximal aggregation of AYPGKF (n = 10–11 per group) and thrombin (n = 4–8 per group). Significance was determined by 2-way ANOVA with Bonferroni’s multiple-comparison test (K) and a mixed-effects analysis with Bonferroni’s multiple-comparisons test (L).