STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets

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Abstract

Ca2+ entry, particularly store-operated Ca2+ entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca2+ content of the stores, where the intraluminal Ca2+ sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca2+ stores to the store-operated Ca2+-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25–139) antibody, directed towards the Ca2+-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288–301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557–571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca2+ entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

Introduction

Cytosolic free Ca2+ concentration ([Ca2+]c) plays a key role in a wide variety of cellular functions. Agonists increase [Ca2+]c either by stimulation of Ca2+ release from intracellular Ca2+ stores or inducing Ca2+ entry through plasma membrane Ca2+-permeable channels. Conversely, there is a fine machinery to remove Ca2+ from the cytosol, including Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA)1 and the Na+/Ca2+ exchanger, and Ca2+ sequestration into agonist-releasable compartments by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) or mitochondria [1]. Agonist-evoked Ca2+ release from finite intracellular stores is sometimes insufficient to induce full activation of certain cellular process and extracellular Ca2+ entry is required. Among the mechanisms for Ca2+ influx, store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the intracellular Ca2+ stores, is a major event in non-excitable cells [2]. The STromal Interaction Molecule 1 (STIM1), a transmembrane protein mostly located in the membrane of the Ca2+ stores, although it has also been found in the plasma membrane [3], [4], [5], has recently been identified as the intraluminal Ca2+ sensor that communicates the amount of stored Ca2+ to the plasma membrane channels [6], [7], [8], [9], [10]. Although the nature of the channels that mediate SOCE has been a matter of intense investigation and debate for over the last decades, the Ca2+-permeable channel protein Orai1 (also named CRACM1 for CRAC modulator) and the transient receptor potential (TRP) channels have been presented as candidates to conduct Ca2+ entry through this mechanism [7], [8], [11], [12], [13], [14].

Human platelets are anucleated and non-excitable blood cells that are known to undergo marked and rapid shape changes and cellular aggregation upon agonist stimulation. Platelet aggregation at sites of vascular injury is a complex mechanism that requires the activation of a number of intracellular signaling events, including protein phosphorylation, cytoskeletal rearrangement and Ca2+ mobilization [15], [16], [17]. In human platelets STIM1 interacts with endogenously expressed canonical human transient receptor potential 1 (hTRPC1) protein and Orai1 after store depletion and agonist stimulation [4], [14]. In these cells, STIM1 has been reported to be present both in the internal Ca2+ stores, where, in addition, it regulates the function of Ca2+-handling proteins such as SERCA3 [18], and the plasma membrane, with enhanced expression in the plasma membrane upon agonist stimulation [4]. Since intracellular Ca2+ homeostasis, and Ca2+ entry, has been reported to be relevant for platelet aggregation, we have investigated the functional role of the intraluminal Ca2+ sensor STIM1 and the store-operated Ca2+ channels Orai1 and hTRPC1 in the regulation of agonist-induced platelet aggregation, which might shed new light on the molecular basis underlying thrombotic disorders associated to platelet Ca2+ homeostasis.

Section snippets

Materials

Fura-2/AM was from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), thapsigargin (TG), rabbit anti-Orai1 antibody (288–301) and thrombin were from Sigma (Madrid, Spain). Mouse anti-STIM1 antibody (25–139) was from BD Transduction Laboratories (Frankin Lakes, NJ, USA). Rabbit anti-hTRPC1 polyclonal antibody (557–571) was obtained from Alomone Laboratories (Jerusalem, Israel). Horseradish peroxidase-conjugated goat

Role of intra and extracellular Ca2+ on agonist-induced platelet aggregation

In a medium containing 1 mM Ca2+, treatment of human platelets with 0.5 U/mL thrombin-induced rapid aggregation characterized by an initial and rapid change in cell shape, indicated by a small decrease in light transmission, followed by a large increase in light transmission as platelets aggregate (Fig. 1A and Table 1; n = 6). Similarly, stimulation with 10 μM ADP-induced platelet aggregation, although the extent of the changes in light transmission was smaller than those observed in

Discussion

It is now well established that Ca2+ entry, and particularly SOCE, plays an important role in a variety of cellular events, from refilling of the internal stores and the maintenance of Ca2+ oscillations [35] to the activation of distinct cell functions. Concerning the immune system, a mutation in Orai1 has been reported as responsible for the hereditary severe combined immune deficiency (SCID) syndrome [11], and STIM1 has been shown to be important for Fcgamma receptors activation on

Acknowledgments

Supported by M.E.C.–FEDER (BFU2007-60104/BFI), Fundesalud-Junta de Extremadura (PRI08A003) and Ministerio de Asuntos Exteriores y Cooperación (A/016208/08). We thank Dr. Hernández-Cruz for blood samples and staff training and Dr. Gómez-Arteta for helpful discussion.

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