Portal de Periódicos da CAPES

Serpins in thrombosis, hemostasis and fibrinolysis

2007; Elsevier BV; Volume: 5; Linguagem: Inglês

10.1111/j.1538-7836.2007.02516.x

1538-7933

Jill C. Rau, Lea M. Beaulieu, James A. Huntington, Frank Church,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Journal of Thrombosis and HaemostasisVolume 5, Issue s1 p. 102-115 Free Access Serpins in thrombosis, hemostasis and fibrinolysis J. C. RAU, J. C. RAU Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA These authors contributed equally to this review.Search for more papers by this authorL. M. BEAULIEU, L. M. BEAULIEU Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA These authors contributed equally to this review.Search for more papers by this authorJ. A. HUNTINGTON, J. A. HUNTINGTON Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC, Cambridge, UKSearch for more papers by this authorF. C. CHURCH, F. C. CHURCH Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USASearch for more papers by this author J. C. RAU, J. C. RAU Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA These authors contributed equally to this review.Search for more papers by this authorL. M. BEAULIEU, L. M. BEAULIEU Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA These authors contributed equally to this review.Search for more papers by this authorJ. A. HUNTINGTON, J. A. HUNTINGTON Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC, Cambridge, UKSearch for more papers by this authorF. C. CHURCH, F. C. CHURCH Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USASearch for more papers by this author First published: 09 July 2007 https://doi.org/10.1111/j.1538-7836.2007.02516.xCitations: 207 Frank C. Church, Division of Hematology-Oncology/Department of Medicine, 932 Mary Ellen Jones Building, Campus Box 7035, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599-7035, USA.Tel.: + 1 919 966 3313; fax: + 1 919 966 7639; e-mail: fchurch@email.unc.edu AboutSectionsPDF 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 Abstract Summary. Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin–protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure–function relationships is imperative for the development of state-of-the-art pharmaceuticals for the selective fine-tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z-dependent protease inhibitor, α1-protease inhibitor, protein C inhibitor, α2-antiplasmin and plasminogen activator inhibitor-1. We focus on the biological function, the important structural elements, their known non-hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins. Introduction Blood flow is maintained by the proper balance of hemostasis and fibrinolysis, an interdependent network of physiological processes and succession of proteolytic reactions. Hemostasis, the physiological cessation of bleeding, involves the interaction of vasoconstriction, platelet aggregation and coagulation. The end result of coagulation is the deposition of cross-linked fibrin polymers to form blood clots. Both the protein C and the fibrinolytic pathways are activated by the coagulation pathway and serve to restrict excessive clot formation or thrombosis. The enzymatic reactions that propel these pathways are dominated by serine proteases and are subject to control by serpins and their local cofactors. Dysfunction, deficiencies or over-expression of serpins can cause either abnormal bleeding or thrombosis. Investigations into the structure and related activities of serpins, their target proteases and cofactors have provided valuable information regarding both serpin-related disease states and potential mechanisms by which medicine can manipulate serpin–protease interactions for the treatment and prevention of thrombosis and bleeding. Hemostasis Coagulation pathway The factors of the coagulation pathway generally circulate in an inactive state until they are activated through proteolysis by an upstream factor. While the end goal of coagulation is fibrin polymerization, the most crucial feature of the coagulation pathway is the generation of thrombin (Fig. 1). Thrombin is responsible for cleaving fibrinogen to fibrin, activating factor (F) XIII to FXIIIa (which cross-links fibrin), activating platelets, and positively feeding back into the cycle by activating upstream factors [1]. Figure 1Open in figure viewerPowerPoint Serpin regulation of coagulation, protein C and fibrinolytic pathways. Serpins and inhibitory functions are shown in red, thrombin activity is shown in cyan. Prothrombinase and tenase complexes are shown in gray boxes. Coagulation is initiated by the exposure of tissue factor to factor VIIa shown in a gray oval. The symbol * indicates degradation. Necessary cofactors, Ca++, phospholipids, proteins S and Z, vitronectin and GAGs are not shown to maintain the simplicity of the schematic. Thrombin generation is initiated when damage to a vessel wall exposes the blood to tissue factor (TF) in the subendothelium [2]. TF is also expressed by activated platelets and leukocytes [3]. Therefore, coagulation can also be initiated by inflammation. TF forms a complex with FVIIa and activates FX. Together, FVa and FXa form the prothrombinase complex, which then cleaves a small amount of prothrombin (FII) to thrombin (FIIa). This small amount of thrombin activates platelets, FV, FVIII and FXI, feeding back into the cycle to increase thrombin formation. Factor IXa, previously activated by either TF-VIIa or by FXIa on the platelet surface, and FVIIIa in the presence of calcium, complex on the platelet surface to form the platelet tenase complex. Platelet tenase activates more FX, which with FVa, generates a 'thrombin burst' (Fig. 1). It is this burst of thrombin rather than the initial thrombin activation that is crucial for the formation of a stable hemostatic plug [2]. In addition to its role in hemostasis, thrombin regulates many proinflammatory processes including leukocyte adhesion molecule expression on the endothelium, platelet activation, leukocyte chemotaxis and endothelial cell production of prothrombotic factors [4]. Thrombin is also a potent growth factor, initiating endothelial, fibroblast and smooth muscle cell proliferation and up-regulating other cytokines and growth factors [5]. These activities have been attributed to proteolytic cleavage of insulin-like growth factor binding proteins [6] and protease activated receptors -1, -3 and -4 (PAR-1, -3, -4) [7] on cell surfaces, and account for thrombin's central role in atherosclerotic lesion formation [8]. Coagulation is regulated predominantly by antithrombin (AT) [9], tissue factor pathway inhibitor (TFPI) [10], the protein C pathway [11] and to a lesser extent heparin cofactor II (HCII) [12] and protein Z-dependent protease inhibitor (ZPI) [13]. Protein C inhibitor (PCI) and plasminogen activator inhibitor-1 (PAI-1) may also contribute by inhibiting thrombin [14, 15]. TFPI is not a member of the serpin family and so will not be discussed in this paper. Protein C pathway The protein C pathway works in hemostasis to control thrombin formation in the area surrounding the clot [16]. The zymogen protein C (PC) is localized to the endothelium by endothelial cell protein C receptor (EPCR) [17]. Thrombin, generated via the coagulation pathway, is localized to the endothelium by binding to the integral membrane protein, thrombomodulin (TM). TM occupies exosite I on thrombin, which is needed for fibrinogen binding and cleavage, thus reducing thrombin's procoagulant activities [18]. However, on the endothelial cell surface TM bound thrombin is able to cleave PC to activated protein C (APC), a serine protease [19]. In the presence of protein S, APC inactivates FVa and FVIIIa [20] (Fig. 1). This limits further thrombin generation on the clot periphery where the endothelium is not damaged [21]. The protein C pathway is also associated with non-hemostatic functions. APC has been shown to be an anti-inflammatory protein [22, 23] and modulates gene expression [24]. It also enhances vascular permeability by signaling through both PAR-1 and sphingosine 1-phosphate receptor-1 [25]. In focal ischemic stroke animal models, APC treatment restored blood flow, and reduced infarct volume and inflammation [26]. These neuroprotective effects of APC were shown to be mediated through EPCR, PAR-1 [27] and PAR-3 [28]. In the PROWESS Study, patients diagnosed with severe sepsis were treated with recombinant human APC, resulting in a mortality reduction of 19.4% [29]. The proteolytic activity of APC is regulated predominantly by protein C inhibitor (PCI) [9]. Additionally, plasminogen activator inhibitor-1 (PAI-1) [30] and α1-protease inhibitor (α1PI) [31] have been shown to inhibit APC, although their role in hemostasis is not well understood. Fibrinolysis Fibrinolytic pathway Fibrinolysis is the physiological breakdown of fibrin to limit and resolve blood clots [32]. Fibrin is degraded primarily by the serine protease, plasmin, which circulates as a zymogen, plasminogen. In an auto-regulatory manner, fibrin serves as both the cofactor for the activation of plasminogen and the substrate for plasmin (Fig. 1). In the presence of fibrin, tissue plasminogen activator (tPA) cleaves plasminogen to plasmin, which proteolyzes the fibrin. Because it is a necessary cofactor for the reaction, the degradation of fibrin limits further activation of plasminogen [33-35]. The serine protease, tPA, is synthesized and released by endothelial cells [32]. In addition to binding fibrin, tPA binds Annexin II (AnII) and other receptors on endothelial cell and platelet surfaces [36]. Thus, plasmin generation and fibrinolysis are restricted to the site of thrombus formation. In addition to its role in fibrinolysis, plasmin has other physiological functions as evidenced by its ability to degrade components of the extracellular matrix [37] and activate matrix metalloproteases 2 and 9 [38, 39]. Plasminogen can also be converted to plasmin by the serine protease, urokinase plasminogen activator (uPA) [37]. Urokinase-catalyzed events are localized on the cell surface through the uPA receptor (uPAR). Complex formation and subsequent reactions are thought to be more important during pericellular proteolysis, cell adhesion and migration than they are for vascular fibrinolysis [32]. These additional functions contribute to the role of the fibrinolytic pathway in cancer [37, 40, 41]. Fibrinolysis is controlled predominantly by α2-antiplasmin (α2AP) [42], PAI-1 [33, 43] and thrombin activatable fibrinolysis inhibitor (TAFI) [44]. PCI can inhibit tPA and uPA [45, 46], but its role in fibrinolysis is unclear. TAFI is not a member of the serpin family and so will not be discussed in this paper. Serpin overview Serpins Serpins are a superfamily of proteins classified into 16 clades (A–P). The systematic name of each serpin is, SERPINXy where X is the clade and y is the number within the clade [47]. Serpins have been identified in the genomes of organisms representing all of the branches of life (Bacteria, Archaea, Eukarya and Viruses), and the genome of humans contains c. 36 serpins [48]. While serpins are named for their ability to inhibit serine proteases (of the chymotrypsin family) (Table 1), some are capable of cross-class inhibition of proteases from the subtilisin, papain and caspase families. In addition, some serpins utterly lack protease inhibitory activity and serve other roles, such as hormone transporters, molecular chaperones or catalysts for DNA condensation. Serpins are typically composed of c. 400 amino acids, but can have large N-, C-terminal or internal insertion loops [47]. Serpins can also be post-translationally modified by glycosylation, sulfation, phosphorylation and oxidation to alter their function. In spite of a low overall primary sequence identity for the family, serpins share a highly conserved three-dimensional fold comprised of a bundle of 9 α-helices (A–I) and a β-sandwich composed of three β-sheets (A–C) (Fig. 2A). It is useful to view a serpin in the 'classic orientation' to illustrate the important structural features (Fig. 2A, left panel). In this view the main β-sheet A is facing and the reactive site loop (RSL) is on top. The RSL is typically composed of 20 amino acids running from P17 at the N-terminus (at the C-terminal end of strand 5A) to P3′ at the C-terminal end (using the nomenclature of Schechter and Berger, where residues are numbered from the scissile P1–P1′ bond). In the normal native state of a serpin, β-sheet A is composed of five strands and the RSL (bridging the C-terminus of strand 5A to the N-terminus of strand 1C) is exposed. This state is, however, not the most stable. An astounding increase in thermodynamic stability (best estimate – 32 kcal mol−1) [49] can be achieved through the incorporation of the RSL into β-sheet A, triggered either through extension of strand 1C (to form the so-called 'latent' state), or through proteolytic nicking anywhere near the scissile bond (the cleaved state). The metastability of the native serpin is critical for its unusual mechanism of protease inhibition [50]. Table 1. Second order rate constants of protease inhibition by serpins in the presence and absence of cofactors* Serpin Systematic name Target protease Cofactor Second order rate k2 (m−1 s−1) Citation AT SERPINC1 Thrombin – 7.5 × 103, 1 × 104 [168], [169] UFH 2 × 107, 4.7 × 107 [168],[170] LMWH 5.3 × 106 [170] Pentasaccharide 2 × 104 [169] FXa – 2.5 × 103, 6 × 103 [171] UFH 5 × 106, 6.6 × 106 [169], [170] LMWH 1.3 × 106 [170] Pentasaccharide 7.5 × 105 [169] FIXa – 1.3 × 102, 5 × 102 [169], [172] UFH 8 × 106, 1.75 × 106 [169], [172] LMWH 3.7 × 105 [172] Pentasaccharide 3 × 104 [169] HCII SERPIND1 Thrombin – 6 × 102 [168] UFH 5 × 106 [168] LMWH ∼5 × 106 [95] Dermatan sulfate 1 × 107 [168] Hexasaccharide 2 × 104 [173] ZPI SERPINA10 FXa – , Ca++, PL 2.3 × 103 [174] Protein Z, Ca++, PL 6.1 × 105 [174] FIXa – 2 × 105 [101] UFH 4 × 105 [101] PCI SERPINA5 Thrombin – 1.7 × 104 [57] UFH ∼2 × 105 [57] Thrombomodulin 2.4 × 106 [57] APC – 3 × 102 [175] UFH 5 × 104 [175] UFH, Ca++ 2.9 × 105 [176] tPA (2-chain) – 8 × 102 [45] UFH 3 × 104 [45] α 1 PI SERPINA1 Thrombin – 4.8 × 101 [177] APC – 4 × 101 [178] α 1 PI Pittsburgh Thrombin – 4.8 × 105 [179] APC – 7 × 104 [179] α 2 AP SERPINF2 Plasmin – 2 × 107 [42, 180] Plasminogen activator inhibitor-1 SERPINE1 Thrombin – 7.9 × 102 [15] UFH 1.6 × 105 [141] Vitronectin 1.9 × 105 [141] APC – 5.7 × 102 [30] Vitronectin 1.8 × 105 [30] tPA (1- , 2-chain) – 4 × 107, 1.5 × 108 [181] *The rate constants indicated here are from selected references and may vary slightly under different experimental conditions. AT, antithrombin; APC, activated protein C; HCII, heparin cofactor II; ZPI, Z-dependent protease inhibitor; PCI, protein C inhibitor; α1-PI, α1-protease inhibitor; t-PA, tissue plasminogen activator; UHF, unfractionated heparin; LMWH, low-molecular-weight heparin; PL, phospholipids. Figure 2Open in figure viewerPowerPoint Serpin structure and mechanism of protease inhibition. (A) The shared serpin fold is illustrated by the structure of the prototypical native serpin α1PI. The 'classic' orientation shown on the left places the RSL (yellow) on top and the main β-sheet A (red) to the front. Sheets B and C are blue and orange, respectively, and helices A, D and H are colored green, cyan and magenta. The accessibility of the RSL is illustrated by rotating the molecule by 110° to the left along the long axis. It shows how the P1–P1′ (rods) scissile bond is exposed for proteolytic attack. Also clearer in this orientation are helices D and H, which are the heparin binding helices. (B) The serpin mechanism of protease inhibition is minimally expressed as a two-step process. In the first step, native serpin (ribbon with the P1 and P1′ residues as magenta balls) interacts reversibly with a protease (surface representation, colored according to temperature factors from blue to red) to form the Michaelis complex (middle). After formation of the acyl-enzyme intermediate the protease is flung to the opposite pole of the serpin and its catalytic architecture is destroyed, and consequently there is a loss of ordered structure (notice the smaller size and increase in temperature factors). The serpin mechanism of protease inhibition The serpin mechanism of protease inhibition has been worked out over the last 20 years through a series of biochemical, fluorescence and structural studies. A minimalist kinetic scheme is composed of two steps: the formation of the encounter complex (also known as the Michaelis complex) where the sequence of the RSL is recognized by the protease as a substrate; and the formation of a final covalent complex where the protease is trapped in an inactive state (Fig. 2B). The rates of formation and dissociation of the reversible Michaelis complex, along with colocalization in tissues, determines the specificity of the serpin–protease interaction [51, 52]. While the obligate RSL-active site contacts contribute significantly to the formation of the Michaelis complexes, exosite interactions may also be involved. As with actual substrates of serine proteases, this step is followed by the nucleophilic attack of the peptide bond between the P1–P1′ residues by the catalytic Ser195 of the protease. This ultimately results in the formation of a covalent ester bond between the P1 residue and Ser195 of the protease (acyl-enzyme intermediate), and then separation of the P′ residues from the active site of the protease. At this stage the serpin rapidly adopts its lowest energy conformation through the incorporation of the N-terminal portion of the RSL into β-sheet A. The tethered protease is thus flung from the top to the bottom of the serpin (c. 70Å), and the resulting pulling force exerted on the catalytic loop results in a conformational distortion of the protease [53]. The acyl-enzyme intermediate is thus trapped, with deacylation prevented, largely because of the destruction of the oxyanion hole. Two structures of final complexes have been solved by X-ray crystallography [54, 55], with one showing an additional distortion of c. 37% of the protease structure [55]. This mechanism is particularly well suited to tightly regulated processes such as hemostasis and fibrinolysis because inhibition is irreversible, and the conformational changes in the serpin and the protease alter cofactor interactions. An example of the physiologic relevance of the conformational change in the protease component of the complex is the complete destruction of thrombin's exosite I in complex with serpins [56]. Thus, when PCI inhibits thrombin bound to thrombomodulin the interaction with thrombomodulin is broken, allowing the serpin–protease complex to diffuse away so that another thrombin molecule can bind [57]. Cofactor interactions Because serpin specificity is determined largely by the rate of formation of the Michaelis complex, cofactors that bind to serpins (and sometimes the protease) can radically alter specificity [51]. Table 1 presents serpin second order rates of protease inhibition in the presence and absence of relevant cofactors. The best understood cofactor for serpins is the glycosaminoglycan (GAG), heparin. It binds to and activates most of the serpins involved in hemostasis and thrombosis [58]. Acceleration of protease inhibition is generally conferred through a template effect where the protease and the serpin bind to the same heparin chain. The hypothesis is that this co-occupation will limit the diffusional freedom from three to one dimension to increase the likelihood (rate) of encounter. In addition, heparin also provides a bridge between the serpin and the protease to help stabilize the Michaelis complex. However, heparin and other GAGs are also capable in some cases of altering the conformation of the serpin to permit more rapid complexation with proteases. The best-characterized examples are AT and HCII, whose activation by heparin is the basis of its therapeutic anticoagulant effect. In the next sections we describe each of the serpins involved in hemostasis and fibrinolysis, their targets, the role of cofactors and available structural data. Serpins in hemostasis and fibrinolysis Antithrombin: SERPINC1 Antithrombin is a 58 kDa, 432 amino acid glycoprotein [59], synthesized in the liver, circulating at approximately 150 μg mL−1 with a half-life of c. 3 days [60]. It is the most important physiological inhibitor of the coagulation pathway [61]. As its name implies, AT inhibits thrombin. In addition, AT is capable of inhibiting all of the other proteolytic coagulation factors (e.g. FIXa, Xa and XIa). The predominance of its anticoagulant activity, however, is focused on the regulation of FXa, FIXa and thrombin. Measurement of thrombin–AT (TAT) complex is used as a marker of hemostatic activation and helps diagnose thrombotic events [62]. Thrombin bound to fibrin, clot-bound thrombin, is protected from inhibition by AT [63]. This may explain the occurrence of rethrombosis after fibrinolytic therapy as clot-bound thrombin is released from the dissolving hemostatic plug [64]. The anticoagulant activity of AT is dependent on its cofactor, heparin. Consisting of variably sulfated repeating disaccharide units, heparin can have a molecular weight ranging from 3 to 40 kDa [65-67]. A unique pentasaccharide sequence in heparin is responsible for the high affinity binding to AT [68]. In vivo, forms of heparin relevant to AT include heparan sulfate, found on the endothelium, and heparin released from endothelium-associated mast cell granules. The interaction of AT with heparan sulfate on the endothelium and subendothelium localizes AT activity to the vessel wall and maintains its normal, non-thrombogenic nature [60]. AT is expressed as both an α-form and a β-form. α-AT represents 90% of AT and is glycosylated at all four positions. While comprising only 10% of AT, β-AT, which is not glycosylated at one position (N135), has a higher affinity for heparin and is thought to exert an overall larger anticoagulant effect [69]. Heparin uses two distinct mechanisms to accelerate protease inhibition by AT. AT undergoes a well-characterized conformational change upon heparin binding, which expels the N-terminus of the RSL from β-sheet A (Fig. 3). This 'liberation' of the RSL is sufficient to confer the majority of the acceleration of FIXa and FXa inhibition, but thrombin inhibition is not appreciably affected. Recently, the structures of the AT-heparin-protease Michaelis complexes have been solved [54, 70] revealing the interactions behind the allosteric and template mechanisms (Fig. 3B). Figure 3Open in figure viewerPowerPoint Native and complexed serpin structures. (A) The native structures of important hemostatic and fibrinolytic serpins are shown as ribbon diagrams, colored essentially as in Fig. 2. The monomeric structure of antithrombin is shown in the left panel, and is similar to that of heparin cofactor II (HCII) with the partial insertion of the N-terminal portion of the reactive site loop (RSL). A modeled position for the N-terminal tail of HCII is shown in magenta although its true position is not known. For AT, HCII and plasminogen activator inhibitor-1 the heparin binding helix (helix D) is shown in cyan, but for protein C inhibitor (PCI) heparin binds to helix H (blue). The increased size and flexibility of the RSL of PCI is also evident from this depiction. (B) Some important serpin complexes are shown. Using S195A proteases it was possible to obtain the structures of the AT Michaelis complexes with thrombin (magenta) and FXa (magenta) with their activating synthetic heparins (SR123781 and fondaparinux, rods). Similarly, the HCII-thrombin (blue) complex was also solved. The somatomedin (SMB) domain of VN (magenta) binds to s1A and helix E to prevent the latent transition through expansion of sheet A. In addition to its anticoagulant activity, AT has been shown to have anti-inflammatory and anti-angiogenic functions. These properties are independent of AT's inhibitory activity. AT regulates inflammation by signaling through heparan sulfate on endothelial and leukocyte cell surfaces [71]. Latent and cleaved AT exert anti-angiogenic effects [72] by binding cell surface heparan sulfate. This blocks fibroblast growth factor-2 and vascular endothelial cell growth factor from forming pro-angiogenic ternary signaling complexes with their protein receptors and the heparin sulfate co-receptors [73]. Antithrombin in disease Inherited and acquired AT deficiency predisposes individuals to different degrees of thrombotic disease. The severity of thromophilia can be exacerbated by other risk factors for thrombosis. Inherited AT is classified as type I or type II. Type I deficiencies, which generally confer a higher thrombotic risk, are caused by genetic mutations that impair the synthesis and secretion of AT. Type II deficiencies are caused by genetic mutations that functionally impair AT. Variations in the degree of thrombophilia in inherited AT deficiencies can be attributed to homozygosity vs. heterozygosity and where the mutation lies in the AT structure [74]. An up-to-date database of AT mutations can be found online at http://www1.imperial.ac.uk/medicine/about/divisions/is/haemo/coag/antithrombin [75]. Some research suggests that certain mutations predispose AT to convert to its latent form, which preferentially dimerizes with native β-AT. Dimerization reduces the presence of highly active AT monomers, thus increasing thrombogenicity [76]. Concern has been raised that therapeutic preparations of AT-concentrates (contain > 10% latent AT) might have thrombotic effects. However, a recent study demonstrated that the addition of latent AT alone does not decrease the activity of AT in plasma [77]. Other mutated forms of AT do not show impaired activity or decreased AT levels in the standard hospital laboratory assays despite associated thrombophilia. In particular, the AT Cambridge II (A384S) variant was undetected by some protocols, but estimated to be the most frequent cause of AT deficiency in Caucasian populations [78]. These results suggest a need for alternative methods for detection of AT deficiencies [79]. Antithrombin-related treatments for coagulation disorders Therapeutic unfractionated heparin (UFH), derived from porcine mucosa, is one of the most commonly used anticoagulant agents administered for treatment and prophylaxis of thrombotic events. Additionally, UFH is used to coat blood collection tubes and surgical devices to prevent clotting on their surfaces. UFH's primary mechanism of action is to accelerate AT's inhibition of thrombin, FXa and FIXa. UFH also accelerates thrombin inhibition by other circulating serpins. It has a very short half-life and optimal dosing of heparin is notoriously difficult to achieve, therefore requiring frequent monitoring [80]. Still in trials, an orally available form of heparin, sodium N-(8-[2-hydroxybenzoyl] amino) caprylate bound heparin or SNAC-heparin, has dose-dependent antithrombotic effects, and has an efficacy comparable with low-molecular weight heparin in reducing venous thrombosis in patients undergoing hip replacement surgery [81]. Contra-intuitively, heparin can cause a dangerous thrombotic condition called heparin-induced thrombocytopenia (HIT). In this autoimmune reaction antibodies develop against platelets [82]. Currently in development stages, synthetic oligosaccharide heparin mimetics show thrombin and FXa inhibition comparable with UFH without inducing HIT and with far fewer side ef