RESUMEN
Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes C3, CFB, and C5 and decreased expression of CFD This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (CD46, THBD, CD55, CFI, and CFH) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases C3AR1 expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.
Asunto(s)
Encéfalo/irrigación sanguínea , Activación de Complemento , Vía Alternativa del Complemento/fisiología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Glomérulos Renales/metabolismo , Microvasos/metabolismo , Síndrome Hemolítico Urémico Atípico/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Vía Alternativa del Complemento/genética , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Expresión Génica , Humanos , Inflamación/metabolismo , Glomérulos Renales/citología , Microvasos/citología , Microvasos/efectos de los fármacos , Modelos Biológicos , Proteínas Musculares/genética , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/genética , Microangiopatías Trombóticas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de von Willebrand/metabolismoRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1ß or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1ß had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.
Asunto(s)
Vía Alternativa del Complemento/inmunología , Células Endoteliales/inmunología , Glomérulos Renales/inmunología , Proteína C/metabolismo , Antígenos CD/análisis , Antígenos CD/biosíntesis , Síndrome Hemolítico Urémico Atípico/inmunología , Síndrome Hemolítico Urémico Atípico/metabolismo , Células Endoteliales/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoensayo , Glomérulos Renales/metabolismo , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr(1605)-Met(1606) peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Toxina Shiga I/metabolismo , Factor de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Escherichia coli Enterohemorrágica/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Femenino , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.
Asunto(s)
Modelos Biológicos , Factor de von Willebrand/química , Agregación Plaquetaria , Conformación Proteica , Estructura Terciaria de Proteína , Desplegamiento Proteico , TermodinámicaRESUMEN
Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.
Asunto(s)
Factor B del Complemento , Factor de von Willebrand , Humanos , Factor B del Complemento/metabolismo , Factor de von Willebrand/metabolismo , Células Endoteliales/metabolismo , Calor , Factor H de Complemento/metabolismo , Inflamación/metabolismo , Activación de Complemento , Complemento C3b/metabolismoRESUMEN
Human umbilical vein endothelial cell (HUVEC)-released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca(2+)/Zn(2+)-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y(1605)-M(1606) decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.
Asunto(s)
Proteínas ADAM/metabolismo , Células Endoteliales/metabolismo , Factor de von Willebrand/metabolismo , Proteína ADAMTS13 , Humanos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.
Asunto(s)
Factores de Coagulación Sanguínea/biosíntesis , Factores de Coagulación Sanguínea/genética , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Trombina/biosíntesis , Antitrombina III/genética , Antitrombina III/metabolismo , Carbohidrato Epimerasas/biosíntesis , Carboxiliasas/genética , Línea Celular , Factor V/genética , Factor V/metabolismo , Factor Xa/metabolismo , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Cetona Oxidorreductasas/biosíntesis , Modelos Biológicos , Fragmentos de Péptidos/metabolismo , Proteolisis , Protrombina/biosíntesis , Protrombina/genética , Protrombina/metabolismo , Trombomodulina/genética , Trombomodulina/metabolismo , Vitamina K Epóxido Reductasas/genéticaRESUMEN
Human endothelial cells (ECs) synthesize, store, and secrete von Willebrand factor multimeric strings and coagulation factor (F) VIII. It is not currently known if ECs produce other coagulation factors for active participation in coagulation. We found that 3 different types of human ECs in primary culture produce clotting factors necessary for FX activation via the intrinsic (FVIII-FIX) and extrinsic (tissue factor [TF]-FVII) coagulation pathways, as well as prothrombin. Human dermal fibroblasts were used as comparator cells. TF, FVII, FIX, FX, and prothrombin were detected in ECs, and TF, FVII, FIX, and FX were detected in fibroblasts. In addition, FVII, FIX, FX, and prothrombin were detected by fluorescent microscopy in EC cytoplasm (associated with endoplasmic reticulum and Golgi proteins). FX activation occurred on human umbilical vein EC surfaces without the addition of external coagulation proteins, proteolytic enzymes, or phospholipids. Tumour necrosis factor, which suppresses the generation of activated protein C and increases TF, augmented FX activation. Fibroblasts also produced TF, but (in contrast to ECs) were incapable of activating FX without the exogenous addition of FX and had a marked increase in FX activation following the addition of both FX and FVII. We conclude that human ECs produce their own coagulation factors that can activate cell surface FX without the addition of exogenous proteins or phospholipids.
Asunto(s)
Coagulación Sanguínea , Factor X/metabolismo , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Línea Celular , Citoplasma/metabolismo , Factor IX/análisis , Factor IX/metabolismo , Factor VII/análisis , Factor VII/metabolismo , Factor VIII/análisis , Factor VIII/metabolismo , Fibroblastos/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Microscopía Fluorescente , Cultivo Primario de Células , Protrombina/análisis , Protrombina/metabolismo , Piel/citología , Tromboplastina/análisis , Tromboplastina/metabolismoRESUMEN
The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, ß-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.
Asunto(s)
Factor VIII/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Complejos Multiproteicos/metabolismo , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/metabolismo , Regulación de la Expresión Génica/fisiología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Complejos Multiproteicos/ultraestructura , Receptores de Vasopresinas/biosíntesis , Cuerpos de Weibel-Palade/ultraestructuraRESUMEN
It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF--but not factor H.
Asunto(s)
Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/inmunología , Citoplasma/metabolismo , Células Endoteliales/inmunología , Fibrinógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunoensayo , Microscopía Fluorescente , Cuerpos de Weibel-Palade/metabolismoRESUMEN
Third-passage human umbilical vein endothelial cells (HUVECs) or fifth-passage human aortic endothelial cells (HAECs) were subjected to 25 dynes/cm(2) for 24 h in a parallel-plate flow system. Matched control cells were maintained in static conditions. Total RNA was isolated and pooled from six to eight slides per experiment. Changes in gene expression were analyzed by Northern blots and reverse transcriptase-polymerase chain reaction. Fold changes were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) values. In HUVECs, arterial levels of shear stress increased mRNA expression of Cytochrome P450 1A1 (CYP1A1) 10.8 +/- 2.1-fold, and CYP1B1 23.1 +/- 3.7-fold; whereas connective tissue growth factor (CTGF) expression was unchanged and endothelin-1 (ET-1) mRNA expression was decreased 0.7 +/- 0.05-fold. The authors determined whether these changes were induced by beta-naphthoflavone, a polyaromatic hydrocarbon, and whether they occurred in HAECs. beta-Naphthoflavone up-regulated CYP1A1 18.3 +/- 4.2-fold, and CYP1B1 4.1 +/- 0.3-fold in HUVECs. Shear stress up-regulated CYP1A1 6.3 +/- 0.4-fold and CYP1B1 51.1 +/- 2.1-fold in HAECs. In addition, the authors examined CYP1A1 and CYP1B1 proteins translated from these genes. Experiments identical to those described above were performed and the cells harvested for protein identification by Western blot of CYP1A1 and CYP1B1. Protein levels of CYP1A1 in HUVECs were up-regulated under shear stress, whereas protein levels of CYP1B1 were not.
Asunto(s)
Citocromo P-450 CYP1A1/genética , Sistema Enzimático del Citocromo P-450/genética , Células Endoteliales/metabolismo , Flujo Sanguíneo Regional/fisiología , Regulación hacia Arriba/fisiología , Hidrocarburo de Aril Hidroxilasas , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Medios de Cultivo Condicionados/farmacología , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelina-1/genética , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Inmediatas-Precoces/genética , Recién Nacido , Péptidos y Proteínas de Señalización Intercelular/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Estrés Mecánico , Regulación hacia Arriba/efectos de los fármacos , beta-naftoflavona/farmacologíaRESUMEN
BACKGROUND: Binding of platelet P-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) is an initial event in the interactions between platelets and monocytes. Platelet-monocyte complexes (PMCs) have been implicated in several vascular disease processes, including acute coronary syndromes (ACS) and complications after percutaneous coronary intervention (PCI). We investigated the effect of ex vivo blockade of PSGL-1, alone and in combination with blockade of the alphaMbeta(2) (Mac-1) and alpha(IIb)beta(3) (GP IIb/IIIa) integrins, on PMC formation. METHODS AND RESULTS: Dual-label flow cytometry was used to detect PMCs in the blood of 10 volunteers and 10 patients undergoing PCI who received intravenous GP IIb/IIIa antagonists. PSGL-1 blockade, both prior to and after platelet stimulation, markedly reduced the formation of PMCs. Concomitant ex vivo blockade of the alphaMbeta(2) and alpha(IIb)beta(3) integrins did not result in further decreases of PMCs compared to PSGL-1 blockade alone. Antagonism of PSGL-1 also led to near elimination of leukocyte-platelet interactions under flowing conditions. CONCLUSION: Blockade of PSGL-1 alone is sufficient to inhibit and reverse the formation of PMCs following platelet stimulation. Concurrent antagonism of PSGL-1 and the alpha(IIb)beta(3) and alphaMbeta(2) integrins was not more effective than inhibition of PSGL-1 alone. These results suggest that platelet-monocyte complex formation is mostly dependent on PSGL-1.
Asunto(s)
Plaquetas/metabolismo , Antígeno de Macrófago-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Monocitos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Adulto , Anticuerpos Monoclonales/metabolismo , Citometría de Flujo , Humanos , Inflamación , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Selectina-P/metabolismo , Activación Plaquetaria , Agregación Plaquetaria , Factores de TiempoRESUMEN
BACKGROUND: Vascular endothelial cells (ECs) express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF) multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP) is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome) or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura). Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs) by real-time PCR: C3 and C5; complement factor (CF) B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative) complement pathways. We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition). We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb) on ULVWF strings. CONCLUSIONS/SIGNIFICANCE: AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric strings. Our findings provide one possible molecular mechanism for clinical linkage between different types of thrombotic and complement-mediated disorders.
Asunto(s)
Vía Alternativa del Complemento , Proteínas del Sistema Complemento/inmunología , Hemostasis/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Trombosis/inmunología , Factor de von Willebrand/inmunología , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Proteína ADAMTS13 , Células Cultivadas , Vía Clásica del Complemento , Lectina de Unión a Manosa de la Vía del Complemento , Proteínas del Sistema Complemento/genética , Expresión Génica , Calor , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Unión Proteica , Multimerización de Proteína , Trombosis/patología , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.
Asunto(s)
Proteínas ADAM/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Síndrome Hemolítico-Urémico/metabolismo , Toxinas Shiga/farmacología , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/farmacología , Proteína ADAMTS13 , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células Cultivadas , Histamina/farmacología , Humanos , Peso Molecular , Adhesividad Plaquetaria , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Cordón Umbilical/citología , Cordón Umbilical/efectos de los fármacos , Cordón Umbilical/metabolismoRESUMEN
This study was undertaken to compare the platelet binding characteristics and anti-platelet efficacy of a nonpeptide glycoprotein IIb/IIIa antagonist roxifiban with orbofiban in static and dynamic adhesion and aggregation assays. The results indicate that roxifiban binds with higher affinity to glycoprotein IIb/IIIa receptors and exhibits slower dissociation rates than orbofiban. Furthermore, the platelet inhibitory effects of roxifiban, but not orbofiban, were unaffected by changes in plasma calcium concentrations. Both agents reduced, in a concentration-dependent manner, the size of platelet thrombi deposited onto collagen I upon perfusion of heparinized blood at a shear rate of 1,500/s. At a clinically achievable concentration of 60 nM, roxifiban abrogated the formation of thrombi containing > 20 platelets per thrombus, thereby displaying comparable in vitro efficacy to that achieved by the theoretical maximal abciximab blood concentration (3.5 microg/ml) produced after standard treatment. In contrast, orbofiban, even at 500 nM, was only effective in inhibiting the formation of larger platelet thrombi (> or =150 platelets per thrombus). Pretreatment of surface-anchored platelets with roxifiban (100 nM), but not orbofiban (500 nM), inhibited monocytic THP-1 cell attachment under flow. However, this heterotypic adhesion process was also suppressed when orbofiban (500 nM) was maintained in the perfusion buffer during the entire course of flow experiment. These findings demonstrate roxifiban (unlike orbofiban) is a potent glycoprotein IIb/IIIa antagonist with a long receptor-bound lifetime and prolonged anti-platelet efficacy and may thus be beneficial for the treatment and prevention of acute ischemic syndromes.