BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

【摘要】
研究背景：蛇毒是一类由蛋白质、有机与无机化合物组成的复杂混合物。其中部分酶类或非酶类蛋白质可与血小板受体结合，引发止血功能紊乱。具有抗血小板活性的毒素所具备的潜在治疗价值，有望引起药理学领域的关注。本研究旨在从交替矛头蝮（Bothrops alternatus）蛇毒中纯化并鉴定一种抗血小板DC蛋白。
研究方法：该蛋白命名为BaltDC（即来自B. alternatus蛇毒的DC蛋白），通过DEAE-Sephacel柱离子交换层析与Sephadex G-75凝胶过滤联用的方式完成纯化。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估算其表观分子量；采用埃德曼降解法（Edman degradation）测定该蛋白的N端氨基酸序列；以人富血小板血浆（PRP）为实验样本开展血小板聚集检测；采用红外光谱（IR）解析BaltDC与血小板膜之间的相互作用模式。
研究结果：SDS-PAGE检测显示，BaltDC呈现单一条蛋白带，在还原与非还原条件下的表观分子量均为32 kDa。该蛋白的N端氨基酸序列为IISPPVCGNELLEVGEECDCGTPENCQNECCDA，与其他蛇毒金属蛋白酶（SVMPs）具有序列同源性。BaltDC不具备蛋白水解、出血、去纤维蛋白原及凝血活性，但可特异性抑制人富血小板血浆中由瑞斯西丁素（ristocetin）与肾上腺素（epinephrine）诱导的血小板聚集。红外光谱分析结果强烈表明，BaltDC所含的PO₃²⁻基团可与血小板膜非脂质组分中的PO₂⁻基团形成氢键。
研究结论：BaltDC可有效抑制血小板聚集，因此具备潜在的医学研究与应用价值。