The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

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 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).

Human plasma contains an inhibitor of tissue factor-initiated coagulation known as the lipoprotein-associated coagulation inhibitor (LACI) or also known as the extrinsic pathway inhibitor (EPI). A competitive fluorescent immunoassay was developed to measure the plasma concentration of LACI in samples from normal individuals and patients with a variety of diseases. The LACI concentration in an adult control population varied from 60% to 160% of the mean with a mean value corresponding to 89 ng/mL or 2.25 nmol/L. Plasma LACI levels were not decreased in patients with severe chronic hepatic failure, warfarin therapy, primary pulmonary hypertension, thrombosis, or the lupus anticoagulant. Plasma LACI antigen was decreased in some, but not all patients with gram-negative bacteremia and evidence for disseminated intravascular coagulation. Plasma LACI levels were elevated in women undergoing the early stages of labor (29%), in patients receiving intravenous tissue-type plasminogen activator (45%), and in patients receiving intravenous heparin (375%). A radioligand blot of the pre- and post-heparin plasma samples shows the increase to be in a 40-Kd form of LACI. Very low levels of plasma LACI antigen were found in patients with homozygous abetalipoproteinemia and hypobetalipoproteinemia, diseases associated with low plasma levels of apolipoprotein B containing lipoproteins. Following the injection of heparin into one patient with homozygous abetalipoproteinemia, the plasma LACI antigen level increased to a level comparable with that in normal individuals after heparin treatment.