Title:Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of
the Blood Coagulation Cascade and Platelet Aggregation
Volume: 25
Issue: 5
Author(s): Sujatha M. Hanumegowda, Chandramma Srinivasa, Ashwini Shivaiah, Manjula M. Venkatappa, Rohith L. Shankar, Ramesh K. Lakshmaiah, Sathisha J. Gonchigar and Devaraja Sannaningaiah*
Affiliation:
- Department of Studies and Research in Biochemistry, Tumkur University-572102, Tumkur, Karnataka, India
Keywords:
Anticoagulant, antithrombotic, clot hydrolysis, electrophoresis, kenaf seed cysteine protease, platelet aggregation.
Abstract:
Background: Thrombosis is the key event that obstructs the flow of blood throughout
the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications.
Currently, available antithrombotic drugs trigger several life-threatening side effects.
Introduction: Antithrombotic agents from natural sources devoid of adverse effects are grabbing
high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet
properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization
of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic
activity was undertaken.
Methods : Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation
and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS
PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase
High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption
Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization.
Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification
time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin
Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic
activity of KSCP.
Result : A single sharp band of KSCP was observed under both reduced and non-reduced conditions,
having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and
69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity
was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature
of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the
blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and
platelet aggregation.
Conclusion : KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it
could be a better candidate for the management of thrombotic complications.
