Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V.
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.
D001792:Blood Platelets / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D005779:Immunodiffusion; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification*; D008970:Molecular Weight; D012794:Sialic Acids / Q000032:analysis; D013917:Thrombin
D006023:Glycoproteins; D008565:Membrane Proteins; D012794:Sialic Acids; D013917:Thrombin