Fitusiran reduces bleeding in factor X-deficient mice.

ABSTRACT: Factor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 µL to 590 ± 335 µL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

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.

Functional inhibition of c-Myc using novel inhibitors identified through "hot spot" targeting.

Protein-protein interactions drive various biological processes in healthy as well as disease states. The transcription factor c-Myc plays a crucial role in maintaining cellular homeostasis, and its deregulated expression is linked to various human cancers; therefore, it can be considered a viable target for cancer therapeutics. However, the structural heterogeneity of c-Myc due to its disordered nature poses a major challenge to drug discovery. In the present study, we used an in silico alanine scanning mutagenesis approach to identify "hot spot" residues within the c-Myc/Myc-associated factor X interface, which is highly disordered and has not yet been systematically analyzed for potential small molecule binding sites. We then used the information gained from this analysis to screen potential inhibitors using a conformation ensemble approach. The fluorescence-based biophysical experiments showed that the identified hit molecules displayed noncovalent interactions with these hot spot residues, and further cell-based experiments showed substantial in vitro potency against diverse c-Myc-expressing cancer/stem cells by deregulating c-Myc activity. These biophysical and computational studies demonstrated stable binding of the hit compounds with the disordered c-Myc protein. Collectively, our data indicated effective drug targeting of the disordered c-Myc protein via the determination of hot spot residues in the c-Myc/Myc-associated factor X heterodimer.

Human coagulation factor X and CD5 antigen-like are potential new members of the zonulin family proteins.

Zonulin is a physiologic epithelial and endothelial permeability modulator. Zonulin increases antigen trafficking from the gut lumen into the bloodstream and in between body compartments, a mechanism linked to many chronic inflammatory diseases. Upon its initial discovery, it was noted that zonulin was not a single protein, but rather a family of structurally and functionally related proteins referred to as the zonulin family proteins (ZFPs). ZFPs are members of the mannose associated serine proteases (MASP) family and are the result of high mutation rates leading to many zonulin polymorphisms. Pre-haptoglobin 2, the precursor of haptoglobin 2, was identified as the first eukaryotic member of the ZFPs, and properdin, a key positive regulator of the alternative pathway, as a second member. In this study, we report two additional proteins that are likely ZFPs. Human coagulation factor X (FX) and CD5 antigen-like (CD5L). Both FX and CD5L recombinant proteins were detected by anti-zonulin antibody in Western immunoblot analysis, and both proteins decreased epithelial barrier competency of Caco-2 cell monolayers as established by the Trans Epithelial Electrical Resistance (TEER) assay. These results indicate that FX and CD5L have structural and functional similarities with previously identified ZFPs and, therefore, can be considered new members of this family of proteins.

Assembly of alternative prothrombinase by extracellular histones initiates and disseminates intravascular coagulation.

Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)- and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.

Identification of non-neutralizing anti-factor X autoantibodies in three Japanese cases of autoimmune acquired factor X deficiency.

INTRODUCTION: Autoimmune factor X (FX or F10) deficiency (AiF10D) is an extremely rare acquired haemorrhagic disorder characterized by a severe reduction in FX activity due to autoantibodies against FX. AIM: Anti-FX autoantibodies were investigated in four patients with suspected AiF10D, and their properties were analysed. METHODS AND RESULTS: Anti-FX auto antibodies in plasma were detected by ELISA with three of four cases. One case of anti-FX autoantibody negativity was later diagnosed as AL-amyloidosis. IgG1 and IgG3 coexisted in all anti-FX autoantibodies of the three patients with AiF10D (cases X1, X2, and X3). Western blot analysis showed that the antibodies were bound to the FX light chain for cases X2 and X3, but the binding was weak for case X1. When the fusion proteins of a secretory luciferase with full-length FX or its γ-carboxylated glutamic acid (Gla) domain were added to the plasma of the three patients, both fusion proteins were immunoprecipitated as antigen-antibody complexes. Contrarily, the latter fusion protein produced in the presence of warfarin demonstrated a decrease in the collection rate, suggesting that their autoantibodies recognized the light chain and regions containing Gla residues. Since all three patients were essentially negative for FX inhibitors, it was concluded that the anti-FX autoantibodies for these cases were predominantly non-neutralizing. The concentration of the FX antigen also significantly reduced in these patients, suggesting that anti-FX autoantibodies promote the clearance of FX. CONCLUSION: Immunological anti-FX autoantibody detection is highly recommended to ensure that AiF10D cases are not overlooked, and to start necessary immunosuppressive therapies.

Adenovirus serotype 5 hexon mediates liver gene transfer.

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Integrated microarray for identifying the hub mRNAs and constructed miRNA-mRNA network in coronary in-stent restenosis.

As a major complication after percutaneous coronary intervention (PCI) in patients who suffer from coronary artery disease, in-stent restenosis (ISR) poses a significant challenge for clinical management. A miRNA-mRNA regulatory network of ISR can be constructed to better reveal the occurrence of ISR. The relevant data set from the Gene Expression Omnibus (GEO) database was downloaded, and 284 differentially expressed miRNAs (DE-miRNAs) and 849 differentially expressed mRNAs (DE-mRNAs) were identified. As predicted by online tools, 65 final functional genes (FmRNAs) were overlapping DE-mRNAs and DE-miRNAs target genes. In the biological process (BP) terms of gene ontology (GO) functional analysis, the FmRNAs were mainly enriched in the cellular response to peptide, epithelial cell proliferation, and response to peptide hormone. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the FmRNAs were mainly enriched in breast cancer, endocrine resistance, and Cushing syndrome. Jun proto-oncogene, activator protein-1 (AP-1) transcription factor subunit (JUN), insulin-like growth factor 1 receptor (IGF1R), member RAS oncogene family (RAB14), specificity protein 1 (SP1), protein tyrosine phosphatase nonreceptor type 1 (PTPN1), DDB1 and CUL4 associated factor 10 (DCAF10), retinoblastoma-binding protein 5 (RBBP5), and eukaryotic initiation factor 4A-I (EIF4A1) were hub genes in the protein-protein interaction network (PPI network). The miRNA-mRNA network containing DE-miRNAs and hub genes was built. Hsa-miR-139-5p-JUN, hsa-miR-324-5p-SP1 axis pairs were found in the miRNA-mRNA network, which could promote ISR development. The aforementioned results indicate that the miRNA-mRNA network constructed in ISR has a regulatory role in the development of ISR and may provide new approaches for clinical treatment and experimental development.

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.

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.

Structure and function of factor X: properties, activation, and activity in prothrombinase. A retrospective in a historical context.

The evolution of our understanding of the formation of thrombin from the postulated thrombokinase of Morawitz to activated Factor X and prothrombinase occurred during a period of nearly 100 years. During this time structure-function relationships have emerged and the roles of phospholipid surfaces, the accessory factor, Factor V and its activated form have been clarified. This paper summarizes this story with particular acknowledgement of the seminal contributions of Haskell Milstone.

Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX ( n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 × 1013 vg boluses. AAV5-hFX animals ( n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients ( n = 3; 2.56 × 1013 vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX ( n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.-Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Direct Amplification of Tissue Factor:Factor VIIa Procoagulant Activity by Bile Acids Drives Intrahepatic Coagulation.

OBJECTIVE: Regulation of TF (tissue factor):FVIIa (coagulation factor VIIa) complex procoagulant activity is especially critical in tissues where plasma can contact TF-expressing cells. One example is the liver, where hepatocytes are routinely exposed to plasma because of the fenestrated sinusoidal endothelium. Although liver-associated TF contributes to coagulation, the mechanisms controlling the TF:FVIIa complex activity in this tissue are not known. Approach and Results: Common bile duct ligation in mice triggered rapid hepatocyte TF-dependent intrahepatic coagulation coincident with increased plasma bile acids, which occurred at a time before observable liver damage. Similarly, plasma TAT (thrombin-antithrombin) levels increased in cholestatic patients without concurrent hepatocellular injury. Pathologically relevant concentrations of the bile acid glycochenodeoxycholic acid rapidly increased hepatocyte TF-dependent procoagulant activity in vitro, independent of de novo TF synthesis and necrotic or apoptotic cell death. Glycochenodeoxycholic acid increased hepatocyte TF activity even in the presence of the phosphatidylserine-blocking protein lactadherin. Interestingly, glycochenodeoxycholic acid and taurochenodeoxycholic acid increased the procoagulant activity of the TF:FVIIa complex relipidated in unilamellar phosphatidylcholine vesicles, which was linked to an apparent decrease in the Km for FX (coagulation factor X). Notably, the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a bile acid structural analog, did not increase relipidated TF:FVIIa activity. Bile acids directly enhanced factor X activation by recombinant soluble TF:FVIIa complex but had no effect on FVIIa alone. CONCLUSIONS: The results indicate that bile acids directly accelerate TF:FVIIa-driven coagulation reactions, suggesting a novel mechanism whereby elevation in a physiological mediator can directly increase TF:FVIIa procoagulant activity.

A candidate activation pathway for coagulation factor VII.

The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway.

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f10-/- zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f10-/- mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions.

Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 µM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K-dependent proteins was observed. Short hairpin RNA-mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI-deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti-vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.

Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII?

During the last decade, the development of improved and novel approaches for the treatment of hemophilia A has expanded tremendously. These approaches include factor VIII (FVIII) with extended half-life (eg, FVIII-Fc and PEGylated FVIII), monoclonal antibodies targeting tissue factor pathway inhibitor, small interfering RNA to reduce antithrombin expression and the bispecific antibody ACE910/emicizumab. Emicizumab is a bispecific antibody recognizing both the enzyme factor IXa and the substrate factor X. By simultaneously binding enzyme and substrate, emicizumab mimics some part of the function exerted by the original cofactor, FVIII, in that it promotes colocalization of the enzyme-substrate complex. However, FVIII and the bispecific antibody are fundamentally different proteins and subject to different modes of regulation. Here, we will provide an overview of the similarities and dissimilarities between FVIII and emicizumab from a biochemical and mechanistical perspective. Such insight might be useful in the clinical decision making for those who apply emicizumab in their practice now or in the future, particularly in view of the thrombotic complications that have been reported when emicizumab is used in combination with FVIII-bypassing agents.

Missense changes in the catalytic domain of coagulation factor X account for minimal function preventing a perinatal lethal condition.

INTRODUCTION: Inherited deficiencies in the coagulation pathway provide diversified models to investigate the molecular bases of perinatal lethality associated with null-like variants. Differently from X-linked haemophilias, homozygous/doubly heterozygous null variants in the rare autosomally inherited deficiency of factor X (FX) might be incompatible with perinatal survival. AIM: To provide experimental evidence about the null/close-to-null FX function. METHODS: The residual secreted (ELISA) and functional (thrombin generation assays) protein levels associated with the novel nonsense (c.1382G>A; p.Trp461Ter) and missense (c.752T>C; p.Leu251Pro) variants, found in the proposita with life-threatening symptoms at birth, were characterized through recombinant (r)FX expression. RESULTS: The rFX-461Ter showed very low secretion and undetectable function. Expression and function of the predicted readthrough-deriving missense variants (rFX-461Tyr, rFX-461Gln) were also severely impaired. These unfavourable features, due to nucleotide and protein sequence constraints, precluded functional readthrough over the 461 stop codon. Differently, the poorly secreted rFX-251Pro variant displayed residual function that was characterized by anti-TFPI aptamer-based amplification or selective inhibition of activated FX function by fondaparinux in plasma and found to be reduced by approximately three orders of magnitude. Similarly to the rFX-251Pro, a group of catalytic domain missense variants cause poorly secreted molecules with modest function in FX-deficient patients with life-threatening symptoms. CONCLUSIONS: Our data, contributing to the knowledge of the very severe FX deficiency forms, support life-saving requirement of trace FX function, clearly exemplified by the dysfunctional but not completely inactive rFX-251Pro variant that, albeit with severely reduced function, is compatible with a residual activity ensuring minimal haemostasis and permitting perinatal survival.

Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis.

Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors. This review will focus on the roles of TF in protective hemostasis and pathological thrombosis.