Biomedical subjects

W F Novotny

Publications and source records attributed to W F Novotny.

24 records · Page 2Linked to original sources

PubMed1990

Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein.

Lipoprotein-associated coagulation inhibitor (LACI) appears to inhibit tissue factor (TF)-induced blood coagulation by forming a quaternary inhibitory complex containing factor Xa, LACI, factor VIIa, and TF. A genetically engineered hybrid protein consisting of the light chain of factor Xa and the first Kunitz-type inhibitor domain of LACI is shown to directly inhibit the activity of the factor VIIa-TF catalytic complex. Unlike inhibition of factor VIIa-TF activity by native LACI, inhibition by the hybrid protein is not dependent on factor Xa. In an assay of TF-induced coagulation, 50% TF inhibition occurs with hybrid protein at 35 nanograms per milliliter, whereas LACI at 2.5 micrograms per milliliter is required for an equivalent effect. gamma-Carboxylation of glutamic acid residues in the factor Xa light chain portion of the hybrid protein is required for inhibitory activity, indicating that the first Kunitz-type domain of LACI alone is not sufficient for inhibition of factor VIIa-TF.

1-Carboxyglutamic Acid↗

PubMed1989

Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma.

The lipoprotein-associated coagulation inhibitor (LACI) has been isolated from human plasma using a combination of hydrophobic, ion-exchange, and affinity chromatography. The final purification required was greater than 500,000-fold with a yield of 13%. Plasma LACI, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains major bands at 40 and 46 kDa and minor bands at 55, 65, 75, 90, and approximately 130 kDa. All of the molecular weight forms are recognized by antibodies to LACI's amino and carboxyl termini and are able to inhibit the factor VII(a)-tissue factor complex and factor Xa. Plasma LACI, reduced with beta-mercaptoethanol, migrates on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis as a doublet at 42 kDa and has an amino-terminal sequence essentially identical to that of HepG2 LACI. The difference in size between reduced plasma LACI (42 kDa) and HepG2 LACI (47 kDa) may be related to differing degrees of N-linked glycosylation. The 46-kDa and larger forms of unreduced plasma LACI are associated with apolipoprotein A-II (apoA-II) in mixed disulfide linkages. Studies using isolated lipoproteins show that low density lipoprotein (LDL) contains primarily the 40-kDa form of LACI, whereas high density lipoprotein (HDL) contains primarily the 46-kDa form of LACI (LACI/apoA-II complexes). Gel filtration of a fresh plasma sample showed approximately 50% of plasma LACI to be associated with LDL/very low density lipoprotein, 44% with HDL, and the remaining 6% to not be associated with lipoproteins.

Amino Acid Sequence↗

PubMed1989

Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein.

Lipoprotein-Associated Coagulation Inhibitor (LACI) is a factor Xa dependent inhibitor of the factor VII(a)/Tissue Factor catalytic complex. Deduced from partial cDNA sequence, LACI's amino acid sequence has recently been reported. Northern blot analysis showed LACI cDNA hybridizes to RNAs of 1.4 and 4.0 kb in size. To complete the characterization of the LACI message(s), overlapping LACI cDNAs were isolated from a human endothelial cell library. Sequence analysis revealed the clones' inserts span 4023 bases of sequence, consisting of 381 bases of 5' untranslated sequence, an open reading frame of 912 bases, 2682 bases of 3' untranslated sequence and 48 bases of poly(A) sequence. In addition, a short 1.4 kb insert which encodes for LACI was found to contain 49 bases of 3' untranslated sequence and a 3' poly(A) tail. The 1.4 kb of sequence is contained in the 4.0 kb sequence, except for 14 bases of 5' sequence, suggesting that the LACI messages arise by the use of alternative termination and polyadenylation signals during processing. Northern blot analysis of RNA isolated from cells treated with actinomycin D showed both RNA species appear to be relatively stable. Using a bovine papilloma virus vector, LACI cDNA was transfected into mouse C127 fibroblasts. The recombinant LACI is recognized by polyclonal anti-LACI IgG, binds to factor Xa and inhibits VII(a)/Tissue Factor activity in a similar fashion as LACI purified from HepG2 cell conditioned media.

Amino Acid Sequence↗

PubMed1989

Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor.

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.

Humans↗

PubMed1988

Platelets secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein associated coagulation inhibitor.

Stimulation with thrombin or the calcium ionophore, A23187 caused human platelets to release a coagulation inhibitor similar to the Lipoprotein Associated Coagulation Inhibitor (LACI). This was documented functionally, with clotting assays measuring tissue factor inhibition and factor Xa inhibition, as well as immunologically, in a competitive immunoassay. The total amount of LACI released by 3 x 10(8) platelets after two hours stimulation was 7% to 8% of the amount found in 1 mL of serum. Half of the LACI was released by five minutes. The LACI was present in the platelet supernatant and was not associated with the platelet membrane or shed vesicles. The tissue factor and factor Xa inhibitory activities that were released were neutralized by preincubating the platelet supernatants with specific rabbit polyclonal anti-LACI IgG. On Western blot, platelet LACI appeared to run as a doublet with a molecular weight (mol wt) 45,000 to 47,000. Blood samples obtained from the site of a wound (template bleeding time) demonstrated a progressive increase in LACI concentration. A cDNA probe, derived from endothelial cell LACI cDNA, hybridized selectively to 4.0 and 1.4 kb transcripts in a preparation of platelet mRNA.

Blood Platelets↗

PubMed1988

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action.

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 VIIa, 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-2222) and bioassays, but no complex between Xa and LACI(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.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticoagulants↗