title: Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor.
abstract: Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.
authors.affiliation: Division of Hematology/Oncology, Jewish Hospital, Washington University Medical Center, St Louis, Missouri 63110.
authors.forename: T J
authors.initials: TJ
authors.lastname: Girard
authors.forename: L A
authors.initials: LA
authors.lastname: Warren
authors.forename: W F
authors.initials: WF
authors.lastname: Novotny
authors.forename: K M
authors.initials: KM
authors.lastname: Likert
authors.forename: S G
authors.initials: SG
authors.lastname: Brown
authors.forename: J P
authors.initials: JP
authors.lastname: Miletich
authors.forename: G J
authors.initials: GJ
authors.lastname: Broze
mesh_terms: D006801:Humans; D008074:Lipoproteins; D008958:Models, Molecular; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D012697:Serine Endopeptidases / Q000235:genetics; D013925:Thromboplastin / Q000037:antagonists & inhibitors
chemical_list: D008074:Lipoproteins; D009363:Neoplasm Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D012697:Serine Endopeptidases