Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V.

摘要: Treatment of human platelets with alpha-thrombin leads to the selective hydrolysis only one membrane protein, glycoprotein V. To determine whether V was directly cleaved by and permit further characterization this as potential functional thrombin receptor, purified > 98% homogeneity. Washed were prepared from concentrates within 18 h venipuncture, since clinically expired (> 72 venipuncture) shown contain little or no detectable Glycoprotein eluted platelet equilibrating (4 X 10(9)/ml) at 37 degrees C for 24 in 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.6 (1 mM EDTA, 0.3 NaCl). Purification achieved initially performing ammonium sulfate fractionation, followed chromatography on Sephacryl S-200, hydroxylapatite, DEAE- CM-cellulose, yielded 0.5 1.0 mg per 100 units concentrate (approximately 6 10(12) platelets). Purified (Mr = 82,000) a substrate, major fragment, GPVf1 69,500), identical molecular weight that observed previously supernatant thrombin-treated, periodate-labeled platelets. existed least eight distinct isoelectric forms pI values ranging 5.85 +/- 0.05 6.55 0.05. The contained approximately 48% carbohydrate weight, consisting neutral hexose, hexosamine, sialic molar ratio 8:2:1. Rabbit antiglycoprotein antibody gave single precipitin line against V, which showed complete identity Triton-solubilized washed availability will be useful delineating role activation