RESUMEN
BACKGROUND: Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia. OBJECTIVES: To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A. METHODS: Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice. RESULTS AND CONCLUSIONS: FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.
Asunto(s)
Factor Va/genética , Hemostasis/genética , Mutación , Ingeniería de Proteínas/métodos , Animales , Coagulación Sanguínea , Disulfuros/química , Factor Va/metabolismo , Hemofilia A/genética , Humanos , Ratones , Ratones Transgénicos , Tiempo de Tromboplastina Parcial , Proteína C/química , Tiempo de Protrombina , Proteínas Recombinantes/química , Trombina/química , Tromboplastina/química , Tromboplastina/genéticaAsunto(s)
Anticoagulantes/metabolismo , Proteína C/genética , Activador de Tejido Plasminógeno/metabolismo , Alanina/genética , Coagulación Sanguínea , Relación Dosis-Respuesta a Droga , Variación Genética , Humanos , Isquemia/patología , Isquemia/terapia , Mutación , Tiempo de Tromboplastina Parcial , Plasminógeno/metabolismo , Proteína C/metabolismo , Proteínas Recombinantes/metabolismo , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/terapia , Trombina/metabolismo , Terapia Trombolítica , Factores de TiempoRESUMEN
BACKGROUND: Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)-bound protein C is activated by thrombomodulin-bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease-activated receptor-1 (PAR-1). Binding of APC to EPCR both modifies and induces PAR-1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. AIM: To determine whether platelets possess receptors for protein C that can generate intracellular signals. RESULTS: Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca(2+). Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2' (ApoER2') and by receptor-associated protein (RAP), an inhibitor of the low-density lipoprotein receptor family. Under shear, surface-bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibalpha-dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2' or RAP. APC bound to purified recombinant ApoER2' or GPIbalpha. CONCLUSIONS: Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbalpha and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbalpha. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbalpha.
Asunto(s)
Plaquetas/metabolismo , Regulación de la Expresión Génica , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Proteína C/metabolismo , Receptores de Lipoproteína/metabolismo , Calcio/metabolismo , Adhesión Celular , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Proteínas Relacionadas con Receptor de LDL , Modelos Biológicos , Adhesividad Plaquetaria , Receptores de LDL/metabolismo , Transducción de SeñalAsunto(s)
Proteínas Sanguíneas/deficiencia , Deficiencia de Proteína S/complicaciones , Trombosis/etiología , Adulto , Proteínas Sanguíneas/genética , Niño , Preescolar , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Heterocigoto , Humanos , Lactante , Masculino , Mutación , Proteína S , Deficiencia de Proteína S/genética , Trombosis/genéticaRESUMEN
Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.
Asunto(s)
Proteína C/metabolismo , Anticoagulantes/metabolismo , Humanos , Modelos Moleculares , Proteína C/químicaRESUMEN
BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.
Asunto(s)
Coagulación Sanguínea , Coagulantes/metabolismo , Disulfuros , Factor VIII/metabolismo , Factor VIIIa/metabolismo , Hemofilia A/sangre , Coagulación Sanguínea/efectos de los fármacos , Línea Celular , Coagulantes/química , Coagulantes/farmacología , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Factor VIII/química , Factor VIII/genética , Factor VIII/farmacología , Semivida , Hemofilia A/metabolismo , Humanos , Técnicas In Vitro , Mutación , Proteínas Recombinantes/metabolismo , Tromboelastografía , Trombina/metabolismo , Tiempo de Trombina , Transfección , Tiempo de Coagulación de la Sangre TotalRESUMEN
BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.
Asunto(s)
Factor IXa/metabolismo , Factor VIII/metabolismo , Factor VIIIa/química , Factor VIIIa/metabolismo , Factor de von Willebrand/metabolismo , Factor VIII/química , Factor VIII/uso terapéutico , Factor VIIIa/genética , Factor VIIIa/uso terapéutico , Hemofilia A/tratamiento farmacológico , Mutación , Unión Proteica , Proteína C/metabolismo , Conformación Proteica , Desnaturalización Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 microg kg(-1) i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 microg kg(-1) loading + 5 microg kg(-1) h(-1) infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL(-1) to 86 ng mL(-1) by 40 min, then declined, but remained elevated at 34 ng mL(-1) at 240 min. A 22-fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL(-1) at 40 min and decreased to 20 ng mL(-1) within 120 min despite continued activator infusion. Co-infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low-grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.
Asunto(s)
Proteína C/metabolismo , Trombina/farmacología , Sustitución de Aminoácidos , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/farmacología , Hemostasis/efectos de los fármacos , Humanos , Infusiones Intravenosas , Inyecciones Intravenosas , Mutagénesis Sitio-Dirigida , Papio , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Trombina/administración & dosificación , Trombina/genéticaRESUMEN
Oral contraceptive (OC) use increases venous thrombosis (VTE) risk and causes activated protein C (APC) resistance. Plasma glucosylceramide (GlcCer) deficiency is associated with VTE and GlcCer functions as an APC anticoagulant cofactor. Because estradiol decreases GlcCer in cultured cells, we hypothesized OC use would decrease plasma GlcCer and contribute to APC resistance. In a pilot study, seven female adults alternatively took second and third generation OCs and plasma samples were analyzed for GlcCer using high performance liquid chromatography and for APC sensitivity using modified prothrombin time assays. Second and third generation OC usage decreased the APC sensitivity ratio by 8.1% +/- 4.7% (P = 0.004) and 11.7% +/- 8.2% (P = 0.013) and plasma GlcCer levels by 10.1% +/- 6.8% (P = 0.008) and 11.0% +/- 5.1% (P = 0.002), respectively. The plasma GlcCer level correlated with the sensitivity of plasma to APC (P = 0.017, r = 0.51, n = 21 plasma samples). Thus, both second and third generation OC usage decreased plasma GlcCer which could cause a reduction in the plasma sensitivity to APC/protein S, thereby potentially increasing VTE risk.
Asunto(s)
Resistencia a la Proteína C Activada/inducido químicamente , Anticonceptivos Orales/farmacología , Glucosilceramidas/sangre , Proteína C/metabolismo , Resistencia a la Proteína C Activada/sangre , Cromatografía Líquida de Alta Presión , Anticonceptivos Orales Combinados/administración & dosificación , Desogestrel/farmacología , Estradiol/metabolismo , Etinilestradiol/farmacología , Femenino , Glucosilceramidas/deficiencia , Humanos , Levonorgestrel/farmacología , Proyectos Piloto , Proteína S/biosíntesis , Tiempo de Protrombina , Riesgo , Sensibilidad y Especificidad , Trombosis de la Vena/sangre , Trombosis de la Vena/inducido químicamenteRESUMEN
The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities.
Asunto(s)
Coagulación Sanguínea , Colesterol/fisiología , Proteína C/fisiología , Anticoagulantes , Pruebas de Coagulación Sanguínea , Factor Va/metabolismo , Humanos , Cinética , Liposomas , Microdominios de Membrana , Fosfolípidos/fisiologíaRESUMEN
BACKGROUND: Venous and arterial thromboses occur in patients with Behçet's disease and are associated with significant morbidity and mortality. Studies on a possible association between the occurrence of thrombosis and thrombophilia in patients with this disease have been controversial. OBJECTIVE: To determine the prevalence of the most common thrombophilias and dyslipidaemia in patients with Behçet's disease with and without thrombosis. METHODS: Blood samples from 107 patients with Behçet's disease who had or did not have thrombosis were analysed for factor V Leiden, prothrombin G20210A polymorphism, methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, factor VIII level, homocysteine and C reactive protein concentrations, dyslipidaemia, and plasma glucosylceramide. RESULTS: There was no difference between patients with and without thrombosis in the prevalence of prothrombin G20210A polymorphism, factor V Leiden, homozygous MTHFR C677T, or plasma concentrations of homocysteine, C reactive protein, or glucosylceramide. In contrast, patients with thrombosis were found to have significantly higher mean levels of factor VIII, total cholesterol, triglycerides, VLDL cholesterol, and apolipoproteins B-100, C-II, and C-III than those without thrombosis. Multistepwise logistic regression analysis showed that triglyceride concentration was the best marker associated with thrombosis (p = 0.008), with an estimated odds ratio of 1.58 (95% confidence interval, 1.09 to 2.30) for a difference of 40 mg/dl. CONCLUSIONS: Thrombophilia does not seem to play a major role in the tendency to thrombosis in Behçet's disease. However, dyslipidaemia, predominantly hypertriglyceridaemia, might be a risk factor.
Asunto(s)
Síndrome de Behçet/complicaciones , Trombofilia/complicaciones , Trombosis/etiología , Adolescente , Adulto , Anciano , Apolipoproteínas/análisis , Síndrome de Behçet/sangre , Biomarcadores/sangre , Niño , Colesterol/sangre , VLDL-Colesterol/sangre , Factor VIII/análisis , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Prevalencia , Trombofilia/sangre , Trombosis/sangre , Triglicéridos/sangreRESUMEN
AIMS: We compared the effects of hirudin and heparin on thrombin generation and activity among 350 patients with acute coronary syndromes enrolled in the GUSTO-IIb trial. METHODS AND RESULTS: We obtained blood at baseline; at 4, 8, and 24h into infusion; at drug termination; and 6 and 24h after termination. We assayed for thrombin activity (fibrinopeptide A, activated protein C, thrombin-antithrombin complex), thrombin generation (prothrombin fragment 1.2), and platelet activation (platelet factor 4). Median baseline fibrinopeptide A and platelet factor 4 levels were elevated. Thrombin formation tended to increase with hirudin and decrease with heparin; by 8h into infusion, thrombin formation was significantly less for heparin (P<0.01). Most patients showed reduced thrombin activity and platelet activation during infusion of either agent. Hirudin-assigned patients had significantly lower fibrinopeptide A levels during infusion. Six h post-termination, both groups had increased thrombin activity. Thrombin formation was increased in heparin patients (P<0.0001), significantly more than with hirudin (P=0.005). Higher values of haemostasis markers tended to be associated with poorer 30-day outcomes. CONCLUSION: Although hirudin did not prevent generation of new thrombin, it appeared to inhibit thrombin activity more than did heparin and produced slower increases in thrombin formation after discontinuation. The reelevation of thrombotic markers after stopping intravenous antithrombin therapy and the tendency toward increased thrombotic events with post-treatment increases in marker levels suggest an ongoing, clinically significant prothrombotic state. These results raise the possibility of improving on current antithrombotics by preventing thrombin generation and thrombin activity and by sustained suppression of the prothrombotic state.
Asunto(s)
Fibrinolíticos/farmacología , Hemostasis/efectos de los fármacos , Heparina/farmacología , Hirudinas/farmacología , Enfermedad Aguda , Anciano , Coagulación Sanguínea/efectos de los fármacos , Método Doble Ciego , Femenino , Fibrinolíticos/uso terapéutico , Cardiopatías/sangre , Cardiopatías/tratamiento farmacológico , Heparina/uso terapéutico , Terapia con Hirudina , Humanos , Masculino , Persona de Mediana Edad , Síndrome , Trombina/metabolismo , Resultado del TratamientoRESUMEN
Regulation of hemostasis and thrombosis involves numerous plasma factors that contribute to procoagulant and anticoagulant pathways. Lipid-containing surfaces provide sites where both procoagulant and anticoagulant enzymes, cofactors and substrates are assembled to express their activities. Plasma and lipoproteins can contribute to either procoagulant or anticoagulant reactions. Procoagulant lipids/lipoproteins include triglyceride-rich particles in plasma and oxidized low density lipoprotein (LDL) which can accelerate activation of prothrombin, factor X and factor VII. Potentially anticoagulant lipids and lipoproteins, each of which enhances inactivation of factor Va by activated protein C, include phosphatidylethanolamine, cardiolipin, the neutral glycosphingolipids glucosylceramide and Gb3 ceramide (CD77), and high density lipoprotein (HDL). Remarkably, treatment of hyperlipidemia with statins not only lowers lipids but also provides antithrombotic effects whose mechanisms remain to be clarified. We hypothesize that procoagulant and anticoagulant lipids and lipoproteins in plasma may contribute to a Yin-Yang balance that helps influence the up-regulation and down-regulation of thrombin generation.
Asunto(s)
Hemostasis/efectos de los fármacos , Lipoproteínas/sangre , Lipoproteínas/farmacología , Trombosis/etiología , Animales , Factores de Coagulación Sanguínea/efectos de los fármacos , Humanos , Protrombina/efectos de los fármacos , Protrombina/metabolismo , Trombosis/sangreRESUMEN
To assess possible interactions between inflammation and activation of the anticoagulant protein C system during post-ischemic reperfusion protein C, APC, neutrophil L-selectin expression and myocardial myeloperoxidase activity (MPO) were measured in 19 patients undergoing cardiopulmonary bypass. After reperfusion for 10 min, APC to protein C ratio (APC/PC) increased from pre-reperfusion value 1.43 +/- 0.12 (mean +/- SEM) to 2.25 +/- 0.29, p = 0.015. Negative correlations were observed between APC/PC and MPO activity (Spearman r -0.64, p = 0.007) and APC/PC and neutrophil L-selectin expression (r = -0.7, p = 0.007, demonstrating that post-ishemic protein C activation was associated with decreased neutrophil tissue sequestration. Thus, physiological protein C activation may be involved in regulation of the inflammatory injury during reperfusion of human ischemic coronary circulation.
Asunto(s)
Puente Cardiopulmonar/efectos adversos , Daño por Reperfusión Miocárdica/sangre , Miocarditis/etiología , Proteína C/análisis , Adulto , Anciano , Activación Enzimática , Paro Cardíaco Inducido/efectos adversos , Humanos , Selectina L/análisis , Masculino , Persona de Mediana Edad , Daño por Reperfusión Miocárdica/etiología , Neutrófilos/enzimología , Peroxidasa/análisisAsunto(s)
Factor V/genética , Pruebas Genéticas , Mutación , Anticoagulantes/metabolismo , Genotipo , Heterocigoto , Homocigoto , Humanos , Factores de RiesgoRESUMEN
BACKGROUND: Activated protein C (APC) contributes to systemic anticoagulant and anti-inflammatory activities. APC may reduce organ damage by inhibiting thrombin generation and leukocyte activation. Neutrophils and cerebrovascular thrombosis contribute to ischemic neuronal injury, suggesting that APC may be a potential protective agent for stroke. METHODS AND RESULTS: We examined the effects of APC in a murine model of focal ischemia. After middle cerebral artery occlusion/reperfusion, the average survival time in controls was 13.6 hours. Animals that received purified human plasma-derived APC 2 mg/kg IV either 15 minutes before or 10 minutes after stroke induction survived 24 hours and were killed for neuropathological analysis. APC 2 mg/kg given before or after onset of ischemia restored cerebral blood flow, reduced brain infarct volume (59% to 69%; P:<0.003) and brain edema (50% to 61%; P:<0.05), eliminated brain infiltration with neutrophils, and reduced the number of fibrin-positive cerebral vessels by 57% (P:<0.05) and 25% (nonsignificant), respectively. The neuroprotective effect of APC was dose-dependent and associated with significant inhibition of ICAM-1 expression on ischemic cerebral blood vessels (eg, 61% inhibition with 2 mg/kg APC). Intracerebral bleeding was not observed with APC. CONCLUSIONS: APC exerts anti-inflammatory, antithrombotic, and neuroprotective effects in stroke. Central effects of APC are likely to be related to improved maintenance of the blood-brain barrier to neutrophils and to reduced microvascular obstructions and fibrin deposition.
Asunto(s)
Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Fibrinolíticos/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Proteína C/uso terapéutico , Animales , Antiinflamatorios/farmacología , Edema Encefálico/metabolismo , Edema Encefálico/patología , Isquemia Encefálica/sangre , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Circulación Cerebrovascular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática , Fibrina/metabolismo , Fibrinolíticos/farmacología , Hemoglobinas/análisis , Humanos , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Molécula 1 de Adhesión Intercelular/análisis , Antígeno de Macrófago-1/análisis , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Quiasma Óptico/química , Quiasma Óptico/metabolismo , Proteína C/farmacología , Desempeño Psicomotor/efectos de los fármacos , Daño por Reperfusión/sangre , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Tasa de SupervivenciaRESUMEN
The dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin-thrombomodulin or alpha-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to approximately 65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.