Berndt M C, Phillips D R
J Biol Chem. 1981 Jan 10;256(1):59-65.
Treatment of human platelets with alpha-thrombin leads to the selective hydrolysis of only one membrane protein, glycoprotein V. To determine whether glycoprotein V was directly cleaved by alpha-thrombin and to permit further characterization of this glycoprotein as the potential functional thrombin receptor, glycoprotein V was purified to > 98% homogeneity. Washed platelets were prepared from concentrates within 18 h of venipuncture, since clinically expired platelets (> 72 h from venipuncture) were shown to contain little or no detectable glycoprotein V. Glycoprotein V was eluted from the platelet membrane by equilibrating the platelets (4 X 10(9)/ml) at 37 degrees C for 24 h in 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.6 (1 mM in EDTA, 0.3 M in NaCl). Purification was achieved by initially performing ammonium sulfate fractionation, followed by chromatography on Sephacryl S-200, hydroxylapatite, and DEAE- and CM-cellulose, and yielded 0.5 to 1.0 mg of purified glycoprotein per 100 units of platelet concentrate (approximately 6 X 10(12) platelets). Purified glycoprotein V (Mr = 82,000) was a thrombin substrate, and on hydrolysis yielded a major fragment, GPVf1 (Mr = 69,500), identical in molecular weight with that observed previously in the supernatant of thrombin-treated, periodate-labeled platelets. Glycoprotein V existed as at least eight distinct isoelectric forms with pI values ranging from 5.85 +/- 0.05 to 6.55 +/- 0.05. The purified glycoprotein contained approximately 48% carbohydrate by weight, consisting of neutral hexose, hexosamine, and sialic acid in a molar ratio of approximately 8:2:1. Rabbit antiglycoprotein V antibody gave a single precipitin line against purified glycoprotein V, which showed a line of complete identity with Triton-solubilized washed platelets. The availability of purified glycoprotein V and antiglycoprotein V antibody will be useful in delineating the role of this glycoprotein in thrombin activation of platelets.
用α-凝血酶处理人血小板会导致仅一种膜蛋白糖蛋白V的选择性水解。为了确定糖蛋白V是否被α-凝血酶直接裂解，并进一步将这种糖蛋白鉴定为潜在的功能性凝血酶受体，将糖蛋白V纯化至>98%的同质性。在静脉穿刺后18小时内从浓缩物中制备洗涤过的血小板，因为临床过期的血小板（静脉穿刺后>72小时）显示几乎不含或无法检测到糖蛋白V。通过在37℃下将血小板（4×10⁹/ml）在0.01M 4-（2-羟乙基）-1-哌嗪乙磺酸（Hepes），pH 7.6（1mM EDTA，0.3M NaCl）中平衡24小时，从血小板膜上洗脱糖蛋白V。通过首先进行硫酸铵分级分离，然后在Sephacryl S-200、羟基磷灰石、DEAE-和CM-纤维素上进行色谱分离来实现纯化，每100单位血小板浓缩物（约6×10¹²个血小板）产生0.5至1.0mg纯化的糖蛋白。纯化的糖蛋白V（Mr = 82,000）是一种凝血酶底物，水解后产生一个主要片段GPVf1（Mr = 69,500），其分子量与先前在经凝血酶处理、高碘酸盐标记的血小板上清液中观察到的相同。糖蛋白V以至少八种不同的等电形式存在，pI值范围为5.85±0.05至6.55±0.05。纯化的糖蛋白按重量计含有约48%的碳水化合物，由中性己糖、己糖胺和唾液酸组成，摩尔比约为8:2:1。兔抗糖蛋白V抗体针对纯化的糖蛋白V产生单一沉淀线，该沉淀线与Triton溶解的洗涤血小板显示出完全相同的线。纯化的糖蛋白V和抗糖蛋白V抗体的可用性将有助于阐明这种糖蛋白在血小板凝血酶激活中的作用。
