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1.
J Lipid Res ; 65(1): 100484, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38103786

RESUMEN

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.


Asunto(s)
Enfermedad de la Arteria Coronaria , Glicerofosfolípidos , Treonina/análogos & derivados , Tromboplastina , Animales , Humanos , Tromboplastina/metabolismo , Fosfatidilserinas/metabolismo , Liposomas/metabolismo , Plaquetas/metabolismo , Trombina/metabolismo
2.
Haemophilia ; 26(1): 6-16, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31846168

RESUMEN

Assay discrepancies can occur with laboratory monitoring of FVIII and FIX replacement therapy, particularly for the extended half-life products. This guideline collates current published data and provides advice on appropriate choice of assays for laboratory measurement of replacement therapy for patients with Haemophilia A and B without inhibitors. It is recommended that each haemophilia centre should ensure that appropriate laboratory assays are available for FVIII and FIX products in local clinical use. Patient samples should be assayed against calibrators traceable to WHO Plasma International Standards. Assay discrepancies are common especially for the extended half-life FVIII and FIX products, and assays of these products may need to be verified with the specific CFC being used.


Asunto(s)
Factores de Coagulación Sanguínea/uso terapéutico , Técnicas de Laboratorio Clínico , Hemofilia A/tratamiento farmacológico , Guías de Práctica Clínica como Asunto , Factor IX/uso terapéutico , Factor VIII/uso terapéutico , Humanos , Reino Unido
3.
Haemophilia ; 26(1): 151-155, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31859415

RESUMEN

INTRODUCTION: The factor VIII mimetic emicizumab (Hemlibra, Hoffman-la Roche, Basel, Switzerland) has a novel mode of action that affects the laboratory monitoring of patients receiving this treatment. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors Organisation (UKHCDO) aims to provide advice for clinical and laboratory staff on appropriate use of laboratory assays in patients with Haemophilia A treated with emicizumab. METHODOLOGY: The guideline was prepared by a review of the available literature and discussion and revision by the authors. RESULTS: The guideline describes the effect of emicizumab on commonly used coagulations tests and provides recommendations on the use of assays for measurement of factor VIII and factor VIII inhibitor in the presence of emicizumab. The guideline also provides recommendations on measurement of emicizumab. CONCLUSION: Knowledge of the effect of emicizumab on coagulation tests and factor assays is required to ensure appropriate testing and monitoring of therapy in patients receiving this drug.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Pruebas de Coagulación Sanguínea , Hemofilia A/tratamiento farmacológico , Guías de Práctica Clínica como Asunto , Anticuerpos/análisis , Anticuerpos Biespecíficos/análisis , Anticuerpos Monoclonales Humanizados/análisis , Factor VIII/análisis , Factor VIII/antagonistas & inhibidores , Factor VIII/uso terapéutico , Humanos , Reino Unido
4.
Blood ; 114(8): 1658-65, 2009 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-19531655

RESUMEN

Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.


Asunto(s)
Factor V/antagonistas & inhibidores , Protaminas/farmacología , Trombina/metabolismo , Animales , Anticoagulantes/farmacología , Tiempo de Sangría , Coagulación Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Factor V/metabolismo , Factor VIIIa/metabolismo , Heparina/farmacología , Ratones , Ratones Endogámicos BALB C , Proteína C/metabolismo , Proteína C/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos
5.
Sci Rep ; 10(1): 18366, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33110150

RESUMEN

ABO blood group is associated with cardiovascular disease, with significantly lower risk in blood group O individuals. ABO(H) blood group determinants are expressed on different glycoproteins on platelet surfaces. In addition, ABO(H) structures are also present on VWF glycans. These ABO(H) carbohydrates influence both platelet and VWF function. Previous studies have reported that approximately 5-10% of normal blood donors express abnormally high or low levels of A or B blood group antigens on their platelet surfaces (high expresser phenotype, HXP or low expresser phenotype, LXP respectively). In this study, the biological effects of the ABO Expresser phenotype were investigated. ABO(H) expression on platelets and plasma VWF was studied in a series of 541 healthy blood donors. Overall, 5.6% of our study cohort were classified as HXP, whilst 4.4% satisfied criteria for LXP. We demonstrate that genotype at the ABO blood group locus plays a critical role in modulating the platelet HXP phenotype. In particular, A1A1 genotype is a major determinant of ABO high-expresser trait. Our data further show that ABH loading on VWF is also affected by ABO expresser phenotype. Consequently, A antigen expression on VWF was significantly elevated in HXP individuals and moderately reduced in LXP subjects (P < 0.05). Collectively, these findings suggest that ABO expresser phenotype influences primary hemostasis though several different pathways. Further studies will be required to define whether inter-individual variations in ABO(H) expression on platelets and/or VWF (particularly HXP and LXP) impact upon risk for cardiovascular disease.


Asunto(s)
Sistema del Grupo Sanguíneo ABO/metabolismo , Plaquetas/metabolismo , Fenotipo , Factor de von Willebrand/metabolismo , Sistema del Grupo Sanguíneo ABO/genética , Sistema del Grupo Sanguíneo ABO/inmunología , Alelos , Donantes de Sangre , Estudios de Cohortes , Genotipo , Hemostasis , Humanos
6.
Thromb Haemost ; 99(3): 570-5, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18327406

RESUMEN

In addition to their anticoagulant activity, unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) have important immunomodulatory properties. However, different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. It was the objective of this study to determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide-binding protein (LBP) in mediating this effect. Whole blood was pre-treated with UFH or LMWH (0.1-200 IU/ml), prior to stimulation with LPS (10 ng/ml). After six hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1beta, IL-6, IL-8, and TNF-alpha) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of LBP (50-100 nM). Under serum-free conditions, heparin demonstrated dose-dependent anti-inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1beta, IL-6, IL-8, and TNF-alpha) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1beta and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10-200 IU/ml). Our data demonstrate that in the absence of LBP, the direct effect of heparin on LPS-stimulated monocytes is anti-inflammatory. However in whole blood, the immunomodulatory effects of heparin are significantly more complex, with either pro- or anti-inflammatory effects dependent upon heparin concentration.


Asunto(s)
Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Heparina/farmacología , Factores Inmunológicos/farmacología , Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/metabolismo , Línea Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo
7.
Thromb Haemost ; 99(5): 916-24, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18449422

RESUMEN

Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.


Asunto(s)
Proteínas ADAM/sangre , Desamino Arginina Vasopresina/administración & dosificación , Hemostáticos/administración & dosificación , Mutación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Enfermedades de von Willebrand/tratamiento farmacológico , Factor de von Willebrand/metabolismo , Proteína ADAMTS13 , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Glicosilación , Semivida , Humanos , Infusiones Intravenosas , Masculino , Fenotipo , Lectinas de Plantas/metabolismo , Unión Proteica , Precursores de Proteínas/sangre , Resultado del Tratamiento , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética
8.
JCI Insight ; 3(6)2018 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-29563336

RESUMEN

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.


Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Hemorragia/enzimología , Hemorragia/metabolismo , Fosfolípidos/metabolismo , Trombina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Coagulación Sanguínea , Factores de Coagulación Sanguínea/genética , Plaquetas , Puente Cardiopulmonar/efectos adversos , Proteínas Portadoras , Cisteína Endopeptidasas , Factor IX/genética , Factor VIII/genética , Factor VIIa/metabolismo , Factor X/genética , Hemofilia A , Hemorragia/prevención & control , Hemostasis , Humanos , Ácidos Hidroxieicosatetraenoicos , Lipoproteínas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas de Neoplasias , Resonancia por Plasmón de Superficie , Tromboplastina/antagonistas & inhibidores , Tromboplastina/metabolismo
9.
Biochem J ; 396(2): 355-62, 2006 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-16503879

RESUMEN

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.


Asunto(s)
Arginina/química , Factor VIIIa/metabolismo , Proteína C/metabolismo , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Animales , Arginina/genética , Sitios de Unión , Catálisis , Células Cultivadas , Cricetinae , Factor VIIIa/genética , Humanos , Riñón/citología , Riñón/metabolismo , Cinética , Mutación , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Blood Rev ; 19(1): 15-27, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15572214

RESUMEN

Factor VIII, a metal ion-dependent heterodimer, circulates in complex with von Willebrand factor. At sites of vessel wall damage, this procofactor is activated to factor VIIIa by limited proteolysis and assembles onto an anionic phospholipid surface in complex with factor IXa to form the intrinsic factor Xase; an enzyme complex that efficiently converts factor X to factor Xa during the propagation phase of coagulation. Factor Xase activity is down-regulated by mechanisms that include self-dampening by dissociation of a critical factor VIIIa subunit and proteolytic inactivation by the activated protein C pathway. Recent studies identify putative metal ion coordination sites as well as ligands involved in the catabolism of the activated and procofactor forms of the protein. Our knowledge of these multiple intra- and inter-molecular interactions has been facilitated by the application of naturally occurring and site-directed mutations to study factor VIII structure and function. In this review, we document important and novel contributions following this line of investigation.


Asunto(s)
Factor VIII , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Factor VIII/antagonistas & inhibidores , Factor VIII/química , Factor VIII/metabolismo , Factor VIIIa/química , Factor VIIIa/metabolismo , Hemofilia A/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína
11.
Br J Haematol ; 133(1): 62-7, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16512830

RESUMEN

Unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti-inflammatory properties; however, their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)-kappaB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 microg and 1 microg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6 and IL-1beta release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-kappaB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-alpha, IL-8, IL-6 and IL-1beta as well as NF-kappaB translocation. These results indicate equivalent and significant heparin anti-inflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-kappaB activation certainly represents one of the mechanisms by which heparin exerts its anti-inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.


Asunto(s)
Anticoagulantes/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Citocinas/inmunología , Heparina/uso terapéutico , Monocitos/inmunología , FN-kappa B/metabolismo , Infecciones Bacterianas/inmunología , Células Cultivadas , Citocinas/análisis , Regulación hacia Abajo , Escherichia coli , Heparina de Bajo-Peso-Molecular/uso terapéutico , Humanos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , FN-kappa B/análisis , Translocación Genética/efectos de los fármacos
14.
Blood ; 100(2): 501-8, 2002 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12091341

RESUMEN

The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factor VIIIa/genética , Hemofilia A/genética , Mutación , Proteínas de Neoplasias , Secuencia de Bases , Catálisis , Cisteína Endopeptidasas/genética , Factor IXa/metabolismo , Factor VIIIa/química , Factor VIIIa/metabolismo , Polarización de Fluorescencia , Hemofilia A/sangre , Hemofilia A/enzimología , Humanos , Cinética , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína
15.
Biochemistry ; 43(17): 5094-101, 2004 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-15109268

RESUMEN

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factor IXa/metabolismo , Factor VIIIa/metabolismo , Proteínas de Neoplasias/metabolismo , Unión Competitiva , Factor VIIIa/química , Factor VIIIa/genética , Humanos , Cinética , Membranas Artificiales , Mutagénesis Sitio-Dirigida , Fosfolípidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Especificidad por Sustrato
16.
Br J Haematol ; 116(1): 187-92, 2002 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11841416

RESUMEN

This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen-binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (< or = 0.7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0.7), but the VWF:RCo/VWF:CBA ratios were discordant (< or = 0.7) (P = 0.001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high-molecular-weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet-binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.


Asunto(s)
Enfermedades de von Willebrand/clasificación , Factor de von Willebrand/análisis , Estudios de Casos y Controles , Colágeno Tipo III/metabolismo , Humanos , Peso Molecular , Agregación Plaquetaria , Valor Predictivo de las Pruebas , Unión Proteica , Ristocetina , Estadísticas no Paramétricas , Enfermedades de von Willebrand/inmunología , Factor de von Willebrand/inmunología
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