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1.
J Thromb Haemost ; 16(10): 2044-2049, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30007049

RESUMO

Essentials Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model. FIX-deficient mice displayed a hemostatic defect and FXI-deficient mice were similar to wild type mice. Infusion of FXI or over-expression of FXI in FIX-deficient mice improved hemostasis. FXI may affect the phenotype of FIX-deficiency (hemophilia B). SUMMARY: Background In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B). Methods Wild-type mice and mice lacking either FIX (F9- ) or FXI (F11-/- ) were tested in the SVB model. The plasma levels of FXI in F11-/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9- mice showed a significant defect in the SVB model, whereas F11-/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9- mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9- mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11-/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9- mice through FIX-independent pathways.


Assuntos
Deficiência do Fator XI/tratamento farmacológico , Fator XI/administração & dosagem , Hemofilia B/tratamento farmacológico , Hemostasia/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Fator IX/genética , Fator IX/metabolismo , Fator XI/genética , Fator XI/metabolismo , Deficiência do Fator XI/sangue , Deficiência do Fator XI/genética , Predisposição Genética para Doença , Hemofilia B/sangue , Hemofilia B/genética , Hemostasia/genética , Infusões Intravenosas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
3.
J Thromb Haemost ; 14(10): 1941-1952, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27430959

RESUMO

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease. SUMMARY: Introduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis 'steady state' and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood.


Assuntos
Anemia Falciforme/sangue , Eritrócitos/citologia , Fosfatidilserinas/química , Deficiência de Proteína S/sangue , Trombina/biossíntese , Adulto , Negro ou Afro-Americano , Antitrombina III/metabolismo , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/biossíntese , Humanos , Masculino , Fosfatidilserinas/sangue , Proteína S/metabolismo , Protrombina/metabolismo , Trombomodulina/sangue , Trombofilia/complicações , Trombose/metabolismo , Adulto Jovem , Talassemia beta/sangue
4.
J Thromb Haemost ; 14(6): 1249-54, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26952654

RESUMO

UNLABELLED: Essentials Disorders of hemostasis can lead to delayed and defective wound healing. In hemophilia B (HB) mice, 7 days of Factor (F)IX or VIIa are needed to normalize wound healing. One dose of a highly active FVIIa variant (DVQ) restored normal wound closure time in HB mice. Coagulation factors with enhanced activity may acquire biological effects not due to hemostasis. SUMMARY: Introduction We have previously reported that hemophilia B (HB) mice have delayed healing of cutaneous wounds and alterations in wound histology. Administration of a single dose of either factor IX or recombinant activated FVII (rFVIIa) (NovoSeven) prior to wounding did not improve wound closure time or histology. The FVIIa analog DVQ (V158D, E296V and M298Q mutations) was designed to have higher tissue factor-independent activity than rVIIa. We hypothesized that a single dose of DVQ would be more effective in restoring wound healing in HB mice. Methods Cutaneous punch wounds were made on the backs of HB and wild-type mice, and the time to wound closure was monitored. HB mice were treated with a dose of rFVIIa (10 mg kg(-1) ) or DVQ (1 mg kg(-1) ) that corrected the tail bleeding time. Skin samples were taken at various time points after wounding, fixed, and stained, and the histology was examined. Results As previously reported, wound closure times in HB mice given one dose of rFVIIa were not improved over those in untreated HB mice. Surprisingly, healing times in HB mice treated with an equally hemostatic dose of DVQ were normalized to that in wild-type mice. However, DVQ did not correct all histologic abnormalities in HB mice. Conclusions As the doses of DVQ and rFVIIa were chosen to support comparable levels of hemostasis, our data suggest that the improved healing seen with DVQ is not solely attributable to its hemostatic activity. It is possible that the improved wound healing arises through the effect of DVQ on cell signaling mechanisms.


Assuntos
Fator VIIa/administração & dosagem , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Tromboplastina/metabolismo , Cicatrização , Administração Tópica , Animais , Tempo de Sangramento , Modelos Animais de Doenças , Fator IX/genética , Fator VIIa/genética , Variação Genética , Hemostasia , Humanos , Camundongos , Mutação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética
6.
Haemophilia ; 22(1): 148-51, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26561343

RESUMO

INTRODUCTION: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype. AIM: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype. METHODS: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry. RESULTS: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001). CONCLUSION: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers.


Assuntos
Plaquetas/fisiologia , Hemofilia A/complicações , Hemofilia A/fisiopatologia , Hemorragia/complicações , Fenótipo , Adolescente , Adulto , Criança , Humanos , Trombose/complicações , Adulto Jovem
8.
Haemophilia ; 19(6): 926-32, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23879625

RESUMO

Previous work has shown that normalized haemostasis only at the time of an injury is not sufficient to promote optimal wound healing in haemophilia B (HB) mice. However, the duration of treatment required for optimal healing has not been established. The goal of these studies was to determine the effect of different durations of replacement or bypassing therapy [factor IX(FIX) or factor VIIa (FVIIa)] on wound healing parameters in a mouse model of HB. A dermal wound was placed on the back of HB mice. Animals were either untreated or pretreated and then subsequently treated for 3 days, 5 days, or 7 days with FIX or FVIIa. Wound area, time to wound healing, haematoma formation and iron deposition were measured. All treated animals showed shortened time to healing relative to untreated animals. Haematoma formation was prevented by treatment and bleeding into the wounds, measured by iron scores, was reduced by treatment. In addition, there was a progressive improvement in healing with 7 days of treatment more effective than 5 days which was more effective than 3 days. Replacement therapy with FIX had slightly shorter healing times than bypassing therapy with FVIIa. HB mice treated with FIX had slightly smaller wound area than untreated animals; by contrast, FVIIa-treated animals had much smaller wound areas that were close to the wound areas seen in wild-type animals. The data suggest that sustained therapy is required for normal wound healing.


Assuntos
Coagulantes/uso terapêutico , Fator IX/uso terapêutico , Fator VIIa/uso terapêutico , Hemofilia B/tratamento farmacológico , Cicatrização , Animais , Esquema de Medicação , Fator IX/genética , Fator IX/metabolismo , Hematoma/prevenção & controle , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
J Thromb Haemost ; 11(6): 1043-52, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23581252

RESUMO

OBJECTIVE: Bleeding is the main complication of warfarin therapy, even patients with an international normalized ratio (INR) in the target range can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. We hypothesized the INR might underestimate the level of anticoagulation in a subject with a lower factor (F) IX level than average. METHODS AND RESULTS: We modeled warfarin anticoagulation in our in vitro thrombin generation (TG) model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2-3. Variation in FIX had a marked effect on TG but had no effect on the prothrombin time (PT)-INR. A prospective observational, cross-sectional clinical study including 341 consecutive patients admitted to the emergency department with an INR between 2 and 3, showed a statistically lower FIX activity in bleeders (P = 0.004) compared with others. No correlation was found between TG capacity and PT-INR results (P = 0.36). However, in patients, presenting with a warfarin-related hemorrhage, TG was significantly lower (P < 0.001) than others. A correlation on the boundary of significance was observed between TG capacity and FIX levels (P = 0.09). CONCLUSION: These data demonstrates that patients who bleed when their PT-INR is in the target range 2-3 might have defective TG related to a lower level of FIX than expected.


Assuntos
Anticoagulantes/uso terapêutico , Fator IX/metabolismo , Hemorragia/induzido quimicamente , Coeficiente Internacional Normatizado , Varfarina/uso terapêutico , Idoso , Anticoagulantes/química , Testes de Coagulação Sanguínea , Plaquetas/citologia , Estudos Transversais , Feminino , Hemorragia/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Estudos Prospectivos , Protrombina/química , Risco , Trombina/química , Vitamina K/química , Varfarina/química
10.
J Thromb Haemost ; 10(5): 870-80, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22385910

RESUMO

BACKGROUND: The conversion of prothrombin to thrombin is one of two non-duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti-thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D-14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. OBJECTIVES: We sought to explore the structure of R9D-14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA(R9D-14T)), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer's anticoagulant activity. METHODS AND RESULTS: RNA(R9D-14T) binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I-mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA(R9D-14T) significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC-183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA(R9D-14T) anticoagulation in vitro. CONCLUSIONS: Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA(R9D-14T) may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.


Assuntos
Anticoagulantes/farmacologia , Antídotos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Protrombina/metabolismo , Trombina/metabolismo , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Antídotos/química , Antídotos/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Ligação Competitiva , Domínio Catalítico , Bovinos , Cães , Estabilidade de Medicamentos , Ativação Enzimática , Fator Va/metabolismo , Meia-Vida , Humanos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Tempo de Tromboplastina Parcial , Ativação Plaquetária/efeitos dos fármacos , Ligação Proteica , Tempo de Protrombina , Coelhos , Ratos , Ribonucleases/metabolismo , Técnica de Seleção de Aptâmeros , Ovinos , Especificidade da Espécie , Relação Estrutura-Atividade , Suínos , Tempo de Trombina
11.
Thromb Haemost ; 107(4): 717-25, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22318669

RESUMO

Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.


Assuntos
Heparina/uso terapêutico , Fator Plaquetário 4/metabolismo , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Biofísica/métodos , Bovinos , Relação Dose-Resposta a Droga , Heparina/análogos & derivados , Heparina/química , Heparina/metabolismo , Heparina/farmacologia , Humanos , Imunoensaio/métodos , Cinética , Protaminas/metabolismo , Trombina/metabolismo , Trombocitopenia/metabolismo
12.
J Thromb Haemost ; 9(4): 759-66, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21294824

RESUMO

BACKGROUND AND OBJECTIVES: Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)-independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF-bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. OBJECTIVES: We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild-type FVIIa. METHODS/RESULTS: FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. CONCLUSIONS: While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.


Assuntos
Plaquetas/citologia , Fator VIIa/metabolismo , Tromboplastina/metabolismo , Adulto , Humanos , Pessoa de Meia-Idade
14.
Haemophilia ; 16 Suppl 3: 13-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20586796

RESUMO

Our group has been studying how haemostasis interacts with repair processes and also how to optimize treatment of bleeding disorders in a mouse model of haemophilia B. We have found that cutaneous wounds heal more slowly in haemophilic mice than in wild-type mice, and also exhibit histological abnormalities, even after closure of the skin defect. The haemophilic wounds showed reduced influx of inflammatory cells and increased angiogenesis. Even after surface closure, the haemophilic animals experienced repeated episodes of re-bleeding and progressive accumulation of iron in the wound bed and deeper tissues. A dose of replacement or bypassing therapy sufficient to establish initial haemostasis did not normalize wound healing. In fact, daily dosing for 7 days was required to normalize wound closure. Thus, normal healing requires adequate haemostatic function for an extended period of time. We have hypothesized that this is because angiogenesis during healing predisposes to bleeding, especially in the setting where haemostasis is impaired. Thus, normalizing haemostasis, until the process of angiogenesis has resolved, may be required to prevent re-bleeding and additional tissue damage.


Assuntos
Hemofilia B/fisiopatologia , Cicatrização/fisiologia , Ferimentos e Lesões/fisiopatologia , Animais , Hemostasia , Inflamação/complicações , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Coelhos , Pele/irrigação sanguínea , Pele/patologia , Ferimentos e Lesões/patologia
16.
J Thromb Haemost ; 5(8): 1577-83, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17663728

RESUMO

BACKGROUND: We recently reported that wound healing is abnormal in hemophilia B (HB) mice [1]. The wounds show abnormal histology: s.c. hematoma formation; delayed re-epithelialization; delayed macrophage influx; and an increase in wound site angiogenesis. OBJECTIVE: To test the hypothesis that restoring a hemostatic level of thrombin generation at the time of wounding would allow formation of an adequate platelet/fibrin plug and correct abnormalities of wound healing in HB. METHODS: We placed a 3-mm cutaneous wound on the back of each HB or wild-type (WT) mouse. Some HB mice were treated just prior to wounding with either human factor IX (FIX) or FVIIa in a dose sufficient to normalize bleeding in a tail bleed model. RESULTS: The average wound size over time in treated HB animals was intermediate between those in WT and untreated HB mice. However, the time to complete skin closure was not improved by treatment. Hematoma formation was decreased and macrophage influx began earlier in treated than in untreated HB animals. However, treated HB mice had evidence of ongoing low-level bleeding near the wound site, even after closure of the skin defect. Treatment also did not normalize the increased angiogenesis observed in HB mice. CONCLUSIONS: Restoring initial hemostasis can modulate some of the parameters of wound healing. However, an extended period of adequate hemostatic function is necessary to achieve normal healing, probably because the risk of hemorrhage is increased by vascular remodeling and angiogenesis during the healing process.


Assuntos
Fator IX/genética , Hemofilia B/terapia , Hemostasia , Trombina/biossíntese , Cicatrização , Animais , Fator IX/metabolismo , Fator VIIa/metabolismo , Hemofilia A/metabolismo , Hemofilia B/metabolismo , Hemorragia/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Pele/efeitos dos fármacos , Trombina/metabolismo
17.
J Thromb Haemost ; 5(6): 1097-105, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17567444

RESUMO

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.


Assuntos
Coagulação Sanguínea/fisiologia , Fator VIIa/fisiologia , Tromboplastina/fisiologia , Fator VIIa/química , Hemostasia , Humanos , Inflamação/sangue , Inflamação/etiologia , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/fisiologia , Modelos Moleculares , Complexos Multiproteicos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Solubilidade , Tromboplastina/química , Trombose/sangue , Trombose/etiologia
18.
J Thromb Haemost ; 5(7): 1403-8, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17425666

RESUMO

BACKGROUND: 'Idling' or ongoing low-level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. OBJECTIVE: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. METHODS: Immunostaining of mouse skin with antibodies against a 15-residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. RESULTS: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site-inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. CONCLUSIONS: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.


Assuntos
Fator VII/metabolismo , Pele/irrigação sanguínea , Pele/metabolismo , Tromboplastina/metabolismo , Animais , Coagulação Sanguínea/fisiologia , Fator VII/química , Fator VIIa/química , Fator VIIa/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Ligação Proteica , Tromboplastina/química , Tromboplastina/imunologia , Distribuição Tecidual
20.
J Thromb Haemost ; 4(1): 60-7, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16409451

RESUMO

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.


Assuntos
Endotélio Vascular/metabolismo , Trombina/biossíntese , Aorta/citologia , Fatores de Coagulação Sanguínea/farmacologia , Cloreto de Cálcio/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Cinética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Plasma , Tromboplastina/farmacologia
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