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1.
Bioconjug Chem ; 30(3): 775-784, 2019 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-30676028

RESUMEN

A nonvolatile fluorine-18 aldehyde prosthetic group was developed from [18F]SFB, and used for site-specific labeling of active site inhibited factor VII (FVIIai). FVIIai has a high affinity for tissue factor (TF), a transmembrane protein involved in angiogenesis, proliferation, cell migration, and survival of cancer cells. A hydroxylamine N-glycan modified FVIIai (FVIIai-ONH2) was used for oxime coupling with the aldehyde [18F]2 under mild and optimized conditions in an isolated RCY of 4.7 ± 0.9%, and a synthesis time of 267 ± 5 min (from EOB). Retained binding and specificity of the resulting [18F]FVIIai to TF was shown in vitro. TF-expression imaging capability was evaluated by in vivo PET/CT imaging in a pancreatic human xenograft cancer mouse model. The conjugate showed exceptional stability in plasma (>95% at 4 h) and a binding fraction of 90%. In vivo PET/CT imaging showed a mean tumor uptake of 3.8 ± 0.2% ID/g at 4 h post-injection, a comparable uptake in liver and kidneys, and low uptake in normal tissues. In conclusion, FVIIai was labeled with fluorine-18 at the N-glycan chain without affecting TF binding. In vitro specificity and a good in vivo imaging contrast at 4 h postinjection was demonstrated.


Asunto(s)
Aldehídos/química , Factor VII/antagonistas & inhibidores , Radioisótopos de Flúor/química , Oximas/química , Animales , Sitios de Unión , Dominio Catalítico , Ciclización , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tromboplastina/metabolismo , Distribución Tisular , Agua
2.
Bioconjug Chem ; 29(1): 117-125, 2018 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-29206443

RESUMEN

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.


Asunto(s)
Azidas/química , Química Clic/métodos , Radioisótopos de Cobre/química , Factor VII/química , Neoplasias Pancreáticas/diagnóstico por imagen , Serina Proteasas/química , Tromboplastina/análisis , Animales , Dominio Catalítico , Línea Celular Tumoral , Factor VII/farmacología , Femenino , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos con 1 Anillo , Humanos , Marcaje Isotópico/métodos , Ratones , Ratones Desnudos , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Serina Proteasas/farmacología
3.
Blood ; 130(14): 1661-1670, 2017 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-28729433

RESUMEN

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.


Asunto(s)
Coagulación Sanguínea , Factor VIII/metabolismo , Factor VIIa/metabolismo , Factor Xa/metabolismo , Tromboplastina/metabolismo , Factor VIIIa/metabolismo , Humanos , Trombina/metabolismo
4.
J Nucl Med ; 57(7): 1112-9, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27013699

RESUMEN

UNLABELLED: Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression. METHODS: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. RESULTS: Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue. CONCLUSION: (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.


Asunto(s)
Factor VII/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Tromboplastina/metabolismo , Animales , Línea Celular Tumoral , Radioisótopos de Cobre , Humanos , Marcaje Isotópico , Imagen por Resonancia Magnética , Ratones , Imagen Multimodal , Trasplante de Neoplasias , Tomografía de Emisión de Positrones , Radiometría , Radiofármacos , Distribución Tisular
5.
J Nucl Med ; 57(1): 89-95, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26383146

RESUMEN

UNLABELLED: Tissue factor (TF) is upregulated in many solid tumors, and its expression is linked to tumor angiogenesis, invasion, metastasis, and prognosis. A noninvasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII is the natural ligand to TF. Here we report the development of a new PET tracer for specific imaging of TF using an (18)F-labeled derivative of factor VII. METHODS: Active site-inhibited factor VIIa (FVIIai) was obtained by inactivation with phenylalanine-phenylalanine-arginine-chloromethyl ketone. FVIIai was radiolabeled with N-succinimidyl 4-(18)F-fluorobenzoate and purified. The corresponding product, (18)F-FVIIai, was injected into nude mice with subcutaneous human pancreatic xenograft tumors (BxPC-3) and investigated using small-animal PET/CT imaging 1, 2, and 4 h after injection. Ex vivo biodistribution was performed after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by enzyme-linked immunosorbent assay and correlated with the uptake of (18)F-FVIIai in the tumors measured in vivo by PET imaging. RESULTS: The PET images showed high uptake of (18)F-FVIIai in the tumor regions, with a mean uptake of 2.5 ± 0.3 percentage injected dose per gram (%ID/g) (mean ± SEM) 4 h after injection of 7.3-9.3 MBq of (18)F-FVIIai and with an average maximum uptake in the tumors of 7.1 ± 0.7 %ID/g at 4 h. In comparison, the muscle uptake was 0.2 ± 0.01 %ID/g at 4 h. At 4 h, the tumors had the highest uptake of any organ. Blocking with FVIIai significantly reduced the uptake of (18)F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P < 0.001). The uptake of (18)F-FVIIai measured in vivo by PET imaging correlated (r = 0.72, P < 0.02) with TF protein level measured ex vivo. CONCLUSION: (18)F-FVIIai is a promising PET tracer for specific and noninvasive imaging of tumor TF expression. The tracer merits further development and clinical translation, with potential to become a companion diagnostics for emerging TF-targeted therapies.


Asunto(s)
Dominio Catalítico , Factor VII/metabolismo , Radioisótopos de Flúor , Regulación Neoplásica de la Expresión Génica , Tomografía de Emisión de Positrones/métodos , Tromboplastina/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Femenino , Humanos , Ligandos , Ratones , Radioquímica , Distribución Tisular , Tomografía Computarizada por Rayos X
6.
J Labelled Comp Radiopharm ; 58(5): 196-201, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25820758

RESUMEN

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress.


Asunto(s)
Clorometilcetonas de Aminoácidos/química , Factor VII/química , Radiofármacos/síntesis química , Clorometilcetonas de Aminoácidos/farmacología , Animales , Dominio Catalítico , Factor VII/antagonistas & inhibidores , Radioisótopos de Flúor/química , Ratones , Radiofármacos/química , Radiofármacos/farmacocinética , Distribución Tisular
7.
J Drug Target ; 23(3): 257-65, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25510254

RESUMEN

We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.


Asunto(s)
Carbocianinas/análisis , Factor VIIa/análisis , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Imagen Óptica/métodos , Tromboplastina/inmunología , Clorometilcetonas de Aminoácidos/química , Animales , Carbocianinas/química , Células Cultivadas , Factor VIIa/química , Xenoinjertos/inmunología , Humanos , Ratones , Neoplasias/inmunología , Neoplasias/patología , Paclitaxel/química
8.
Thromb Res ; 134(1): 132-7, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24787991

RESUMEN

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI 1-161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays. MATERIALS AND METHODS: LDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4 µM phospholipids or 0.5 nM factor Xa (FXa) and 4 µM phospholipids, respectively. RESULTS: TFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI 1-161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height. CONCLUSIONS: Our results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI 1-161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.


Asunto(s)
Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Lipoproteínas LDL/farmacología , Lipoproteínas/metabolismo , Tromboplastina/antagonistas & inhibidores , Tromboplastina/metabolismo , Hemostasis , Humanos
9.
J Drug Deliv ; 2014: 865732, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25574399

RESUMEN

Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs). TF plays a central role in cancer angiogenesis, growth, and metastasis and, as such, is a target for therapy and drug delivery. TF is the cognate receptor of factor VIIa (fVIIa). We have coupled PTX (paclitaxel, also named Taxol) with a tripeptide, phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) and conjugated it with fVIIa. The key aim of the work is to evaluate the antiangiogenic effects of PTX-FFRck-fVIIa against a PTX-resistant breast cancer cell line. Matrigel mixed with VEGF and MDA-231 was injected subcutaneously into the flank of athymic nude mice. Animals were treated by tail vein injection of the PTX-FFRck-fVIIa conjugate, unconjugated PTX, or PBS. The PTX-FFRck-fVIIa conjugate significantly reduces microvessel density in matrigel (p < 0.01-0.05) compared to PBS and unconjugated PTX. The breast cancer lung metastasis model in athymic nude mice was developed by intravenous injection of MDA-231 cells expressing luciferase. Animals were similarly treated intravenously with the PTX-FFRck-fVIIa conjugate or PBS. The conjugate significantly inhibits lung metastasis as compared to the control, highlighting its potential to antagonize angiogenesis in metastatic carcinoma. In conclusion, PTX conjugated to fVIIa is a promising therapeutic approach for improving selective drug delivery and inhibiting angiogenesis.

10.
Thromb Res ; 130(3): e163-70, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22683021

RESUMEN

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 µg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 µg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.


Asunto(s)
Melanoma/tratamiento farmacológico , Melanoma/secundario , Proteínas y Péptidos Salivales/uso terapéutico , Tromboplastina/antagonistas & inhibidores , Animales , Aumento de la Célula , Línea Celular Tumoral , Proliferación Celular , Humanos , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Resultado del Tratamiento
11.
Thromb Res ; 129 Suppl 2: S44-5, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22405586

RESUMEN

Bleeds in hemophilia are treated either on demand or prophylactically by intravenous replacement therapy with FVIII or FIX. However, there is a call for subcutaneous and less frequent drug administration, and this need may be met by administration of a suitable antibody. Pioneering studies in vitro] and in a rabbit hemophilia model suggest that blockage of tissue factor pathway inhibitor (TFPI) provides a potential alternative approach to current therapy of hemophilia patients. TFPI down-regulates the initiation of coagulation by inhibition of FVIIa/TF/FXa and blockage of TFPI enhances FXa and thrombin generation. In line with these discoveries, TFPI targeting reagents with different potential benefits are currently evaluated as possible novel therapeutic agents. The development and testing of these agents in in vitro and in vivo hemophilia models provide new information about the mode of action of TFPI and its role in hemostasis. Blockage of TFPI with various antagonists has been shown to effectively enhance FX activation by TF/FVIIa and improve clot formation in hemophilia blood and plasma. The monoclonal antibody, mAb 2021, is one such antagonist directed towards the Kunitz-type protease inhibitor (KPI) 2 domain of TFPI which is now being tested in preclinical and clinical trials. Using mAb 2021, we have confirmed the original findings, and further characterized the pro-haemostatic effect of this specific anti-KPI-2 mAb in preclinical studies.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Hemofilia A/tratamiento farmacológico , Hemostáticos/farmacología , Lipoproteínas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Hemofilia A/sangre , Hemostáticos/uso terapéutico , Humanos , Lipoproteínas/sangre
12.
Nature ; 483(7391): 627-31, 2012 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-22407318

RESUMEN

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.


Asunto(s)
Intestino Delgado/irrigación sanguínea , Intestino Delgado/microbiología , Neovascularización Fisiológica , Receptor PAR-1/metabolismo , Tromboplastina/metabolismo , Alelos , Angiopoyetina 1/metabolismo , Animales , Enterocitos/metabolismo , Enterocitos/microbiología , Femenino , Glicosilación , Intestino Delgado/citología , Ratones , Fosforilación , Estructura Terciaria de Proteína/genética , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transducción de Señal , Trombina/metabolismo , Tromboplastina/química , Tromboplastina/genética
13.
Proc Natl Acad Sci U S A ; 109(10): 3927-31, 2012 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-22355108

RESUMEN

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8(-/-)) did not rescue the embryonic death of TFPI null (Tfpi(-/-)) mice. Tfpi(+/-) did not alter the bleeding phenotype of F8(-/-) mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8(-/-) mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi(+/-);F8(-/-) mice with hematopoietic Tfpi(-/-) cells compared with Tfpi(+/-);F8(-/-) mice with Tfpi(+/+) hematopoietic cells. Additionally, following femoral vein injury, Tfpi(+/-);F8(-/-) mice with Tfpi(-/-) hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi(+/+) hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.


Asunto(s)
Hemofilia A/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Femenino , Prueba de Complementación Genética , Genotipo , Células Madre Hematopoyéticas/citología , Hemofilia A/tratamiento farmacológico , Hemofilia A/genética , Hemorragia , Hígado/embriología , Trasplante de Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo
14.
Thromb Haemost ; 106(4): 712-23, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21800005

RESUMEN

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Tromboplastina/metabolismo , Animales , Coagulación Sanguínea , Línea Celular Tumoral , Transformación Celular Neoplásica , Micropartículas Derivadas de Células/patología , Coagulantes/metabolismo , Humanos , Melanocitos/patología , Melanocitos/trasplante , Melanoma/patología , Melanoma/fisiopatología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Plasma/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/fisiopatología , Trombofilia , Trombosis , Microambiente Tumoral
15.
Br J Haematol ; 152(1): 99-107, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21083658

RESUMEN

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.


Asunto(s)
Proteínas Antitrombina/fisiología , Coagulantes/farmacocinética , Factor VII/farmacocinética , Hemofilia A/sangre , Animales , Coagulación Sanguínea/efectos de los fármacos , Inhibidores de Factor de Coagulación Sanguínea/fisiología , Coagulantes/antagonistas & inhibidores , Modelos Animales de Enfermedad , Perros , Factor VII/antagonistas & inhibidores , Factor VIIa/antagonistas & inhibidores , Semivida , Masculino , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/sangre , Tromboelastografía/métodos
16.
J Biol Chem ; 286(7): 5756-67, 2011 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-21149441

RESUMEN

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.


Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Tromboplastina/metabolismo , Animales , Antígenos CD/genética , Células Cultivadas , Receptor de Proteína C Endotelial , Factor VIIa/genética , Factor VIIa/metabolismo , Factor X/genética , Factor X/metabolismo , Glicoproteínas/genética , Humanos , Ratones , Ratones Noqueados , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Miocitos del Músculo Liso/citología , Proteína C/genética , Proteína C/metabolismo , Estructura Terciaria de Proteína , Receptor PAR-1/genética , Receptor PAR-2/genética , Receptores de Superficie Celular/genética , Trombina/genética , Trombina/metabolismo , Tromboplastina/genética
17.
Blood ; 116(26): 6106-13, 2010 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-20861457

RESUMEN

Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized ß-arrestin recruitment site. Similar to PAR2(-/-) mice, TF cytoplasmic domain-deleted (TF(ΔCT)) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TF(ΔCT) mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TF(ΔCT) and TF(ΔCT)/PAR2(-/-) mice, and increased tumor vessel diameters of TF(ΔCT) mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.


Asunto(s)
Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/patología , Neovascularización Patológica , Receptor PAR-2/fisiología , Tromboplastina/fisiología , Animales , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal
18.
Thromb Res ; 126(5): 418-25, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20723970

RESUMEN

INTRODUCTION: Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay. RESULTS: Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIß. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells. CONCLUSIONS: Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.


Asunto(s)
Lipoproteínas/inmunología , Monocitos/inmunología , Tromboplastina/inmunología , Adulto , Citometría de Flujo , Humanos , Lipoproteínas/biosíntesis , Lipoproteínas/genética , Lipoproteínas/metabolismo , Microscopía Confocal , Persona de Mediana Edad , Monocitos/citología , Monocitos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tromboplastina/biosíntesis , Tromboplastina/genética , Tromboplastina/metabolismo , Trombosis/sangre
19.
J Biol Chem ; 285(26): 19959-66, 2010 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-20388709

RESUMEN

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF.FVIIa complex and by FXa in the ternary TF.FVIIa.FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF.FVIIa-Xa complex normally but were severely impaired in binary TF.FVIIa.PAR2 signaling. The residues identified were located in the model-predicted S2' pocket of FVIIa, and complementary PAR2 P2' Leu-38 replacements demonstrated that the P2' side chain was indeed crucial for PAR2 cleavage by TF.FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF.FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.


Asunto(s)
Factor VIIa/metabolismo , Receptor PAR-2/metabolismo , Transducción de Señal , Tromboplastina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Línea Celular , Línea Celular Tumoral , Factor VIIa/química , Factor VIIa/genética , Humanos , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Receptor PAR-2/genética , Especificidad por Sustrato , Tromboplastina/genética , Transfección
20.
Thromb Haemost ; 101(5): 818-26, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19404533

RESUMEN

The mechanism for the elimination of factor VII (FVII) from the circulation is unknown, just as it is unclear how activation of FVII to FVIIa and subsequent complex formation with antithrombin III (AT) or alpha2-macroglobulin (alpha2M) affects clearance. The possibility that the clearance mechanism involves activation and inhibitor complex formation as obligatory intermediate reactions is examined in this study. Human and murine sera were spiked with human FVIIa in the absence and presence of heparin and analysed for complex formation. Complex formation in vivo was studied after intravenous injection of (125)I-VIIa in mice; and the pharmacokinetics (PK) of human and murine FVIIa was studied in normal mice. Furthermore, comparative PK studies were performed with FVII, FVIIa, active site blocked FVIIa and a preformed FVIIa-AT complex in normal and alpha2M-deficient mice. The data demonstrated that FVIIa-AT complexes and to a much lesser extent FVIIa-alpha2M-complexes accumulated in vivo after FVIIa administration. FVIIa-AT accounted for about 50% of total FVIIa antigen left in the circulation after 3 hours. All FVII derivatives studied including FVII, FVIIa and FVIIa-AT were cleared with similar rates suggesting an elimination kinetics which is unaffected by FVII activation and subsequent inactivation by plasma inhibitors.


Asunto(s)
Antitrombina III/metabolismo , Factor VII/farmacocinética , Factor VIIa/farmacocinética , Seroglobulinas/metabolismo , alfa-Macroglobulinas/metabolismo , Animales , Factor VIIa/administración & dosificación , Heparina/sangre , Humanos , Inyecciones Intravenosas , Radioisótopos de Yodo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Seroglobulinas/deficiencia , Seroglobulinas/genética , alfa-Macroglobulinas/deficiencia , alfa-Macroglobulinas/genética
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