Functional relationship between tetraspanin CD151 and TSSC6 with ADP purinergic receptor, P2Y12 in platelets regulates thrombus formation and stability

© 2015 Dr. Mohammed MakkawiBackground: Platelets play a critical role in maintaining normal haemostasis and thrombosis. However, they also participate actively in fatal pathological conditions. Tetraspanins are present in many cell types and are involved in a number of cellular processes. Of these, CD151 and tumor-suppressing subchromosomal transferable fragment cDNA 6 (TSSC6) are cell surface proteins that are structurally linked to this group. A range of cells and tissue types has been demonstrated to express CD151, such as vascular, immune and haematopoietic compartments, including platelets while TSSC6 expression is restricted to haematopoietic lineage. The adenosine diphosphate (ADP) purinergic receptor P2Y12 is mainly expressed by platelets and is known to be one of the most important targets for several different clinically approved oral and intravenous antithrombotic agents. All three receptors have been shown to influence the maintenance of the sustained activation of the integrin αIIbβ3 and the regulation of growing thrombi. Aim: Preliminary observations have shown that CD151 and TSSC6 act in conjunction with binding partners, including P2Y12 receptor in platelets. Therefore, the aim of this study was to investigate if close physical proximity of CD151 and TSSC6 with P2Y12 receptor translates into a functional relationship to regulate thrombus growth and stability. Method and Results: In this study, a mouse model deficient in both receptors was achieved by genetically knocking out the CD151 and TSSC6 receptors and functionally blocking P2Y12 receptor. The latter was achieved using the clinically approved treatment Clopidogrel, which specifically blocks P2Y12 receptor through frequent oral doses. Different in vitro, ex vivo, and in vivo approaches were used to examine these relationships. The data demonstrate that CD151 and TSSC6 form functional relationships with the purinergic ADP receptor, P2Y12 but not P2Y1 receptor in the regulation of ‘outside-in’ integrin αIIbβ3–mediated platelet aggregation, clot retraction and cytoskeletal reorganisation. In addition, to examine whether treating CD151 or TSSC6 (knockout) KO mice with Clopidogrel would affect the interaction between platelets and type I collagen, the interaction was tested using an in vitro arterial physiological flow condition with a wall shear rate of 1800 seconds-1. Using this model, it was observed that platelet-collagen interactions were significantly reduced further compared to CD151 or TSSC6 KO mice or wild-type (WT) mice treated with Clopidogrel. These results confirmed the functional relationship between CD151 and TSSC6 with P2Y12 receptor. This was additionally supported by in vivo experiments using a ferric chloride (FeCl3) vascular injury model of mesenteric arterioles showing that CD151 or TSSC6 KO mice treated with Clopidogrel exhibited smaller thrombi that were very unstable with a tendency to embolise in comparison to CD151 or TSSC6 KO mice treated with sham control or WT mice treated with Clopidogrel. Conclusion: This study demonstrates a functional association between the tetraspanin superfamily members, CD151 and TSSC6 with the ADP purinergic receptor P2Y12 in platelets. In addition, it shows that P2Y12 receptor blockade in CD151 or TSSC6 KO mice leads to a further ‘outside-in’ signalling defect of integrin αIIbβ3–mediated events compared to mice with CD151 or TSSC6 deficiency or P2Y12 receptor blockade alone. Further studies will examine the interaction of P2Y12 receptor with other tetraspanin superfamily members and whether similar functional relationships exist