Thrombin binding to platelets and their activation in plasma.
The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.
Liu
L
L
Canadian Red Cross Society Blood Services, Hamilton, Ontario, Canada.
Freedman
J
J
Hornstein
A
A
Fenton
J W
JW
Ofosu
F A
FA
D001792:Blood Platelets / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006629:Hirudins; D006801:Humans; D019007:P-Selectin; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
D006629:Hirudins; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D018179:Receptors, Thrombin; D011516:Prothrombin; D013917:Thrombin