An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa.

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 Trp215 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.

Intermediate molecular weight-fucosylated chondroitin sulfate from sea cucumber Cucumaria frondosa is a promising anticoagulant targeting intrinsic factor IXa.

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 (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-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 FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-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 FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.

Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Protein S antibody as an adjunct therapy for hemophilia B.

ABSTRACT: Hemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Molecular Dynamics Simulation Study of the Selective Inhibition of Coagulation Factor IXa over Factor Xa.

Thromboembolic disorders, arising from abnormal coagulation, pose a significant risk to human life in the modern world. The FDA has recently approved several anticoagulant drugs targeting factor Xa (FXa) to manage these disorders. However, these drugs have potential side effects, leading to bleeding complications in patients. To mitigate these risks, coagulation factor IXa (FIXa) has emerged as a promising target due to its selective regulation of the intrinsic pathway. Due to the high structural and functional similarities of these coagulation factors and their inhibitor binding modes, designing a selective inhibitor specifically targeting FIXa remains a challenging task. The dynamic behavior of protein-ligand interactions and their impact on selectivity were analyzed using molecular dynamics simulation, considering the availability of potent and selective compounds for both coagulation factors and the co-crystal structures of protein-ligand complexes. Throughout the simulations, we examined ligand movements in the binding site, as well as the contact frequencies and interaction fingerprints, to gain insights into selectivity. Interaction fingerprint (IFP) analysis clearly highlights the crucial role of strong H-bond formation between the ligand and D189 and A190 in the S1 subsite for FIXa selectivity, consistent with our previous study. This dynamic analysis also reveals additional FIXa-specific interactions. Additionally, the absence of polar interactions contributes to the selectivity for FXa, as observed from the dynamic profile of interactions. A contact frequency analysis of the protein-ligand complexes provides further confirmation of the selectivity criteria for FIXa and FXa, as well as criteria for binding and activity. Moreover, a ligand movement analysis reveals key interaction dynamics that highlight the tighter binding of selective ligands to the proteins compared to non-selective and inactive ligands.

Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

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) (IC50; 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 KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax 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.

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa.

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.

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa.

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.

Site-specific functional roles of the Factor X activation peptide in the intrinsic tenase-mediated Factor X activation.

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.

A Structure Based Study of Selective Inhibition of Factor IXa over Factor Xa.

Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein-ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than -8.0 kcal/mol in the FIXa-grids and higher than -7.5 kcal/mol in the FXa-grids.

Probing activation-driven changes in coagulation factor IX by mass spectrometry.

BACKGROUND: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering). OBJECTIVE: The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site. METHODS: Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop. RESULTS: HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa. CONCLUSION: In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site.

The components and activities analysis of a novel anticoagulant candidate dHG-5.

Intrinsic Xase (iXase), the last and rate-limiting enzyme complex in the intrinsic coagulation pathway, may be an ideal target for antithrombotic treatment. A depolymerized fraction of fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata, dHG-5 (Mw 5.2 kDa), showed potent and selective inhibition of iXase (IC50, 14 nM). In this work, the series of oligosaccharides contained in dHG-5 were purified and their precise structures were confirmed by 2D NMR and MS spectra. The relationships between anti-iXase, f.IXa-binding, anticoagulant and antithrombotic activities (y) and molecular weight (x) could be approximately expressed as the power function (y = a × xb), and these activity potencies of dHG-5 were approximately equivalent to the weighted average sum of that of its oligosaccharides. Given the prominent pharmacological properties, well-defined chemical composition and explicable relationships between dHG-5 and its oligosaccharides in pharmacological behaviors, dHG-5 is expected to be an ideal novel anticoagulant medicine.

Exosite binding drives substrate affinity for the activation of coagulation factor X by the intrinsic Xase complex.

Factor X activation by the intrinsic Xase complex, composed of factor IXa bound to factor VIIIa on membranes, is essential for the amplified blood coagulation response. The biological significance of this step is evident from bleeding arising from deficiencies in factors VIIIa or IXa in hemophilia. Here, we assess the mechanism(s) that enforce the distinctive specificity of intrinsic Xase for its biological substrate. Active-site function of IXa was assessed with a tripeptidyl substrate (PF-3688). The reversible S1 site binder, 4-aminobenzamidine (pAB), acted as a classical competitive inhibitor of PF-3688 cleavage by Xase. In contrast, pAB acted as a noncompetitive inhibitor of factor X activation. This disconnect between peptidyl substrate and protein substrate cleavage indicates a major role for interactions between factor X and extended sites on Xase in determining substrate affinity. Accordingly, an uncleavable factor X variant, not predicted to engage the active site of IXa within Xase, acted as a classical competitive inhibitor of factor X activation. Fluorescence studies confirmed the binding of factor X to Xase assembled with IXa with a covalently blocked active site. Our findings suggest that the recognition of factor X by the intrinsic Xase complex occurs through a multistep "dock-and-lock" pathway in which the initial interaction between factor X and intrinsic Xase occurs at exosites distant from the active site, followed by active-site docking and bond cleavage.

Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry.

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.

Discovery of hydroxy pyrimidine Factor IXa inhibitors.

The synthesis and structure activity relationship development of a pyrimidine series of heterocyclic Factor IXa inhibitors is described. Increased selectivity over Factor Xa inhibition was achieved through SAR expansion of the P1 element. Select compounds were evaluated in vivo to assess their plasma levels in rat.

Plasmin-mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer.

BACKGROUND: Factor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. OBJECTIVE: Investigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. METHODS: NH2 -terminal sequencing, mass spectrometry analysis, and functional assays. RESULTS: Plasmin in the presence of Ca2+ /phospholipid (PL) rapidly cleaved FIXaß at Lys316↓Gly317 to yield FIXaγ followed by a slow cleavage at Lys413↓Leu414 to yield FIXaδ. FIXaγ/FIXaδ migrated indistinguishably from FIXaß in nondenaturing gel system indicating that C-terminal residues 317-415/317-413 of heavy chain remain noncovalently associated with FIXaγ/FIXaδ. However, as compared with FIXaß, FIXaγ or FIXaγ/FIXaδ (25-75 mixture, 8-hour/24-hour incubation analysis by mass spectrometry) was impaired ~ 10-fold in hydrolyzing synthetic substrate CBS 31.39 (CH3-SO2-D-Leu-Gly-Arg-pNA), ~ 30-fold (~ 5-fold higher Km , ~ 6-fold lower kcat ) in activating FX in a system containing Ca2+ /PL, and ~ 650-fold in a system containing Ca2+ /PL and FVIIIa. Further, FIXaγ or FIXaγ/FIXaδ bound FVIIIa with ~ 60-fold reduced affinity compared with FIXaß. Additionally, in ligand blots, plasminogen or diisopropylfluorophosphate-inhibited plasmin (DIP-plasmin) bound FIXaγ and FIXaδ but not FIXaß. This interaction was prevented by ε-aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP-plasmin binds to FIXaγ/FIXaδ through newly generated C-terminal Lys316 and Lys413. Importantly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tissue plasminogen activator (tPA)-mediated plasminogen activation in a concentration dependent manner. Similarly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tPA-induced clot lysis in FIX-depleted plasma. CONCLUSION: Plasmin cleavage at Lys316↓Gly317 abrogates FIXaß coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer.