Immunophilins are involved in the altered platelet aggregation observed in patients with type 2 diabetes mellitus.

UNLABELLED: Platelet hyperaggregability might contribute to vascular complications associated with type 2 diabetes mellitus (DM2).Experimental evidence supports a direct link between altered Ca(2+) entry and hyperaggregability in DM2 patients. OBJECTIVES: We aimed to investigate whether altered immunophilin expression and function are involved in the abnormal Ca(2+) entry observed in platelets from DM2 patients. RESULTS: Inhibition of immunophilins by tacrolimus (FK506) and sirolimus (rapamycin) reduced Ca(2+) entry in platelets from healthy donors and DM2 patients. Similarly, immunophilin inhibitors reduced platelet degranulation in both healthy and DM2 subjects. Nevertheless, α-granule secretion reduction was greater than that observed for dense granules in platelets from DM2 patients. However, no difference was observed in the inhibition of secretion in platelets from healthy subjects. Additionally, altered expression of FK506 binding protein-52 (FKBP52) and coupling to Ca(2+) channels were found in platelets from DM2 patients compared to healthy subjects. Finally, reduction in platelet function from healthy subjects and DM2 patients in the presence of immunophilin antagonists was observed, being this dysfunction more evident in platelets from DM2 patients. CONCLUSIONS: We suggest that, among others, FKBP52 expression and function are altered in platelets from DM2 patients, contributing to the altered Ca(2+) entry and hyperaggregability in these cells.

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation.

BACKGROUND: All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP(3)Rs)-binding (CIRB) site involved in the regulation of TRPC channel function. OBJECTIVES: To assess the basis of CaM/IP(3)Rs binding to the CIRB site of TRPC6 and its role in platelet physiology. METHODS: Protein association was detected by co-immunoprecipitation and Western blotting, Ca(2+) mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry. RESULTS: Co-immunoprecipitation of TRPC6 with CaM or the IP(3)Rs at different cytosolic free Ca(2+) concentrations ([Ca(2+)](c)) indicates that the association between these proteins is finely regulated by cytosolic Ca(2+) via association of CaM and displacement of the IP(3)Rs at high [Ca(2+)](c). Thrombin-stimulated association of TRPC6 with CaM or the IP(3)Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca(2+)-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP(3)Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca(2+) entry in the presence of low or high, respectively, extracellular Ca(2+) concentrations, as well as altered thrombin-evoked platelet aggregation. CONCLUSIONS: Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca(2+) entry and aggregation through its interaction with CaM and IP(3)Rs.