Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively.
Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.
Yun
Thomas H
TH
Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
Baglia
Frank A
FA
Myles
Timothy
T
Navaneetham
Duraiswamy
D
López
José A
JA
Walsh
Peter N
PN
Leung
Lawrence L K
LL
D000838:Anions; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D016264:Dextran Sulfate / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005172:Factor XI / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D012685:Sepharose; D005172:Factor XI; D016264:Dextran Sulfate; D012964:Sodium; D013917:Thrombin