RESUMO
BACKGROUND: Amotosalen/UVA pathogen-reduced platelet components (PRPCs) with storage up to 7 days are standard of care in France, Switzerland, and Austria. PRPCs provide effective hemostasis with reduced risk of transfusion-transmitted infections and transfusion-associated graft versus host disease, reduced wastage and improved availability compared with 5-day-stored PCs. This study evaluated the potency of 7-day PRPCs by in vitro characterization and in vivo pharmacokinetic analysis of autologous PCs. STUDY DESIGN AND METHODS: The in vitro characteristics of 7-day-stored apheresis PRPCs suspended in 100% plasma or 65% platelet additive solution (PAS-3)/35% plasma, thrombin generation, and in vivo radiolabeled post-transfusion recovery and survival of 7-day-stored PRPCs suspended in 100% plasma were compared with either 7-day-stored or fresh autologous conventional platelets. RESULTS: PRPCs after 7 days of storage maintained pH, platelet dose, in vitro physiologic characteristics, and thrombin generation when compared to conventional 7-day PCs. In vivo, the mean post-transfusion survival was 151.4 ± 20.1 h for 7-day PRPCs in 100% plasma (Test) versus 209.6 ± 13.9 h for the fresh autologous platelets (Control), (T-ΔC: 72.3 ± 8.8%: 95% confidence interval [CI]: 68.5, 76.1) and mean 24-h post-transfusion recovery 37.6 ± 8.4% for Test versus 56.8 ± 9.2% for Control (T-ΔC: 66.2 ± 11.2%; 95% CI: 61.3, 71.1). DISCUSSION: PRPCs collected in both 100% plasma as well as 65% PAS-3/35% plasma and stored for 7 days retained in vitro physiologic characteristics. PRPCs stored in 100% plasma for 7 days retained in vivo survival. Lower in vivo post-radiolabeled autologous platelet recovery is consistent with reported reduced count increments for allogenic transfusion.
Assuntos
Furocumarinas , Trombocitopenia , Reação Transfusional , Plaquetas , Preservação de Sangue , Furocumarinas/farmacologia , Humanos , Transfusão de Plaquetas , Plaquetoferese , Trombina/farmacologia , Raios UltravioletaRESUMO
In many patients with deep vein thrombosis and pulmonary embolism (venous thromboembolism, VTE), biomarkers or genetic risk factors have not been identified. To discover novel plasma metabolites associated with VTE risk, we employed liquid chromatography-mass spectrometry-based untargeted metabolomics, which do not target any specific metabolites. Using the Scripps Venous Thrombosis Registry population for a case-control study, we discovered that 10:1 and 16:1 acylcarnitines were low in plasmas of the VTE patient group compared with matched controls, respectively. Data from targeted metabolomics studies showed that several long-chain acylcarnitines (10:1, 12:0, 12:2, 18:1, and 18:2) were lower in the VTE group. Clotting assays were used to evaluate a causal relationship for low acylcarnitines in patients with VTE. Various acylcarnitines inhibited factor Xa-initiated clotting. Inhibition of factor Xa by acylcarnitines was greater for longer acyl chains. Mechanistic studies showed that 16:0 acylcarnitine had anticoagulant activity in the absence of factor Va or phospholipids. Surface plasmon resonance investigations revealed that 16:0 acylcarnitine was bound to factor Xa and that binding did not require the γ-carboxy glutamic acid domain. In summary, our study identified low plasma levels of acylcarnitines in patients with VTE and showed that acylcarnitines have anticoagulant activity related to an ability to bind and inhibit factor Xa.
Assuntos
Carnitina/análogos & derivados , Inibidores do Fator Xa/metabolismo , Fator Xa/metabolismo , Trombose Venosa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticoagulantes/uso terapêutico , Carnitina/sangue , Carnitina/química , Carnitina/metabolismo , Estudos de Casos e Controles , Fator Xa/química , Inibidores do Fator Xa/sangue , Inibidores do Fator Xa/química , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Risco , Trombose Venosa/sangue , Trombose Venosa/tratamento farmacológico , Trombose Venosa/etiologia , Varfarina/uso terapêuticoRESUMO
Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.
Assuntos
Anticorpos Monoclonais/farmacologia , Hemofilia A/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Inibidor da Proteína C/farmacologia , Proteína C/antagonistas & inibidores , Animais , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Tempo de Sangramento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Hemofilia A/sangue , Hemorragia/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Proteína C/química , Proteína C/imunologia , Proteína C/metabolismo , Inibidor da Proteína C/sangue , Inibidor da Proteína C/farmacocinéticaRESUMO
The hypothesis that prothrombin (FII) protects coagulation factor Va (FVa) from proteolytic inactivation by activated protein C (APC) was tested using purified proteins. FII dose-dependently protected FVa from APC proteolysis under conditions where competition of proteins for binding to negatively-charged phospholipid surface was not relevant (i.e. either at high phospholipid vesicle concentrations or using soluble dicaproylphosphatidylserine at levels below its critical micellar concentration). Cleavages in FVa at both Arg(506) and Arg(306) by APC were inhibited by FII. FII did not alter the amidolytic activity of APC towards chromogenic oligopeptide substrates or inhibit FVIIIa inactivation by APC, implying that the FII-mediated protection of FVa from APC proteolysis was due to the ability of FII to inhibit protein-protein interactions between FVa and APC. FII also protected FVa from inactivation by Gla-domainless APC, ruling out a role for the APC Gla domain for these observations. To identify domains of FII responsible for the observed phenomenon, various forms or fragments of FII were employed. Biotin-Phe-ProArg-CMK-inhibited meizothrombin and fII-fragment 1*2 protected FVa from proteolysis by APC. In contrast, no significant protection of FVa from APC cleavage was observed for Gladomainless-FII, prethrombin-1, prethrombin-2, FII fragment 1 or active site inhibited-thrombin (DEGR-thrombin). Overall, these data demonstrate that the Gla domain of FII linked to kringle 1 and 2 is necessary for the ability of FII to protect FVa from APC cleavage and support the general concept that assembly of the FII activation complex (FXa*FVa*FII*lipid surface) protects FVa from APC inactivation so that the procoagulant, thrombin generating pathway can act unhindered by APC. Only following FII activation and dissociation of the FII Gla domain fragments from the FII-ase complex, can APC inactivate FVa and down-regulate thrombin generation.
Assuntos
Coagulação Sanguínea , Fator Va/metabolismo , Proteína C/metabolismo , Protrombina/metabolismo , Animais , Ligação Competitiva , Precursores Enzimáticos/metabolismo , Fator VIIIa/metabolismo , Fator Va/genética , Fator Xa/metabolismo , Humanos , Kringles , Mutação , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Trombina/metabolismo , Fatores de TempoRESUMO
FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Membrana Celular/genética , Ativação Enzimática/genética , Fator Xa/química , Fator Xa/isolamento & purificação , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Deleção de Sequência/genéticaRESUMO
The multiprotein complex of factor Xa, factor Va, and prothrombin efficiently generates the blood-clotting agent, thrombin. Here, the formation of the factor Xa*prothrombin complex and the effects of factor Va on this complex were examined using a photoactivable thiol-specific fluorescent probe (LWB), which was synthesized and incorporated into the active site of factor Xa. The use of fluorescent LWB illustrated that factor Xa has an increased affinity for prothrombin in the presence of factor Va. Further exposure of these components to UV light resulted in a specific photocrosslinking of LWB-factor Xa to prothrombin, suggesting a physical association between these proteins. These data demonstrate that LWB can successfully function both as a spectroscopic probe and as a photocrosslinking reagent for studying protein-protein interactions.
Assuntos
Fator Va/farmacologia , Fator Xa/metabolismo , Protrombina/metabolismo , Reagentes de Ligações Cruzadas/efeitos da radiação , Polarização de Fluorescência , Corantes Fluorescentes/efeitos da radiação , Humanos , Métodos , Marcadores de Fotoafinidade/efeitos da radiação , Ligação Proteica/efeitos dos fármacos , Compostos de Sulfidrila , Raios UltravioletaAssuntos
Hidrazonas/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Biotina/química , Biotina/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
Blood coagulation reactions are strongly influenced by phospholipids, but little is known about the influence of sphingolipids on coagulation mechanisms. Lysosulfatide (lyso-SF) (sulfogalactosyl sphingosine) prolonged factor Xa (fXa) 1-stage plasma clotting assays, showing it had robust anticoagulant activity. In studies using purified clotting factors, lyso-SF inhibited >90% of prothrombin (II) activation for reaction mixtures containing fXa/factor Va (fVa)/II, and also inhibited II activation generation by fXa/ phospholipids and by Gla-domainless-fXa/fVa/phospholipids. When lyso-SF analogs were tested, results showed that N-acetyl-sulfatide was not anticoagulant, implying that the free amine group was essential for the anticoagulant effects of lyso-SF. Lyso-SF did not inhibit fXa enzymatic hydrolysis of small peptide substrates, showing it did not directly inhibit the fXa activity. In surface plasmon resonance studies, lyso-SF bound to immobilized inactivated fXa as well as inactivated Gla-domainless-fXa. Confirming this lyso-SF:fXa interaction, fluorescence studies showed that fluorescently-labeled-fXa in solution bound to lyso-SF. Thus, lyso-SF is an anticoagulant lipid that inhibits fXa when this enzyme is bound to either phospholipids or to fVa. Mechanisms for inhibition of procoagulant activity are likely to involve lyso-SF binding to fXa domain(s) that are distinct from the fXa Gla domain. This suggests that certain sphingolipids, including lyso-SF and some of its analogs, may down-regulate fXa activity without inhibiting the enzyme's active site or binding to the fXa Gla domain.
Assuntos
Fator V/metabolismo , Fator Xa/metabolismo , Psicosina/análogos & derivados , Trombina/biossíntese , Coagulação Sanguínea/efeitos dos fármacos , Fator Va/metabolismo , Fator Xa/química , Fator Xa/farmacologia , Humanos , Complexos Multiproteicos/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Protrombina/antagonistas & inibidores , Psicosina/química , Psicosina/metabolismo , Psicosina/farmacologia , Trombina/antagonistas & inibidoresRESUMO
Vascular hyperpermeability contributes to morbidity in inflammation. Current methodologies for in vivo assessment of permeability based on extravasation of Evans Blue (EB)-bound albumin are cumbersome and often lack sensitivity. We developed a novel infrared fluorescence (IRF) methodology for measurement of EB-albumin extravasation to quantify vascular permeability in murine models. Vascular permeability induced by endotoxaemia was examined for all solid organs, brain, skin and peritoneum by IRF and the traditional absorbance-based measurement of EB in tissue extracts. Organ IRF increased linearly with increasing concentrations of intravenous EB (2.5-25 mg/kg). Tissue IRF was more sensitive for EB accumulation compared to the absorbance-based method. Accordingly, differences in vascular permeability and organ EB accumulation between lipopolysaccharide-treated and saline-treated mice were often significant when analysed by IRF-based detection but not by absorbance-based detection. EB was detected in all 353 organs analysed with IRF but only in 67% (239/353) of organs analysed by absorbance-based methodology, demonstrating improved sensitivity of EB detection in organs with IRF. In contrast, EB in plasma after EB administration was readily measured by both methods with high correlation between the two methods (n=116, r2=0.86). Quantitation of organ-specific EB-IRF differences due to endotoxin was optimal when IRF was compared between mice matched for weight, gender, and age, and with appropriate corrections for organ weight and EB plasma concentrations. Notably, EB-IRF methodology leaves organs intact for subsequent histopathology. In summary, EB-IRF is a novel, highly sensitive, rapid, and convenient method for the relative quantification of EB in intact organs of treatment versus control mice.
Assuntos
Permeabilidade Capilar/fisiologia , Azul Evans/metabolismo , Albumina Sérica/metabolismo , Animais , Corantes/administração & dosagem , Corantes/metabolismo , Modelos Animais de Doenças , Azul Evans/administração & dosagem , Feminino , Hemoglobinas/metabolismo , Inflamação/sangue , Inflamação/etiologia , Raios Infravermelhos , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Imagem Óptica , Especificidade de ÓrgãosRESUMO
To test the hypothesis that factor Xa (fXa) interacts with protein S, fXa was labeled active-site specifically with a dansyl (D) dye via a Glu-Gly-Arg (EGR) tether to yield DEGR-fXa(i). When protein S was added to phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicle-bound DEGR-fXa(i), the anisotropy of the dansyl moiety was altered from 0.219 +/- 0.002 to 0.245 +/- 0.003. This change in dansyl anisotropy was not observed when DEGR-Xa(i) was titrated with protein S in the absence of PC/PS vesicles, or in the presence of 100% PC vesicles, or when PC/PS vesicle-bound DEGR-fXa(i) was titrated with thrombin-cleaved protein S. The protein S-dependent dansyl fluorescence change was specific for fXa because it was not observed for two homologous and similarly labeled DEGR-fIXa(i) and DEGR-fVIIa(i). Furthermore, protein S specifically and saturably altered the fluorescence anisotropy of PC/PS-bound active site-labeled LWB-FPR-fXa(i) (Kd = 33 nm) and was photocross-linked to PC/PS-bound LWB-FPR-fXa(i) analog, independently confirming the above results. Chemically synthesized microprotein S, comprising residues 1-116 of protein S and including the gamma-carboxyglutamic-rich domain, the thrombin-sensitive region (TSR), and the first epidermal growth factor-like domain (EGF1) of protein S, altered the anisotropy of PC/PS-bound DEGR-fXa(i) from 0.219 to 0.242, similar to the effect of the protein S titration (Kd = 303 nm), suggesting that microprotein S binds to DEGR-fXa(i). To identify individual protein S domain(s) that binds DEGR-fXa(i), the EGF1 and TSR domains were chemically synthesized and studied. The TSR altered the anisotropy of DEGR-fXa(i) by approximately 16% (Kd = 3.9 microm), but the EGF1 domain had no effect on the signal. In controls, the TSR domain did not alter the anisotropy of DEGR-fIXa(i) and DEGR-fVIIa(i), respectively. These data demonstrate that membrane-bound fXa binding to protein S involves the TSR of protein S.
Assuntos
Fator Xa/química , Membranas Artificiais , Fosfatidilcolinas/química , Fosfatidilserinas/química , Proteína S/química , Trombina/química , Fator Xa/metabolismo , Corantes Fluorescentes , Humanos , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica/fisiologia , Proteína S/metabolismo , Estrutura Terciária de Proteína/fisiologia , Trombina/metabolismoRESUMO
Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with residues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat) and K(m)) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499-505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.
Assuntos
Fator Va/metabolismo , Fator Xa/metabolismo , Proteína C/metabolismo , Substituição de Aminoácidos , Animais , Arginina , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Fator Va/química , Fator Va/genética , Fator Xa/química , Humanos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Protrombina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tromboplastina/metabolismoRESUMO
Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.
Assuntos
Fator Va/química , Fator Xa/química , Peptídeos/química , Proteína C/química , Substituição de Aminoácidos , Sítios de Ligação/genética , Ativação Enzimática , Fator Va/genética , Fator Xa/genética , Humanos , Mutação de Sentido Incorreto , Peptídeos/genética , Proteína C/genética , Protrombina/química , Protrombina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membrane-anchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k(cat). The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex.
Assuntos
Coagulação Sanguínea/fisiologia , Fator VII/metabolismo , Fator VIIa/química , Fator VIIa/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tromboplastina , Animais , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Fator VIIa/genética , Fator X/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Selectina-P/química , Selectina-P/genética , Selectina-P/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Tromboplastina/química , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
Sphingolipids contribute to modulation of two opposing cell processes, cell growth and apoptotic cell death; ceramide and sphingosine promote the latter and sphingosine-1-phosphate triggers the former. Thrombin, a pro-inflammatory protease that is regulated by the blood coagulation cascade, exerts similar effects depending on cell type. Here we report a new mechanism for cross-talk between sphingolipid metabolism and thrombin generation. Sphingosine and sphinganine, but not ceramide or sphingosine-1-phosphate, down-regulated thrombin generation on platelet surfaces (IC(50) = 2.4 and 1.4 microm for sphingosine and sphinganine, respectively) as well as in whole plasma clotting assays. Thrombin generation was also inhibited by glucosylsphingosine, lysosphingomyelin, phytosphingosine, and primary alkylamines with >10 carbons. Acylation of the amino group ablated anticoagulant activities. Factor Va was required for the anticoagulant property of sphingosine because prothrombin activation was inhibited by sphingosine, sphinganine, and stearylamine in the presence but not in the absence of factor Va. Sphingosine did not inhibit thrombin generation when Gla-domainless factor Xa was used in prothrombinase assays, whereas sphingosine inhibited activation of Gla-domainless prothrombin by factor Xa/factor Va in the absence of phospholipids (IC(50) = 0.49 microm). Fluorescence spectroscopy studies showed that sphingosine binds to fluorescein-labeled factor Xa and that this interaction required the Gla domain. These results imply that sphingosine disrupts interactions between factor Va and the Gla domain of factor Xa in the prothrombinase complex. Thus, certain sphingolipids may be bioactive lipid mediators of thrombin generation such that certain sphingolipid metabolites may modulate proteases that affect cell growth and death, blood coagulation, and inflammation.
Assuntos
Regulação da Expressão Gênica , Fosforilcolina/análogos & derivados , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Trombina/biossíntese , Anticoagulantes/farmacologia , Sítios de Ligação , Plaquetas/metabolismo , Carbono/química , Relação Dose-Resposta a Droga , Regulação para Baixo , Fator Va/química , Fator Xa/química , Humanos , Concentração Inibidora 50 , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Micelas , Fosforilcolina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Protrombina/metabolismo , Psicosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Trombina/metabolismoRESUMO
To identify amino acid sequences in factor Xa (fXa) and prothrombin (fII) that may be involved in prothrombinase complex (fXa.factor Va.fII.phospholipids) assembly, synthetic peptides based on fXa and fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. One fII peptide (PT557-571 homologous to chymotrypsin (CHT) residues 225-239) and two fXa peptides (X404-418, CHT231-244, and X415-429, CHT241-252C) potently inhibited plasma clotting and prothrombinase activity with 50% inhibition between 41 and 115 microM peptide. Inhibition of prothrombinase by PT557-571 and X415-429 was fVa-independent, whereas the inhibition by X404-418 was fVa-dependent. X404-418 inhibited the binding of fVa to fluorescein-labeled, inhibited fXai in the presence of phosphatidylcholine/phosphatidylserine vesicles, whereas X415-429 inhibited binding of fII to phospholipid-bound fluorescein-labeled, inhibited fXai. PT557-571 altered the fluorescence emission of fluorescein-labeled fXai, showing that PT557-571 binds to fXai. These data suggest that residues 404-418 in fXa provide fVa binding sites, whereas residues 557-571 in fII and 415-429 in fXa mediate interactions between fXa and fII in the prothrombinase complex.
Assuntos
Fator Xa/química , Tromboplastina/química , Sequência de Aminoácidos , Quimotripsina/química , Relação Dose-Resposta a Droga , Fator Va/química , Fibrinogênio/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Proteína C/química , Estrutura Terciária de Proteína , Protrombina/química , EspectrofotometriaRESUMO
The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.
Assuntos
Glucosilceramidas/farmacologia , Fosfatidilserinas/metabolismo , Proteína C/metabolismo , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Bovinos , Transferência Ressonante de Energia de Fluorescência , Humanos , Lipossomos/química , Lipossomos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Tempo de ProtrombinaRESUMO
Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells.
Assuntos
Anticoagulantes/metabolismo , Coagulação Sanguínea , Citoproteção , Variação Genética/genética , Proteína C/genética , Proteína C/metabolismo , Apoptose , Sobrevivência Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotelinas/metabolismo , Humanos , Mutação/genética , Receptor PAR-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
To identify sequences in prothrombin (fII) involved in prothrombinase complex (fXa.fVa.fII.phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473-487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity, with 50% inhibition of 12 and 10 microm peptide, respectively. Prothrombinase inhibition by PT473-487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473-487SCR, was not inhibitory. Peptide PT473-487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473-487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473-487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC(50) at 3 microm peptide), whereas the control peptide PT473-487SCR did not alter the anisotropy, suggesting a direct binding interaction between PT473-487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473-487 provide fVa-binding sites and mediate interactions between fVa and fII in the prothrombinase complex.