Excessive cytosolic calcium ion (Ca²⁺) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca²⁺ entry (CCE) is a process whereby depletion of intracellular Ca²⁺ stores causes the activation of plasma membrane Ca²⁺ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca²⁺ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca²⁺ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca²⁺ accumulation in neurons. Neurons from Stim2^−/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2^−/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, α_IIbβ₃ integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate–binding protein or immunoreceptor tyrosine-based activation motif–coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1^−/− mice displayed impaired α_IIbβ₃ integrin activation in response to major agonists and defective glycoprotein Ib–dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.