RESUMEN
Nitric oxide (NO) is known to modulate the action of several phytohormones. This includes the gaseous hormone ethylene, but the molecular mechanisms underlying the effect of NO on ethylene biosynthesis are unclear. Here, we observed a decrease in endogenous NO abundance during apple (Malus domestica) fruit development and exogenous treatment of apple fruit with a NO donor suppressed ethylene production, suggesting that NO is a ripening suppressor. Expression of the transcription factor MdERF5 was activated by NO donor treatment. NO induced the nucleocytoplasmic shuttling of MdERF5 by modulating its interaction with the protein phosphatase, MdPP2C57. MdPP2C57-induced dephosphorylation of MdERF5 at Ser260 is sufficient to promote nuclear export of MdERF5. As a consequence of this export, MdERF5 proteins in the cytoplasm interacted with and suppressed the activity of MdACO1, an enzyme that converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The NO-activated MdERF5 was observed to increase in abundance in the nucleus and bind to the promoter of the ACC synthase gene MdACS1 and directly suppress its transcription. Together, these results suggest that NO-activated nucleocytoplasmic MdERF5 suppresses the action of ethylene biosynthetic genes, thereby suppressing ethylene biosynthesis and limiting fruit ripening.
Asunto(s)
Malus , Transporte Activo de Núcleo Celular , Etilenos/metabolismo , Factor V/genética , Factor V/metabolismo , Factor V/farmacología , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Malus/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
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
INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.
Asunto(s)
Deficiencia del Factor V/tratamiento farmacológico , Factor V/uso terapéutico , Plasma/metabolismo , Adulto , Anciano , Pruebas de Coagulación Sanguínea , Factor V/farmacología , Deficiencia del Factor V/metabolismo , Deficiencia del Factor V/fisiopatología , Femenino , Hemostasis/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Trombina/biosíntesisRESUMEN
Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase, which are largely used in patients with or at risk of cardiovascular disease. Available data on thromboembolic disease include primary and secondary prevention as well as bleeding and mortality rates in statin users during anticoagulation for VTE. Experimental studies indicate that statins alter blood clotting at various levels. Statins produce anticoagulant effects via downregulation of tissue factor expression and enhanced endothelial thrombomodulin expression resulting in reduced thrombin generation. Statins impair fibrinogen cleavage and reduce thrombin generation. A reduction of factor V and factor XIII activation has been observed in patients treated with statins. It is postulated that the mechanisms involved are downregulation of factor V and activated factor V, modulation of the protein C pathway and alteration of the tissue factor pathway inhibitor. Clinical and experimental studies have shown that statins exert antiplatelet effects through early and delayed inhibition of platelet activation, adhesion and aggregation. It has been postulated that statin-induced anticoagulant effects can explain, at least partially, a reduction in primary and secondary VTE and death. Evidence supporting the use of statins for prevention of arterial thrombosis-related cardiovascular events is robust, but their role in VTE remains to be further elucidated. In this review, we present biological evidence and experimental data supporting the ability of statins to directly interfere with the clotting system.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , Trombosis , Tromboembolia Venosa , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Trombina/farmacología , Tromboembolia Venosa/tratamiento farmacológico , Factor V/farmacología , Factor V/uso terapéutico , Coagulación Sanguínea , Trombosis/tratamiento farmacológico , Anticoagulantes/farmacologíaRESUMEN
OBJECTIVES: This study is aimed to investigate the role of nuclear factor of activated T cells 5 (NFAT5), originally known as the osmosensitive mammalian transcription factor, in the pathogenesis of osteoarthritis (OA) in mice. METHODS: OA was induced in male C57BL/6 (wild-type) and NFAT5 haplo-insufficient (NFAT5+/-) mice via destabilization of the medial meniscus (DMM) surgery. OA severity and synovial inflammation were histologically assessed. Expression of CCL2, inflammatory cytokines, cartilage degrading enzymes was determined in the knee joints and cultured chondrocytes from wild-type and NFAT5+/- mice. RESULTS: NFAT5 expression was significantly upregulated in the knee joint of a mouse after DMM surgery. NFAT5 deficiency decreased the severity of synovial inflammation and osteoarthritic changes in cartilage and subchondral bone. Moreover, NFAT5 deficiency also decreased the expression of CCL2, IL-1ß, MMP-13, ADMATS-5, and macrophage infiltration in the joint. In cultured chondrocytes, hyperosmolar or IL-1ß stimulation significantly enhanced the expression of NFAT5, CCL2, IL-1ß, IL-6, and MMP-13, and this effect was abolished in chondrocytes from NFAT5+/- mice. Hyperosmolarity or IL-1ß-induced NFAT5 and CCL2 downregulated by inhibiting p38 MAPK, JNK, and ERK pathways. CONCLUSIONS: Our results indicate that NFAT5 is a crucial regulator of OA pathogenesis by upregulating CCL2 expression and macrophage recruitment. In chondrocyte, NFAT5 plays an important role in the response to hyperosmolar or IL-1ß stimulation. Thus, NFAT5 could be an attractive therapeutic target for OA treatment.
Asunto(s)
Cartílago Articular , Osteoartritis , Factores de Transcripción/metabolismo , Animales , Cartílago Articular/patología , Células Cultivadas , Condrocitos , Factor V/metabolismo , Factor V/farmacología , Factor V/uso terapéutico , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Interleucina-1beta/uso terapéutico , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteoartritis/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Activated protein C (APC) binds to its substrates activated factor V (FVa) and activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma serpins. Charged residues of the autolysis loop were individually mutated to alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations. Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the autolysis loop. Together, these results confirmed that the autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa.
Asunto(s)
Autólisis , Factor VIIIa/metabolismo , Proteína C/fisiología , Autólisis/genética , Factor V/farmacología , Humanos , Mutación , Proteína C/genética , Proteína S/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC-cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex-hormone-binding globulin (SHBG)-like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed. It comprised the amino-terminal half of protein S and the SHBG-like region of Gas6, a structurally similar protein having no known anticoagulant properties. The protein S/Gas6 chimera expressed 40-50%, APC-cofactor activity in plasma as compared to wild-type protein S. In the degradation of FVa by APC, the protein S/Gas6 chimera was only slightly less efficient than wild-type protein S. In contrast, the protein S/Gas6 chimera expressed no FV-dependent APC-cofactor activity in a FVIIIa-degradation system. This demonstrates the SHBG-like region to be important for expression of APC-cofactor activity of protein S and suggests that the SHBG-like region of protein S interacts with FV during the APC-mediated inactivation of FVIIIa.
Asunto(s)
Factor V/farmacología , Péptidos y Proteínas de Señalización Intercelular , Proteína C/fisiología , Proteína S/química , Proteína S/fisiología , Globulina de Unión a Hormona Sexual/química , Factor VIIIa/metabolismo , Factor Va/metabolismo , Humanos , Tiempo de Tromboplastina Parcial , Proteína S/genética , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.
Asunto(s)
Arginina/química , Factor V/farmacología , Mutagénesis Sitio-Dirigida , Trombina/biosíntesis , Tromboplastina/metabolismo , Pruebas de Coagulación Sanguínea , Factor V/genética , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.
Asunto(s)
Factores de Coagulación Sanguínea/antagonistas & inhibidores , Coagulación Sanguínea/fisiología , Cisteína Endopeptidasas/metabolismo , Factor IXa/antagonistas & inhibidores , Factor VIIIa/antagonistas & inhibidores , Factor V/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína C/metabolismo , Proteína S/farmacología , Animales , Sitios de Unión , Bovinos , Sinergismo Farmacológico , Humanos , Especificidad de la EspecieRESUMEN
Carriership of the factor V (FV) R2 haplotype is associated with mild APC-resistance, moderately reduced FV levels and a relative increase of the more thrombogenic FV isoform, FV1. Since low FV levels and increased FV1 can theoretically cause APC-resistance, we investigated whether these alterations can quantitatively account for R2-associated APC-resistance. In order to determine the effect of FV concentration and FV isoform composition on the APC-response, we reconstituted FV-deficient plasma with purified FV1 and FV2 in different molar ratios and to varying FV concentrations. APC sensitivity ratios (APCsr) were determined with the Coatest APC Resistance V, which probes the effect of APC on both FVa- and FVIIIa-inactivation, and with the Immunochrom APC response test, which only quantifies the effect of APC on FVIII(a)-inactivation. In both assays, low FV concentrations and/or high relative amounts of FV1 rendered plasma samples more resistant to APC. APCsr were also determined in FV-deficient plasma reconstituted with purified FV at levels and isoform ratios observed in R2-homozygotes (98% FV, 42% FV1) and age-matched controls (119% FV, 26% FV1). In both tests the APCsr of reconstituted control plasma was the same as that of plasma from controls, whereas reconstituted R2-plasma was less APC-resistant than plasma from homozygous carriers of the R2 haplotype. We conclude that the low FV levels and altered FV isoform ratio cannot fully explain R2-associated APC-resistance.
Asunto(s)
Resistencia a la Proteína C Activada/etiología , Factor V/genética , Factor V/farmacología , Resistencia a la Proteína C Activada/diagnóstico , Estudios de Casos y Controles , Relación Dosis-Respuesta a Droga , Factor VIII/farmacología , Haplotipos , Heterocigoto , Homocigoto , Humanos , Isoformas de Proteínas/farmacología , Proteína S/farmacología , Juego de Reactivos para DiagnósticoRESUMEN
Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme, that after activation down regulates fibrinolysis. TAFI is activated by thrombin in the presence of the cofactor thrombomodulin (TM). By stimulation of TAFI activation TM down regulates fibrinolysis, however TM is also a cofactor in the activation of protein C. Activated protein C (APC) can up regulate fibrinolysis by limitine the activation of TAFI via the attenuation of thrombin production. We studied these counteracting fibrinolytic properties of TM in plasma by measuring the activation of TAFI during tissue factor induced coagulation. TAFI activation was stimulated at low concentrations of TM but decreased at higher concentrations of TM. Similarly, the clot lysis times increased at low concentrations of TM but decreased at higher concentrations of TM. The reduction of TAFI activation at high TM concentrations was found to be dependent on a functional protein C pathway. The concentration of TM is therefore an important factor in the regulation of TAFI activation and in the regulation of fibrinolysis. High concentrations of TM result in up regulation of fibrinolysis, whereas low concentrations of TM have a down regulatory effect on fibrinolysis. These results suggest that fibrinolysis might be differentially regulated by TM in different parts of the body depending on the local TM concentration in the vasculature.
Asunto(s)
Carboxipeptidasas/farmacología , Fibrinólisis/efectos de los fármacos , Proteína C/farmacología , Trombomodulina/sangre , Coagulación Sanguínea/efectos de los fármacos , Carboxipeptidasa B2 , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/química , Endotelio Vascular/citología , Factor V/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Tromboplastina/farmacologíaRESUMEN
Blood coagulation factor V (FV) circulates in the blood in two forms designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.
Asunto(s)
Factor V/análisis , Adulto , Pruebas de Coagulación Sanguínea , Calibración , Factor V/metabolismo , Factor V/farmacología , Factor Va/efectos de los fármacos , Femenino , Haplotipos , Humanos , Cinética , Liposomas/farmacología , Masculino , Plasma/química , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Protrombina/efectos de los fármacos , Protrombina/metabolismo , Valores de Referencia , Reproducibilidad de los Resultados , Trombina/farmacologíaRESUMEN
Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.
Asunto(s)
Factor VIIIa/antagonistas & inhibidores , Factor V/farmacología , Integrina alfaXbeta2/farmacología , Proteína C/farmacología , Proteína S/antagonistas & inhibidores , Anticoagulantes/antagonistas & inhibidores , Anticoagulantes/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Factor VIIIa/metabolismo , Humanos , Integrina alfaXbeta2/metabolismo , Cinética , Membranas Artificiales , Fosfolípidos/metabolismo , Unión Proteica , Proteína C/metabolismo , Proteína S/efectos de los fármacos , Proteína S/metabolismo , Proteína S/farmacologíaRESUMEN
It is a long-known principle that tumour cells tend to exploit the host's physiologic systems in order to get support in terms of, for example, nutrition, growth or metastasis. One of these physiologic systems is the blood coagulation cascade, which has been found activated in many tumour patients. The mechanisms of the activation of coagulation have been assessed in numerous animal and in vitro experiments, and the results appeared to point to several distinct activators. The present study used a large panel of different cultivated human lung cancer cell lines and experimental systems involving normal plasma, plasmas deficient of factors V, VII or X, purified coagulation factors II and X, recombinant tissue factor (TF), and specific inhibitory antibodies against factor VII and TF. The results provide strong evidence that there is no activator of coagulation besides TF in the wide array of lung cancer cells examined. However, this work reveals a striking variability of TF content among the cell lines. This might explain ambiguous results of clinical trials of anticoagulation as an adjunct to antineoplastic therapy in lung cancer. By sensitive diagnostic tools like the plasma thrombin-antithrombin complex levels it might be possible to select patients with activated coagulation, who might benefit from anticoagulation.
Asunto(s)
Coagulación Sanguínea/fisiología , Neoplasias Pulmonares/fisiopatología , Tromboplastina/farmacología , Anticuerpos/inmunología , Factor V/farmacología , Factor VII/farmacología , Factor X/farmacología , Humanos , Neoplasias Pulmonares/inmunología , Trombina/análisis , Células Tumorales CultivadasRESUMEN
When thrombin, tissue thromboplastin or Russell's viper venom was added to a suspension of either lymphocytes or neutrophils containing normal plasma, aggregation of these cells ensued. The aggregate formed one gelatinous mass which was readily separable from the cell free supernatant, an aliquot of which caused platelet aggregation. This leukocyte derived platelet aggregatory substance had characteristics similar to thrombin but not AGEPC. When plasma deficient in Factor V was substituted for normal plasma, the platelet stimulatory substance was not produced. Substitution with plasma deficient in Factor VII, VIII, IX, X or XI was without effect. Thrombin clotting time measurements indicated a generation of activity, relative to thrombin, of about 3.0 U/5 X 10(6) cells.
Asunto(s)
Leucocitos/metabolismo , Trombina/biosíntesis , Agregación Celular , Factor V/farmacología , Humanos , Técnicas In Vitro , Leucocitos/efectos de los fármacos , Linfocitos/metabolismo , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Tromboplastina/farmacologíaRESUMEN
During thrombosis, vascular wall cells are exposed to clotting factors, including the procoagulant proteases thrombin and factor Xa (FXa), both known to induce cell signaling. FXa shows dose-dependent induction of intracellular Ca(2+) transients in vascular wall cells that is active-site-dependent, Gla-domain-independent, and enhanced by FXa assembly into the prothrombinase complex. FXa signaling is independent of prothrombin activation as shown by the lack of inhibition by argatroban, hirudin and the sulfated C-terminal peptide of hirudin (Hir(54-65)(SO3(-))). This peptide binds to both proexosite I in prothrombin and exosite I in thrombin. In contrast, signaling is completely blocked by the FXa inhibitor ZK-807834 (CI-1031). No inhibition is observed by peptides which block interaction of FXa with effector cell protease 1 receptor (EPR-1), indicating that this receptor does not mediate signaling in the cells assayed. Receptor desensitization studies with thrombin or peptide agonists (PAR-1 or PAR-2) and experiments with PAR-1-blocking antibodies indicate that signaling by FXa is mediated by both PAR-1 and PAR-2. Potential pathophysiological responses to FXa include increased cell proliferation, increased production of the proinflammatory cytokine IL-6 and increased production of prothrombotic tissue factor. These cellular responses, which may complicate vascular disease, are inhibited by ZK-807834.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Factor Xa/farmacología , Receptores de Trombina/fisiología , Transducción de Señal/efectos de los fármacos , Amidinas/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Factor V/farmacología , Inhibidores del Factor Xa , Humanos , Proteínas Inhibidoras de la Apoptosis , Piridinas/farmacología , Receptor PAR-1 , Receptor PAR-2 , Receptores de Superficie Celular , Inhibidores de Serina Proteinasa/farmacología , Survivin , Trombina/farmacologíaRESUMEN
Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.
Asunto(s)
Expresión Génica , Protrombina/genética , Útero/metabolismo , Animales , Western Blotting , Endometrio/química , Estro , Factor V/farmacología , Factor Xa/farmacología , Femenino , Heparina/farmacología , Hirudinas/farmacología , Inmunohistoquímica , Músculo Liso/química , Miometrio/química , Placenta/química , Embarazo , Proestro , Protrombina/análisis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venenos de Serpiente/farmacología , Distribución Tisular , Contracción Uterina/efectos de los fármacos , Útero/químicaRESUMEN
Increased plasma FVIII:C concentrations occur in several conditions, including diabetes, but whether this leads to clinically relevant hypercoagulability is uncertain. Accordingly, we investigated the effects of increasing FVIII:C levels on coagulation in vitro using a computer-assisted measurement of thrombin activity. Defibrinated plasma was activated with kaolin, thrombin activity measured using the chromogenic substrate S2238 and time to generate 50% maximal thrombin activity (T50) recorded in seconds. Increasing FVIII:C levels from 100 to 350% significantly reduced T50 (mean +/- SD) from 91 +/- 3 to 64 +/- 6.2 s (P < 0.001, n = 6), and T50 correlated inversely with FVIII:C (r = -0.884, P < 0.001, n = 36). Increasing FV concentrations resulted in an additive effect with high FVIII:C levels on the rate of thrombin generation. The results showed that increasing plasma FVIII:C and FV concentrations accelerate rate of generation of thrombin activity independently, and in an additive manner.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor VIII/farmacología , Factor V/farmacología , Trombina/biosíntesis , Dipéptidos/metabolismo , Humanos , Caolín/farmacología , Cinética , Microcomputadores , Estimulación QuímicaRESUMEN
Coagulation factor V has been at the centre of investigation for several years. In addition to factor V Leiden, various other polymorphisms are becoming the object of interest. Different results have been published about the association of the HR2 haplotype with decreased factor V levels and with reduced response to activated protein C (APC). Due to the central position of factor V in the clotting process, its activity can be determined in both thromboplastin-based and activated partial thromboplastin time (aPTT)-based assays. A multitude of assays are known for the determination of APC response. The aim of our study was to investigate whether different methods disclose genotype-dependent differences in factor V activity as well as APC response. Three wild-type carriers, three carriers homozygous for the R2 allele (4070G), and three carriers homozygous for the G allele (2391G, 2663G, 2684G, 2863G) were investigated. For each individual plasma sample, the factor V activity was determined using 12 different reagent combinations of three different thromboplastins, three different aPTT reagents, and two different factor V deficient plasma sources. The determination of factor V activity in the thromboplastin system revealed differences between the genotypes. These differences were independent of the thromboplastin reagent and the factor V-deficient plasma. The aPTT system exhibited a dependency on the aPTT reagent and the factor V-deficient plasma. Analysis of APC response disclosed genomic differences in specific test systems only. One type of assay could be more appropriate than other types in dependence of the position of genomic variations. Therefore, the applied assay is an important influential factor in investigations of functional consequences of genomic variations.