Biomedical subjects
Publications and source records attributed to Jeffrey I Weitz.
The limitations of traditional anticoagulants, heparin and warfarin, have prompted the development of new anticoagulant drugs for prevention and treatment of both venous and arterial thromboembolism. After a brief review of thrombogenesis and its regulation, this study focuses on new anticoagulant agents in more advanced stages of clinical testing.
Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.
BACKGROUND: Because clinical diagnosis is inaccurate, objective testing is usually considered necessary when patients present with suspected deep venous thrombosis (DVT). OBJECTIVE: To determine whether a negative result on a quantitative latex D dimer assay eliminates the need for further investigation in patients with a low or moderate pretest probability of DVT. DESIGN: Prospective cohort study. SETTING: Three tertiary care hospitals in Canada. PATIENTS: 556 consecutive outpatients with suspected first DVT. INTERVENTION: Patients were categorized as having a low, moderate, or high pretest probability of DVT and then underwent D-dimer testing. Patients with low or moderate pretest probability and a negative D-dimer result had no further diagnostic testing and received no anticoagulant therapy. Serial compression ultrasonography was performed in all other patients. Patients who did not receive a diagnosis of DVT were followed for symptomatic venous thromboembolism. MEASUREMENTS: Objectively confirmed symptomatic venous thromboembolic events during 3 months of follow-up. RESULTS: 283 patients (51%) had low or moderate pretest probability and a negative D-dimer result. One of these patients had DVT during follow-up (negative likelihood ratio, 0.05 [CI, 0.01 to 0.23]). The negative likelihood ratio of the d -dimer test in all patients was 0.03 (CI, 0.01 to 0.16). CONCLUSION: A negative result on a quantitative latex d -dimer assay safely eliminates the need for further testing in patients with low or moderate pretest probability of DVT.
Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although approximately 30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT + H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (<or=12 monosaccharides) resulted in active unbound (40%) and bound fractions (190 and 560 units/mg, respectively). Factor Xa-ATH or thrombin-ATH inhibitor complexes gave chromatograms on Sepharose-AT with more unbound material compared with that of free ATH. Also, ATH did not bind to Sepharose-heparin, and the intrinsic fluorescence due to activation of AT in ATH by its heparin chain was reversed at higher [NaCl] than that required to dissociate non-covalent AT.H complexes. Thus, exogenous AT can compete with the AT moiety of ATH for binding to the covalently linked heparin chain, leading to catalytic inhibition of factor Xa or thrombin. These data may suggest that access to pentasaccharide units in non-covalent AT.H complexes by free AT may be facile.
Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.
Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.
The limitations of heparin and warfarin have prompted the search for new anticoagulants for the prevention and treatment of venous and arterial thromboembolism. Although many such agents are in development, only a few have reached phase II or higher levels of clinical testing. This article reviews venous and arterial thrombogenesis, discusses the regulation of coagulation, identifies the molecular targets for new anticoagulants currently under development, describes the agents in more advanced stages of clinical testing, and provides clinical perspective on the opportunities for new anticoagulant drugs.
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Most acute coronary syndromes (ACS) are triggered by platelet-rich thrombus superimposed on disrupted atherosclerotic plaque. Thrombin and platelets both play a role in this process. Whereas unfractionated heparin and aspirin have served as cornerstones in the treatment of ACS, several limitations of heparin provide the impetus to seek out better anticoagulants. Direct thrombin inhibitors such as bivalirudin, hirudin, and argatroban offer several pharmacologic advantages over heparin. Additionally, bivalirudin also appears to provide clinical advantages over unfractionated heparin therapy in ACS patients and those undergoing percutaneous coronary intervention.
Arterial and venous thrombosis are a major cause of morbidity and mortality. Anticoagulants are a cornerstone of treatment in patients with these disorders. The two most frequently used anticoagulants, heparin and warfarin, have pharmacological and/or biophysical limitations that make them difficult to use in day-to-day clinical practice. Development of new anticoagulants, which were designed to overcome these limitations, has been facilitated by an increased understanding of the coagulation cascade, the advent of molecular modeling and structure-based drug design, and the realization that the treatment of thrombosis and its complications consumes billions of dollars in annual healthcare expenditures. New anticoagulants target various steps in the coagulation pathway. Coagulation is triggered by the factor VIIa/tissue factor complex and propagated by factors Xa and IXa, together with their activated cofactors, factor Va and VIIIa, respectively. Thrombin, the final effector in coagulation, then converts soluble fibrinogen into insoluble fibrin, the major matrix protein of the clot. New anticoagulation drugs that target each of these clotting enzymes have been developed. This review will focus on those drugs in more advanced stages of clinical evaluation. These include inhibitors of initiation of coagulation (tissue factor pathway inhibitor, nematode anticoagulant peptide and active-site blocked factor VIIa), inhibitors of propagation of coagulation (active-site blocked factor IXa, antibodies against factor IX/IXa, fondaparinux sodium, direct factor Xa inhibitors, protein C derivatives and soluble thrombomodulin), and thrombin inhibitors (hirudin, bivalirudin, argatroban and ximelagatran).
A variety of new anticoagulants for the prevention and treatment of venous and arterial thromboembolism are under development. Designed to overcome the limitations of heparin and vitamin K antagonists, these new agents are at various stages of clinical testing. This paper reviews venous and arterial thrombogenesis, discusses the regulation of coagulation, identifies the molecular targets for new anticoagulants, describes new anticoagulants in more advanced stages of clinical testing, and provides perspective on how these agents will impact on practice.
Venous thromboembolism is the most common preventable cause of death in surgical patients. Thromboprophylaxis, using mechanical methods to promote venous outflow from the legs and antithrombotic drugs, provides the most effective means of reducing morbidity and mortality in these patients. Despite the evidence supporting thromboprophylaxis, it remains underused because surgeons perceive that the risk of venous thromboembolism is not high enough to justify the potential hemorrhagic complications of anticoagulant use. The risk of venous thromboembolism is determined by patient characteristics and by the type of surgery that is performed. In this paper we identify the risk factors for venous thromboembolism and provide a scheme for stratifying surgical patients according to their risk. We describe the mechanism of action of the various forms of thromboprophylaxis and outline the evidence supporting thromboprophylaxis in different surgical settings. Finally, we recommend optimal forms of thromboprophylaxis in patients who undergo various types of surgery. Intermittent pneumatic compression, with or without elastic stockings, can be used for thromboprophylaxis in patients who undergo neurosurgical procedures; for patients who undergo vascular or cardiovascular procedures, long-term acetylsalicylic acid should be used for thromboprophylaxis. Low-molecular-weight heparin (LMWH) or warfarin is the choice for patients with spinal cord operations and all patients with major trauma who do not have contraindications to anticoagulation should receive thromboprophylaxis with LMWH.
Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.
Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH(2)-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TM(LeD/LeD)), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1-dependent and -independent pathways through the suppression of extracellular signal-regulated kinase (ERK)(1/2) activation. TM(LeD/LeD) mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor kappaB and activation of ERK(1/2), and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.
Low-molecular weight heparin (LMWH) is increasingly used in place of unfractionated heparin (UFH) in patients with unstable angina, and phase II clinical trials using fondaparinux for this indication are underway. Because unstable angina patients often require urgent percutaneous coronary interventions (PCI) or aortocoronary bypass surgery, a point-of-care test is needed to monitor the anticoagulant effect of these agents. The activated clotting time (ACT) and activated partial thromboplastin time (aPTT) are the tests most often used to monitor heparin. The purpose of this in vitro study was to determine whether the ACT or the aPTT can be used to monitor the anticoagulant effect of low-molecular weight heparin and fondaparinux. The ACT and aPTT were measured after heparin, enoxaparin or fondaparinux was added to the blood of healthy volunteers, in doses with equivalent inhibitory activity against activated factor X (factor Xa). To mimic the clinical scenario where an unstable angina patient, who has already received enoxaparin, is urgently taken for PCI or bypass surgery, the ACT was determined after heparin was added to blood containing clinically relevant doses of enoxaparin. We determined that enoxaparin produced significantly less prolongation of both the ACT and the aPTT than heparin, whereas fondaparinux had no effect on either of these tests. Addition of enoxaparin to heparin-containing plasma did not prolong the ACT beyond that produced by heparin alone. The ACT and aPTT therefore cannot be used to monitor low-molecular weight heparin or fondaparinux, highlighting the need for a point-of-care anti-factor Xa assay.
Thrombin plays a central role in thrombosis. Consequently, most current antithrombotic treatment strategies are aimed at blocking the activity of thrombin, or preventing its generation. Although heparin has been a cornerstone of treatment, it has limitations. Thus, the anticoagulant response to heparin is unpredictable, the heparin/antithrombin complex is unable to inhibit fibrin-bound thrombin, an important trigger of thrombus growth, and heparin is neutralized by platelet factor 4. Direct thrombin inhibitors were developed to overcome these limitations. Unlike heparin, direct thrombin inhibitors produce a predictable anticoagulant response that is unaffected by platelet factor 4, and they inhibit fibrin-bound thrombin. Three parenteral direct thrombin inhibitors--hirudin, bivalirudin and argatroban--are currently licensed for use in North America, and orally available direct thrombin inhibitors are under investigation. This review summarizes the clinical trial data with direct thrombin inhibitors and provides perspective on the role of direct thrombin inhibitors in the face of other new anticoagulants currently under development.
BACKGROUND: We studied whether aspirin resistance, defined as failure of suppression of thromboxane generation, increases the risk of cardiovascular events in a high-risk population. METHODS AND RESULTS: Baseline urine samples were obtained from 5529 Canadian patients enrolled in the Heart Outcomes Prevention Evaluation (HOPE) Study. Using a nested case-control design, we measured urinary 11-dehydro thromboxane B2 levels, a marker of in vivo thromboxane generation, in 488 cases treated with aspirin who had myocardial infarction, stroke, or cardiovascular death during 5 years of follow-up and in 488 sex- and age-matched control subjects also receiving aspirin who did not have an event. After adjustment for baseline differences, the odds for the composite outcome of myocardial infarction, stroke, or cardiovascular death increased with each increasing quartile of 11-dehydro thromboxane B2, with patients in the upper quartile having a 1.8-times-higher risk than those in the lower quartile (OR, 1.8; 95% CI, 1.2 to 2.7; P=0.009). Those in the upper quartile had a 2-times-higher risk of myocardial infarction (OR, 2.0; 95% CI, 1.2 to 3.4; P=0.006) and a 3.5-times-higher risk of cardiovascular death (OR, 3.5; 95% CI, 1.7 to 7.4; P<0.001) than those in the lower quartile. CONCLUSIONS: In aspirin-treated patients, urinary concentrations of 11-dehydro thromboxane B2 predict the future risk of myocardial infarction or cardiovascular death. These findings raise the possibility that elevated urinary 11-dehydro thromboxane B2 levels identify patients who are relatively resistant to aspirin and who may benefit from additional antiplatelet therapies or treatments that more effectively block in vivo thromboxane production or activity.
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