Dematin Regulates Calcium Mobilization and Signaling in Platelets

Dematin is a broadly expressed membrane cytoskeletal protein that has been well characterized in erythrocytes and to a lesser extent in non-erythroid cells. However, dematin’s function in platelets is not known. Here, we show that dematin is abundantly expressed in both human and mouse platelets. Platelets harvested from the dematin headpiece knock-out (HPKO) mouse model exhibit a striking defect in the mobilization of calcium in response to multiple agonists of platelet activation. The reduced calcium mobilization in HPKO platelets is associated with concomitant inhibition of platelet aggregation and granule secretion. Integrin αIIBβ3 activation in response to agonists is attenuated in the HPKO platelets. The mutant platelets show nearly normal spreading on fibrinogen and an unaltered basal cAMP level; however, the clot retraction was compromised in the mutant mice. Immunofluorescence analysis indicated that dematin is present both at the dense tubular system and plasma membrane fractions of platelets. Proteomic analysis of dematin-associated proteins in human platelets identified inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) as a binding partner, which was confirmed by immunoprecipitation analysis. IP3KB, a dense tubular system protein, is a major regulator of calcium homeostasis. Loss of the dematin headpiece resulted in a decrease of IP3KB at the membrane and increased levels of IP3KB in the cytosol. Collectively, these findings unveil dematin as a novel regulator of internal calcium mobilization in platelets affecting multiple signaling and cytoskeletal functions. Implications of a conserved role of dematin in the regulation of calcium homeostasis in other cell types will be discussed

Dual Mechanism of Integrin alpha(IIb)beta(3) Closure in Procoagulant Platelets

Aggregation of platelets via activated integrin α(IIb)β(3) is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that α(IIb)β(3) inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β(3) chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed α(IIb)β(3) inactivation. Integrin α(IIb)β(3) inactivation was unchanged in platelets from Capn1(−/−) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced α(IIb)β(3) inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(−/−) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and α(IIb)β(3) inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of α(IIb)β(3) inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation

Targeted Gene Inactivation of Calpain-1 Suppresses Cortical Degeneration Due to Traumatic Brain Injury and Neuronal Apoptosis Induced by Oxidative Stress*

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role