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Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp 215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain., Competing Interests: Competing interests statement:Duke University has issued patents on the anti-FIXa aptamer used in the manuscript. B.A.S. is listed as an inventor on these patents. Other authors have no competing interests.

Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCS cf ) and depolymerized into a series of lower molecular weights (FCS cf s). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCS cf s with different molecule weights demonstrated that 10 kDa FCS cf (FCS cf -10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCS cf -10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCS cf -10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCS cf -10 K is a promising candidate for the development of novel anticoagulant drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa., Aim: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798., Methods and Results: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC 50 ; 19.2 and 42.9 μM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater K D compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the K m by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the K m was increased by 3.4-fold and the V max was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction., Conclusion: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masahiro Takeyama reports financial support was provided by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Keiji Nogami reports financial support was provided by Japan Agency for Medical Research and Development (AMED). Keiji Nogami reports financial support was provided by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits., (© 2023 by The American Society of Hematology.)

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52-residue long activation peptide (AP). Through site-directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn-39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A non-negligible proportion of human pathogenic variants are known to be present as wild type in at least some non-human mammalian species. The standard explanation for this finding is that molecular mechanisms of compensatory epistasis can alleviate the mutations' otherwise pathogenic effects. Examples of compensated variants have been described in the literature but the interacting residue(s) postulated to play a compensatory role have rarely been ascertained. In this study, the examination of five human X-chromosomally encoded proteins (FIX, GLA, HPRT1, NDP and OTC) allowed us to identify several candidate compensated variants. Strong evidence for a compensated/compensatory pair of amino acids in the coagulation FIXa protein (involving residues 270 and 271) was found in a variety of mammalian species. Both amino acid residues are located within the 60-loop, spatially close to the 39-loop that performs a key role in coagulation serine proteases. To understand the nature of the underlying interactions, molecular dynamics simulations were performed. The predicted conformational change in the 39-loop consequent to the Glu270Lys substitution (associated with hemophilia B) appears to impair the protein's interaction with its substrate but, importantly, such steric hindrance is largely mitigated in those proteins that carry the compensatory residue (Pro271) at the neighboring amino acid position., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII., Competing Interests: None declared., (Thieme. All rights reserved.)

The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system., (© 2020 by The American Society of Hematology.)

COVID-19 can be associated with coagulopathy (CAC, COVID-19-associated coagulopathy) with a high prothrombotic risk based on an intense inflammatory response to viral infection leading to immunothrombosis through different procoagulant pathways. Emerging evidence suggests that the use of heparin in these patients could be associated with lower mortality. Emicizumab is a bispecific humanized monoclonal antibody that bridges activated factor IX and factor X, thereby restoring the function of missing factor VIIIa in hemophilia A. The use of emicizumab has been associated with thrombotic events in patients who also received high cumulative amounts of activated prothrombin complex concentrates. Although this risk is extremely low, there is a lack of evidence on whether CAC increases the thrombotic risk in patients on emicizumab prophylaxis. We present the case of a patient with severe hemophilia A in prophylaxis treatment with emicizumab; due to the potential thrombotic risk we decided to administer low molecular weight heparin as prophylaxis treatment without any thrombotic or bleeding complications., (© 2020 International Society on Thrombosis and Haemostasis.)

Blood coagulation involves extrinsic and intrinsic pathways, which merge at the activation step of blood coagulation factor X to factor Xa. This step is catalysed by the extrinsic or intrinsic Xase, which consists of a complex of factor VIIa and its cofactor tissue factor or factor IXa (FIXa) and its cofactor coagulation factor VIIIa (FVIIIa). Upon complex formation with FVIIIa, FIXa is conformationally activated to the Xase complex. However, the mechanistic understanding of this molecular recognition is limited. Here, we examined FVIIIa-FIXa binding in the context of FIXa's activation status. Given the complexity and the labile nature of FVIIIa, we decided to employ two FVIII-derived peptides (558-loop, a2 peptide) to model the cofactor binding of FIX(a) using biosensor chip technology. These two FVIII peptides are known to mediate the key interactions between FVIIIa and FIXa. We found both of these cofactor mimetics as well as full-length FVIIIa bind more tightly to zymogenic FIX than to proteolytically activated FIXa. Consequently and surprisingly, we observed that the catalytically inactive FIX zymogen can outcompete the activated FIXa from the complex with FVIIIa, resulting in an inactive, zymogenic Xase complex. By contrast, the thrombophilic Padua mutant FIXa-R170 in complex with the protein-substrate analogue BPTI bound tighter to FVIIIa than to the zymogen form FIX-R170L, suggesting that the active Xase complex preferentially forms in the Padua variant. Together, these results provide a mechanistic basis for the thrombophilic nature of the FIX-R170L mutant and suggest the existence of a newly discovered safety measure within the coagulation cascade., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Essentials Consensus sequence and biochemical data suggest a Na + -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37 Å reveals a Na + -site not observed earlier. Molecular dynamics simulations data support that Na +  ± Ca 2+ promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na + -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na + -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na + ; perform molecular dynamics (MD) simulations to explore the role of Na + in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-Å resolution revealed that Na + is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na + -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na + reduced fluctuations in residues 217-225 (Na + -loop) and 70-80 (Ca 2+ -loop), whereas Ca 2+ reduced fluctuations only in residues of the Ca 2+ -loop. Ca 2+ and Na + together reduced fluctuations in residues of the Ca 2+ -loop and Na + -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na + -site in association with Ca 2+ contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin., (© 2019 International Society on Thrombosis and Haemostasis.)

Human Coagulation Factor IXa (FIXa), specifically inhibited at the initiation stage of the blood coagulation cascade, is an excellent target for developing selective and safe anticoagulants. To explore this inhibitory mechanism, 86 FIXa inhibitors were selected to generate pharmacophore models and subsequently SAR models. Both best pharmacophore model and ROC curve were built through the Receptor-Ligand Pharmacophore Generation module. CoMFA model based on molecular docking and PLS factor analysis methods were developed. Model propagations values are q 2  = 0.709, r 2  = 0.949, and r 2 pred  = 0.905. The satisfactory q 2 value of 0.609, r 2 value of 0.962, and r 2 pred value of 0.819 for CoMSIA indicated that the CoMFA and CoMSIA models are both available to predict the inhibitory activity on FIXa. On the basis of pharmacophore modeling, molecular docking, and 3D-QSAR modeling screening, six molecules are screened as potential FIXa inhibitors.

Introduction: The thrombin generation test (TGT) is used both as a global haemostasis assay, and to compare activities of coagulation factor concentrates that have been spiked into patient plasma. However, TGT has not been systematically optimized to evaluate factor VIII (FVIII) product potency., Aims: To improve the sensitivity of TGT to FVIII and allow a comparative analysis of the thrombin generating capacities of FVIII concentrates against reference preparations with known FVIII activity., Methods: Concentrations of TGT components (analytical variables) were assessed to maximize the linearity and range of responses to the concentration of FVIII., Results: We optimized the range and sensitivity of the TGT assay with respect to FVIII through the addition of FXIa to the assay. Other parameters that were adjusted, i.e. tissue factor (TF), procoagulant lipids and plasma concentrations, did not improve the ability of the assay to measure both high and very low levels of FVIII. In the optimized TF/FXIa-activated TGT assay, all thrombin generation curve parameters were suitable for FVIII quantification, but thrombin peak height and maximal velocity demonstrated better linearity in the desired FVIII range. We found that the optimized TF/FXIa-activated TGT has a wider range of sensitivity to FVIII than a commercially available TGT. Additionally, we demonstrated that the TF/FXIa-activated assay performs adequately by comparing potency measurements of five commercially available FVIII products using TGT and traditional chromogenic and one-stage clotting assays., Conclusions: The optimized TGT assay can be used to quantify and compare the thrombin generating capacities of FVIII concentrates., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Factor IXa (activated Factor IX) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of Factor IXa, its substrate Factor X and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by Factor IXa is enhanced 200000-fold; however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood. A multifaceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity-enhanced Factor IXa variants has demonstrated a delicately balanced bidirectional network. Essential molecular interactions across multiple regions of the Factor IXa molecule co-operate in the maturation of the active site. This maturation is specifically facilitated by long-range communication through the Ile(212)-Ile(213) motif unique to Factor IXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys(168)-Cys(182) disulfide bond. Ultimately, the network consists of compensatory brakes (Val(16) and Ile(213)) and accelerators (Tyr(99) and Phe(174)) that together allow for a subtle fine-tuning of enzymatic activity., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The biophysics of blood flow can dictate the function of molecules and cells in the vasculature with consequent effects on hemostasis, thrombosis, embolism, and fibrinolysis. Flow and transport dynamics are distinct for (i) hemostasis vs. thrombosis and (ii) venous vs. arterial episodes. Intraclot transport changes dramatically the moment hemostasis is achieved or the moment a thrombus becomes fully occlusive. With platelet concentrations that are 50- to 200-fold greater than platelet-rich plasma, clots formed under flow have a different composition and structure compared with blood clotted statically in a tube. The platelet-rich, core/shell architecture is a prominent feature of self-limiting hemostatic clots formed under flow. Importantly, a critical threshold concentration of surface tissue factor is required for fibrin generation under flow. Once initiated by wall-derived tissue factor, thrombin generation and its spatial propagation within a clot can be modulated by γ'-fibrinogen incorporated into fibrin, engageability of activated factor (FIXa)/activated FVIIIa tenase within the clot, platelet-derived polyphosphate, transclot permeation, and reduction of porosity via platelet retraction. Fibrin imparts tremendous strength to a thrombus to resist embolism up to wall shear stresses of 2400 dyne cm(-2) . Extreme flows, as found in severe vessel stenosis or in mechanical assist devices, can cause von Willebrand factor self-association into massive fibers along with shear-induced platelet activation. Pathological von Willebrand factor fibers are A Disintegrin And Metalloprotease with ThromboSpondin-1 domain 13 resistant but are a substrate for fibrin generation due to FXIIa capture. Recently, microfluidic technologies have enhanced the ability to interrogate blood in the context of stenotic flows, acquired von Willebrand disease, hemophilia, traumatic bleeding, and drug action., (© 2016 International Society on Thrombosis and Haemostasis.)

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The coagulopathy in cirrhosis is associated with thrombosis and bleeding., Objectives: To gain better insights into the coagulopathy in patients with cirrhosis, we evaluated plasma thrombin generation and whole blood clot formation in a cross-sectional study., Methods: Blood was collected from 73 patients with all-cause cirrhosis (Child-Pugh-A n = 52, B n = 15, C n = 6) and 20 healthy controls. Activity of the coagulation pathways was measured with assays for factor (F) VIIa and FIXa-antithrombin and FXa-antithrombin complexes, respectively. Thrombin generation by calibrated automated thrombography was determined in platelet-poor plasma using a 1 or 5 pm tissue factor trigger with/without thrombomodulin. ROTEM measurements were performed in whole blood triggered with 35 pm tissue factor without/with 175 ng mL(-1) tissue plasminogen activator (the latter refered to as 'tPA-ROTEM')., Results: We observed an increased generation of FVIIa and a moderately elevated amount of FIXa (in complex with antithrombin) without apparent increase in FX activation in patients with cirrhosis. In accordance with this prothrombotic state, markers of thrombin generation potential were also increased upon increasing severity of cirrhosis. In the whole blood clotting assay we observed delayed clot formation and decreased clot strength associated with increased severity of cirrhosis. No significant differences were found for tPA-ROTEM parameters of clot degradation., Conclusion: These results indicate that cirrhosis patients have an overall procoagulant plasma milieu but a decreased whole blood clot formation capacity with an apparently unaltered resistance to clot lysis., (© 2014 International Society on Thrombosis and Haemostasis.)

Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein-protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293-Lys301 and Asp332-Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3-C2 domain interface of FVIIIa., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131-136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and (1)H NMR spectra. Heparin binding gives only ∼3-4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIIIC2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (~11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIIIC2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (~2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIIIC2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIIIC2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (~10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIIIC2C2 variant (~4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIIIC2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.

Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 ± 1.6 nm) that was similar to the A3C1C2 subunit (Kd = 3.6 ± 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. Vmax values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent Kd relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys(707), Asp(712), and Lys(713) demonstrated the greatest increases in apparent Kd (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.

The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.

Factor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10-fold and the catalytic efficiency of the mutant protease-FVIIIa-PCPS complex in the activation of FX has been decreased ~100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Å from 75 ± 2 Å for FIXa to 65 ± 2 Å for FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic tenase complex.

An aptamer targeting factor IXa has been evaluated in animal models and several clinical studies as a potential antithombotic therapy. We elucidate the molecular mechanism by which this aptamer acts as an anticoagulant. The aptamer binds tightly to factor IXa and prolongs the clotting time of human plasma. The aptamer completely blocks factor IXa activation of factor X regardless of the presence of factor VIIIa. However, the aptamer does not completely block small synthetic substrate cleavage, although it does slow the rate of cleavage. These data are consistent with the aptamer binding to the catalytic domain of factor IXa in such a way as to block an extended substrate-binding site. Therefore, unlike small molecule inhibitors, aptamers appear to be able to bind surfaces surrounding an active site and thereby sterically interfere with enzyme activity. Thus, aptamers may be useful agents to probe and block substrate-binding sites outside of the active site of an enzyme.

Background: A key feature of factor IXa is its allosteric transformation from an enzymatically latent form into a potent procoagulant. Although several small molecules have been found to be capable of partially affecting FIXa function (i.e. ethylene glycol, Ca(2+), and low molecular weight heparin [LMWH]), the resulting modest changes in peptidolytic activity have made the study of their mechanisms of action challenging. As these effects provide hints about potential regulatory forces that may be operational in the full expression of FIXa coagulant activity, their description remains of great interest. Studies of crystal structures have yielded insights into the structural changes induced by these effectors, but there remains a paucity of information to correlate any given structural change with specific consequences for FIXa function., Objectives: To correlate structural changes induced by these modulators with defined consequences for FIXa substrate discrimination and function., Methods: A peptidomics-based mass spectrometry (MS) approach was used to examine the patterns of hydrolysis of four combinatorial chemistry-derived pentapeptide libraries by FIXa under various conditions in a soluble, active enzyme system., Results: Ethylene glycol specifically altered the S3 subsite of FIXa to render it more tolerant to side chains at the P3 substrate position, whereas Ca(2+) enhanced tolerance at the S2 subsite. In contrast, LMWH altered both the S2 and S1' subsites., Conclusions: These results demonstrate the role of plasticity in regulating FIXa function with respect to discrimination of extended substrate sequences, as well as providing crucial insights into active site modulations that may be capitalized on by various physiologic cofactors of FIXa and in future drug design., (© 2011 International Society on Thrombosis and Haemostasis.)

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 domain (residues 2228-2240) interacts with the FIXa Gla domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228-2240. The peptide 2228-2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804-1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228-2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228-2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 domain (residues 2228-2240) with the FIXa Gla domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.

Treatment of acute ischemic stroke with intravenous tissue-type plasminogen activator is underutilized partly due to the risk of life-threatening hemorrhage. In response to the clinical need for safer stroke therapy, we explored using an aptamer-based therapeutic strategy to promote cerebral reperfusion in a murine model of ischemic stroke. Aptamers are nucleic acid ligands that bind to their targets with high affinity and specificity, and can be rapidly reversed with an antidote. Here we show that a Factor IXa aptamer administered intravenously after 60 minutes of cerebral ischemia and reperfusion improved neurological function and was associated with reduced thrombin generation and decreased inflammation. Moreover, when the aptamer was administered in the setting of intracranial hemorrhage, treatment with its specific antidote reduced hematoma volume and improved survival. The ability to rapidly reverse a pharmacologic agent that improves neurological function after ischemic stroke should intracranial hemorrhage arise indicates that aptamer-antidote pairs may represent a novel, safer approach to treatment of stroke.

Supersulfated low molecular weight heparin (ssLMWH) inhibits the intrinsic tenase (factor IXa-factor VIIIa) complex in an antithrombin-independent manner. Recombinant factor IXa with alanine substitutions in the protease domain (K126A, N129A, K132A, R165A, R170A, N178A, R233A) was assessed with regard to heparin affinity in solution and ability to regulate protease activity within the factor IXa-phospholipid (PL) and intrinsic tenase complexes. In a soluble binding assay, factor IXa K126A, K132A, and R233A dramatically (10-20-fold) reduced ssLMWH affinity, while factor IXa N129A and R165A moderately (5-fold) reduced affinity relative to wild type. In the factor IXa-PL complex, binding affinity for ssLMWH was increased 4-fold, and factor X activation was inhibited with a potency 7-fold higher than predicted for wild-type protease-ssLMWH affinity in solution. In the intrinsic tenase complex, ssLMWH inhibited factor X activation with a 4-fold decrease in potency relative to wild-type factor IXa-PL. The mutations increased resistance to inhibition by ssLMWH in a similar fashion for both enzyme complexes (R233A > K126A > K132A/R165A > N129A/N178A/wild type) except for factor IXa R170A. This protease had ssLMWH affinity and potency for the factor IXa-PL complex similar to wild-type protease but was moderately resistant (6-fold) to inhibition in the intrinsic tenase complex based on increased cofactor affinity. These results are consistent with conformational regulation of the heparin-binding exosite and macromolecular substrate catalysis by factor IXa. An extensive overlap exists between the heparin and factor VIIIa binding sites on the protease domain, with residues K126 and R233 dominating the heparin interaction and R165 dominating the cofactor interaction.

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited approximately 5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with approximately 4-fold decreased k(cat) and approximately 2-fold decreased K(m), although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the extrinsic pathway, leading to an optimum combination of efficacy and safety with low incidence of bleeding. We have identified 2-amindinobenzothiophene template as a lead scaffold for FIXa inhibiton based on its homology with urokinase plasminogen activator (uPA). Subsequent SAR work on the template revealed a number of highly potent FIXa inhibitors, though with moderate selectivity against FXa. X-ray study with one of the analogues demonstrated active site binding interaction with the induced opening of the S1 beta pocket and a secondary binding at the S2-S4 sites, which is in direct contrast with the previous finding.

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-A 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 beta-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.

Human coagulation factor IX serves both to maintain and to control blood coagulation. The dual function of this hemophilic factor is implemented by a tiered activation mechanism. Processed two-chain factor IXa is catalytically silent; only together with its cofactor VIIIa does factor IXa form the highly potent Xase complex. The detailed mechanism of this secondary activation has remained elusive so far. Here we present the crystal structures of Xase-like factor IXa mutants with several-thousand-fold activity enhancement that mimic the secondary activation by Xase formation. The structures reveal how cofactor-triggered and substrate-assisted modulations in the factor IXa 99- and 60-loops cooperate in S4 through S2' formation, allowing for productive substrate recognition. We could further physically map and visualize a distinct communication line, along which agonists such as Ca(2+) direct their effects to the active site and vice versa.

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cell-expressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K(d) for A2. The second assay determined the K(d) for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K(d) and k(cat) values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K(d) values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K(d) for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k(cat) for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC(50)) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mechanism. In the presence of excess TF, the EC(50) for DHG was increased 13- to 27-fold, suggesting inhibition was dependent on intrinsic tenase (factor IXa-factor VIIIa) components. In factor VIII-deficient plasma supplemented with 700 pM factor VIII or VIIIa, and factor IX-deficient plasma supplemented with plasma-derived factor IX or 100 pM factor IXa, the EC(50) for DHG was similar. Thus, cofactor and zymogen activation did not contribute to DHG inhibition of thrombin generation. Factor IX-deficient plasma supplemented with mutant factor IX(a) proteins demonstrated resistance to DHG inhibition of thrombin generation [factor IX(a) R233A > R170A > WT] that inversely correlated with protease-heparin affinity. These results replicate the effect of these mutations with purified intrinsic tenase components, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition by DHG. Glycosaminoglycan-mediated intrinsic tenase inhibition is a novel antithrombotic mechanism with physiologic and therapeutic applications.

Blood haemostasis is accomplished by a complex network of coagulatory and fibrinolytic processes. These processes have to be delicately balanced, as clinically manifested by bleeding disorders, such as haemophilia A and B. These disorders are caused by defects in coagulation factor VIII and factor IX, respectively. Following a dual strategy, we emphasise on the one hand principles conserved in most coagulation enzymes, thus mirroring much of the underlying complexity in haemostasis; on the other hand, we identify enzymatic properties of the factor IXa-factor VIIIa system (Xase) that distinguish this proteolytic machine from other components of the coagulation system. While the exact mechanisms of its activity modulation remain baffling until today, superactive factor IX mutants significantly improve our current understanding and serve as a specific and testable model of Xase action.

We have recently reported that plasmin likely associates with the factor VIII light chain to proteolyze at Lys36 within the A1 domain. In this study, we determined that the rate of plasmin-catalyzed inactivation on the forms of factor VIIIa containing A1-(1-336) and 1722A3C1C2, reflecting Lys36 cleavage, was reduced by approximately 60%, compared with those containing 1649A3C1C2 and 1690A3C1C2. SDS-PAGE analysis revealed that Lys36 cleavage of factor VIIIa with 1722A3C1C2 was markedly slower than those with 1649A3C1C2 and 1690A3C1C2. Surface plasmon resonance-based assays, using active site-modified anhydro-plasmin (Ah-plasmin) showed that 1722A3C1C2 bound to Ah-plasmin with an approximately 3-fold lower affinity than 1649A3C1C2 or 1690A3C1C2 (Kd, 176, 68.2, and 60.3 nM, respectively). Recombinant A3 bound to Ah-plasmin (Kd, 44.2 nM), whereas C2 failed to bind, confirming the presence of a plasmin-binding site within N terminus of A3. Furthermore, the Glu-Gly-Arg active site-modified factor IXa also blocked 1722A3C1C2 binding to Ah-plasmin by approximately 95%, supporting the presence of another plasmin-binding site overlapping the factor IXa-binding site in A3. In keeping with a major contribution of the lysine-binding sites in plasmin for interaction with the factor VIII light chain, analysis of the A3 sequence revealed two regions involving clustered lysine residues in 1690-1705 and 1804-1818. Two peptides based on these regions blocked 1649A3C1C2 binding to Ah-plasmin by approximately 60% and plasmin-catalyzed Lys36 cleavage of factor VIIIa with A1-(1-336) by approximately 80%. Our findings indicate that an extended surface, centered on residues 1690-1705 and 1804-1818 within the A3 domain, contributes to a unique plasmin-interactive site that promotes plasmin docking during cofactor inactivation by cleavage at Lys36.

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by approximately 95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and factor IXa when the serpin is conformationally activated by heparin. Here we show that functional exosites can be engineered at homologous positions in a P1 Arg variant of the serpin alpha1-proteinase inhibitor (alpha1PI) that does not require heparin for activation. The combined effect of the two exosites increased the association rate constant for the reactions of alpha1PI with factors Xa and IXa 11-14-fold, comparable with their rate-enhancing effects on the reactions of heparin-activated antithrombin with these proteases. The effects of the engineered exosites were specific, alpha1PI inhibitor reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abrogated the ability of the engineered exosites in alpha1PI to promote factor Xa inhibition. Binding studies showed that the exosites enhance the Michaelis complex interaction of alpha1PI with S195A factor Xa as they do with the heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP reactive loop residues in the alpha1PI exosite variant with a preferred IEG substrate sequence for factor Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by approximately 2-fold but greatly increased the selectivity of alpha1PI for inhibiting factor Xa over thrombin by approximately 1000-fold. Together, these results show that a specific and selective inhibitor of factor Xa can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a serpin.

The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the protease-factor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4- to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2- to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl(3)-induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

The drawbacks and limitations of existing anticoagulant therapy which may result in serious adverse effects and a high mortality rate, have given rise to many anticoagulant development programmes in the last decade, focusing mainly at development of thrombin and FXa low-molecular weight inhibitors. A detailed understanding of blood coagulation pathways, functioning of the serine proteases thrombin, FXa, FVIIa and FIXa and elucidation of their crystal structures resulted in many potent compounds, among which some have entered the clinical phase or have been approved for use in clinical practice. Recently, the focus of anticoagulant research turned to inhibition of the TF:FVIIa complex, with some promising clinical candidates on the horizon. This article provides an overview of the current development status of serine protease inhibitors as anticoagulants, including new trends such as dual coagulation factor inhibitors.

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.

Sodium plays an important role in modulating both the amidolytic and proteolytic activities of thrombin. By contrast, while the optimal amidolytic activity of factor Xa requires Na(+), the proteolytic activity of factor Xa in the prothrombinase complex is minimally affected by the monovalent cation. In this study, we analyzed the effect of Na(+) on the amidolytic and proteolytic activity of factor IXa in the absence and presence of factor VIIIa. Factor IXa exhibited normal activity towards a fIXa-specific chromogenic substrate and antithrombin in the presence of physiological concentrations of Ca(2+) with no obvious requirement for Na(+) in either reaction. Further studies revealed that factor IXa binds to its cofactor factor VIIIa with a normal affinity in the absence of Na(+) and that the catalytic function in the intrinsic Xase complex is also independent of Na(+) in the presence of physiological concentrations of Ca(2+). These results suggest that unlike the important role that Na(+) plays in modulating the macromolecular substrate specificity of thrombin, the monovalent cation is not required for the physiological function of factor Ixa in the intrinsic Xase complex.

Synthesis and biological data of a novel selective and efficacious factor IXa inhibitor are described along with its crystal structure in factor VIIa.