Changes In Antithrombin Research Articles

Crystal structure of antithrombin in a heparin-bound intermediate state.

Antithrombin is activated as an inhibitor of the coagulation proteases through its specific interaction with a heparin pentasaccharide. The binding of heparin induces a global conformational change in antithrombin which results in the freeing of its reactive center loop for interaction with target proteases and a 1000-fold increase in heparin affinity. The allosteric mechanism by which the properties of antithrombin are altered by its interactions with the specific pentasaccharide sequence of heparin is of great interest to the medical and protein biochemistry communities. Heparin binding has previously been characterized as a two-step, three-state mechanism where, after an initial weak interaction, antithrombin undergoes a conformational change to its high-affinity state. Although the native and heparin-activated states have been determined through protein crystallography, the number and magnitude of conformational changes render problematic the task of determining which account for the improved heparin affinity and how the heparin binding region is linked to the expulsion of the reactive center loop. Here we present the structure of an intermediate pentasaccharide-bound conformation of antithrombin which has undergone all of the conformational changes associated with activation except loop expulsion and helix D elongation. We conclude that the basis of the high-affinity state is not improved interaction with the pentasaccharide but a lowering of the global free energy due to conformational changes elsewhere in antithrombin. We suggest a mechanism in which the role of helix D elongation is to lock antithrombin in the five-stranded fully activated conformation.

Physiologic Inhibitors of Coagulation in Patients on Chronic Hemodialysis

Patients on hemodialysis are at increased risk for bleeding and thromboses. The intriguing balance between these risks is more complex than once thought, as endogenous clotting factors and their regulators come into contact with bioincompatible dialyzer membranes, in the setting of an extracorporeal circuit of blood flow, in the face of the uremic state. In this review, we summarize the current data on the interaction between the physiologic inhibitors of coagulation and hemodialysis. Data sources and study selection were obtained from research and review articles related to the endogenous anticoagulation pathway published in English on MEDLINE from 1972 to 2002. While protein C activity and protein S antigen concentrations are increased, there is no change in antithrombin III levels during hemodialysis in relation to predialysis levels. Plasma protein Z, which has only recently been studied in uremic subjects, is increased as well. In addition, hemodialysis leads to elevated tissue factor plasminogen inhibitor, thrombomodulin, tissue plasminogen activator, and plasminogen activator inhibitor-1 activities. The potential functional significance of these observations is discussed. Finally, as erythropoietin is commonly prescribed to uremic patients and is recognized to be prothrombotic, an appraisal of its interaction with the naturally occurring anticoagulants is presented. It is apparent that we are only beginning to realize the complexity of the interplay between this myriad of serum factors and hemodialysis. Further research is needed to shed light on this underexplored area of hemodialysis.

The antithrombin III–saving effect of reduced systemic heparinization and heparin-coated circuits

Objective: To investigate the perioperative changes of antithrombin III (AT-III) activity using reduced systemic heparinization and the possible role of AT-III in determining a better postoperative outcome. Design: Prospective randomized study. Setting: University hospital. Participants: Patients undergoing elective coronary revascularization with cardiopulmonary bypass (n = 90). Interventions: Of patients, 30 were treated with heparin-coated circuits and reduced systemic heparinization; 30, with heparin-coated circuits and full systemic heparinization; 30, with conventional circuits and full systemic heparinization. Measurements and Main Results: Heparin-coated circuits with full systemic heparinization did not exert any effect on coagulation parameters. Low systemic heparinization resulted in a significantly (p < 0.01) higher hematocrit value on arrival in the intensive care unit and in significantly higher values of AT-III activity during cardiopulmonary bypass (66 ± 12% v 57.4 ± 13% and 59.1 ± 12% in the full systemic heparinization groups; p < 0.05), on arrival in the intensive care unit (69.7 ± 13% v 60.7 ± 13% and 60.8 ± 11% in the full systemic heparinization groups; p < 0.01), and on the first postoperative day (81.3 ± 15% v 67.4 ± 13% and 70.2 ± 12% in the full systemic heparinization groups; p < 0.01). No differences were observed in the clinical outcome. Conclusion: Reducing systemic heparinization determines an AT-III-saving effect that could be responsible for the decrease in thromboembolic complications already observed by other authors. It induces higher hematocrit levels immediately after the operation, probably reducing the unmeasurable intraoperative blood loss. Copyright 2002, Elsevier Science (USA). All rights reserved.

Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

Changes in antithrombin III (AT-III) activity in women with singleton pregnancies

International Journal of Gynecology & ObstetricsVolume 70, Issue S4 p. D131-D132 Medical and surgical complications of pregnancy Changes in antithrombin III (AT-III) activity in women with singleton pregnancies H. Minakami, H. MinakamiSearch for more papers by this authorA. Ohkuchi, A. OhkuchiSearch for more papers by this authorT. Koike, T. KoikeSearch for more papers by this authorA. Izumi, A. IzumiSearch for more papers by this authorS. Matsubara, S. MatsubaraSearch for more papers by this authorI. Sato, I. SatoSearch for more papers by this author H. Minakami, H. MinakamiSearch for more papers by this authorA. Ohkuchi, A. OhkuchiSearch for more papers by this authorT. Koike, T. KoikeSearch for more papers by this authorA. Izumi, A. IzumiSearch for more papers by this authorS. Matsubara, S. MatsubaraSearch for more papers by this authorI. Sato, I. SatoSearch for more papers by this author First published: 11 December 2003 https://doi.org/10.1016/S0020-7292(00)84622-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume70, IssueS42000Pages D131-D132 RelatedInformation

How Serpins Are Shaping Up

The serpins are a versatile family of serine protease inhibitors that are involved in coagulation, fibrinolysis, and many other physiological processes. In a Perspective, [Carrell][1] discusses new findings that reveal that small conformational changes in a serpin after cleavage of its reactive site by a target protease can result in acquisition of new functions. For example, [ O'Reilly et al .][2] demonstrate that small conformational changes in antithrombin III result in a switch from its job as an inhibitor of clotting enzymes to a new function as an inhibitor of blood vessel formation and tumor progression. [1]: http://www.sciencemag.org/cgi/content/full/285/5435/1861 [2]: http://www.sciencemag.org/cgi/content/short/285/5435/1926

Antithrombin activity during the period of percutaneous coronary revascularization: Relation to heparin use, thrombotic complications and restenosis

OBJECTIVESThis study evaluated changes in antithrombin (AT) activity around the time of percutaneous transluminal coronary revascularization (PTCR) with unfractionated heparin anticoagulation and the effects these changes had on major thrombotic complications of PTCR.BACKGROUNDHeparin is used during PTCR to prevent thrombosis. However, heparin, a cofactor for AT, causes AT activity to fall. AT activity <70% is associated with thrombosis. There is a prothrombotic state after heparin discontinuation that has not been well explained.METHODSAntithrombin activity was sampled at the start and end of PTCR and the next two mornings in 250 consecutive patients. We recorded occurrence of major thrombotic events, defined as 1) major thrombotic complications of PTCR; 2) major in-lab thrombus formation; or 3) subacute occlusion. Discriminant analysis was employed to evaluate the relationship of AT activity to these events. Change in AT activity and its relationship to heparin was evaluated. Evidence of restenosis at six months was obtained.RESULTSThere were 14 major thrombotic events. Antithrombin activity <70% was strongly (p = 0.006) associated with these events. The AT activity fell significantly through the morning after PTCR when 21% of patients had AT activity <70%; AT activity did not normalize until >20 h after heparin discontinuation. Pre-PTCR use of heparin led to lower AT activity in proportion to duration of heparin use. There was no relationship between AT activity and restenosis.CONCLUSIONSLow AT activity may contribute to major thrombotic complications of PTCR. The way heparin is used before and after PTCR is important to development of low AT activity.

Differential Effects of Low Molecular Weight Heparin and Unfractionated Heparin on Circulating Levels of Antithrombin and Tissue Factor Pathway Inhibitor (TFPI): A Possible Mechanism for Difference in Therapeutic Efficacy

There is growing evidence on the superior efficacy and safety of low molecular weight heparins (LMWHs) over unfractionated heparin (UFH) for the treatment of both venous and arterial thromboembolism. Heparin exerts its function by potentiating antithrombin and by mobilizing tissue factor pathway inhibitor (TFPI) into the circulation. The present study was conducted to compare the effect of subcutaneous LMWH and infusion of UFH on these two anticoagulants in plasma. Eighteen healthy male volunteers were randomly allocated to therapy with continuous intravenous (iv) UFH ( n=6) (initial infusion rate 450 IU/kg/day) or low molecular weight heparin (LMWH) (enoxaparin, 1.5 mg/kg/day) subcutaneously (sc) once daily for 72 hours. Free TFPI antigen, measured by a solid-phase two-site enzyme immunoassay, and antithrombin and protein C activities, measured by chromogenic assays, were assessed in plasma samples before, during, and after anticoagulant treatment. Infusion of UFH, but not subcutaneous LMWH, was found to attenuate the antithrombotic defense by a selective decrease of both circulating antithrombin (−21±7%, p<0.0001) and of free and endothelial-bound TFPI. The changes in antithrombin and TFPI by LMWH and UFH were statistically different between groups ( p<0.001). The differential effect of UFH and LMWH on antithrombin and TFPI may explain the superior efficacy of subcutaneous LMWH compared with conventional intravenous UFH for treatment of both arterial and venous thrombosis.

Hydration Structure of Antithrombin Conformers and Water Transfer during Reactive Loop Insertion

The serine protease inhibitor antithrombin undergoes extensive conformational changes during functional interaction with its target proteases. Changes include insertion of the reactive loop region into a β-sheet structure in the protein core. We explore the possibility that these changes are linked to water transfer. Volumes of water transferred during inhibition of coagulation factor Xa are compared to water-permeable volumes in the x-ray structure of two different antithrombin conformers. In one conformer, the reactive loop is largely exposed to solvent, and in the other, the loop is inserted. Hydration fingerprints of antithrombin (that is, water-permeable pockets) are analyzed to determine their location, volume, and size of access pores, using α shape-based methods from computational geometry. Water transfer during reactions is calculated from changes in rate with osmotic pressure. Hydration fingerprints prove markedly different in the two conformers. There is an excess of 61–76 water molecules in loop-exposed as compared to loop-inserted conformers. Quantitatively, rate increases with osmotic pressure are consistent with the transfer of 73 ± 7 water molecules. This study demonstrates that conformational changes of antithrombin, including loop insertion, are linked to water transfer from antithrombin to bulk solution. It also illustrates the combined use of osmotic stress and analytical geometry as a new and effective tool for structure/function studies.

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin.

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.

Antithrombin III in hematopoietic stem cell transplantation.

Many of the serious, potentially fatal complications of hematopoietic stem cell transplantation have similarities to the multiple organ dysfunction syndrome (MODS) in critically ill nontransplant patients. One of these similarities is the alteration in the hemostatic system in such a way as to lower the levels of the naturally occurring anticoagulant proteins, especially antithrombin III. As in MODS, the outcome of transplant patients with these complications correlates with the degree of change in antithrombin III levels. Preliminary studies suggest that antithrombin III concentrate in pharmacologic doses along with intensive supportive care efforts can improve the clinical outcome of patients with these transplant-related complications. Further work to confirm these findings and, it is hoped, provide insight into the mechanism of action of antithrombin III in this setting is obviously warranted. Until such studies are completed, however, the preponderance of evidence suggests that when subjected to a risk-benefit analysis, patients in the early stages of transplant-related complications would be better off receiving antithrombin III supplementation than not.

Is there a need for antithrombin III substitution early after burn injury?

The changes in antithrombin III (AT-III) levels in the blood and restitution in congulation parameters between patients receiving and not receiving AT-III substitution were examined early after burn injury. The study was divided into two parts with a total of 14 consecutive patients (per cent total body surface area (TBSA) ≥20 per cent). The first six patients were given AT-III substitution when AT-III levels fell below 50 per cent. The second part examined the restitution of the coagulation parameters when the patients ( n = 8) obtained AT-III substitution only at extremely low values of AT-III. The decline in AT-III observed occurred in parallet to the permeability changes and the haemodilution normally seen secondarily to the initial fluid rescucitation. The observed changes in the coagulation parameters were modest and no hepatic dysfunction was noted. In addition, no differences of the restitution in these coagulation parameters were noted between the substituted and non-substituted groups. These results suggest that changes in AT-III early after burn injury depend mainly on factors other than an ongoing disseminated coagulation process. Probable causes are increased capillary leak and haemodilution. Our results suggest that substitution of AT-III in the early postburn period, on the assumption that low levels alone indicate ongoing coagulation, is not warranted.

Role of Arginine 132 and Lysine 133 in Heparin Binding to and Activation of Antithrombin

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.

Antithrombin and Atherosclerosis in the Rotterdam Study

Antithrombin is a potent inhibitor of thrombotic tendency. Whether atherosclerotic disease is associated with high or low antithrombin is unclear. Studies of the relation between antithrombin and presence of arterial disease have shown contrasting results. In the Rotterdam Study, a single-center, population-based cohort study of 7983 subjects aged 55 years and older, the association between atherosclerosis and antithrombin was evaluated. The ratio of ankle to arm blood pressure is a graded marker for atherosclerosis and provides the opportunity to investigate nonlinear associations. In the first 1427 participants of the Rotterdam Study who did not use anticoagulants, both antithrombin and the ratio of ankle to arm blood pressure were measured. In men the association between the two was quadratic: antithrombin activity was increased in men with moderate peripheral arterial atherosclerosis compared with those without, and in men with more severe atherosclerosis it was decreased. In women the association was linear: a decreased ratio of ankle to arm pressure was associated with increased antithrombin activity. These associations were independent of smoking, body mass index, serum lipids, fibrinogen, and factor VIIc. We propose that antithrombin activity rises in response to increased risk of cardiovascular disease and also in response to the presence of atherosclerosis, whereas antithrombin may decrease with increasing severity of the atherosclerotic process in men. This may explain the contrasting results found in previous studies. Changes in antithrombin over time might be useful in predicting the risk of cardiovascular disease and progression.

Disseminated Intravascular Coagulation Is a Frequent Complication of Systemic Inflammatory Response Syndrome

To evaluate the role of disseminated intravascular coagulation (DIC) and to determine the influence of antithrombin, protein C, and plasminogen activator inhibitor 1 on multiple organ dysfunction syndrome (MODS) and outcome in patients with systemic inflammatory response syndrome (SIRS), we made a prospective cohort study. The study subjects consisted of thirty-five patients who exhibited two or more of the conditions of SIRS for more than three consecutive days. They were classified into subgroups of survivors (n = 13) and nonsurvivors (n = 22). The global coagulation and fibrinolytic markers, antithrombin, protein C, and plasminogen activator inhibitor 1 were measured on the day of the diagnosis of SIRS, and also on the 1st, 3rd, and 5th days. The results of these measurements, demographic data, criteria of severity, incidence of MODS were compared between the subgroups. For prediction of patient's death, a receiver operating characteristic (ROC) curve analysis was made. DIC was frequently associated with SIRS patients (29/35, 82.9%). A significant decrease in the DIC score was found in the survivors (p = 0.0001). None of them suffered from DIC on the 5th day. In the nonsurvivors, low levels of protein C and antithrombin and markedly high values of plasminogen activator inhibitor 1 continued up to the 5th day, no improvement of the DIC was observed during the study period and the number of the dysfunctioning organs were significantly higher than in the survivors. Plasminogen activator inhibitor 1 on the 5th day had prognostic value for the prediction of death on the SIRS patients. In conclusion, DIC occurs commonly in patients with SIRS and may be the main determinant for the outcome of these patients. Changes in antithrombin, protein C, and plasminogen activator inhibitor 1 are one of the aggravating factors of MODS. Furthermore, plasminogen activator inhibitor 1 is a good predictor of death in these patients.

Antithrombin III deficiency, deep venous thrombosis, and oral contraceptive use

Oral contraceptive use is a risk factor for the development of deep venous thromboses in adolescents. Most patients with venous thromboses have no well-defined disorder of hemostasis. Identifying such abnormalities in some patients, however, will allow the best decisions to be made concerning contraceptive practices, the treatment of acute thromboses, and long-term prophylaxis. For example, studies have established a relationship between oral contraceptive use and changes in antithrombin III. A case is presented which highlights the multifaceted nature of venous thromboses and the need for clinicians to be aware of potential hypercoagulable states and their implications for patient care. A 16-year-old Black female had a five-day history of rapidly progressive left leg swelling and pain. Five weeks earlier she was diagnosed with dysfunctional uterine bleeding and placed upon a triphasic oral contraceptive. This patient had multiple risks for thrombosis including obesity, prolonged periods of bed rest, oral contraceptive use, and possibly hypercoagulability related to markedly decreased antithrombin III levels. The patient was initially felt to be at high risk for a congenital antithrombin III deficiency because of her profoundly low plasma level and thus treated with antithrombin III concentrates. Based on the subsequent normalization of her antithrombin III levels, however, it appears that her acquired deficiency resulted from either accelerated utilization associated with an extensive thrombus or oral contraceptive use or the combination of both. Decreases in antithrombin III of this magnitude have not been previously reported in association with either oral contraceptives or extensive thromboses. The clinical importance of acquired antithrombin III deficiency for an increased risk of venous thromboembolism remains uncertain.

Conformational Change in Antithrombin Induced by Heparin, Probed with a Monoclonal Antibody against the 1C/4B Region

A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining the heparin-binding domain of antithrombin

Antithrombin is a serine protease inhibitor that participates in the inactivation and removal from the circulation of thrombin and a variety of other procoagulant serine proteases. Antithrombin is also the major plasma cofactor of heparin which exerts its therapeutic effect primarily through its ability to substantially increase the rate of inactivation by antithrombin of the procoagulant serine proteases. Binding of heparin to antithrombin is thus believed to be a prerequisite for this rate enhancement effect. Heparin binding to antithrombin is mediated by a well-defined unique heparin pentasaccharide sequence. Interaction between this pentasaccharide sequence and antithrombin induces a conformational change in antithrombin, an alteration that appears to be sufficient to explain the enhanced ability of antithrombin to inhibit factor Xa and related serine proteases, but not thrombin. Heparin species with longer polysaccharide chains appear to be required in order to enhance the inhibition of thrombin by antithrombin. This may be because the enhancement of this reaction requires that heparin interacts simultaneously with both the antithrombin and the thrombin molecules. This review describes the interactions between heparin and antithrombin, focusing on the antithrombin residues which are involved in the binding of heparin. The role of the heparin-induced conformational change in enhancing serine protease inhibition by antithrombin is also explored. Then, based on available data, an hypothesis is proposed to explain the mechanisms by which heparin accelerates the rate of inactivation by antithrombin of the various serine proteases.

Transmission of conformational change from the heparin binding site to the reactive center of antithrombin

Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.

Platelet, antithrombin, and fibrinolytic activities in taurine-deficient and taurine-replete cats

Summary Cats with cardiomyopathy, especially dilated cardiomyopathy associated with taurine deficiency, often develop systemic thrombi. To investigate the relation of taurine deficiency to formation and persistence of thrombi, cats were made taurine-deficient by consumption of a casein-based taurine-deficient diet, then were evaluated for anticoagulant and profibrinolytic activities and platelet function. The cats served as their own controls in the taurine-replete state; then, values were compared for the taurine-deficient state. Plasma (P &lt; 0.01), blood (P &lt; 0.05), and platelet (P &lt; 0.05) taurine concentrations were decreased markedly after cats consumed the taurine-deficient diet for 6 weeks, compared with baseline concentrations before diet. Compared with the taurine-replete state, taurine deficiency induced significantly (P &lt; 0.05) increased mean antithrombin III activity, no significant change in plasminogen and fibrinolytic activities, and similar clot retraction/lysis test results. Decreased (P &lt; 0.01) adenosine diphosphate (adp)-induced platelet aggregation and [14C]serotonin release, and slightly increased (P &lt; 0.05) collagen-induced platelet [14C]serotonin release, but unchanged collagen-induced platelet aggregation were observed in taurine-deficient cats, compared with taurine-replete cats. Changes in antithrombin III activity most likely reflected hepatocellular acute-phase reaction, which indicates that taurine deficiency may induce a stress-responsive state. Results of platelet function testing indicate that taurine may modulate platelet responsiveness to physiologic agonists, but not in consistent manner. That platelets from the taurine-deficient cats had decreased responsiveness to adp, but increased responsiveness to collagen is surprising, because irreversible aggregation is mediated by release of granule-associated ADP after sufficient initial stimulus. All cats had normal clot retraction in dilute blood, which indicated adequate platelet numbers and function; however, clots failed to lyse in vitro. To the authors knowledge, this observation, at present, lacks adequate explanation. Development of marked taurine deficiency and altered in vitro results of anticoagulant activities and some platelet function tests did not result in clinical manifestations in our cats. Results of our study do not conclusively document a pathophysiologic role of taurine depletion in the formation or persistence of thrombi.