Thrombin mutant W215A/E217A acts as a platelet GPIb antagonist.
OBJECTIVE
Thrombin containing the mutations Trp215Ala and Glu217Ala (WE) selectively activates protein C and has potent antithrombotic effects in primates. The aim of this study was to delineate the molecular mechanism of direct WE-platelet interactions under static and shear conditions.


RESULTS
Purified platelets under static conditions bound and spread on immobilized wild-type but not WE thrombin. In PPACK-anticoagulated blood under shear flow conditions, platelets tethered and rolled on both wild-type and WE thrombin, and these interactions were abrogated by the presence of a glycoprotein Ib (GPIb)-blocking antibody. Platelet deposition on collagen was blocked in the presence of WE, but not wild-type thrombin or prothrombin. WE also abrogated platelet tethering and rolling on immobilized von Willebrand factor in whole blood under shear flow.


CONCLUSIONS
These observations demonstrate that the thrombin mutant WE, while not activating platelets, retains the ability to interact with platelets through GPIb, and inhibits GPIb-dependent binding to von Willebrand factor-collagen under shear.
Berny
Michelle A
MA
Department of Biomedical Engineering, Oregon Health & Science University, 3303 SW Bond Ave, Portland, OR 97239, USA.
White
Tara C
TC
Tucker
Erik I
EI
Bush-Pelc
Leslie A
LA
Di Cera
Enrico
E
Gruber
András
A
McCarty
Owen J T
OJ
D000328:Adult; D000818:Animals; D001777:Blood Coagulation / Q000502:physiology*; D001792:Blood Platelets / Q000502:physiology; D004800:Enzymes, Immobilized; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000502:physiology*; D015539:Platelet Activation / Q000502:physiology*; D010973:Platelet Adhesiveness / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000502:physiology*; D011994:Recombinant Proteins; D012212:Rheology; D013917:Thrombin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D013927:Thrombosis / Q000503:physiopathology; D014841:von Willebrand Factor / Q000502:physiology
D004800:Enzymes, Immobilized; D050505:Mutant Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; C023592:glycocalicin; D014841:von Willebrand Factor; D013917:Thrombin