Selective inhibition of Panx1 channels decreases hemostasis and thrombosis in vivo

Abstract

Background: Hemostasis is a tightly regulated physiological process to rapidly induce hemostatic plugs at sites of vascular injury. Inappropriate activation of this process may lead to thrombosis, i.e. pathological blood clot formation in uninjured vessels or on atherosclerotic lesions. ATP release through Pannexin1 (Panx1) membrane channels contributes to collagen-induced platelet aggregation in vitro. Objective: To investigate the effects of genetic and pharmacological inhibition of Panx1 on hemostasis and thrombosis in vivo. Results: Bleeding time after tail clipping was increased by 2.5-fold in Panx1 −/− mice compared to wild-type controls, suggesting that Panx1 deficiency impairs primary hemostasis. Wire myography on mesenteric arteries revealed diminished vasoconstriction in response to phenylephrine or U446619 in Panx1 −/− mice. Mice with platelet-specific deletion of Panx1 (Panx1 PDel) displayed 2-fold longer tail bleeding times than Panx1 fl/fl controls. Moreover, venous thromboembolism (VTE) after injection of collagen/epinephrine in the jugular vein was reduced in Panx1 −/− and Panx1 PDel mice. Panx1 PDel mice also showed reduced FeCl 3-induced thrombosis in mesenteric arteries. BrilliantBlue-FCF, a Panx1 channel inhibitor, decreased collagen-induced platelet aggregation in vitro, increased tail bleeding time and reduced VTE in wild-type mice. Furthermore, we developed a specific Panx1 blocking antibody targeting a Panx1 extracellular loop, which reduced ATP release from platelets in vitro. Treating wild-type mice with this antibody increased tail bleeding time and decreased VTE compared to control antibody. Conclusions: Panx1 channel deletion or inhibition diminishes clot formation during hemostasis and thrombosis in vivo. Blocking Panx1 channels may be an attractive strategy for modulating platelet aggregation in thrombotic disease.

Mind the gap: connexins and pannexins in platelet function

“…A common platelet phenotype was observed with each connexin knockout mouse whereby fibrinogen binding, P -selectin exposure and ATP release were all reduced [ 6 , 7 ]. In vivo studies demonstrate that Panx1 contributes to both hemostasis and thrombosis with significantly elevated tail bleeding times and reduced mortality in a thromboembolic model in Panx1 −/- mice [ 25 ]. Extensive evidence therefore supports the notion that connexins, specifically Cx37 5 [ 18 ], 40 6 and 62 7 , and Panx1 21 [ 25 , 26 ], perform important regulatory roles in platelet function in hemostasis and thrombosis.…”

“…In the case of Panx1, several putative phosphorylation sites have been reported for Src Family Kinases (SFKs), protein Kinase A (PKA) and Protein Kinase C (PKC) [ 31 , 36 , 37 ]. Collagen-evoked platelet activation leads to Panx1 phosphorylation at Y308, which is inhibited by preincubation with the SFK inhibitor PP2 [ 25 ]. The functional relevance of predicted PKA and PKC sites on Panx1 are not yet clear.…”

“…Ex vivo analyses demonstrated that mouse platelets treated with probenecid, reduced murine platelet aggregation [ 25 , 51 ]. In vitro thrombus formation assays also demonstrate reduced thrombus formation in the presence of carbenoxolone or probenecid [ 18 , 21 ].…”

“…Indeed, megakaryocytes have been shown to reduce deposition of type I collagen by osteoclasts, through Cx43-mediated interactions [ 61 ]. The direct roles of connexins and pannexins during megakaryocyte development and thrombopoiesis have not been evaluated, but data suggest that platelet counts are normal in both Cx37 and panneixn-1 knockout mice [ 5 , 25 ].…”

“…However, the impact of this model upon platelet function and thrombus formation was not assessed. In vivo thrombosis studies with Cx40 −/- [ 6 ] and Panx1 −/- [ 25 ] mice both report reduced thrombus formation, indicating therapeutic potential in targeting these channels.…”