Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein ib

Abstract Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib α-chain (GPIba, 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 (αβ-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived μ-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 μM 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 GPIbα was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIbα at one point and released glycocalicin.