Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease

Abstract

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5’-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in the hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in the steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa/αIIbβ3 integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice

“…Similar to other studies of PH in Townes or Berkeley SS mice, 38,43,44 Figure 4. Three weeks of SMC CYB5R3 knockdown has no effect on cardiopulmonary hemodynamics or cardiac morphology in AS chimeras.…”

Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease

“…Previously, Nwankwo et al reported lower platelet counts in SS mice under normoxia than in healthy (AA) controls. 40 Here, we found that, compared to treatment with vehicle, rADAMTS13 treatment ameliorated the H/R-induced thrombocytopenia in SS mice (Figure 1E). Since a functional connection between platelets and ADAMTS13 activity has been reported in other models of inflammatory vasculopathy associated with H/R stress, we conducted preliminary experiments of platelet adhesion to immobilized collagen in a perfusion chamber with or without rADAMTS13.…”

Innate immune receptors in platelets and platelet-leukocyte interactions

“…Platelet activation is elevated in patients under steady‐state conditions and can be further enhanced during acute vaso‐occlusive crisis 212 . High platelet activation is mainly related with the hemolysis of sickle RBCs that release classical platelet agonists as ADP and ATP and erythrocyte‐derived DAMPs such as hemoglobin, which blocks the inhibitory effects of NO 87,212,213 . In addition, free heme is a known DAMP that activates TLR4 in nucleated cells, 214 but the role of heme in platelet TLR4 activation remains unknown.…”

“…212 High platelet activation is mainly related with the hemolysis of sickle RBCs that release classical platelet agonists as ADP and ATP and erythrocyte-derived DAMPs such as hemoglobin, which blocks the inhibitory effects of NO. 87,212,213 In addition, free heme is a known DAMP that activates TLR4 in nucleated cells, 214 but the role of heme in platelet TLR4 activation remains unknown. SCD patients exhibit high levels of tissue factor, thrombin, and fibrin degradation products, characterizing an hypercoagulability state.…”

“…[215][216][217][218] Thrombin activates protease-activated receptors (PAR) in different cell types, including platelets, inducing inflammatory cytokines and platelet-leukocyte aggregation in SCD. [219][220][221] Platelets from patients or animal models of SCD are more sensitive to PAR4 agonists, 212,213 and PAR4 hyperstimulation is enhanced in patients presenting pulmonary hypertension. 212 Activated platelets promote sickle RBC adhesion to endothelium and multicellular aggregation with leukocytes, supporting vascular occlusion, pulmonary hypertension, and lung injury.…”