Blockade of Human PAR4 in Novel Humanized Mouse Strains Supports PAR4 As a Potential Target in Stroke: Ex Vivo Demonstration of Platelet Hyperreactivity of the Thr120 Variant

Human platelets express two thrombin-activated G-protein coupled receptors, PAR1 and PAR4, that activate upon protease cleavage of the amino-terminus of the receptor. Thrombin induces rapid and transient platelet activation through PAR1, while PAR4 mediates slower and sustained activation, as well as greater phosphatidylserine (PS) exposure and thrombin generation under shear stress. Because PAR1 blockade is contraindicated in treating stroke, PAR4 has been considered as a novel potential anti-platelet target. Human PAR4 has a common Ala120Thr variant that is much more common in individuals of sub-Saharan African ancestry. The Thr120 variant induces platelet hyperreactivity, and is associated with increased stroke risk and less bleeding. Because human PAR4 (hPAR4) differs significantly from mouse Par4 (mPar4), in vivo studies of hPAR4 and the Ala120Thr variant have not been possible. The goals of the current study were to (1) express and characterize hPAR4 in vivo and (2) determine specifically if the PAR4 Thr120 variant results in platelet hyperactivity. We generated humanized PAR4 Ala120 (hPAR4^Ala) and PAR4 Thr120 (hPAR4^Thr) mouse lines using CRISPR/Cas9 to replace the mouse F2rl3 with either human variant gene (F2RL3). Both hPAR4^Ala and hPAR4^Thr lines are viable, display germline transmission of hPAR4 and produce expected numbers of offspring. Genomic DNA from both strains contained human F2RL3 and not murine F2rl3, and whole genome sequencing confirmed the successful insertion of a single copy of F2RL3 in the proper location. hPAR4 mRNA was abundantly expressed in platelets from hPAR4^Ala and hPAR4^Thr, but not wild type mice. Our engineering strategy retained murine F2rl3 regulatory sequences, and hPAR4 transcripts were readily detected in brain, liver and lung in hPAR4^Ala and hPAR4^Thr mice. Importantly for our goals, hPAR4 mRNA and protein was expressed at similar levels in both hPAR4^Ala and hPAR4^Thr lines. Platelet counts and mean platelet volume from hPAR4^Ala (635±149 K/μL and 4.2±0.1) and hPAR4^Thr (617±145 K/μL and 4.0±0.3) mice were similar to wild type mPar4 mice (705±142 K/μL and 4.2±0.1). When washed platelets were stimulated with PAR4 activation peptides (APs) AYPGKF (artificial) or GYPGQV (natural tethered ligand), a dose-dependent activation of integrin aIIbb3 (by Jon/A binding) was observed that was significantly higher on hPAR4^Thr platelets compared to hPAR4^Ala platelets (p<0.05; n=8 mouse pairs). hPAR4^Thr platelets also showed greater Jon/A binding after activation with human thrombin. Platelets from hPAR4 mice responded similarly to human and murine thrombin, demonstrating for the first time hPAR4 is responsive to murine thrombin. Compared to hPAR4^Ala mice, hPAR4^Thr mice displayed greater platelet aggregation to AYPGKF (p<0.05, n=4 mouse pairs) and 1.7-fold greater PS exposure (p<0.0001) after dual stimulation with AYPGKF and a GPVI agonist. In vivo administration of a potent human PAR4 inhibitor (BMS-986120) resulted in ex vivo reduction in platelet Jon/A binding (p<0.05) in response to AYPGKF, but not a GPVI agonist, further supporting PAR4 specificity. Lastly, compared to vehicle, pre-treatment with BMS-986120 prior to transient middle cerebral artery occlusion (tMCAO) reduced infarct volume (p = 0.021, n=10) and improved neurological (p=0.0037) and motor (p=0.0007) function. In summary, we have generated humanized PAR4 mouse strains that directly demonstrate the role of human PAR4 in stroke and support PAR4 blockade as a potential therapeutic option. In addition, because these novel hPAR4^Ala or hPAR4^Thr mouse strains differ only by the Ala120 or Thr120 variant, our results directly demonstrate the PAR4 Thr120 variant causes increased platelet reactivity. The development of this new model will allow for novel insights into PAR4 biology in both platelets and other tissues, and provides a system to test future antithrombotic therapies.