A complementary role for tetraspanin superfamily member CD151 and ADP purinergic P2Y12 receptor in platelets

Abstract

P2Y12 receptor is required for sustained activation of integrin αIIbβ3, irreversible platelet aggregation and thrombus stabilisation. Tetraspanin superfamily member CD151 associates with integrin αIIbβ3 and plays critical roles in regulation of thrombus growth and stability in vivo. The possible functional relationship between P2Y12 and CD151 in a molecular cluster in platelets may affect thrombus formation. Hence our aim was to investigate the physical and functional requirements for this association in platelets. Our investigations reveal a specific and constitutive association between CD151 and P2Y12 receptor in human platelets shown by immunoprecipitation/western blot studies and by flow cytometry. Specifically, the prominent association involves CD151 with P2Y12 oligomers, and to a lesser extent P2Y12 monomers. This association is not altered by platelet aggregation induced by different agonists. There is also a distinct complex of tetraspanin CD151 with ADP purinergic receptor P2Y12 but not P2Y1. P2Y12 oligomer interaction with CD151 is selective as compared to other tetraspanins. To investigate the functional relationship between these receptors in platelets we used wild-type or CD151 knockout (KO) mice treated with either PBS or 50 mg/kg clopidogrel. CD151 KO mice treated with clopidogrel exhibited synergy in delayed kinetics of clot retraction, in PAR-4 and collagen-mediated platelet aggregation, platelet spreading on fibrinogen and without restricting cAMP inhibition. Clopidogrel treated CD151 KO arterioles showed smaller and less stable thrombi with increased tendency to embolise ex vivo and in vivo. These studies demonstrate a complementary role between CD151 and P2Y12 receptor in platelets in regulating thrombus growth and stability.

Platelet integrin αIIbβ3: signal transduction, regulation, and its therapeutic targeting

“…Impaired platelet aggregation and platelet spreading on fibrinogen, delayed kinetics of clot retraction, restricted cytoskeletal reorganization. Increased bleeding time and volume and rebleeding, but without spontaneous bleeding complications[150, 156, 157]CEACAM-1NullEnhanced aggregation, enhanced platelet adhesion on type I collagen but not fibrinogen, elevated granule secretion, larger and more stable thrombi[137]CIB1Normal soluble fibrinogen bindingNormal aggregation and α-granule secretion, increased tail bleeding time and rebleeding, formation of unstable thrombi, impaired spreading on immobilized fibrinogen, reduced tyrosine phosphorylation of the integrin β3 tail[93, 101]cPLA2αImpaired fibrinogen binding in response to CRP or the lower concentration of PAR4 peptideImpaired collagen-induced aggregation, spreading on fibrinogen, platelet aggregation. Prolonged bleeding time[224, 225]Dab2Impaired soluble fibrinogen bindingSelectively defective in thrombin-induced aggregation, platelet spreading on fibrinogen and clot retraction.…”

“…Studies using murine CD151 −/− platelets have demonstrated that deletion of CD151 is capable of inhibiting the outside-in signaling properties of αIIbβ3, including reducing agonist-induced platelet aggregation, delaying clot retraction, diminishing platelet spreading on fibrinogen, and reducing formation of filopodia. However, CD151 −/− platelets display normal αIIbβ3 inside-out signaling properties, as evidenced by standard agonist-induced binding of soluble fibrinogen or JON/A antibody [150, 156, 157]. Recent studies by Orlowski et al that used three different models for thrombus formation have confirmed that platelet CD151 is required for regulating thrombus formation in vivo [149].…”

“…Recent studies by Orlowski et al that used three different models for thrombus formation have confirmed that platelet CD151 is required for regulating thrombus formation in vivo [149]. CD151 forms a CD151/P2Y 12 receptor complex and participates in integrin αIIbβ3 outside-in signaling [157]. TSSC6 regulates integrin αIIbβ3 outside-in signaling by physically associating with the P2Y 12 receptor [158].…”