Binding Of Thrombin By Plasma Inhibitors

Our study was undertaken with the purpose of clarifying the role of plasma inhibitors other than antithrombin III (ATIII) in binding thrombin in the presence and absence of heparin. In the clotting system, several antiproteinases are involved in neutralizing active factors. The capacity to destroy thrombin is attributed to ATIII whose concentration in plasma is ten times less than α1 antitrypsin (α1A) . If heparin is added to plasma, ATIII becomes an immediate inhibitor of thrombin. Several studies have indicated that a modest depression in ATIII activity leads to thrombosis, inferring that other plasma antiproteinases play a minor role. Experimental approach was as follows: Defibrinated platelet poor citrated human plasma was depleted of ATIII and α1 by means of monospecific insolubilized antibodies. Purified thrombin, labelled by Chloramine T incorporation of 125I in NaOH, had a specific activity of 1.3 × 105 CPM/μg. Total antiproteinase thrombin binding capacity of defibrinated plasma was estimated by incubating 4 × 10-33 μM thrombin with plasma (100 μl) for 2 hours and 24 hours in the absence of heparin and for 5 minutes in the presence of heparin (20 units). Free from bound *thrombin was separated by gel filtration on Sephacryl S200 and chromatograms monitored on gamma analyzer. Following the same protocol, ATIII deficient plasma was used to evaluate thrombin binding capacity not attributable to ATIII, and role of ATIII in the absence of α1A was assessed in α1A depleted plasma. As a result, ATIII binding capacity was found to be 62 μg *thrombin/100 μl of normal and α1A deficient plasma, in the presence and absence of heparin, eluting at a volume corresponding to 9 × 104 daltons. Binding capacity of other plasma inhibitors in ATIII deficient plasma amounted to 24 μg *thrombin in the absence of heparin and 7 μg in its presence. This confirms the role of ATIII as the major antithrombin, heparin cofactor. In its absence inactivation of thrombin is reduced by 61%.