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 2

hPAR4Thr/Thr mice have worse stroke outcomes than hPAR4Ala/Ala mice in a moderate tMCAO model.

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hPAR4Thr/Thr mice have worse stroke outcomes than hPAR4Ala/Ala mice in a...
Mice were subjected to 40 minutes of tMCAO followed by 23 hours of reperfusion (moderate). (A) Brain infarct volumes were quantified by staining with 2,3,5-triphenyl-tetrazolium chloride (TTC). Dead tissue is outlined with black dotted lines. Representative images from 12 to 15 mice per group. (B) Quantification of brain infarct volumes. Neurological and motor function were assessed by the Bederson (C) and grip (D) tests (10). Normality was determined by Shapiro-Wilk test, while significance was determined by an unpaired t test (B) and Mann-Whitney U test (C and D). n = 12–15 per group. (E) Average number of whole-blood PNAs based on Ly6G-CD41–positive events. n = 9 per group. (F) Representative images of neutrophils in infarcted brain tissue identified by myeloperoxidase (MPO) (red) and DNA (DAPI; blue) from n = 5 per group. (G) Number of neutrophils per infarcted brain area. n = 5 per group. (H) Representative images of NETs in brain tissue identified by MPO (red), citrullinated histone H3 (H3cit) (green), and DNA (DAPI; blue) from n = 5 per group. Images in H are magnified insets of images presented in F. (I) Percentages of NET-forming, H3cit-positive neutrophils. n = 5 per group. Normality was determined by Shapiro-Wilk test, while significance was determined by an unpaired t test (G and I). (J) Platelets and neutrophils were activated with AYPGKF, CVX, or vehicle (baseline). PNAs were measured by flow cytometry. Significance was determined by 2-way ANOVA with an uncorrected Fisher’s least significant difference (LSD). n = 5 per group. (K) Platelets and neutrophils from hPAR4Ala/Ala and hPAR4Thr/Thr mice activated with AYPGKF, CVX, or vehicle (baseline). NETs were quantified using an MPO-DNA ELISA. Significance was determined by 2-way ANOVA with Šidák’s multiple-comparisons test. n = 3 per group.