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Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-03-31 , DOI: 10.1002/bab.1912 Timsy Bhando 1 , Satish Singh 1 , Mangesh Dattu Hade 1 , Jagdeep Kaur 2 , Kanak L Dikshit 1, 2
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-03-31 , DOI: 10.1002/bab.1912 Timsy Bhando 1 , Satish Singh 1 , Mangesh Dattu Hade 1 , Jagdeep Kaur 2 , Kanak L Dikshit 1, 2
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Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α‐2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK‐30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C‐terminal end of SAK with a RGD (Arg–Gly–Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK‐30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat/Km, of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2‐ antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin‐specific manner and its ability to clear platelet‐rich plasma clot faster than SAK.
中文翻译：
VEK-30肽的整合增强了葡萄激酶激酶的纤溶特性
葡激酶（SAK）是一种具有纤溶蛋白特性的136个氨基酸的细菌蛋白，由于其纤维蛋白特异性和减少的α-2抗纤溶酶抑制作用，已成为一种重要的溶栓剂。为了增强SAK的凝块溶解能力，将源自纤溶酶原（Pg）结合蛋白（PAM）的30个氨基酸的肽（VEK-30）与RGD（Arg融合）融合在SAK的C末端。 –Gly–Asp）链接器。嵌合蛋白SAKVEK在大肠杆菌中表达，并纯化为可溶性蛋白。SAKVEK和SAK与纤溶酶的等摩尔复合物激活Pg表明，VEK-30肽的融合显着增强了SAK的催化活性。动力学常数，k cat / K m底物Pg的SAKVEK的出现时间比SAK高2.7倍，血纤蛋白和富含血小板的血块溶解所需的时间分别缩短了30％和50％。SAKVEK与纤溶酶的二元激活复合物被α2-抗纤溶酶抑制，但在纤维蛋白存在下仍受保护，这与SAK非常相似。因此，本研究表明，SAKVEK作为血栓溶解剂更有效，因为它以血纤蛋白特异的方式对Pg活化具有更高的催化活性，并且具有比SAK更快的清除富含血小板的血凝块的能力。
更新日期：2020-03-31
中文翻译：
VEK-30肽的整合增强了葡萄激酶激酶的纤溶特性
葡激酶（SAK）是一种具有纤溶蛋白特性的136个氨基酸的细菌蛋白，由于其纤维蛋白特异性和减少的α-2抗纤溶酶抑制作用，已成为一种重要的溶栓剂。为了增强SAK的凝块溶解能力，将源自纤溶酶原（Pg）结合蛋白（PAM）的30个氨基酸的肽（VEK-30）与RGD（Arg融合）融合在SAK的C末端。 –Gly–Asp）链接器。嵌合蛋白SAKVEK在大肠杆菌中表达，并纯化为可溶性蛋白。SAKVEK和SAK与纤溶酶的等摩尔复合物激活Pg表明，VEK-30肽的融合显着增强了SAK的催化活性。动力学常数，k cat / K m底物Pg的SAKVEK的出现时间比SAK高2.7倍，血纤蛋白和富含血小板的血块溶解所需的时间分别缩短了30％和50％。SAKVEK与纤溶酶的二元激活复合物被α2-抗纤溶酶抑制，但在纤维蛋白存在下仍受保护，这与SAK非常相似。因此，本研究表明，SAKVEK作为血栓溶解剂更有效，因为它以血纤蛋白特异的方式对Pg活化具有更高的催化活性，并且具有比SAK更快的清除富含血小板的血凝块的能力。
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