Cav 3.2 T-type calcium channel regulates mouse platelet activation and arterial thrombosis

Abstract

Background: Ca_v 3.2 is a T-type calcium channel that causes low-threshold exocytosis. T-type calcium channel blockers reduce platelet granule exocytosis and aggregation. However, studies of the T-type calcium channel in platelets are lacking.

Objective: To examine the expression and role of Ca_v 3.2 in platelet function.

Methods: Global Ca_v 3.2^-/- and platelet-specific Ca_v 3.2^-/- mice and littermate controls were used for this study. Western blot analysis was used to detect the presence of Ca_v 3.2 and activation of the calcium-responsive protein extracellular signal-regulated kinase (ERK). Fura-2 dye was used to assess platelet calcium. Flow cytometry and light transmission aggregometry were used to evaluate platelet activation markers and aggregation, respectively. FeCl₃ -induced thrombosis and a microfluidic flow device were used to assess in vivo and ex vivo thrombosis, respectively.

Results: Ca_v 3.2 was expressed in mouse platelets. As compared with wild-type controls, Ca_v 3.2^-/- mouse platelets showed reduced calcium influx. Similarly, treatment with the T-type calcium channel inhibitor Ni²⁺ decreased the calcium influx in wild-type platelets. As compared with controls, both Ca_v 3.2^-/- and Ni²⁺ -treated wild-type platelets showed reduced activation of ERK. ATP release, P-selectin exposure, and α_IIb β3 activation were reduced in Ca_v 3.2^-/- and Ni²⁺ -treated wild-type platelets, as was platelet aggregation. On in vivo and ex vivo thrombosis assay, Cav3.2 deletion caused delayed thrombus formation. However, tail bleeding assay showed intact hemostasis.

Conclusion: These results suggest that Ca_v 3.2 is required for the optimal activation of platelets.

Keywords: calcium; platelet; platelet aggregation; thrombosis; voltage-gated.