Increased calpain activity after hypoxia exposure in rats and human DVT patients, and the antithrombotic effect of calpain inhibition in vivo and ex vivo. Platelets were isolated from indicated groups and processed for either florescence-based calpain activity or intracellular calcium assays, as described in the supplemental Methods. (A) Quantitation of calpain activity in platelets demonstrating higher proteolytic activity of calpain in exposed animals. (B) Intraplatelet calcium levels measured by Fura-2 based fluorogenic assay showed increased platelet iCa²⁺ in exposed animals. (C-D) Representative aggregation curves showing reversal of hypoxia-induced platelet aggregation by preincubation of PRP with calpain inhibitor PD150606 (50 µM) ex vivo (C) and by preinfusion of PD150606 (1 mg/kg body weight) in vivo (D). (E-I) The antithrombotic and platelet inhibitory effects of preinfusion of PD150606 via tail vein in rat model of stasis-induced thrombosis, as described in the supplemental Methods. (E) Representative images of extracted portions of IVC with thrombus (top) from thrombotic animals with their heat maps (bottom), showing smaller thrombus in the case of PD150606 preinfusion. (F) Quantitations of the size of the thrombus isolated from the IVC portions of thrombotic animals (n = 8). (G) Decreased calpain activity in platelets isolated from thrombotic animals preinfused with PD150606. (H) Representative platelet aggregation curves showing a strong negative effect of PD150606 preinfusion in thrombotic rats compared with vehicle control or no infusion. Data are presented as mean ± SEM (n = 8) and analyzed by unpaired t test. *P < .05, **P < .01 vs thrombotic + vehicle. (I) Hematoxylin-eosin-stained sections of thrombus with vessel wall, showing morphological differences (×200 original magnification). (J-K) The plasma samples from human DVT patients from high-altitude regions and from control individuals were analyzed for soluble P selectin levels and calpain activity, (J) Higher sP-selectin levels in plasma samples (data presented as box and whiskers plot) indicated hyperactive platelets in high-altitude-induced DVT patients. (K) Calpain activity in human plasma samples of high-altitude-induced DVT patients compared with age- and sex-matched healthy controls, shown as a scatter plot (n = 10). The activity in patients was significantly (P = .0014) higher in comparison with controls. Data are presented as mean ± SEM (n = 10 in each group) and analyzed by t-test compared with respective controls.