The current study explores the anticoagulant and fibrin clot-hydrolyzing properties of Momordica charantia seed extract (MCSE). MCSE hydrolyzed casein with the specific activity of 0.780 units/mg per min. Interestingly, it enhanced the clot formation process of citrated human plasma from control 146 to 432 s. In addition, the intravenous injection of MCSE significantly prolonged the bleeding time in a dose-dependent manner from control 150 to more than 800 s, and strengthened its anticoagulant activity. Interestingly, MCSE specifically prolonged the clotting time of only activated partial thromboplastin time, but not prothrombin time, and revealed the participation of MCSE in the intrinsic pathway of the blood coagulation cascade. Furthermore, MCSE completely hydrolyzed both Aα and Bβ chains of the human fibrinogen and partially hydrolyzed the γ chain. However, it hydrolyzed all the chains (α polymer, α chain, β chain and γ-γ dimmers) of partially cross-linked human fibrin clot. The proteolytic activity followed by the anticoagulant effect of the MCSE was completely abolished by the 1,10-phenanthroline and phenyl methyl sulphonyl fluoride, but iodoacetic acid, EDTA, and ethylene glycol-N,N,N',N'-tetra acetic acid did not. Curiously, MCSE did not hydrolyze any other plasma proteins except the plasma fibrinogen. Moreover, MCSE was devoid of RBC lysis, edema and hemorrhagic properties, suggesting its nontoxic nature. Taken together, MCSE may be a valuable candidate in the treatment of blood clot/thrombotic disorders.

Objective: The current study assesses the fibrinogen and fibrin clot hydrolyzing activities of aqueous seed extract of Jackfruit (AqSEJ). Methods: The protein banding pattern of AqSEJ (100 μg) was analyzed on SDS-PAGE. The proteolytic activity of AqSEJ was confirmed by spectrophotometer and zymography experiments. Fibrinogen, fibrin and plasma protein hydrolyzing activities of AqSEJ were analyzed on SDS-PAGE under reduced conditions. Plasminogen activation and indirect hemolytic activities was analyzed using spectrophotometer. The non-toxic property of AqSEJ was tested by edema, hemorrhage in experimental mice. Results: AqSEJ exhibited proteolytic activity and the specific activity was found to be 1.04 units/mg/min. Furthermore, AqSEJ non-specifically hydrolyzed Aα, followed by Bβ and γ chains of human fibrinogen and specifically hydrolyzed α polymer and α chain of partially cross linked human fibrin clot without affecting β chain and γ-γ dimer even up to the tested dose of 30 μg for the incubation period of 8 hours. Importantly, AqSEJ did not hydrolyze other plasma proteins and devoid of plasminogen activation property. The proteolytic activity of AqSEJ was completely neutralized by PMSF and IAA, while EDTA, EGTA, 1,10-Phenanthroline did not, suggesting the presence of serine and cysteine family proteases. Moreover, AqSEJ did not cause edema and hemorrhage in experimental mice up to the tested dose of 200 μg and non-toxic to RBC cells. Conclusion: AqSEJ hydrolyzes fibrinogen and fibrin clot and non-toxic in nature. Hence, this work showcases the potential applications of Jack fruit seed proteases in the treatment of thrombotic disorders.