title: Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.
abstract: Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Heather K
authors.initials: HK
authors.lastname: Kroh
authors.forename: Peter
authors.initials: P
authors.lastname: Panizzi
authors.forename: Svetlana
authors.initials: S
authors.lastname: Tchaikovski
authors.forename: T Regan
authors.initials: TR
authors.lastname: Baird
authors.forename: Nancy
authors.initials: N
authors.lastname: Wei
authors.forename: Sriram
authors.initials: S
authors.lastname: Krishnaswamy
authors.forename: Guido
authors.initials: G
authors.lastname: Tans
authors.forename: Jan
authors.initials: J
authors.lastname: Rosing
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Furie
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001777:Blood Coagulation; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013927:Thrombosis / Q000201:enzymology* / Q000235:genetics
chemical_list: D011516:Prothrombin; D013925:Thromboplastin