Effect of covalent serpin-heparinoid complexes on plasma thrombin generation on fetal distal lung epithelium

Extravascular coagulation within the lung airspace is a hallmark of respiratory distress syndrome (RDS) in premature infants. We previously showed that covalent antithrombin-heparin complex (ATH) is superior to noncovalent antithrombin (AT) + heparin (H) mixtures at inhibiting plasma thrombin generation on rat fetal distal lung epithelium (FDLE) in vitro. However, heparin cofactor II (HC) has been shown to selectively inhibit thrombin, which may be advantageous if other enzyme activities are present in the airspace. We compared the abilities of ATH, covalent HC-heparin complex (HCH), and covalent HC-dermatan sulfate (HCD) to inhibit thrombin generation on FDLE in plasmas from either adults or newborns. In the presence of ATH, peak free thrombin generation in adult plasma on the cell surface was reduced by 92% compared with controls (no anticoagulant). However, whereas HCH reduced peak free thrombin generation in adult plasma by 81%, HCD was only able to reduce activity by 33%. All covalent complexes caused a greater decrease in thrombin activity compared with that with the corresponding noncovalent serpin + heparinoid mixtures. Experiments in plasma from newborns resulted in peak free thrombin that was less than or equal to that in adult plasma when covalent conjugates were added. Relative peak free thrombin was proportional to rate of prothrombin consumption and amount of thrombin-inhibitor complexes formed. In vivo, experiments in newborn rats showed that a greater percentage of intratracheally instilled ATH and HCH could be recovered in lung lavage fluid compared withwith that for HCD. In summary, ATH, HCH, and HCD are inhibitors of thrombin generation on FDLE superior to the corresponding noncovalent mixtures, with ATH and HCH being more potent than HCD. Covalent conjugates of AT or HC with H may be preferred in treatment of extravascular coagulation.