Covalent Antithrombin-Heparin Complexes with High Anticoagulant Activity

Abstract

Although heparin has been used clinically for prophylaxis and treatment of thrombosis, it has suffered from problems such as short duration within compartments in vivo that require long term anticoagulation. A covalent antithrombin-heparin complex has been produced with high anticoagulant activity and a long half-life relative to heparin. The product had high anti-factor Xa and antithrombin activities compared with noncovalent mixtures of antithrombin and heparin (861 and 753 units/mg versus 209 and 198 units/mg, respectively). Reaction with thrombin was rapid with bimolecular and second order rate constants of 1.3 ؋ 10 9 M ؊1 s ؊1 and 3.1 ؋ 10 9 M ؊1 s ؊1 , respectively. The intravenous half-life of the complex in rabbits was 2.6 h as compared with 0.32 h for similar loads of heparin. Subcutaneous injection of antithrombin-heparin resulted in plasma levels (peaking at 24 -30 h) that were still detectable 96 h post-injection. Given the increased lifetime in these vascular and intravascular spaces, use of the covalent complex in the lung was investigated. Activity of antithrombin-heparin instilled into rabbit lungs remained for 48 h with no detection of any complex systemically. Thus, this highly active agent has features required for pulmonary sequestration as a possible treatment for thrombotic diseases such as respiratory distress syndrome.

Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung

“…ATH was prepared as described previously (11,21). Briefly, 1 mg/ml of human AT (Affinity Biologicals, Ancaster, ON, Canada) and 56 mg/ml of aldose-containing unfractionated heparin (UFH; Sigma, Mississauga, ON, Canada) were heated in PBS at 37°C for 14 days, followed by purification involving hydrophobic chromatography on butyl-sepharose (Amersham Biosciences, Uppsala, Sweden) before anion exchange on DEAE-sepharose (Amersham Biosciences).…”

“…Briefly, 1 mg/ml of human AT (Affinity Biologicals, Ancaster, ON, Canada) and 56 mg/ml of aldose-containing unfractionated heparin (UFH; Sigma, Mississauga, ON, Canada) were heated in PBS at 37°C for 14 days, followed by purification involving hydrophobic chromatography on butyl-sepharose (Amersham Biosciences, Uppsala, Sweden) before anion exchange on DEAE-sepharose (Amersham Biosciences). ATH product was assessed with or without exhaustive heparinase treatment (2 l of ATH incubated with 2 l of heparinase and 10 l of 0.15 M NaCl at 37°C for 2 h) by SDS-PAGE separation on gradient NuPage Gel (4 -12% Bis Tris gel) under reducing conditions, with staining for protein (Coomassie blue) and heparin (alcian blue-silver) (21). To confirm anticoagulant function, anti-factor Xa assays were performed [by standard methods described previously (21)] that determine the specific activity of ATH heparin to catalyze factor Xa inhibition by AT.…”

“…Thus, ATH that contains an AT moiety in the latent or inactivated state still possesses catalytic anticoagulant activity through its heparin chain. Inhibition by ATH of thrombin on fibrin surfaces and thrombin generation on fetal distal lung epithelial cells was significantly more potent than equivalent doses of AT plus heparin (21,22). Furthermore, intratracheal instillation of ATH resulted in high anticoagulant activity in lung lavage fluid for over 48 h, with no measurable activity systemically (12).…”

“…ATH reacts directly with thrombin 4 -10 times faster (11,21) than non-covalent AT plus heparin mixtures. Additionally, the heparin chains of ATH can potently catalyze thrombin neutralization by free plasma AT (21). Thus, ATH that contains an AT moiety in the latent or inactivated state still possesses catalytic anticoagulant activity through its heparin chain.…”

“…A latent form of AT (L-AT), in which the reactive center loop is inserted into the main ␤-sheet, strongly inhibits growth factorstimulated proliferation, migration, and capillary tube formation in cultured endothelial cells and induces tumor regression in a mouse model (51,53,61). AT, particularly in the latent form, inhibits angiogenesis during development (21,51,61). L-AT exerts its antiangiogenic effects by inducing cell apoptosis (53,67), downregulating endothelial proangiogenic heparan sulfate perlecan proteoglycan expression (87) and global genetic reprogramming of systems involving endothelial ligandreceptor signaling mechanisms (86,89).…”