In human platelets, depletion of the intracellular Ca²⁺ stores by treatment with thapsigargin (TG) or thrombin results in the de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP₃RII) to the putative store-operated Ca²⁺ channel, hTrp1 (Rosado & Sage, 2000a). This has led us to suggest that store-mediated Ca²⁺ entry (SMCE) in platelets may be activated by this coupling event. An essential requirement of this hypothesis is that protein coupling occurs rapidly enough to account for the observed Ca²⁺ entry. Here we have examined the latencies of divalent cation entry and the coupling of IP₃RII to hTrp1 in human platelets stimulated with thrombin.

Blood was drawn from healthy, drug free volunteers with local ethical committee approval and fura-2-loaded platelets prepared as previously described (Rosado & Sage, 2000a). The latency of thrombin-evoked Mn²⁺ entry was determined using a single mix stopped flow system with an excitation wavelength of 360 nm and emission at 500 nm. Mixing cells with agonist-free buffer was without effect on fura-2 fluorescence. To investigate coupling of IP₃RII to hTrp1, platelets were stimulated with thrombin for varying times (at 100 ms intervals) and then quenched with a lysis buffer (Rosado & Sage, 2000a) using a rapid quench flow system. Lysed samples were then subjected to immunoprecipitation using either an anti-hTrp1 or anti-IP₃RII antibody and co-immunoprecipitation probed for by Western blotting with the reciprocal antibody following SDS-PAGE (Rosado & Sage, 2000a). Coupling was absent in resting platelet preparations, and samples mixed with agonist-free buffer. All experiments were conducted at 37 °C.

Thrombin at final concentrations of 0.1 or 1 unit ml^-1 evoked Mn²⁺ entry (in the presence of 100 µM Mn²⁺ and 1 mM EGTA) with a delay of 1.36 ± 0.09 or 0.81 ± 0.07 s, respectively (means ± S.E.M., n = 7). At thrombin concentrations of 0.1 or 1.0 unit ml^-1, co-immunoprecipitation of IP₃RII with hTrp1was first detected at 1.4 s (n = 4) or 0.9 s (n = 8), respectively. We have found that thrombin at high concentrations (10 units ml^-1) can evoke Ca²⁺ entry independently of Ca²⁺ store depletion, but dependent on the activation of protein kinase C (PKC; Rosado & Sage, 2000b). To exclude the possibility that this PKC-dependent pathway contributed to the divalent cation entry evoked by 1 unit ml^-1 thrombin, the experiments were repeated using cells with or without a 5 min pre-treatment with the PKC inhibitor Ro-31-8220 (5 µM, a concentration sufficient to block divalent cation entry evoked by 1 µM phorbol myristate acetate). The delay in the onset of Mn²⁺ entry (in the presence of 1 mM extracellular Ca²⁺) evoked by 1 unit ml^-1 thrombin was 0.83 ± 0.02 s in the absence of Ro-31-8220 and 0.85 ± 0.00 s in its presence (n = 10). This difference was not significant (Student’s paired t test, P = 0.39). Coupling of IP₃RII to hTrp1 was first detected at 0.9 s in the presence or absence of Ro-31-8220.

These results indicate that thrombin evokes the coupling of IP₃RII to hTrp1 rapidly enough for this to mediate the activation of SMCE in human platelets.

This work was supported by the Wellcome Trust (064070).