Pasteurization of antithrombin without generation of the prelatent form of antithrombin

Human antithrombin (AT) is the major inhibitor of blood coagulation and has also been shown to exert anti-inflammatory and anti-angiogenic effects. Pasteurization of pharmaceutical AT products is usually performed at 60 degrees C for 10h in the presence of sodium citrate as stabilizer, sometimes in combination with sucrose. These stabilizers significantly decrease the aggregation and denaturation of AT, but during the pasteurization, a small amount of latent AT (LAT), a partially denatured form, is usually generated, as is an equal amount of another latent form of AT, the so-called prelatent AT (PLAT). The LAT formed during pasteurization has a rather low affinity to heparin and is easily removed by using a second heparin affinity chromatography step in the production process. This is in contrast to the PLAT, which has a slightly lower affinity to heparin than does native AT, which makes it hard to remove. Hence, four commercial products of pasteurized AT were previously shown to contain about 4% of PLAT. In the present work, an alternative pasteurization method is presented, where 2M ammonium sulfate and 50% sucrose are used as stabilizers. During this pasteurization, no, or trace amounts ( < 0.5%), of PLAT may be generated with no formation of aggregates. Moreover, the pasteurized AT has the same specific thrombin-inhibiting activity when compared to incubation in the presence of citrate and sucrose. Heparin affinity high-performance liquid chromatography was used for the determination of PLAT, LAT, and AT.     

Proteolytic modulation of factor Xa–antithrombin complex enhances fibrinolysis in plasma

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13 kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~ 13 kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys–Met339 and Lys–Asp389. Ligand blots demonstrated ¹²⁵I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~ 16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~ 16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.     

Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells

Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.     

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.     

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.     

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.     

Coagulation inhibitors and glycaemic control in non-insulin-dependent diabetes mellitus

The relationship between long-term glycaemic control and the activity of coagulation inhibitors was investigated in 60 non-insulin-dependent diabetes mellitus (NIDDM) patients not on insulin therapy. Overall, the activities of antithrombin III (AT III) (median 96%, range 65–133%), protein C (127%, 24–190%) and protein S (130%, 54–163%) were not reduced. Patients in poor long-term glycaemic control as verified by increased glycated haemoglobin (HbA1c) demonstrated significantly decreased median AT III activity in comparison with patients in good glycaemic control (92% vs 101%,P=0.016). However, individual values for AT III activity were not below the critical limit of 60%. An inverse correlation between AT III activity and long-term glycaemic control (HbA1c) was calculated (r=–0.378,P=0.0029). As AT III concentrations were found to be normal, we propose that non-enzymatic glycation leads to reduced activity of AT III without affecting its concentration.     

Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography

Latent antithrombin (LAT) is a partially denatured form of human antithrombin (AT). LAT does not inhibit clotting of the blood, but has previously been shown to inhibit angiogenesis and carcinogenesis. Another probably partially denatured form is the so-called prelatent AT (P-LAT), described by Larsson et al. [J. Biol. Chem. 276 (2001) 11996]. In the present work, an analytical heparin affinity chromatography method is described that separates an AT form, which is formed during the pasteurization process and which we believe to be identical to the previously described P-LAT, from native AT and LAT. Non-pasteurized AT was shown to contain no P-LAT, while four, heat-treated commercial AT products all contained P-LAT (1-6%, mean=4%). P-LAT has a slightly lower affinity to heparin than does native AT, but exhibits a much stronger heparin affinity when compared to LAT. P-LAT and native AT were shown to have very similar thrombin inhibiting activity, while LAT lacks such activity.     

Copper concentrations are prevalently associated with antithrombin III,but also with prothrombin time and fibrinogen in patients with liver cirrhosis: A cross-sectional retrospective study

BackgroundConcerning the link between copper excess and the pathogenesis of chronic liver diseases, its retention is reckoned to develop as a complication of cholestasis. Recently, it has been found that cholestatic liver injury involves largely inflammatory cell-mediated liver cell necrosis, with consequent reduced hepatic mass, more than occurring through direct bile acid-induced apoptosis. On the other hand, interference with protein synthesis could be expected to result, ending in an altered ability of the liver to retain copper. Little is known about the association between serum copper and clotting factors in cirrhotics. We aimed at studying a possible relationship between increased levels of copper and an aspect of the haemostatic process in liver cirrhosis patients, assessing an index of protein synthesis (albumin) and parameters of protein synthesis/coagulation/fibrinolysis, such as prothrombin time (PT), antithrombin (AT) III and fibrinogen.MethodsRecords from 85 patients suffering from liver cirrhosis of various aetiology and different severity were retrospectively examined. Serum concentrations of copper were determined by atomic absorption spectrophotometer. An index of protein synthesis, such as albumin and parameters of both synthesis and coagulation/hypercoagulation such as PT %, AT III%, levels of fibrinogen were taken into account to study possible correlations to serum copper. The severity of cirrhosis was evaluated by the Child-Pugh (C–P) classification. The relationship among variables were studied by linear regression.ResultsCopper levels of patients suffering from liver cirrhosis were increased respect to those of controls, 102.7+/-28.7 versus 80.4+/-19.5 mcg/dL, (P = .0009), independently from disease severity, and were positively predicted by PT% (P = 0. 017), fibrinogen (P = 0.007) and AT III% (P = 0.000), at linear regression. Among the previous parameters, to which serum albumin was added, the unique predictor of copper levels was AT III%, at multiple regression (P = 0. 010); AT III% was negatively predicted by the C–P classification (P = 0.000); copper levels, adjusted for C–P classification, were predicted by AT III% (P = 0.020) and fibrinogen concentrations, but not by PT% (P = 0.09).ConclusionThe copper concentration is reckoned as responsible for production of the hydroxyl radicals. On the basis that oxidants may enhance the activity of the extrinsic coagulation cascade, ultimately leading to thrombin formation, via their combined effects on stimulation of tissue factor activity and inhibition of fibrinolytic pathways, the positive relationship of copper to coagulation/hypercoagulation parameters (mainly AT III) in our research could find a plausible interpretation.     

Monogenetische Risikofaktoren einer Thrombophilie

Inherited thrombophilias are a group of hereditary conditions that predispose to thrombotic events. The most important monogenetic causes of the venous thromboembolic (VTE) phenotype are mutations in the genes for the coagulation inhibitors antithrombin (AT), protein C (PC), and protein S (PS). Their mutation profiles show high heterogeneity in loss-of-function defects. The frequencies of AT, PC, and PS deficiencies in VTE patients are estimated at 1.9%, 2.3%, 3.7%, respectively. The rate of recurrence in that group is 48.4%. The composite risk of recurrence for VTE patients with AT, PC or PS deficiency is estimated at 1.5 times that for VTE patients without inhibitor deficiency, for those with AT deficiency alone up to 1.9 times. The detection rates for inhibitor deficiency and symptomatic VTE are about 70% for AT, 60% for PC, and 30% for PS. These results demonstrate the problems with the varying accuracy of phenotype diagnostics and differential diagnosis of inherited and intrinsic inhibitor deficiencies.     

Dynamic properties of the native free antithrombin from molecular dynamics simulations: computational evidence for solvent- exposed Arg393 side chain

While antithrombin (AT) has small basal inhibitory activity, it reaches its full inhibitory potential against activated blood coagulation factors, FXa, FIXa, and FIIa (thrombin), via an allosteric and/or template (bridging) mechanism by the action of heparin, heparan sulfate, or heparin-mimetic pentasaccharides (PS). From the numerous X-ray structures available for different conformational states of AT, only indirect and incomplete conclusions can be drawn on the inherently dynamic properties of AT. As a typical example, the basal inhibitory activity of AT cannot be interpreted on the basis of “non-activated” free antithrombin X-ray structures since the Arg393 side chain, playing crucial role in antithrombin-proteinase interaction, is not exposed. In order to reveal the intrinsic dynamic properties and the reason of basal inhibitory activity of antithrombin, 2 μs molecular dynamics simulations were carried out on its native free-forms. It was shown from the simulation trajectories that the reactive center loop which is functioning as “bait” for proteases, even without any biasing potential can populate conformational state in which the Arg393 side chain is solvent exposed. It is revealed from the trajectory analysis that the peptide sequences correspond to the helix D extension, and new helix P formation can be featured with especially large root-mean-square fluctuations. Mutual information analyses of the trajectory showed remarkable (generalized) correlation between those regions of antithrombin which changed their conformations as the consequence of AT–PS complex formation. This suggests that allosteric information propagation pathways are present even in the non-activated native form of AT.     

Preparative conversion of native human antithrombin to the latent form

Human native antithrombin (AT) can be converted to a partially denaturated form of AT, known as latent AT (L-AT). This latent form of AT has been shown to exhibit strong antiangiogenic activity and also to suppress tumor growth in mice models. In the present work, a method is presented which induces the conversion of native AT to L-AT, using incubation at 60 degrees C, for 16 h, with 0.9 M ammonium sulfate, in 5mM Hepes buffer, pH 7.4, giving a recovery of more than 70%. L-AT was determined by integration of the low heparin affinity peak when analyzed by the affinity chromatography method. Native polyacrylamide gel electrophoresis was used to show that the preparation contained no aggregates. Hydrophobic interaction chromatography was also used for the separation of AT and L-AT.     

Heterogeneity of mammalian antithrombin III

Affinity chromatography on heparin-Sepharose was used to isolate two forms of antithrombin III(AT) from human, bovine, rabbit and rat blood plasma. The two isolated forms of AT are the major form. AT alpha, making up to 90% of the whole inhibitor molecule, and the minor form, AT beta (10% of AT). The molecular mass of AT beta in all mammalian species under study is by 3-5 kDa lower than that of AT alpha. The isoelectric point for bovine AT alpha lies within the range of 4.95-4.5, whereas that for AT beta--at 5.28-4.76. No significant differences in the progressive antithrombin activity of the major and minor forms of the bovine inhibitor were observed. In contrast, the heparin-cofactor activity of the AT beta-heparin complex exceeds that of the AT alpha-heparin complex--3-fold. The functional differences in the AT forms are due to the differences in their affinities for heparin. It was shown that AT beta exhibits a higher affinity for free and bound heparin.     

Separation of native and latent forms of human antithrombin by hydrophobic interaction high-performance liquid chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.     

Mitochondrial protection by the thioredoxin-2 and glutathione systems in an in vitro endothelial model of sepsis

The Streptococcus pyogenes cysteine protease SpeB (streptococcal pyrogenic exotoxin B) is important for the invasive potential of the bacteria, but its production is down-regulated following systemic infection. This prompted us to investigate if SpeB potentiated the host immune response after systemic spreading. Addition of SpeB to human plasma increased plasma-mediated bacterial killing and prolonged coagulation time through the intrinsic pathway of coagulation. This effect was independent of the enzymatic activity of SpeB and was mediated by a non-covalent medium-affinity binding and modification of the serpin A1AT (α-1 antitrypsin). Consequently, addition of A1AT to plasma increased bacterial survival. Sequestration of A1AT by SpeB led to enhanced contact system activation, supported by increased bacterial growth in prekallikrein deficient plasma. In a mouse model of systemic infection, administration of SpeB reduced significantly bacterial dissemination. The findings reveal an additional layer of complexity to host-microbe interactions that may be of benefit in the treatment of severe bacterial infections.     

Some haemostatic parameters in patients with deep and superficial venous thrombosis.

Some haemostatic parameters (AT III, alpha 2-AP, C1-INH, kallikrein, F.XII, fibrinogen, plasminogen, euglobulin lysis time, FDP and ethanol test) were studied in patients with deep (DVT) and superficial (SVT) venous thrombosis. The patients with DVT revealed significantly decreased AT III activity, increased alpha 2-AP, C1-INH activity, fibrinogen and FDP concentrations and prolongation of euglobulin lysis time. Ethanol gelation test was positive in 61% in DVT group. Plasminogen level was unchanged in patients with DVT. No significant changes in these parameters were found in SVT group. Only the ethanol gelation test was positive in 21% in this group. These results show a markedly expressed phenomenon of hypercoagulability in the group of patients with DVT and suggest that in the treatment different therapeutic procedures should be considered which influence these specific changes in these coagulation parameters.     

Coagulation factor mutations and thrombosis

The coagulation system is governed by a subtle balance between clotting activators and inhibitors. Many genes can contribute to the overall phenotype, and polymorphisms may act to up regulate or down regulate the generation of thrombin, the coagulation-key enzyme. An increase in coagulation factor (gain function) or/and a decrease in coagulation inhibitors (loss of function) may favor venous thromboembolism (VTE). It has been observed since a long time that VTE may be a familial disease, but it was only in 1965 that Egeberg published the first case of inherited antithrombin (AT) deficiency. This was followed by similar reports of protein C (PC) and protein S (PS) deficiencies. Hereditary thrombophilia was thus initially considered as a rare monogenic disorder with incomplete penetrance. AT, PC and PS deficiencies are due to multiple and mostly private mutations of the corresponding genes. Most patients are heterozygous and experience VTE at adult age. Homozygosity associated with severe thrombosis at birth has been observed in newborns with undetectable PC or PS concentrations. The discovery of factor (F) V Leiden and F2 g.20210 G>A, two gain of function mutations, challenged the view of thrombophilia as a rare monogenic disorder. FV Leiden and F2 g.20210 G>A are due to a founder effect and affect populations of European descent with frequencies at 5% and 3% respectively. These two mutations are moderate of risk factor for thrombosis and paved the way for gene-gene and gene-environment interactions. Patients carrying more than one genetic risk factor are at higher risk to develop VTE. The exposition to acquired risk factors such as estrogen based oral contraception may also have a synergistic effect favoring thrombosis in patients with FV Leiden or other genetic risk factors.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

THE BOOK CORNER

In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs.     

Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.