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Introduction: Antithrombin, the principal inhibitor of coagulation proteases, requires allosteric activation by its physiological cofactor, heparin or heparin sulfate to achieve physiologically permissible rates. This forms the basis of heparin's use as a clinical anticoagulant. However, heparin therapy is beset with severe complications, giving rise to the need to search new non-heparin activators of antithrombin, devoid of these complications and with favorable safety profiles. Materials and Methods: We chose some representative organic compounds that have been shown to be involved in coagulation modulation by affecting antithrombin and applied a blind docking protocol to find the binding energy and interactions of the modified (sulfated) versus unmodified organic scaffolds. Results and Conclusion: Increased sulfation plays a key role in shifting the specificity of organic compounds like quercetin, diosmin, rutin, mangiferin, isomangostin, Trapezifolixanthone and benzofuran towards the heparin binding site (HBS). However, in hesperetin and tetrahydroisoquinoline, sulfation shifts the specificity away from HBS. We have further tried to elucidate changes in the binding affinity of quercetin on account of gradual increase in the number of hydroxyl groups being substituted by sulfate groups. The results show gradual increase in binding energy with increase in sulfation. A theoretical screening approach is an ideal mechanism to predict lead molecules as activators of antithrombin and in determining the specificity for antithrombin. [ABSTRACT FROM AUTHOR]

The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A β-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17 A away from the RCL insertion. Crystallographic structures show that the Tyr131 ring is buried in native ATIII and then becomes exposed when pentasaccharide binds to the inhibitor and activates it. This change suggested that Tyr131 might serve as a switch for ATIII conformational activation. The hypothesis is supported by results from this study, which progressively removed atoms from the Tyr131 side chain. Rates of heparin-independent Y131L and Y131A factor Xa inhibition were 25 and 29 times faster than for the control and Y131F, suggesting that Tyr131 ring interactions with neighboring helix D and strand 2A residues shift the uncatalyzed native-to-activated conformational equilibrium toward the RCL-inserted state. Thermal denaturation experiments showed Y131A and Y131L were less stable than the control and Y131F, implying an increased tendency toward A-sheet mobility in these genetically activated molecules. Thus, the tight Tyr131-Asn127-Leu130-Leu140-Ser142 cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anti-coagulant activity. [ABSTRACT FROM AUTHOR]

Addresses the roles of antithrombin III phenylalanines 121 and 122 in heparin binding and activation. Proximity of phenlyalanines to positively charged residues of the pentasaccharide binding site; Role in heparin binding and activation of antithrombin by coordinating the P-helix-mediated conformational change; Organization of a network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 μg/ml) showed a 2–4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates. [ABSTRACT FROM AUTHOR]

Abstract Currently available anticoagulants for prevention and treatment of thrombosis have several limitations, thus, small organic scaffolds that can dissolve clots in vivo in a dose dependent manner with lesser side effects are highly desirable. Here we report the synthesis of esculin pentasulfate (EPS) and assessment of its in vitro , in vivo and ex vivo anticoagulant and antithrombotic potential. Assessment of in vitro clotting times showed prolonged activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) in the presence of EPS. EPS also showed remarkable reduction in thrombus formation when administered in occlusion induced thrombotic rats at a low dose (2.5 mg/kg). Further, assessment of clot rate with plasma isolated from EPS treated rats confirmed its anticoagulation potential. EPS at varying concentrations showed no significant cytotoxic effect on HEK293 cell line. Further, molecular docking analysis of EPS with known anticoagulant proteins [(antithrombin (ATIII) and heparin cofactor II (HCF II)] that require heparin revealed good binding affinity (−7.9 kcal/mol) with ATIII but not with HCF II. ATIII when incubated with EPS showed increased fluorescence intensity, with no change in secondary structure. Overall, our results clearly show the in vivo modulation of thrombus formation using a modified natural scaffold EPS. [ABSTRACT FROM AUTHOR]

Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 β-sheets and 7–9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis. [ABSTRACT FROM AUTHOR]

Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell. [ABSTRACT FROM AUTHOR]