Siritapetawee Jaruwan, Talabnin Chutima, Vanichtanankul Jarunee, Songsiriritthigul Chomphunuch, Thumanu Kanjana, Chen Chun-Jung, Komanasin Nantarat
School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Khlong Luang, Pathum Thani, Thailand.
Biotechnol Appl Biochem. 2017 Nov;64(6):862-870. doi: 10.1002/bab.1555. Epub 2017 May 11.
In this study, the binding of a glycosylated serine protease (EuP-82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP-82 to fibrinogen in the conditions with or without the activator (Ca ) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen-EuP-82 in the condition with the inhibitor (Zn ) was an unfavorable endothermic reaction. EuP-82 could not inhibit the platelet activity in citrated whole blood via the ADP-receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP-82 provided multiple fibrinogen-binding sites that were not related to the arginine-glycine-aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP-82 had neither thrombin-like activity nor anticoagulant activity. The SR-FTIR spectra revealed that EuP-82 was a glycoprotein. Deglycosylation of EuP-82 did not affect its proteolytic activity. Moreover, EuP-82 did not exhibit any toxicity to the living cells (NIH-3T3). This study supports that EuP-82 may be useful for wound-healing material through stabilizing the clot via the platelet induction for the first process.
在本研究中，采用等温滴定量热法（ITC）研究了一种糖基化丝氨酸蛋白酶（EuP-82）与人纤维蛋白原的结合情况。ITC分析表明，在有或无激活剂（Ca）的条件下，EuP-82与纤维蛋白原的结合是一个放热反应（主导负焓），该反应倾向于由氢键和范德华相互作用驱动。相比之下，在有抑制剂（Zn）的条件下，纤维蛋白原-EuP-82的结合是一个不利的吸热反应。EuP-82不能通过ADP受体途径（主要是P2Y1和P2Y12）抑制枸橼酸化全血中的血小板活性，但它可以增强血小板聚集。ITC结合全血血小板聚集表明，EuP-82提供了多个与纤维蛋白原的精氨酸-甘氨酸-天冬氨酸（RGD）和十二肽序列无关的纤维蛋白原结合位点。此外，EuP-82既没有类凝血酶活性也没有抗凝活性。SR-FTIR光谱显示EuP-82是一种糖蛋白。EuP-82的去糖基化不影响其蛋白水解活性。此外，EuP-82对活细胞（NIH-3T3）没有任何毒性。本研究支持EuP-82可能通过在第一个过程中通过血小板诱导稳定血凝块而对伤口愈合材料有用。
