Liu C Z, Huang T F
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Arch Biochem Biophys. 1997 Jan 15;337(2):291-9. doi: 10.1006/abbi.1996.9787.
By means of liquid chromatography consisting of gel filtration, anionic exchange, and C8 reverse-phase HPLC, a selective inhibitor of collagen-induced platelet aggregation, named crovidisin, has been purified to homogeneity from the venom of Crotalus viridis snake. Crovidisin is a single-chain, 53-kDa protein with a selective inhibitory activity on collagen-induced aggregation of human washed platelets without affecting those elicited by thrombin, sodium arachidonate, and ADP. Partial sequencing of tryptic digests of crovidisin reveals that partial sequence of crovidisin appears to be identical to that of catrocollastatin, a collagen antagonist occurring in the venom of Crotalus atrox snake. Crovidisin dose-dependently blocked aggregation of human washed platelets triggered by 5 and 10 microg/ml of collagen with IC50 of 0.17 and 0.47 microM, respectively. Not only platelet aggregation but also release reaction, thromboxane formation, and increase of intracellular Ca2+ level of platelets in response to collagen were all completely abolished by crovidisin. In the presence of crovidisin, the Mg2+-dependent adhesion of platelets to collagen was diminished in a dose-dependent manner, while the glycoprotein IIb/IIIa-mediated platelet-fibrinogen interaction was unaffected. When collagen was pretreated with crovidisin and followed by three washes with phosphate-buffered saline, the antiadhesion activity of crovidisin was unaffected. In addition, collagen fibers emitted fluorescence after incubation with fluorescein isothiocyanate-conjugated crovidisin, indicating that crovidisin binds directly to collagen fibers. In conclusion, crovidisin blocks the interaction between platelets and collagen fibers through its binding to collagen fibers, resulting in the blockade of collagen-mediated platelet functions such as adhesion, release reaction, thromboxane formation, and aggregation.
通过由凝胶过滤、阴离子交换和C8反相高效液相色谱组成的液相色谱法，从绿曼巴蛇的毒液中纯化出一种名为crovidisin的胶原诱导血小板聚集的选择性抑制剂，并使其达到同质。Crovidisin是一种单链53 kDa蛋白，对人洗涤血小板的胶原诱导聚集具有选择性抑制活性，而不影响凝血酶、花生四烯酸钠和ADP诱导的聚集。Crovidisin胰蛋白酶消化产物的部分测序表明，crovidisin的部分序列似乎与atrocollastatin相同，atrocollastatin是一种存在于墨西哥矛头蝮蛇毒液中的胶原拮抗剂。Crovidisin剂量依赖性地阻断了5和10 μg/ml胶原触发的人洗涤血小板的聚集，IC50分别为0.17和0.47 μM。Crovidisin不仅完全消除了血小板聚集，还消除了释放反应、血栓素形成以及血小板对胶原的细胞内Ca2+水平升高。在存在crovidisin的情况下，血小板与胶原的Mg2+依赖性粘附以剂量依赖性方式减少，而糖蛋白IIb/IIIa介导的血小板-纤维蛋白原相互作用不受影响。当胶原用crovidisin预处理，然后用磷酸盐缓冲盐水洗涤三次时，crovidisin的抗粘附活性不受影响。此外，胶原纤维在与异硫氰酸荧光素偶联的crovidisin孵育后发出荧光，表明crovidisin直接与胶原纤维结合。总之，crovidisin通过与胶原纤维结合来阻断血小板与胶原纤维之间的相互作用，从而导致胶原介导的血小板功能如粘附、释放反应、血栓素形成和聚集的阻断。
