Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

IF 1.9 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Current protein & peptide science Pub Date : 2024-01-01 DOI:10.2174/0113892037265109231114065204

Sujatha M Hanumegowda, Chandramma Srinivasa, Ashwini Shivaiah, Manjula M Venkatappa, Rohith L Shankar, Ramesh K Lakshmaiah, Sathisha J Gonchigar, Devaraja Sannaningaiah

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引用次数: 0

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.

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红麻种子半胱氨酸蛋白酶(KSCP)抑制凝血级联和血小板聚集的内在途径。

背景:血栓形成是阻碍血液在整个循环系统中流动的关键事件，可导致中风、心肌梗死和严重的心血管并发症。目前，可用的抗血栓药物会引发几种危及生命的副作用。导论:天然来源的抗血栓药物无不良反应，引起了人们的高度关注。在我们之前的研究中，我们报道了红麻种子蛋白提取物的抗氧化、抗凝血和抗血小板性能。因此，在目前的研究中，从红麻种子蛋白提取物中纯化和表征具有潜在抗血栓活性的半胱氨酸蛋白酶。方法:采用凝胶渗透法和离子交换柱层析法纯化红麻种子半胱氨酸蛋白酶。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS PAGE)对酶的纯度进行鉴定。采用反相高效液相色谱法(RP-HPLC)、基质辅助激光解吸电离飞行时间法(MALDI-TOF)和圆二色技术(CD)对其进行表征。利用光谱学对其进行了蛋白水解、纤维蛋白溶解和动力学研究。采用血浆再钙化时间、凝血酶原时间(PT)、凝血酶凝血时间(TCT)、活化部分凝血活素时间(APTT)、出血时间和血小板聚集研究KSCP的抗血栓活性。结果:在还原和非还原条件下均可观察到KSCP单条尖锐带，分子量为24.1667kDa。发现KSCP含有30.3%的螺旋旋转和69.7%的随机线圈，没有β褶板。KSCP消化酪蛋白和纤维蛋白，其活性受到碘乙酸(IAA)的抑制。KSCP在pH 6.0、温度40℃时活性最佳。KSCP通过干扰血凝级联的内在途径表现出抗凝特性。此外，KSCP溶解全血和血浆凝块和血小板聚集。结论:从红麻籽提取物中纯化的KSCP具有抗血栓作用。因此，它可能是一个更好的候选管理血栓性并发症。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Current protein & peptide science 生物-生化与分子生物学

CiteScore

5.20

自引率

0.00%

发文量

审稿时长

6 months

期刊介绍： Current Protein & Peptide Science publishes full-length/mini review articles on specific aspects involving proteins, peptides, and interactions between the enzymes, the binding interactions of hormones and their receptors; the properties of transcription factors and other molecules that regulate gene expression; the reactions leading to the immune response; the process of signal transduction; the structure and function of proteins involved in the cytoskeleton and molecular motors; the properties of membrane channels and transporters; and the generation and storage of metabolic energy. In addition, reviews of experimental studies of protein folding and design are given special emphasis. Manuscripts submitted to Current Protein and Peptide Science should cover a field by discussing research from the leading laboratories in a field and should pose questions for future studies. Original papers, research articles and letter articles/short communications are not considered for publication in Current Protein & Peptide Science.