Anticoagulant, antiplatelet and fibrin clot hydrolyzing activities of flax seed buffer extract

Abstract

Background: Flax seeds possess long array of medicinal qualities as it found to show beneficial effects on cardiovascular, hypertension, diabetes, cancer, inflammatory and autoimmune disorders. While, the therapeutic role of flax seed proteins on thrombotic disorder is least explored. Objective: The current study aims to identify the fibrin clot-dissolving, anticoagulant and antiplatelet properties of flaxseed buffer extract (FSBE) using Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP). Materials and Methods: Flax Seed Buffer Extract (FSBE) proteins were characterized upon subjected to SDS-PAGE. The presence of cysteine protease in the extract was identified using colorimeter and zymography experiments. Anticoagulant activity was established using invitro recalcification time. While, the fibrinogenolytic, fibrinolytic and human plasma proteins degradation activities were proved using SDS-PAGE. Furthermore, antiplatelet activity was confirmed using Chronology dual channel whole blood/optical lumi aggregation system (Model-700). Toxicity studies were assayed using RBC cells and albino mice. Results: FSBE showed divergent protein banding pattern from the molecular mass ranging 200 kDa-14 kDa under both reduced and non-reduced conditions. The FSBE showed proteolytic activity as it hydrolyzed casein and gelatin with the specific activity 0.180 and 0.201 units/mg/min respectively. The proteolytic activity of FSBE was completely abolished only by IAA but PMSF, 1, 10 Phenanthroline and EDTA did not, revealing the presence of cysteine protease in the extract. FSBE showed anticoagulant activity as it enhanced the clotting time from control 220s to 320s. Furthermore, FSBE showed antiplatelet activity by inhibiting ADP and Epinephrine induced platelet aggregation and the observed aggregation inhibition was found to be 63.6% and 16.3% respectively. In addition, it hydrolyzed specifically Aα and Bβ chains of human fibrinogen and all the chains of human fibrin clot. The fibrinogenolytic activity was inhibited by IAA but PMSF, EDTA, 1, 10 Phenanthroline did not, suggesting the role of cysteine proteases. Interestingly, FSBE did not cause edema and hemorrhage in the experimental mice and did not hydrolyze RBC cells suggesting its nontoxic properties. Abbreviations used: FSBE: Flaxseed buffer extract; PMSF: Phenylmethylsulfonyl fluoride; IAA: Iodoacetic acid; EDTA: Ethylenediaminetetraacetic acid; PRP: Platelet-rich plasma; PPP: Platelet-poor plasma; INR: International normalized ratio; MED: Minimum edema dose; MHD: Minimum hemorrhagic dose; APTT: Activated partial thromboplastin time; PT: Prothrombin time; HMWK: High-molecular-weight kininogen.