Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.
We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.
Jandrot-Perrus
M
M
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Clemetson
K J
KJ
Huisse
M G
MG
Guillin
M C
MC
D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011486:Protein C; C023592:glycocalicin; D005340:Fibrinogen; D013917:Thrombin