Key Points• A novel TM mutation results in shedding of active TM into the blood.• Subsequent activation of the protein C anticoagulant system causes bleeding.In this study, we describe a novel thrombomodulin (TM) mutation (c.1611C>A) that codes for a change from cysteine 537 to a premature stop codon (p.Cys537Stop). Three members of a family with a history of posttraumatic bleeding were identified to be heterozygous for this TM mutation. All coagulation screening tests, coagulation factor assays, and platelet function test results were within normal limits. However, the endogenous thrombin potential was markedly reduced at low-tissue factor concentration, and failure to correct with normal plasma indicated the presence of a coagulation inhibitor. Plasma TM levels were highly elevated (433-845 ng/ml, normal range 2-8 ng/ml, equating to 5 to 10 nM), and the addition of exogenous protein C further decreased thrombin generation. The mutation, p.Cys537Stop, results in a truncation within the carboxyl-terminal transmembrane helix. We predict that as a consequence of the truncation, the variant TM is shed from the endothelial surface into the blood plasma. This would promote systemic protein C activation and early cessation of thrombin generation within a developing hemostatic clot, thereby explaining the phenotype of posttraumatic bleeding observed within this family. (Blood. 2014;124(12):1951-1956 Introduction Thrombomodulin (TM) is a 557 amino acid type-1 transmembrane glycoprotein expressed on the surface of endothelial cells, certain epithelial cells, monocytes, and megakaryocytes. It is comprised of an amino-terminal C-type lectin domain, an epidermal growth factor (EGF)-like domain comprising 6 EGF modules, an O-glycosylation domain, a helical transmembrane domain, and a short cytoplasmic domain of 36 amino acid residues.1-3 TM has well-described roles in the regulation of hemostasis. [2][3][4] Central to hemostasis is the role of thrombin, which when formed in trace amounts following tissue factor (TF) exposure, activates platelets by cleavage of protease activated receptors on the platelet surface. [5][6][7] This is an important step for both platelet activation and for provision of the anionic phospholipid membrane surface required for coagulation complex assembly. Thrombin also activates factor VIII and factor V, which are critical for the formation of tenase and prothrombinase membrane complexes to create a burst of thrombin production of sufficient magnitude to cleave fibrinogen, factor XIII and form a stable fibrin clot. 5,7 The ability of TM to bind thrombin with high affinity and transform thrombin from a procoagulant to a facilitator of anticoagulation was noted in 1982 by Esmon et al. 8 The activation of protein C to activated protein C (APC) by the thrombin-TM complex, and the subsequent downregulation of coagulation by cleavage of factors Va and VIIIa, has been widely described. 9,10The thrombin-TM complex also activates thrombin activatable fibrinolysis inhibitor (TAFI) at a 1250-fold higher rate t...
SummaryAntithrombin (AT) is the most important inhibitor of the coagulation proteases. Its activity is stimulated by glycosaminoglycans such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and presence of the pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from β-sheet A, known to be critical for the allosteric activation of AT. Here we present data that reveals an energetically distinct intermediate on the path to full activation, where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native like state, in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulphide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free energy contribution of conformational change is provided by the final step of hinge region expulsion and subsequent closure of the main β-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in β-sheet A.
Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-Å crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on β-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.hemophilia | hemostasis | pentasaccharide | protease | thrombosis B lood coagulation (hemostasis) is traditionally described as two separate cascades of proteolytic activation events, the so-called intrinsic and extrinsic pathways (1, 2) (for a recent review, see ref.3). The extrinsic pathway is initiated by tissue damage that exposes tissue factor (TF) and subendothelial matrix proteins to the blood. Platelets adhere to collagen, and circulating factor (f) VIIa binds to and is activated by TF. The fVIIa-TF complex (extrinsic Xase) activates fX, and fXa in the presence of fVa produces thrombin. Just enough thrombin is generated at this stage for the formation of an initial clot through platelet activation; however, unless much more thrombin is produced in short order, the clot will not persist and bleeding will result. The second stage of hemostasis utilizes components of the intrinsic pathway, factors VIIIa and IXa, and deficiency of these critical proteins is the cause of hemophilia A and B, respectively. The fIXa-fVIIIa complex is known as intrinsic Xase and forms on the surface of activated platelets to efficiently produce fXa, resulting in a burst of thrombin formation.Regulatory proteins guard against overgrowth or dissemination of the clot, and all proteases generated in the cascade must eventually be inhibited. Factors VIIa and Xa are inhibited during the initiation phase by tissue factor pathway inhibitor (4), a triple Kunitz-domain canonical inhibitor, but the principal inhibitor of the coagulation proteases is the aptly named member of the serpin fam...
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 ϳ20 Å , 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 (AT)2 is activated by heparin through two distinct mechanisms depending on the target protease and the length of the heparin chain. Thrombin inhibition is accelerated by a simple template mechanism where heparin serves as a bridge to improve diffusion and to stabilize the Michaelis complex, whereas heparin stimulation of factor IXa and Xa inhibition depends on an AT conformational change involving the reactive center loop (RCL) (for reviews see Refs. 1 and 2). Thus, low molecular weight heparin and synthetic pentasaccharides, representing the minimal high-affinity binding sequence, are capable of stimulating the inhibition of factors IXa and Xa by two orders of magnitude but accelerate thrombin inhibition by only ϳ2-fold. The slow reactivity of the circulating native form of AT is crucial in preventing bleeding, which occurs with the overdosing of therapeutic heparin, whereas the localized activation of AT is critical in preventing venous thrombosis. The molecular basis of the activation of AT is thus a subject of great interest.It is natural when considering the activation of AT to focus on the conformation of the RCL. The crystal structure of native AT revealed the partial incorporation of the very N terminus of the RCL (the hinge region) into -sheet A (3, 4). Both biochemical (5, 6) and structural (7) studies later demonstrated that pentasaccharide binding induced the expulsion of the hinge region from -sheet A (Fig. 1A). Although it was assumed that the expulsion of the hinge region would induce a conformational change in the rest of the RCL, the...
Antithrombin (AT) inhibits most of the serine proteases generated in the blood coagulation cascade, but its principal targets are factors IXa, Xa, and thrombin. Heparin binding to AT, via a specific pentasaccharide sequence, alters the conformation of AT in a way that promotes efficient inhibition of factors IXa and Xa, but not of thrombin. The conformational change most likely to be relevant to protease recognition is the expulsion of the N-terminal portion of the reactive center loop (hinge region) from the main -sheet A. Here we investigate the hypothesis that the exosites on the surface of AT are accessible for interaction with a protease only when the hinge region is fully extended, as seen in the related Michaelis complex between heparin cofactor II and thrombin. We engineered a disulfide bond between residues 222 on strand 3A and 381 in the reactive center loop to prevent the extension of the hinge region upon pentasaccharide binding. The disulfide bond did not significantly alter the ability of the variant to bind to heparin or to inhibit thrombin. Although the basal rate of factor Xa inhibition was not affected, that of factor IXa inhibition was reduced to the limit of detection. In addition, the disulfide bond completely abrogated the pentasaccharide accelerated inhibition of factors Xa and IXa. We conclude that AT hinge region extension is the activating conformational change for inhibition of factors IXa and Xa, and propose models for the progressive and activated AT Michaelis complexes with thrombin, factor Xa, and factor IXa. The serpin antithrombin (AT)1 is capable of inhibiting most of the serine proteases generated in the blood coagulation cascade. Its central role is illustrated by the embryonic lethal phenotype of the AT knock-out mouse (1) and by the success of therapeutic heparin. Heparin exerts its anticoagulant effect primarily through an interaction with AT, due to the presence of a specific pentasaccharide sequence found in one-third of heparin chains (2, 3). The binding of the isolated pentasaccharide to AT catalyzes the inhibition of factors IXa and Xa by ϳ300-fold but does not appreciably affect the rate of thrombin inhibition (4, 5). Thrombin inhibition by AT is accelerated by approximately four orders of magnitude (6) in the presence of heparin chains of at least 18 residues in length (7), due to the obligate co-occupation of thrombin on the same heparin chain. The heparin activation mechanism of AT toward factors IXa and Xa is thus allosteric, but it is not clear which of the heparin-induced conformational changes in AT is responsible for the improvement in rate of inhibition.The AT conformational changes that take place in response to heparin binding are well characterized (8 -11) and are summarized in the first two panels of Fig. 1. They include the Nand C-terminal elongation of helix D in the heparin binding region and the expulsion of the N-terminal portion (hinge region) of the reactive center loop (RCL) from -sheet A. Tertiary structural changes also occur in response to he...
Summary. Allele frequencies for the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene were determined in a large case±control study of 517 unselected patients with venous thromboembolism and 478 blood donors. The D allele frequency was 0´53 [95% confidence interval (CI) 0´50±0´56] in patients and 0´54 (95% CI 0´50±0´57) in controls, giving an odds ratio for the D allele of 0´97 (95% CI 0´81±1´16). In the same population, the odds ratio for the factor V Leiden mutation (F5G1691A) was 6´9 (95% CI 4´0±11´9). Therefore, the ACE I/D polymorphism is not a risk factor in a representative group of unselected patients with venous thromboembolism. The possibility that the I/D polymorphism is a risk factor for venous thromboembolism specifically after hip replacement cannot be excluded.Keywords: thrombosis, angiotensin-converting enzyme, ACE, factor V Leiden.Human angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase present at high concentrations on the luminal surfaces of capillary endothelial cells. It is also present in the kidney, brain and liver and is present at lower concentrations in plasma. ACE cleaves the carboxy-terminal dipeptide of angiotensin I, releasing the physiologically active octapeptide angiotensin II. This potently vasoconstrictive molecule plays a key role in modulating vascular tone. In addition, ACE degrades the vasodilator bradykinin, producing a synergistic effect with angiotensin II (Dzau, 1994). The ACE gene contains a polymorphism which results in the insertion (I) or deletion (D) of a 287-bp fragment of intron 16, producing three genotypes, DD and II homozygotes and ID heterozygotes. The I allele has a sequence similar to a silencer sequence, which may explain why the D allele is associated with higher ACE levels than the I allele. The DD and ID genotypes are associated with ACE levels 1´7 and 1´3 times greater than those observed in subjects with the II genotype. This polymorphism accounts for 47% of the serum ACE level variance (Rigat et al, 1990).
Essentials• An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype.• This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk.• Characterization of the antibody showed that it specifically binds to thrombin exosite I.• A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. Summary. Background:We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L À1 . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. Objectives: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. Methods: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. Results: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. Conclusions: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L À1 , suggesting that the presence of a highaffinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.
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