RESUMEN
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.
Asunto(s)
Factor IXa , Hemofilia B , Animales , Ratones , Factor IXa/química , Coagulación Sanguínea , Anticoagulantes/uso terapéutico , Pruebas de Coagulación Sanguínea , Hemofilia B/genética , Antitrombina III/metabolismoRESUMEN
Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in â¼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.
Asunto(s)
Linfocitos T CD4-Positivos , Factor VIII , Hemofilia A , Hemostáticos , Animales , Ratones , Células Dendríticas/metabolismo , Factor VIII/inmunología , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemostáticos/inmunología , Hemostáticos/uso terapéutico , Ovalbúmina/inmunologíaRESUMEN
Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg306, Arg506, and Arg679). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963-1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg506. Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg506 and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg506. We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation.
Asunto(s)
Factor V , Proteína C , Factor V/genética , Factor V/metabolismo , Proteína C/genética , Proteína C/metabolismo , Anticoagulantes , Péptido Hidrolasas , Factor Va/genética , Factor Va/metabolismo , Trombina/metabolismoRESUMEN
Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.
Asunto(s)
Factor VIII/metabolismo , Hemofilia A/patología , Hemostasis , Proteína C/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Cloruros/toxicidad , Factor VIII/genética , Femenino , Compuestos Férricos/toxicidad , Hemofilia A/inducido químicamente , Hemofilia A/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína C/genética , Proteínas Recombinantes/genéticaRESUMEN
Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd â¼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.
Asunto(s)
Factor V/química , Factor Va/química , Lipoproteínas/química , Trombina/genética , Coagulación Sanguínea/genética , Factor V/genética , Factor Va/genética , Factor Xa/química , Factor Xa/genética , Humanos , Ligandos , Lipoproteínas/genética , Unión Proteica/genética , Dominios Proteicos/genética , Proteolisis/efectos de los fármacos , Trombina/química , Tromboplastina/química , Tromboplastina/genéticaRESUMEN
Blood coagulation factor X/Xa sits at a pivotal point in the coagulation cascade and has a role in each of the three major pathways (intrinsic, extrinsic and the common pathway). Due to this central position, it is an attractive therapeutic target to either enhance or dampen thrombin generation. In this brief review, I will summarize key developments in the molecular understanding of this critical clotting factor and discuss the molecular basis of FX deficiency, highlight difficulties in expressing recombinant factor X, and detail two factor X variants evaluated clinically.
Asunto(s)
Biología Molecular , Coagulación Sanguínea , Factores de Coagulación Sanguínea/genética , Pruebas de Coagulación Sanguínea , Factor X/genética , Factor Xa , Humanos , TrombinaRESUMEN
Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1' bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.
Asunto(s)
Hemofilia B/tratamiento farmacológico , Inhibidor de Proteína C/farmacología , Proteína C/antagonistas & inhibidores , Serpinas/farmacología , Animales , Modelos Animales de Enfermedad , Diseño de Fármacos , Electroforesis en Gel de Poliacrilamida , Humanos , RatonesRESUMEN
Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.
Asunto(s)
Plaquetas/citología , Células de la Médula Ósea/inmunología , Factor V/genética , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Animales , Apoptosis/efectos de los fármacos , Arteriolas/efectos de los fármacos , Arteriolas/inmunología , Arteriolas/lesiones , Biomarcadores/sangre , Plaquetas/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular , Linaje de la Célula/inmunología , Endocitosis , Factor V/inmunología , Factor V/farmacología , Citometría de Flujo , Expresión Génica , Humanos , Inmunofenotipificación , Rayos Láser , Células Progenitoras de Megacariocitos/inmunología , Megacariocitos/inmunología , Ratones , Ratones SCID , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunologíaRESUMEN
Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa. Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV), which has been activated by FXa. Because full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that although FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an â¼8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa also enhanced TFPI-mediated inhibition of FXa â¼12-fold in the presence of protein S. In contrast, neither activated FV nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants, we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not activated FV) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.
Asunto(s)
Anticoagulantes/metabolismo , Coagulación Sanguínea/fisiología , Factor V/metabolismo , Sustitución de Aminoácidos , Factor V/genética , Factor Xa/genética , Factor Xa/metabolismo , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutación Missense , Dominios Proteicos , Proteína S/genética , Proteína S/metabolismo , Protrombina/genética , Protrombina/metabolismoRESUMEN
PURPOSE OF REVIEW: New therapies are needed to control bleeding in a range of clinical conditions. This review will discuss the biochemical properties of zymogen-like factor Xa, its preclinical assessment in different model systems, and future development prospects. RECENT FINDINGS: Underlying many procoagulant therapeutic approaches is the rapid generation of thrombin to promote robust clot formation. Clinically tested prohemostatic agents (e.g., factor VIIa) can provide effective hemostasis to mitigate bleeding in hemophilia and other clinical situations. Over the past decade, we explored the possibility of using zymogen-like factor Xa variants to rapidly improve clot formation for the treatment of bleeding conditions. Compared to the wild-type enzyme, these variants adopt an altered, low activity, conformation which enables them to resist plasma protease inhibitors. However, zymogen-like factor Xa variants are conformationally dynamic and ligands such as its cofactor, factor Va, stabilize the molecule rescuing procoagulant activity. At the site of vascular injury, the variants in the presence of factor Va serve as effective prohemostatic agents. Preclinical data support their use to stop bleeding in a variety of clinical settings. Phase 1 studies suggest that zymogen-like factor Xa is safe and well tolerated, and a phase 1b is ongoing to assess safety in patients with intracerebral hemorrhage. SUMMARY: Zymogen-like factor Xa is a unique prohemostatic agent for the treatment of a range of bleeding conditions.
Asunto(s)
Precursores Enzimáticos/uso terapéutico , Factor Xa/uso terapéutico , Hemorragia/tratamiento farmacológico , Técnicas Hemostáticas , Coagulación Sanguínea/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Factor Va/metabolismo , Hemorragia/sangre , HumanosRESUMEN
There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.
Asunto(s)
Precursores Enzimáticos , Factor Xa , Hemostáticos/aislamiento & purificación , Animales , Coagulación Sanguínea/efectos de los fármacos , Dominio Catalítico , Análisis Mutacional de ADN , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Factor Xa/química , Factor Xa/genética , Factor Xa/metabolismo , Semivida , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Hemostáticos/síntesis química , Hemostáticos/química , Hemostáticos/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/uso terapéutico , Protrombina/metabolismo , Tromboplastina/químicaRESUMEN
The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.
Asunto(s)
Factor V/metabolismo , Proteína C/metabolismo , Sepsis/metabolismo , Transducción de Señal , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus , Tromboplastina/metabolismo , Animales , Factor V/genética , Ratones , Ratones Transgénicos , Proteína C/genética , Proteína S/genética , Proteína S/metabolismo , Sepsis/genética , Sepsis/patología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Tromboplastina/genéticaRESUMEN
The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological milieu to affect hemostatic plug formation remains unaddressed. We used confocal fluorescence microscopy to image the distribution of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster arteriole. These proteins bound to the injury site extend beyond the platelet mass to the surrounding endothelium. Although bound FVa and FXa may have been present on the platelet core at the nidus of the injury, bound proteins were not evident on platelets adherent even a small distance from the injury site. Manipulations to drastically reduce adherent platelets yielded a surprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation. Thus, platelets adherent to the site of vascular injury do not play the presumed preeminent role in supporting prothrombinase assembly and thrombin formation. Rather, the damaged/activated endothelium and possibly other blood cells play an unexpectedly important role in providing a procoagulant membrane surface in vivo.
Asunto(s)
Endotelio Vascular/metabolismo , Factor Va/metabolismo , Factor Xa/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismo , Animales , Arteriolas/metabolismo , Arteriolas/patología , Plaquetas/metabolismo , Plaquetas/patología , Endotelio Vascular/patología , Ratones , Ratones Endogámicos BALB C , Adhesividad PlaquetariaRESUMEN
Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.
Asunto(s)
Coagulación Sanguínea/fisiología , Lipoproteínas/metabolismo , Tromboplastina/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Biología Computacional , Secuencia Conservada/genética , Relación Dosis-Respuesta a Droga , Factor Xa/metabolismo , Polarización de Fluorescencia , Humanos , Lipoproteínas/farmacología , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.
Asunto(s)
Plaquetas/citología , Fibrinólisis/fisiología , Glicoproteínas/metabolismo , Lipoproteínas/metabolismo , Megacariocitos/citología , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Coagulación Sanguínea , Plaquetas/enzimología , Células de la Médula Ósea/citología , Femenino , Sangre Fetal/enzimología , Regulación de la Expresión Génica , Glicoproteínas/genética , Hemostasis , Humanos , Ligandos , Lipoproteínas/genética , Embarazo , Inhibidores de Proteasas/química , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD-injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.
Asunto(s)
Factor VIII/genética , Terapia Genética/métodos , Hemofilia A/genética , Hemofilia A/terapia , Animales , Perros , Factor VIII/química , Factor VIII/inmunología , Variación Genética , Hemostasis/genética , Hemostasis/fisiología , Humanos , Tolerancia Inmunológica , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Especificidad de la EspecieRESUMEN
The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.
Asunto(s)
Factor V/química , Factor Xa/química , Venenos de Serpiente/enzimología , Tromboplastina/química , Animales , Sitios de Unión , Coagulación Sanguínea , Cristalografía por Rayos X , Venenos Elapídicos/química , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , SerpientesRESUMEN
Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg(1545), ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.
Asunto(s)
Factor Va/química , Factor Xa/química , Péptidos/química , Proteolisis , Factor Va/antagonistas & inhibidores , Factor Va/genética , Factor Va/metabolismo , Factor Xa/genética , Factor Xa/metabolismo , Humanos , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.