Adam J. Wieschhaus

Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

Background: Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Results and conclusion: Platelets from patient (Scott) and mouse (Capn1−/− and Ppif−/−) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. Significance: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 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 αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca2+, stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 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 αIIbβ3 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 αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 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.

Background: Calpains play an important role in the regulation of cell death. Results: Calpain-1 inhibition decreases cortical neurodegeneration following TBI by regulating calcium influx and apoptosis of neurons under oxidative stress. Conclusion: Genetic inhibition of calpain-1 reduces neurodegeneration and suppresses neuronal apoptosis. Significance: Targeted inhibition of calpain-1 offers a promising therapeutic approach against TBI and other neurodegenerative diseases. 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.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD‐related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain‐1 in favor of activator in dense sickle cells. Calpain‐1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain‐1, BDA‐410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA‐410 improved RBC morphology, reduced RBC density (D20; from 1106±0.001 to 1100±0.001 g/ml; P<0.05) and increased RBC‐K+ content (from 364±10 to 429±12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca2+‐activated K+channel (Gardos channel), and decreased membrane association of peroxiredoxin‐2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA‐410 treatment, which suggests that calpain‐1 inhibition affects the PKC‐dependent fraction of the Gardos channel. BDA‐410 prevented hypoxia‐induced RBC dehydration and K+ loss in SAD mice. These data suggest a potential role of BDA‐410 as a novel therapeutic agent for treatment of SCD.—De Franceschi, L., Franco, R. S., Bertoldi, M., Brugnara, C., Matté, A., Siciliano, A., Wieschhaus, A. J., Chishti, A. H., Joiner, C. H. Pharmacological inhibition of calpain‐1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J. 27, 750–759 (2013). www.fasebj.org

Targeted gene inactivation reveals a functional role of calpain‐1 in platelet spreading

Summary.  Background:  Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets.

Calpain-1 Contributes to IgE-Mediated Mast Cell Activation

Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. In this study, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow–derived mast cells enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null bone marrow–derived mast cells showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1 deficiency led to specific attenuation of IκB–NF-κB pathway and IKK–SNAP23 pathway, whereas calcium flux, MAPK, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematins actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.

Headpiece domain of dematin regulates calcium mobilization and signaling in platelets

Background: Dematin is a major component of the erythrocyte membrane. Its abundance in platelets suggests a conserved function. Results: Dematin regulates calcium mobilization in platelets. Mice lacking the dematin headpiece domain show multiple defects in platelet function. Conclusion: Dematin emerges as a new regulator of calcium mobilization in platelets. Significance: Inhibition of dematin interactions may offer new therapeutic approaches against platelet activation and cardiovascular diseases. 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, dematins 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.