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Background: This study was designed to determine whether arterial reocclusion after thrombolysis can be prevented by lipoprotein-associated coagulation inhibitor (LACI), a physiological inhibitor of tissue factor-induced coagulation mediated by the extrinsic pathway., Methods and Results: Thrombosis was induced in femoral arteries of anesthetized dogs with the use of anodal current to elicit extensive vascular injury and formation of platelet-rich thrombi in one artery and with thrombogenic copper wire to elicit fibrin-rich thrombi without appreciable vascular injury in the contralateral artery. Recanalization of both vessels was induced with t-PA (1.7 mg/kg i.v. over 1 hour) and verified with Doppler flow probes. Reocclusion occurred within 2 hours in seven of seven arteries with electrical injury-induced thrombosis and in four of seven arteries with copper wire-induced thrombosis in the absence of LACI. In dogs given infusions of recombinant DNA-produced LACI (225 micrograms/kg over 15 minutes, followed by 4 micrograms/kg/min i.v.) after completion of the infusion of t-PA, no reocclusion occurred during the 2-hour interval of observation in any of the five arteries subjected to electrical injury (p less than 0.001), and cyclic partial occlusions were nearly abolished (0.4 +/- 0.4/hr in LACI-treated dogs compared with 13.7 +/- 5.5/hr in saline-treated dogs, p less than 0.0001). In contrast, reocclusion occurred in two of five arteries with indwelling copper wires, and cyclic partial occlusions were unaffected despite LACI. LACI prolonged the partial thromboplastin time modestly (1.7 +/- 0.2 x baseline) but did not affect platelet counts or aggregation assessed ex vivo., Conclusions: Inhibition of the extrinsic pathway of coagulation with LACI prevents thrombotic arterial reocclusion after thrombolysis in vessels subjected to extensive vascular injury. Our results demonstrate that activation of the extrinsic pathway plays a critical role in thrombotic reocclusion and that LACI provides a highly targeted approach to facilitate sustained recanalization without directly inhibiting platelets.

Lipoprotein-associated coagulation inhibitor produces feed-back inhibition of tissue factor (tissue thromboplastin)-induced coagulation in the presence of factor Xa Recombinant lipoprotein-associated coagulation inhibitor (rLACI) was tested for its ability to modify thromboplastin-induced intravascular coagulation in a rabbit model that allows monitoring of iodine-125 fibrin accumulation/disappearance in the lung and sampling of blood for the measurement of coagulation parameters. Infusion of thromboplastin into the rabbit caused a rapid increase of radioactivity over the lungs, possibly due to the accumulation of 125I fibrin in the lungs, followed by a rapid decline of radioactivity, suggestive of removal of fibrin from the lungs. Thromboplastin also caused a rapid decrease of systemic fibrinogen that was accompanied by a lengthening of the activated partial thromboplastin time and prothrombin time. The effect of coinfusion of rLACI with thromboplastin or bolus injection of rLACI before thromboplastin infusion was studied. At a high dose of rLACI (800 micrograms/kg body weight), the thromboplastin-induced radioactivity increase in the lungs and the systemic fibrinogen decrease were completely suppressed. The activated partial thromboplastin time and prothrombin time of the plasma samples lengthened, possibly due to the presence of thromboplastin in circulation. The thromboplastin-induced radioactivity increase over the lungs was not completely suppressed by lower doses of rLACI (135 to 270 micrograms/kg body weight), but these doses of rLACI prevented systemic fibrinogen decrease. At a bolus dose of 23 micrograms/kg body weight, rLACI provided 50% protection of the fibrinogen consumption (fibrinogen decreased to 82% compared with 65% in rabbits treated with thromboplastin alone). These results show that rLACI is effective in the inhibition of thromboplastin-induced coagulation in vivo.

A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.