RESUMO
Coagulation factor IX plays a central role in hemostasis through interaction with factor VIIIa to form the factor X-activating complex at the site of injury. The absence of factor IX activity results in the bleeding disorder hemophilia B. This absence of activity can arise either from a lack of circulating factor IX protein or from mutations that decrease the activity of factor IX. This review focuses on analyzing the structure of factor IX with respect to molecular mechanisms that are at the basis of factor IX function. Proteolytic activation of factor IX to activated factor IX(a) and subsequent structural rearrangements are insufficient to generate fully active factor IXa. Multiple specific interactions between factor IXa, the cofactor VIIIa, and physiological substrate factor X further alter the factor IXa structure to realize the full enzymatic activity of factor IXa. Factor IXa also interacts with inhibitors, extravascular proteins, and cellular receptors that clear factor IX(a) from circulation. Hemophilia B is treated by replacement of the missing factor IX by plasma-derived protein, a recombinant bioequivalent, or via gene therapy. An understanding of how the function of factor IX is tied to structure is leading to modified forms of factor IX that have increased residence time in circulation, higher functional activity, protection from inhibition, and even activity in the absence of factor VIIIa. These modified forms of factor IX have the potential to significantly improve therapy for patients with hemophilia B.
RESUMO
BACKGROUND: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. OBJECTIVES: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. METHODS: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. RESULTS: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. CONCLUSION: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.
Assuntos
Coagulação Sanguínea , Fator X , Inibidores do Fator Xa , Pirazóis , Piridonas , Rivaroxabana , Humanos , Inibidores do Fator Xa/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Pirazóis/farmacologia , Células HEK293 , Fator X/metabolismo , Piridonas/farmacologia , Fator Xa/metabolismo , Piridinas/uso terapêutico , Piridinas/farmacologia , Simulação de Dinâmica Molecular , Tiazóis/farmacologia , Trombina/metabolismo , Trombina/química , Hemorragia , Ligação ProteicaRESUMO
BACKGROUND: Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk. AIM: To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1). METHODS: A case-cohort study was established within the BLEEDS cohort, which includes 16,570 patients who initiated VKAs between 2012 and 2014. We selected all 326 major bleeding cases that occurred during 17,613 years of follow-up and a random subcohort of 978 patients. We determined variants in CYP2C9, CYP4F2, GGCX, VKORC1 and evaluated the interaction between variant genotypes. Hazard ratios for major bleeding with 95% confidence intervals (95% CI) were estimated by weighted Cox regression. RESULTS: Genotype was determined in 256 cases and 783 subcohort members. Phenprocoumon was the most prescribed VKA for both cases and the subcohort (78% and 75%, respectively). Patients with major bleeding were slightly older than subcohort patients. CYP4F2-TT carriership was associated with a 1.6-fold (95% CI 0.9-2.8) increased risk of major bleeding compared with CC-alleles, albeit not statistically significant. For the CYP2C9 and GGCX variants instead, the major bleeding risk was around unity. Carrying at least two variant genotypes in CYP2C9 (poor metabolizer), CYP4F2-TT, and VKORC1-AA was associated with a 4.0-fold (95%CI 1.4-11.4) increased risk, while carriers of both CYP4F2-TT and VKORC1-AA had a particularly increased major bleeding risk (hazard ratio 6.7, 95% CI 1.5-29.8) compared with carriers of CC alleles in CYP4F2 and GG in VKORC1. However, the number of major bleeding cases in carriers of multiple variants was few (8 and 5 patients, respectively). CONCLUSIONS: CYP4F2 polymorphism was associated with major bleeding, especially in combination with VKORC1 genetic variants. These variants could be considered to further personalize anticoagulant treatment.
Assuntos
Anticoagulantes , Hemorragia , Polimorfismo Genético , Vitamina K Epóxido Redutases , Vitamina K , Humanos , Vitamina K/antagonistas & inibidores , Hemorragia/induzido quimicamente , Hemorragia/genética , Hemorragia/epidemiologia , Feminino , Masculino , Idoso , Vitamina K Epóxido Redutases/genética , Estudos de Coortes , Anticoagulantes/efeitos adversos , Pessoa de Meia-Idade , Citocromo P-450 CYP2C9/genética , Genótipo , Família 4 do Citocromo P450/genética , Idoso de 80 Anos ou mais , Carbono-Carbono Ligases/genética , Estudos de Casos e ControlesRESUMO
BACKGROUND: Tamoxifen is an effective treatment for primary breast cancer but increases the risk for venous thromboembolism. Tamoxifen decreases anticoagulant proteins, including antithrombin (AT), protein C (PC) and tissue factor (TF) pathway inhibitor, and enhances thrombin generation (TG). However, the relation between plasma concentrations of both tamoxifen and its active metabolite endoxifen and coagulation remains unknown. METHODS: Tamoxifen and endoxifen were measured in 141 patients from the prospective open-label intervention TOTAM-study after 3 months (m) and 6 m of tamoxifen treatment. Levels of AT and PC, the procoagulant TF, and TG parameters were determined at both timepoints if samples were available (n = 53-135 per analysis). Levels of coagulation proteins and TG parameters were correlated and compared between: 1) quartiles of tamoxifen and endoxifen levels, and 2) 3 m and 6 m of treatment. RESULTS: At 3 m, levels of AT, PC, TF and TG parameters were not associated with tamoxifen nor endoxifen levels. At 6 m, median TF levels were lower in patients in the 3rd (56.6 [33] pg/mL), and 4th (50.1 [19] pg/mL) endoxifen quartiles compared to the 1st (lowest) quartile (76 [69] pg/mL) (P=0.027 and P=0.018, respectively), but no differences in anticoagulant proteins or TG parameters were observed. An increase in circulating TF levels (3 m: 46.0 [15] versus 6 m: 54.4 [39] pg/mL, P < 0.001) and TG parameters was observed at the 6 m treatment timepoint, while AT and PC levels remained stable. CONCLUSIONS: Our results indicate that higher tamoxifen and endoxifen levels are not correlated with an increased procoagulant state, suggesting tamoxifen dose escalation does not further promote hypercoagulability.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Estudos Prospectivos , Neoplasias da Mama/tratamento farmacológico , Citocromo P-450 CYP2D6/metabolismo , Tamoxifeno/farmacologia , Anticoagulantes/uso terapêutico , AntitrombinasAssuntos
Hemofilia A , Mordeduras de Serpentes , Humanos , Venenos de Víboras , Hemofilia A/diagnóstico , AntivenenosRESUMO
BACKGROUND: Recombinant factor (F)IX-FIAV has previously been shown to function independently of activated FVIII (FVIIIa) and ameliorate the hemophilia A (HA) phenotype in vitro and in vivo. OBJECTIVES: The aim of this study was to assess the efficacy of FIX-FIAV in plasma from HA patients using thrombin generation (TG) and intrinsic clotting activity (activated partial thromboplastin time [APTT]) analyses. METHODS: Plasma obtained from 21 patients with HA (>18 years; 7 mild, 7 moderate, and 7 severe patients) was spiked with FIX-FIAV. The FXIa-triggered TG lag time and APTT were quantified in terms of FVIII-equivalent activity using FVIII calibration for each patient plasma. RESULTS: The linear, dose-dependent improvement in the TG lag time and APTT reached its maximum with approximately 400% to 600% FIX-FIAV in severe HA plasma and with approximately 200% to 250% FIX-FIAV in nonsevere HA plasma. The cofactor-independent contribution of FIX-FIAV was therefore suggested and confirmed by the addition of inhibitory anti-FVIII antibodies to nonsevere HA plasma, resulting in a FIX-FIAV response similar to severe HA plasma. Addition of 100% (5 µg/mL) FIX-FIAV mitigated the HA phenotype from severe to moderate (from <0.01% to 2.9% [IQR 2.3%-3.9%] FVIII-equivalent activity), from moderate to mild (3.9% [IQR 3.3%-4.9%] to 16.1% [IQR 13.7%-18.1%] FVIII-equivalent activity), and from mild to normal (19.8% [IQR 9.2%-24.0%] to 48.0% [IQR 34.0%-67.5%] FVIII-equivalent activity). No substantial effects were observed when combining FIX-FIAV with current HA therapies. CONCLUSION: FIX-FIAV is capable of increasing the FVIII-equivalent activity and coagulation activity in plasma from HA patients, thereby mitigating the HA phenotype. Hence, FIX-FIAV could serve as a potential treatment for HA patients with or without inhibitors.
Assuntos
Hemofilia A , Hemostáticos , Humanos , Fator VIII/genética , Fator VIII/uso terapêutico , Fator IX/genética , Tempo de Tromboplastina Parcial , FenótipoRESUMO
Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.
Assuntos
Carbono-Carbono Ligases , Fator IX , Carbono-Carbono Ligases/metabolismo , Fator IX/genética , Fator IX/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
It is unknown how lower-leg injury and knee arthroscopy, both associated with venous thromboembolism (VTE), affect coagulation. To study the effect of (1) lower-leg trauma and (2) knee arthroscopy on coagulation, plasma samples of the Prevention of Thrombosis following CAST immobilization (POT-CAST, #NCT01542762) and Prevention of Thrombosis following Knee Arthroscopy (POT-KAST, #NCT01542723) trials were used, which were collected shortly after lower-leg trauma and before/after (<4 hours) knee arthroscopy. For aim 1, 1204 lower-leg injury patients were compared with preoperative samples of 1001 controls. Mean differences/ratios (if ln-retransformed because of skewedness) were adjusted for sex, age, body mass index, comorbidity, malignancy, and oral contraceptives using linear regression. For aim 2, perioperative mean changes of 715 arthroscopy patients were calculated. Plasma levels of fibrinogen, factor (F)VIII, FIX, FXI, von Willebrand Factor (VWF), and D-dimer were measured in all individuals. Parameters of underlying mechanisms (tissue factor, interleukin-6 [IL-6], myeloperoxidase DNA, cell-free DNA) were measured in random subsets. In lower-leg injury patients, coagulation parameter levels increased, especially FVIII, VWF, and D-dimer, that is, adjusted mean differences: FVIII 26.8% (95% confidence interval [CI], 23.7-29.9), FIX 13.8% (95% CI, 11.9-15.6), FXI 5.1% (95% CI, 3.3-7.0), VWF 29.8% (95% CI, 26.0-33.6), fibrinogen 32.5 mg/dL (95% CI, 25.8-39.2), and D-dimer (mean ratio) 3.3 (95% CI, 3.1-3.6). Remaining parameters were unchanged, except for increased IL-6 levels. After arthroscopy, all parameters decreased. Lower-leg trauma is associated with increased procoagulant factor levels in contrast to knee arthroscopy. This suggests that, in both situations, different pathways are involved in development of VTE.
Assuntos
Traumatismos da Perna , Trombose , Tromboembolia Venosa , Artroscopia/efeitos adversos , Fibrinogênio/metabolismo , Humanos , Interleucina-6 , Traumatismos da Perna/complicações , Tromboembolia Venosa/etiologia , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs). OBJECTIVE: To investigate the potential effect of different DOACs on vascular integrity. METHODS: To model the impact of DOACs on vascular integrity, we utilized platelet-free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro-coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro- and anti-barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics. RESULTS: We provide evidence that the anti-FXa DOAC rivaroxaban and the anti-thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom-made peptide that blocks thrombin's exosite-I. CONCLUSIONS: Proteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease-activated receptor-1 mimicking peptide blocking thrombin's exosite-I.
Assuntos
Fibrilação Atrial , Dabigatrana , Administração Oral , Anticoagulantes/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Dabigatrana/efeitos adversos , Fator Xa/uso terapêutico , Inibidores do Fator Xa/efeitos adversos , Humanos , Receptor PAR-1 , Rivaroxabana/efeitos adversos , Trombina/uso terapêuticoRESUMO
OBJECTIVE: Plasma thrombin generation (TG) provides important information on coagulation status; however, current TG output parameters do not predict major bleeding of patients on anticoagulants. We recently reported that factor V (FV) activation by factor X (FX)a contributes importantly to the initiation phase of TG. Here we investigated how this pathway varies in the normal population and whether FXa-mediated activation of FV is associated with major bleeding in patients on anticoagulant therapy. APPROACH: We employed TIX-5, a specific inhibitor of FV activation by FXa, to estimate the contribution of FXa-mediated FV activation to tissue factor (TF)-initiated TG. RESULTS: We show that the contribution of this pathway to plasma TG varies considerably in the normal population, as measured by the time needed to form the first traces of thrombin (TG lag time; mean prolongation by TIX-5 40%, range 0%-116%). Comparing patients on vitamin K antagonists (VKA) of the BLEED study (263 patients with and 538 patients without major bleeding), showed a marked prolongation in the median TG lag time in the presence of TIX-5 in cases (12.83 versus 11.00 minutes, P = 0.0030), while the TG lag time without TIX-5 only showed a minor although significant difference (5.83 vs. 5.67 minutes, P = 0.0198). The TIX-5 sensitivity (lag time + TIX-5/lag time + vehicle) in the upper quartile was associated with a 1.62-fold (95% confidence interval 1.04-2.52) increased risk of major bleeding compared to the lowest quartile. CONCLUSION: A greater dependence on FXa-mediated activation of FV of TG is associated with increased risk of major bleeding during VKA therapy.
Assuntos
Fator V , Fator Xa , Anticoagulantes/efeitos adversos , Testes de Coagulação Sanguínea , Fator V/metabolismo , Fator Xa/metabolismo , Hemorragia/induzido quimicamente , HumanosRESUMO
Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Fibrinolíticos/farmacologia , Venenos de Serpentes/farmacologia , Venenos de Víboras/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibrinólise/efeitos dos fármacos , Humanos , Inibidores de Serina Proteinase/farmacologiaRESUMO
OBJECTIVE: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). APPROACH AND RESULTS: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. CONCLUSIONS: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.
Assuntos
Venenos Elapídicos/metabolismo , Fator Va/metabolismo , Proteína C/metabolismo , Animais , Linhagem Celular , Cricetinae , Venenos Elapídicos/química , Ativação Enzimática , Fator Va/química , Fator Va/genética , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Proteólise , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. OBJECTIVE: We aimed to unravel the structure-function relationships of TIX-5. METHOD: We used a structure model generated based on homology with the allergen Der F7. RESULTS: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. CONCLUSION: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.
Assuntos
Anticoagulantes , Inibidores do Fator Xa , Coagulação Sanguínea , Fator V , Fator Va , Fator Xa , Inibidores do Fator Xa/farmacologia , Humanos , Protrombina , Trombina , TromboplastinaRESUMO
BACKGROUND: Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while using anticoagulants is soluble thrombomodulin (sTM). METHODS: Plasma was available from 16,570 patients of the BLEEDS cohort that consisted of patients who started treatment with vitamin K antagonists between 2012 and 2014. A case-cohort study was performed including all patients with a major bleed (n = 326) during follow-up and a random sample of individuals selected at baseline (n = 652). Plasma sTM levels were measured and stratified by percentiles. Patients were also categorized by international normalized ratio (INR). Adjusted hazard ratios (for age, sex, hypertension, and diabetes) with 95% confidence intervals (CIs) were estimated by means of Cox regression. RESULTS: Plasma sTM levels were available for 263 patients with a major bleed and 538 control subjects. sTM levels were dose-dependently associated with risk of major bleeding, with a 1.9-fold increased risk (95% CI: 1.1-3.1) for levels above the 85th percentile versus the <25th percentile. A high INR (≥4) in the presence of high (≥70th percentile) sTM levels was associated with a 7.1-fold (95% CI: 4.1-12.3) increased risk of major bleeding, corresponding with a bleeding rate of 14.1 per 100 patient-years. CONCLUSION: High sTM levels at the start of treatment are associated with major bleeding during vitamin K antagonist treatment, particularly in the presence of a high INR.
Assuntos
Anticoagulantes/efeitos adversos , Hemorragia/induzido quimicamente , Trombomodulina/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Hemorragia/sangue , Humanos , Coeficiente Internacional Normatizado , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Vitamina K/antagonistas & inibidoresRESUMO
A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma.
Assuntos
Fator X , Fator Xa , Animais , Testes de Coagulação Sanguínea , Inibidores do Fator Xa , Hemostasia , Humanos , Camundongos , Modelos Animais , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Coelhos , Ratos , Roedores , SuínosRESUMO
OBJECTIVE: Whether hepatic triglyceride content (HTGC) contributes to hypercoagulability beyond total body fat (TBF) and visceral adipose tissue (VAT) is unclear. We, therefore, aimed to investigate the association between HTGC and coagulation factors (F)I (fibrinogen), VIII, IX, and XI while adjusting for TBF and VAT. Approach and Results: In this cross-sectional analysis of the NEO study (Netherlands Epidemiology of Obesity; n=6671), a random subset of participants underwent magnetic resonance imaging and magnetic resonance spectroscopy to assess VAT and HTGC (n=2580). We excluded participants without complete imaging and coagulation assessment, and with history of liver disease, venous thrombosis, or on anticoagulation. Mean differences in coagulation factor levels across HTGC quartiles were estimated by linear regression adjusted for age, sex, ethnicity, education, alcohol intake, physical activity, smoking, estrogen, and menopause, in addition to TBF and VAT. Among the 1946 participants included, median HTGC was 2.66% (interquartile range: 1.34%-6.27%). Coagulation factor levels increased dose-dependently across HTGC quartiles. Mean differences between the fourth and first quartiles were 14.7 mg/dL (95% CI, 2.1-27.2) for fibrinogen, 6.7 IU/dL (95% CI, 0.5-12.9) for FVIII, 26.1 IU/dL (95% CI, 22.4-29.8) for FIX, and 8.6 IU/dL (95% CI, 4.6-12.6) for FXI. With further adjustment for TBF and VAT, the dose-response association of HTGC with FIX persisted, whereas associations with other factors disappeared. CONCLUSIONS: HTGC was associated with various coagulation factors, of which FIX remained associated with HTGC after adjustment for TBF and VAT. HTGC might contribute to venous thrombosis risk beyond total body and visceral fat through FIX levels.
Assuntos
Fator IX/metabolismo , Fígado/metabolismo , Obesidade/epidemiologia , Triglicerídeos/metabolismo , Trombose Venosa/epidemiologia , Adiposidade , Idoso , Estudos Transversais , Feminino , Humanos , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/fisiopatologia , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Medição de Risco , Fatores de Risco , Trombose Venosa/metabolismo , Trombose Venosa/fisiopatologiaRESUMO
The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa-factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor-factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16-Asp194 salt bridge via modification of the activation peptide.