Materials

L-Glutamic acid, CNQX, AICAR, calcium ionophore A23187, CCCP, 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), phalloidin-FITC, EGTA, EDTA, sodium orthovanadate, acetylsalicylic acid, thrombin, fibrinogen, dimethylsulfoxide (DMSO), anti-β-actin antibody (Ab) and Triton X-100, were purchased from Sigma. Fura 2/AM and anti-PAI-1 Ab were acquired from Calbiochem. Calcein-AM was from Life Technologies. Collagen was acquired from Chrono-log. Glycine and reagents for electrophoresis were from Merck. Polyvinylidene fluoride (PVDF) membranes and enhanced chemiluminescence detection kit were from Millipore. Antibodies against phospho(Thr-18/Ser-10)-myosin light chain (pMLC) and phospho(Thr-853)-MLC phosphatase (pMYPT1), phospho(Thr-172)-AMP-activated protein kinase (pAMPK), phospho(ser-79)-acetyl CoA carboxylase (pACC) were procured from Cell Signalling Technology. HIF-2α Ab was obtained from Novus Biochemicals. FITC-labeled PAC-1 and flow cytometry sheath fluid was from BD Biosciences. Sources of other reagents and antibodies used were as follow: Alexa fluor 488-fibrinogen and MitoTracker-Red (Invitrogen), horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Bangalore Genei), and RhoA activation assay Biochem kit (Cytoskeleton). All other reagents were of analytical grade. Type I deionized water (18.2 MΩ⋅cm, Millipore) has been used throughout the experiments. Platelets were isolated from venous blood collected from healthy volunteers under informed consent, strictly as per the recommendations and as approved by the Institutional Ethical Committee of Banaras Hindu University. The study methodologies conformed to the standards set by the Declaration of Helsinki.