Mechanistic Insights and Clinical Implications of the Thrombotic Risks of a Highly Prevalent GRK5 Variant Affecting Platelets

Background: In recent years, the National Center for Health Statistics reported over 13 million doctor visits for chronic ischemic heart disease, over 400,000 hospitalizations for pulmonary embolism, and over 160,000 stroke deaths, highlighting the need for genetic screening to prevent and treat cardiovascular diseases. Two recent genome-wide association studies identified the rs10886430 variant in the G protein-coupled receptor kinase 5 gene (GRK5) as being associated with a higher risk of pulmonary embolism, stroke, and coronary artery disease in over 5 million Americans. However, neither study examined homozygous carrier platelets' change in GRK5 protein expression, platelet hyperactivity, and platelet involvement in pathological thrombus formation in vivo.

Aim: To use preclinical models to investigate the molecular basis of platelet hyperactivity in homozygous carriers and to assess their risk of thrombotic diseases.

Methods: GRK5 regulation in thrombin PAR1/PAR4 signaling was measured using 1) GG (homozygous carrier) vs. AA (non-carrier) donor platelets, 2) a GRK5^-/--induced Pluripotent Stem Cell (iPSC) line, and 3) a GRK5-specific inhibitor. Furthermore, Grk5 was deleted from a humanized PAR4 mouse model in a Par4 knockout mouse background. We assessed the effects of the variant on human platelet function utilizing 1) a microfluidic chamber and 2) an NSG/vWF^R1326H mouse model, which switches murine vWF from binding mice to human GP1bα.

Results: Genetic screening of 217 individuals identified three homozygous GRK5 GG donors in their early twenties with no reported health conditions. Their GG platelets showed ~90% reduction in GRK5 protein levels compared to AA platelets. The reduction in GRK5 expression led to increased platelet responses to thrombin and PAR1 agonist peptide, but not to PAR4 agonist peptide. GG platelets exhibited less PAR1 internalization upon thrombin stimulation, resulting in sustained platelet signaling and hyperactivity. Next, we observed increased thrombin- and PAR1-mediated signaling, but not PAR4-mediated signaling in GRK5^-/- iPSC-derived megakaryocytes (iMKs) compared to wild-type (WT) iMKs. Thrombin-stimulated GRK5^-/- iMKs displayed reduced PAR1 internalization compared to WT iMKs.

To further elucidate GRK5's role in thrombin signaling, we used humanized mice that express human (h), but not mouse (m), Par4 or Grk5. Platelet aggregation and activation markers of these platelets were similar to those of controls, demonstrating that Grk5 does not regulate hPAR4. In addition, pharmacological inhibition of GRK5 in healthy AA donor platelets increased platelet activation and intracellular calcium in response to a PAR1, but not PAR4, agonist peptide.

In the context of vessel pathology, we flowed GG whole blood through microfluidic chambers to examine platelet accumulation on a collagen/tissue factor (TF) coated surface. GG platelets showed enhanced platelet accumulation in a setting mimicking vessel injury compared to AA platelets. Lastly, we utilized an NSG/vWF^R1326H mouse model to assess the effects of the GRK5 GG variant on human platelet function in vivo. These mice, in effect, have a von Willebrand disease-like bleeding diathesis unless infused with human platelets. We observed enhanced carotid artery occlusion in these mice infused with GG platelets compared to those infused with AA platelets.

Conclusions: Our data have demonstrated, for the first time, that GRK5 protein expression is markedly reduced in individuals with the GRK5 GG variant that is found in 5 million individuals in the United States. These platelets are associated with increased thrombin and PAR1 reactivity and greater platelet accumulation on collagen/TF. Knockout studies in iMKs and murine platelets, as well as pharmacologic reduction of GRK5 in AA donor platelets, further confirmed these findings. Lastly, using a murine model that depends on infused human platelets for hemostatic efficacy, we show that infusion of the GRK5 GG platelets leads to enhanced carotid artery occlusion at sites of vascular injury compared to AA platelets. These findings demonstrate that this variant plays a significant role in platelet reactivity for a large subset of the population, potentially impacting their cardiovascular risk. This work also suggests models which could be used to facilitate the development of targeted therapeutics for homozygous carriers of this GRK5 variant.

Poncz:Alexion: Research Funding; Astra Zeneca: Research Funding.