Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein Ib.
Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin.
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000494:pharmacology*; D002645:Chickens; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010641:Phenotype; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins
D006023:Glycoproteins; D007976:Leupeptins; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C023592:glycocalicin; D000001:Calcimycin; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D004492:Edetic Acid; D002154:Calpain; D007930:Leucine; C032854:leupeptin; D002118:Calcium