RESUMO
Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 +/- 53 nmol L(-1), 135 +/- 57 nmol L(-1), 218 +/- 55 nmol L(-1) and 407 +/- 59 nmol L(-1) (mean +/- SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (< or = 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.
Assuntos
Coagulação Sanguínea , Fenômenos Fisiológicos Celulares , Tromboplastina/fisiologia , Trombose/etiologia , Células Cultivadas , Cisteína Endopeptidases/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Fibroblastos/fisiologia , Humanos , Cinética , Macrófagos/fisiologia , Microscopia de Vídeo , Miócitos de Músculo Liso/fisiologia , Proteínas de Neoplasias/fisiologia , Fosfolipídeos/fisiologia , Trombina/biossíntese , Trombose/patologiaRESUMO
Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Anexina A5/metabolismo , Coagulação Sanguínea/fisiologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Coagulantes/farmacologia , Relação Dose-Resposta a Droga , Fator IXa/metabolismo , Fator VIII/metabolismo , Fator X/metabolismo , Humanos , Técnicas In Vitro , Glicoproteínas da Membrana de Plaquetas/análise , Trombina/farmacologiaRESUMO
The coagulation factor VIII is required for normal haemostasis, because deficiency or genetic defects in this molecule cause a life-threatening coagulation disorder known as hemophilia A. While the role of f VIII in the intrinsic pathway of blood coagulation has been extensively studied, the mechanisms responsible for f VIII turnover in circulation have not been characterized until recently. This review focuses on the finding that f VIII catabolism in vitro and in vivo is mediated by low-density lipoprotein receptor-related protein (LRP), representing a hepatic clearance receptor. FVIII interaction with LRP involves two distinct sites localized within the C2 and A2 domains of f VIII. We discuss the contribution of the A2 site (residues 484-509) and the C2 site in f VIII catabolism in the presence and absence of vWf. We present the evidence that LRP-mediated f VIII catabolism is facilitated by cell-surface heparan sulfate proteoglycans (HSPGs), which bind to the A2 residues 558-565 of f VIII. Because both LRP- and HSPGs-binding sites within the A2 domain are potentially exposed in the circulating f VIII/vWf complex, we discuss the possibility of prolongation of the f VIII lifetime in circulation by disrupting these sites employing site-directed mutagenesis. In its turn, generation of a novel recombinant f VIII may be prospective for more efficient hemophilia A therapy.
Assuntos
Fator VIII/metabolismo , Hemofilia A/sangue , Proteínas Relacionadas a Receptor de LDL/metabolismo , Fator VIII/fisiologia , Hemofilia A/genética , Heparitina Sulfato/farmacologia , Humanos , Proteínas Relacionadas a Receptor de LDL/fisiologia , Fator de von Willebrand/fisiologiaRESUMO
Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.
Assuntos
Fator IX/uso terapêutico , Fator VIII/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Fator IX/farmacocinética , Fator VIII/genética , Fator VIII/farmacocinética , Hemofilia A/tratamento farmacológico , Humanos , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Relação Estrutura-AtividadeRESUMO
Monoclonal antibodies (MABs) to a new drug Dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) enhancing ulcer healing have been produced. Dalargin is a synthetic analog of Leu-enkephalin. With about 40 compounds tested in competition radioimmunoassay it has been shown that specificity of the MABs is directed against the N-terminal tetrapeptide of the molecule. The MABs are sensitive to amino acid substitutions in any of the positions of the fragment and have no cross-reactivity with endogenous opioids. Their further application in pharmacokinetic studies in humans and for characterization of opioid receptors is discussed.
Assuntos
Anticorpos Monoclonais , Leucina Encefalina-2-Alanina/análogos & derivados , Encefalina Leucina/análogos & derivados , Sequência de Aminoácidos , Animais , Encefalina Leucina/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologiaRESUMO
At present, computer-assisted molecular modeling and virtual screening have become effective and widely-used tools for drug design. However, a prerequisite for design and synthesis of a therapeutic agent is determination of a correct target in the metabolic system, which should be either inhibited or stimulated. Solution of this extremely complicated problem can also be assisted by computational methods. This review discusses the use of mathematical models of blood coagulation and platelet-mediated primary hemostasis and thrombosis as cost-effective and time-saving tools in research, clinical practice, and development of new therapeutic agents and biomaterials. We focus on four aspects of their application: 1) efficient diagnostics, i.e. theoretical interpretation of diagnostic data, including sensitivity of various clotting assays to the changes in the coagulation system; 2) elucidation of mechanisms of coagulation disorders (e.g. hemophilias and thrombophilias); 3) exploration of mechanisms of action of therapeutic agents (e.g. recombinant activated factor VII) and planning rational therapeutic strategy; 4) development of biomaterials with non-thrombogenic properties in the design of artificial organs and implantable devices. Accumulation of experimental knowledge about the blood coagulation system and about platelets, combined with impressive increase of computational power, promises rapid development of this field.
Assuntos
Coagulação Sanguínea , Adesividade Plaquetária , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Simulação por Computador , Desenho de Fármacos , Humanos , Modelos TeóricosRESUMO
Technologies in molecular biology have greatly advanced the knowledge regarding the origin of haemophilia A and the physiology of the factor VIII (FVIII) protein. A variety of different mutations in the FVIII gene have been identified and their effects on the FVIII protein described. It has been shown that the frequency of haemophilia A is due to a high mutation rate predominantly in male germ cells. A significant proportion is originating de novo in early embryogenesis from somatic mutations, a finding that has implications for genetic counselling. The life-cycle of the FVIII protein and its structure-function relationships are continuously clarified. Most recently it has been shown that FVIII clearance from the circulation is mediated by the low-density lipoprotein receptor-related protein (LRP) and cell-surface heparan sulphate proteoglycans (HSPGs). These findings raise hope for novel recombinant FVIII molecules with prolonged half-life that may improve therapies for haemophlia A.
Assuntos
Hemofilia A/genética , Fator IX/genética , Fator VIII/genética , Fator VIII/metabolismo , Feminino , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Mutação , Receptores de LDL/genética , Relação Estrutura-Atividade , Fator de von Willebrand/genéticaRESUMO
The glycosaminoglycan hyaluronan (HA) and HA-binding proteins (HABPs) serve important structural and regulatory functions during development and in maintaining adult tissue homeostasis. Here we have identified and partially characterized the sequence and expression pattern of three putative novel HABPs. DNA sequence analysis revealed that two of the novel HABPs, WF-HABP and BM-HABP, form a unique HA-binding subfamily, whereas the third protein, OE-HABP, is more closely related to the LINK subfamily of HABPs. Northern blotting experiments revealed that the expression of BM-HABP was highly restricted, with substantial expression detected only in human fetal liver. In contrast, WF-HABP and OE-HABP mRNAs were detected in a number of tissues, with particularly prominent expression in highly vascularized tissues such as the heart, placenta, and lung. Additional studies showed that OE-HABP was expressed by cultured human endothelial cells, smooth muscle cells, and differentiated monocytes. However, only endothelial cells expressed WF-HABP mRNA, and its expression was regulated by growth state, being most prominent in quiescent endothelial cells. We further characterized the expression of WF-HABP in vivo and found that its expression colocalized with CD31-positive cells and was prominently expressed in microvessels in the human aorta and in atherectomy samples. Our data suggest that WF-HABP is an endothelial cell-specific HA receptor and that it may serve a unique function in these cells. The WF-HABP gene was localized to chromosome 3p21.31 and the OE-HABP gene to 15q25.2-25.3.
Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 3 , DNA Complementar/análise , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Adulto , Sequência de Aminoácidos , Mapeamento Cromossômico , DNA Complementar/genética , Humanos , Receptores de Hialuronatos/metabolismo , Dados de Sequência Molecular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
We have demonstrated previously that catabolism of a coagulation factor VIII (fVIII) from its complex with von Willebrand factor (vWf) is mediated by low density lipoprotein receptor-related protein (LRP) (Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999) J. Biol. Chem. 274, 37685-37692). In the present study, we found that this process is facilitated by cell surface heparan sulfate proteoglycans (HSPGs). This was demonstrated by simultaneous blocking of LRP and HSPGs in model cells, which completely prevented fVIII internalization and degradation from its complex with vWf. In contrast, the selective blocking of either receptor had a lesser effect. In vivo studies of clearance of (125)I-fVIII-vWf complex in mice also demonstrated that the simultaneous blocking of HSPGs and LRP led to a more significant prolongation of fVIII half-life (5.5-fold) than blocking of LRP alone (3.5-fold). The cell culture and in vivo experiments revealed that HSPGs are also involved in another, LRP-independent pathway of fVIII catabolism. In both pathways, HSPGs act as receptors providing the initial binding of fVIII-vWf complex to cells. We demonstrated that this binding occurs via the A2 domain of fVIII, since A2, but not other portions of fVIII or isolated vWf, strongly inhibited cell surface binding of fVIII-vWf complex, and the affinities of A2 and fVIII-vWf complex for the cells were similar. The A2 site involved in binding to heparin was localized to the region 558-565, based on the ability of the corresponding synthetic peptide to inhibit A2 binding to heparin, used as a model for HSPGs.
Assuntos
Fator VIII/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Receptores Imunológicos/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , CamundongosRESUMO
Fibroblast growth factor-1 (FGF-1) and lipoproteins play an important role in atherogenesis. In the present study, we explored a possible mechanism by which abnormal lipid metabolism could be linked to the proliferative aspects of the disease. We tested oxidized LDL (oxLDL) as a possible pathophysiological mediator of the release of FGF-1, using FGF-1-transfected mouse NIH 3T3 cells and FGF-1-transfected rabbit smooth muscle cells, and compared it with the release caused by elevated temperature. Immunoblot analysis showed that oxLDL induced the release of FGF-1 in a concentration-dependent manner from 10 to 100 micrograms/mL. The effect correlated with the extent of oxidative modification of LDL and was maximal within 4 hours of exposure of cells to oxLDL. In contrast to the temperature stress-induced FGF-1 secretion pathway, FGF-1 released in response to oxLDL (1) appeared in the conditioned medium as a monomer, (2) appeared independently of the presence of either actinomycin D or cycloheximide, and (3) was neither enhanced nor inhibited by brefeldin A. We did not detect cell loss, significant morphological changes, changes in growth characteristics, or other indications of lethal toxicity in oxLDL-treated cells. Although the level of lactate dehydrogenase activity was elevated after oxLDL exposure, the calculations showed that > 90% of the FGF-1 was released by viable cells. We propose that oxLDL-induced FGF-1 release is mediated by sublethal and apparently transient changes in cell membrane permeability. In the environment of an atherosclerotic lesion, oxLDL-induced FGF-1 release may be among the mediators of endothelial and smooth muscle cell proliferation.
Assuntos
Fator 1 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/metabolismo , Células 3T3 , Animais , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fator 1 de Crescimento de Fibroblastos/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Camundongos , Músculo Liso Vascular/patologia , Coelhos , TransfecçãoRESUMO
Factor VIII (FVIII) is an essential component of the intrinsic pathway of blood coagulation. Normal functioning of FVIII requires its interactions with other components of the coagulation cascade. In the circulation, it exists as a complex with von Willebrand factor (vWF). Upon activation by thrombin or activated factor X (FXa), activated FVIII (FVIIIa) functions as a cofactor for the serine protease factor IXa. Their complex assembled on the phospholipid surface activates FX to FXa, which consequently participates in formation of thrombin, the key protease of the coagulation cascade. Genetic deficiency in FVIII results in a coagulation disorder haemophilia A, which is treated by infusions of FVIII products. Approximately 25-30% of patients develop antibodies inhibiting FVIII activity (FVIII inhibitors). The major epitopes of inhibitors are located within the A2, C2 and A3 domains of the FVIII molecule. The inhibitory effects of antibodies are manifested at various stages of the FVIII functional pathway, including FVIII binding to vWF, activation of FVIII by thrombin, and FVIIIa incorporation into the Xase complex. We summarize the current knowledge of the FVIII sites involved in interaction with its physiological ligands and different classes of inhibitory antibodies and describe their inhibitory mechanisms. We outline the strategies aimed to overcome the effects of inhibitory antibodies such as development of human/porcine FVIII molecules, resistant to inhibitors. We also discuss approaches to modulate the antibody response, as well as efforts to develop a long-term immunotolerance to FVIII protein.
Assuntos
Hemofilia A/imunologia , Animais , Sítios de Ligação , Fator VIII/química , Fator VIII/imunologia , Fator VIII/fisiologia , Terapia Genética , Hemofilia A/terapia , Humanos , Tolerância Imunológica , Imunoterapia , Isoanticorpos/imunologia , Isoanticorpos/farmacologiaRESUMO
Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM alpha-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells. These findings suggest involvement of the stellate cells in a local immune response.