Characterization of the initial alpha-thrombin interaction with glycoprotein Ib alpha in relation to platelet activation.
We have evaluated the properties of alpha-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37 degrees C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha-thrombin interaction site on glycoprotein (GP) Ib alpha. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib alpha antibody. Results were similar at room temperature (22-25 degrees C), whereas the initial characteristics of alpha-thrombin interaction with platelets were preserved for at least 20 min at 4 degrees C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib alpha, completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10(-8) M) and relatively high capacity, rather than "high" affinity (kd on the order of 10(-10) M) and low capacity as currently thought. The parameters of alpha-thrombin binding to intact GP Ib alpha on platelets at 4 degrees C corresponded closely to those measured with isolated GP Ib alpha fragments regardless of temperature. Blocking the alpha-thrombin-GP Ib alpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib alpha contributes to the thrombogenic potential of alpha-thrombin.
Mazzucato
M
M
Servizio Immunotrasfusionale e Analisi Cliniche, Centro di Riferimento Oncologico, Aviano, Italy.
Marco
L D
LD
Masotti
A
A
Pradella
P
P
Bahou
W F
WF
Ruggeri
Z M
ZM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology / Q000378:metabolism*; D013696:Temperature; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors
D000906:Antibodies; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D000255:Adenosine Triphosphate; D013917:Thrombin