THE LIPOPROTEIN-ASSOCIATED COAGULATION INHIBITOR THAT INHIBITS THE FACTOR-VII-TISSUE FACTOR COMPLEX ALSO INHIBITS FACTOR XA - INSIGHT INTO ITS POSSIBLE MECHANISM OF ACTION
- 1 February 1988
- journal article
- research article
- Vol. 71 (2) , 335-343
Abstract
Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VII, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III.alpha. alone had no effect, but antithrombin III.alpha. with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-222) and bioassays, but no complex between Xa and LAC(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its .gamma.-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with .alpha.-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-Xa-LACI complex that requires the GD of Xa. Because the GD contains the .alpha.-carboxyglutamic acids required for the Ca2+-dependent binding of factor Xa to phospholipid surfaces, the results also suggest that Ca2+ may be required for the native Xa-LACI complex to bind to and inhibit VII(A)/TF. LACI is a novel inhibitor that can rapidly affect feedback inhibition of the VIIa-Ca2+-TF enzymatic complex after the generation of small amounts of Xa and probably plays an important role in the regulation of in vivo coagulation.This publication has 27 references indexed in Scilit:
- Studies of anti-Xa activity in human plasma I: Comparison of a fast-acting inhibitor with antithrombin IIIThrombosis Research, 1981
- Kinetics of factor IX activation via the extrinsic pathway. Dependence of Km on tissue factor.Journal of Biological Chemistry, 1980
- Purification and properties of human coagulation factor VII.Journal of Biological Chemistry, 1980
- The N-terminal sequences of blood coagulation factor X1 and X2 light chains. Mass-spectrometric identification of twelve residues of γ-carboxyglutamic acid in their vitamin K-dependent domainsBiochemical Journal, 1978
- Properties of the factor Xa binding site on human platelets.Journal of Biological Chemistry, 1978
- Kinetics of the activation of bovine coagulation factor X by components of the extrinsic pathway. Kinetic behavior of two-chain factor VII in the presence and absence of tissue factorJournal of Biological Chemistry, 1977
- A Simple Method for Preparing FibrinogenThrombosis and Haemostasis, 1966
- DISC ELECTROPHORESIS – II METHOD AND APPLICATION TO HUMAN SERUM PROTEINS*Annals of the New York Academy of Sciences, 1964
- IDENTIFICATION OF A THROMBOPLASTIN INHIBITOR IN SERUM AND IN PLASMA 1Journal of Clinical Investigation, 1953
- THE ACTIVE PRINCIPLE OF PLACENTAL TOXIN: THROMBOPLASTIN; ITS INACTIVATOR IN BLOOD: ANTITHROMBOPLASTINAmerican Journal of Physiology-Legacy Content, 1947