TRPing out platelet calcium – TRPM7 modulates calcium mobilization and platelet function via PLC interactions

Originally identified as coagulation factor IV, calcium (Ca²⁺) is now well established as a key cofactor in the formation of the tenase and prothrombinase complexes on the extracellular surfaces of activated platelets to ultimately mediate fibrin generation and hemostasis.¹ Similarly, Ca²⁺ has long been known to serve an important intracellular role in orchestrating the cell biological responses of platelets in hemostatic plug formation.^2,3,4,5 Over the past several decades, as biochemical efforts have identified and refined roles for Ca²⁺ as an essential second messenger in virtually all cells,³ complementary studies of platelets have similarly detailed how spatiotemporal changes in intracellular Ca²⁺ levels regulate platelet granule secretion, cytoskeletal dynamics, aggregation and other cell biological outputs underying platelet physiology. On a general mechanistic level, changes in intracellular Ca²⁺ concentrations that trigger the cellular responses driving platelet function are solicited downstream of a variety of receptors that differentially activate phospholipase C (PLC) family members, resulting in inositol-1,4,5-triphosphate (IP₃) production and IP₃ receptor (IP₃R)-mediated release of Ca²⁺ from intracellular stores (Figure 1).^4,6,7,8,9

Figure 1. TRPM7 kinase domain interacts with PLC family members to regulate intracellular Ca²⁺ responses and platelet function (model).

Platelet activation downstream of GPVI, CLEC-2, PARs and other receptors results in intracellular signaling events that upregulate phospholipase C (PLCγ2, PLCβ3) phosphorylation and activity to drive the metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) on the cytosolic face of the platelet plasma membrane to produce IP₃ and diacylglycerol (DAG). Rather than serving as a constitutively active Mg²⁺/Ca²⁺ channel or regulating DAG-mediated receptor-operated calcium entry (ROCE) through TRP channels (TRPC) and other associated processes, this study supports a model whereby the TRPM7 kinase domain interacts with PLC family members to modulate PLC phosphorylation and activation, IP₃ production, intracellular Ca²⁺ mobilization and STIM1-Orai1-mediated store operated calcium entry (SOCE), ultimately modulating intracellular Ca²⁺ concentrations and associated Ca²⁺ responses underlying platelet function.¹⁰

Given the critical importance of intracellular Ca²⁺ concentration to platelet function, diverse endeavors have aimed to understand and target the myriad of molecular processes regulating and driven by Ca²⁺ dynamics. In addition to mobilization from internal stores following IP₃ generation, extracellular Ca²⁺ also enters platelets via store- and receptor-operated calcium entry (SOCE and ROCE, respectively) routes involving more recently described players such as Orai1, STIM1 and transient receptor potential (TRP) family channels.³ In this issue of ATVB, Gotru et al. now uncover a novel mechanism by which the TRP subfamily member transient receptor potential melastatin-like 7 (TRPM7) modulates PLC phosphorylation and intracellular calcium mobilization to effect SOCE and platelet function.¹⁰ By taking advantage of a transgenic mouse model with a loss-of-function mutation in the cytosolic TRPM7 C-terminal serine/threonine kinase domain (Trpm7^R/R),¹¹ this study specifies a role for the TRPM7 kinase domain–rather than its constitutive Mg²⁺ and Ca²⁺ channel activity–in modulating the phosphorylation and activation of PLC family members and consequently intracellular calcium responses and platelet functional processes underlying both hemostasis and thrombosis (Figure 1).

Earlier work also from the Braun group suggested a general role for TRPM7 in regulating platelet function through pathways controlling megakaryocyte and platelet cytoskeletal dynamics.¹² In the current study, Gotru et al. now more specifically show altered signaling responses in Trpm7^R/R platelets, including a delay in phosphorylation of Syk, LAT, PLCγ2 and PKCε in response to the platelet GPVI receptor agonist collagen-related peptide (CRP).¹⁰ Similar effects are also found to be associated with rhodocytin→CLEC-2→PLCγ2 as well as thrombin→PAR→PLCβ3 signaling axes. To investigate the physiological consequences associated with these alterations in signaling kinetics in platelets, Gotru et al. also analyze a number of platelet phenotypes and functional responses in Trpm7^R/R mice, revealing defects in granule secretion, integrin activation and platelet aggregation in vitro. In association with these altered platelet responses, Trpm7^R/R mice display prolonged tail bleeding times, as well as significantly less vessel occlusion in response to arteriole injury as compared to wild-type (WT) counterparts, supporting roles for the TRPM7 kinase domain in modulating platelet function in vivo. Perhaps most remarkably, antithrombotic effects of mutating the TRPM7 kinase domain appear to be mediated through marrow derived cells, especially platelets, as WT chimeric mice transplanted with bone marrow from Trpm7^R/R mice, or thrombocytopenic WT mice transfused with platelets from Trpm7^R/R animals were similarly protected from infarct progression and showed overall improved neurological and motor function outcomes relative to matched controls following transient middle cerebral artery occlusion (tMCAO) to model ischemic stroke.

While Gotru et al. have astutely elucidated platelet-associated roles for the TRPM7 kinase domain in complete and proper platelet function, a number of intriguing questions remain regarding how TRPM7 impacts platelet physiology in hemostasis and thrombosis. For instance, it is not clear how mutation of this serine/threonine kinase domain translates into a delay in the tyrosine phosphorylation of receptor-proximal components of platelet signaling systems, especially as physiological substrates of TRPM7 are very limited and have not been yet demonstrated in platelets. Furthermore, these findings remain to be placed into the context of the Braun group’s former studies that made use of megakaryocyte/platelet-lineage specific deletions of TRPM7 that resulted in altered cytoskeletal phenotypes.¹² Along these lines, as platelets from Trpm7^R/R mice exhibit delayed PKC phosphorylation kinetics, a number of other signaling processes around the platelet cytoskeleton may be disrupted, especially given the spatial and temporal link between intracellular calcium mobilization and PKC activation in regulating Rho GTPases and platelet function.^13,14 Finally, from a translational perspective, the rather unique TRPM7 kinase domain may represent a specialized therapeutic or biomarker target; however, validation of the relevance of TRPM7 within human platelet biology remains to be shown. Regardless, whether TRPM7 serves as a kinase, a molecular scaffold, an effector, or some combination thereof, Gotru et al. provide a rich contribution to the ever-evolving and increasingly important understanding of mechanisms regulating intracellular Ca²⁺ dynamics in health and disease in platelet physiology and beyond.