AMINO ACID SEQUENCE OF THE THROMBIN-BINDING SITE ON PLATELET GLYCOPROTEIN Ib

 

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Amino acid sequence of the thrombin-binding site on platelet glycoprotein (GP) Ib was determined and the effects of the synthesized analogous peptides on thrombin-induced aggregation were studied. Crude platelet membrane fraction solubilized with 5mM dithiothreitol and 0.2% Tween 20 was applied to a WGA-Sepharose. The bound fractions were eluted with 0.3M N-actylglucosamine, then applied to a TM60 ( a monoclonal antibody against GP Ib)-Affi-gel column. Only one GP was bound to the column and was eluted with 50mM glycine-HCl, pH3.0, containing 0.2% Tween 20. SDS-PAGE showed a single band of 130kDa, corresponding to GP Ib alpha chain (GP Ibα). When the purified GP Ibα was digested with trypsin, two fragments (94kDa . and 43kDa) were obtained. The 43kDa fragment was shown to bind to both affinity colomns of TM60- and thrombin-Affi-gel, while the 94kDa fragment did not bind to either Affi-gel. When the same experiment was performed using chymotrypsin, three fragments (94kDa, 45kDa and 39kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45kDa and 39kDa) were bound to both columns. However, on. thrombin-Affi-Gel column, 39kDa fragment was found in both unbound and bound fractions. It showed that the 45kDa fragment interacts with thrombin with a higher affinity than the 39kDa fragment. These results indicate that the thrombin-binding site is located on the "tail" portion of GPIba, especially on a chymotryptic cleavage site.

Then 43kDa tryptic fragment was purified and its partial amino acid sequence was analyzed using gas phase amino acid sequence analyzer. Based on its amino acid sequence, several analogous oligopeptides were synthesized. Three peptides (#1-10, #11-23, #17-28) inhibited 0.05 U/ml thrombin-induced aggregations of washed human platelets in dose-dependent manners. IC50 were in the range of L50-550μM for each of these peptides.