RESUMO
The proteolytic conversion of factor V to factor Va is central for amplified flux through the blood coagulation cascade. Heterodimeric factor Va is produced by cleavage at three sites in the middle of factor V by thrombin, yielding an N terminus-derived heavy chain and a C terminus-derived light chain. Here, we show that light chain formation resulting from the C-terminal cleavage is the rate-limiting step in the formation of fully cleaved Va. This rate-limiting step also corresponded to and was sufficient for the ability of cleaved factor V to bind Xa and assemble into the prothrombinase complex. Meizothrombin, the proteinase intermediate in thrombin formation, cleaves factor V more slowly than does thrombin, resulting in a pronounced defect in the formation of the light chain. A â¼100-fold reduced rate of meizothrombin-mediated light chain formation by meizothrombin corresponded to equally slow production of active cofactor and an impaired ability to amplify flux through the coagulation cascade initiated in plasma. We show that this defect arises from the occlusion of anion-binding exosite 2 in the catalytic domain by the covalently retained propiece in meizothrombin. Our findings provide structural insights into the prominent role played by exosite 2 in the rate-limiting step of factor V activation. They also bear on how factor V is converted into a cofactor capable of assembling into prothrombinase.
Assuntos
Precursores Enzimáticos/química , Fator Va/química , Proteólise , Trombina/química , Precursores Enzimáticos/metabolismo , Fator Va/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Trombina/metabolismoRESUMO
Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal γ-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase function. The latter results from the ability of F12 to reversibly bind to the (pro)catalytic domain through K2 with high affinity and highly favorable thermodynamic constants when it is a zymogen in comparison to proteinase. Such discrimination is lost for K2 binding after proteolytic removal of the N-terminal Gla-K1 region of F12. The Ca(2+)-stabilized structure of the Gla domain is not required for F12 to bind the zymogen form more favorably. Enhanced binding to zymogen versus proteinase correlates with the ability of the propiece to enforce zymogen-like character in the proteinase. This is evident in variants of meizothrombin, an intermediate of prothrombin activation that contains the propiece covalently attached. This phenomenon is also independent of the Gla domain. Thus, the presence of K1 in covalent linkage with K2 in the propiece governs the ability of K2 to bind the (pro)catalytic domain in favor of zymogen, thereby enforcing zymogen-like character in the proteinase.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Protrombina/química , Protrombina/metabolismo , Domínio Catalítico , Ativação Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Kringles , Modelos Moleculares , Ligação Proteica , TermodinâmicaRESUMO
Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by prothrombinase. We have examined the action of prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpectedly modest decrease in the rate of thrombin formation for desGla prothrombin but with a major effect on the pathway for substrate cleavage. Using desGla prothrombin variants in which the individual cleavage sites have been singly rendered uncleavable, we find that loss of membrane binding and other Gla-dependent functions in the substrate leads to a decrease in the rate of cleavage at Arg(320) and a surprising increase in the rate of cleavage at Arg(271). These compensating effects arise from a loss in the membrane component of exosite-dependent tethering of substrate to prothrombinase and a relaxation in the constrained presentation of the individual cleavage sites for active site docking and catalysis. Loss of constraint is evident as a switch in the pathway for prothrombin cleavage and the intermediate produced but without the expected profound decrease in rate. Extension of these findings to the action of prothrombinase assembled on platelets and endothelial cells on fully carboxylated prothrombin reveals new mechanistic insights into function on physiological membranes. Cell-dependent enzyme function is probably governed by a differential ability to support prothrombin binding and the variable accumulation of intermediates from the two possible pathways of prothrombin activation.
Assuntos
Membrana Celular/metabolismo , Protrombina/química , Tromboplastina/química , Coagulação Sanguínea , Plaquetas/metabolismo , Fator V/química , Fator Va/química , Fator Xa/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligação Proteica , Especificidade por SubstratoRESUMO
Thrombin is produced by the ordered action of prothrombinase on two cleavage sites in prothrombin. Meizothrombin, a proteinase precursor of thrombin, is a singly cleaved species that accumulates abundantly as an intermediate. We now show that covalent linkage of the N-terminal propiece with the proteinase domain in meizothrombin imbues it with exceptionally zymogen-like character. Meizothrombin exists in a slowly reversible equilibrium between two equally populated states, differing by as much as 140-fold in their affinity for active site-directed ligands. The distribution between the two forms, designated zymogen-like and proteinase-like, is affected by Na(+), thrombomodulin binding, or active site ligation. In rapid kinetic measurements with prothrombinase, we also show that the zymogen-like form is produced following the initial cleavage reaction and slowly equilibrates with the proteinase-like form in a previously unanticipated rate-limiting step before it can be further cleaved to thrombin. The reversible equilibration of meizothrombin between zymogen- and proteinase-like states provides new insights into its ability to selectively exhibit the anticoagulant function of thrombin and the mechanistic basis for its accumulation during prothrombin activation. Our findings also provide unexpected insights into the regulation of proteinase function and how the formation of meizothrombin may yield a long lived intermediate with an important regulatory role in coagulation.
Assuntos
Coagulação Sanguínea/fisiologia , Precursores Enzimáticos/química , Trombina/química , Trombomodulina/química , Precursores Enzimáticos/metabolismo , Humanos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Trombina/metabolismo , Trombomodulina/metabolismoRESUMO
Prothrombinase converts prothrombin to thrombin via cleavage at Arg(320) followed by cleavage at Arg(271). Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg(320) and initial cleavage at this site. Precise positioning of the Arg(320) site for cleavage is implied by essentially normal cleavage at Arg(320) in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg(320) to Gln reveals that prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg(271). In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.
Assuntos
Protrombina/metabolismo , Tromboplastina/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Domínio Catalítico , Linhagem Celular , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Protrombina/química , Especificidade por Substrato , Sus scrofaRESUMO
OBJECTIVE: Plasma high-molecular-weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to HKa with release of bradykinin (BK). We postulated a direct link between HKa and cytokine/chemokine release. METHODS AND RESULTS: HKa, but not BK, releases cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-S-transferase (GST) fusion proteins of kininogen domain 3 (D3), a fragment of domain 3, E7P (aaG255-Q292), HK domain 5 (D5), the D5 recombinant peptides HG (aa K420-D474) and HGK (aa H475-S626) stimulated secretion of IL-1beta from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1beta by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1beta secretion induced by tKa 98%, 89%, 85%, and 62%, respectively. Fractionation of mononuclear cells identified the responsible cell, a blood monocyte. Inhibitors of signaling pathways NFkB, JNK, and p38 but not extracellular signal-regulated kinase (ERK) decreased cytokine release from mononuclear cells. HKa increased the synthesis of IL-1beta as deduced by an increase of IL-1beta mRNA at 1 to 2 hours. CONCLUSIONS: HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1beta from human monocytes using intracellular signaling pathways initiated by uPAR, beta2 integrins and gC1qR.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Cininogênio de Alto Peso Molecular/farmacologia , Antígeno de Macrófago 1/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Complemento/metabolismo , Anticorpos Monoclonais/farmacologia , Antígeno CD11a/imunologia , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-1/genética , Interleucina-1/metabolismo , Cininogênio de Alto Peso Molecular/imunologia , Cininogênio de Alto Peso Molecular/metabolismo , Antígeno de Macrófago 1/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , NF-kappa B/fisiologia , Concentração Osmolar , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Complemento/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fatores de TempoRESUMO
Domain 5 (D5) of cleaved high molecular weight kininogen (HKa) inhibits angiogenesis in vivo and endothelial cell migration in vitro, but the cell signaling pathways involved in HKa and D5 inhibition of endothelial cell migration are incompletely delineated. This study examines the mechanism of HKa and D5 inhibition of two potent stimulators of endothelial cell migration, sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF), that act through the P13-kinase-Akt signaling pathway. HKa and D5 inhibit bovine pulmonary artery endothelial cell (BPAE) or human umbilical vein endothelial cell chemotaxis in the modified-Boyden chamber in response toVEGF or S1P. The inhibition of migration by HKa is reversed by antibodies to urokinase-type plasminogen activator receptor. Both HKa and D5 decrease the speed of BPAE cell migration and alter the morphology in live, time-lapse microscopy after stimulation with S1P or VEGF. HKa and D5 reduce the localization of paxillin to the focal adhesions after S1P and VEGF stimulation. To better understand the intracellular signaling pathways, we examined the effect of HKa on the phosphorylation of Akt and its downstream effector, GSK-3alpha HKa and D5 inhibit phosphorylation of Akt and GSK-3alpha after stimulation withVEGF and S1P. Inhibitors of Akt and P13-kinase, the upstream activator of Akt, block endothelial cell migration and disrupt paxillin localization to the focal adhesions after stimulation with VEGF and S1P. Therefore we suggest that HKa through its D5 domain alters P13-kinase-Akt signaling to inhibit endothelial cell migration through alterations in the focal adhesions.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Cininogênio de Alto Peso Molecular/farmacologia , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Androstadienos/farmacologia , Animais , Bovinos , Células Cultivadas , Cromonas/farmacologia , Células Endoteliais/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Cininogênio de Alto Peso Molecular/química , Lisofosfolipídeos/farmacologia , Morfolinas/farmacologia , Fragmentos de Peptídeos/química , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Estrutura Terciária de Proteína , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/farmacologia , WortmaninaRESUMO
High-molecular-weight kininogen (HK) and its domain 3 (D3) exhibit anticoagulant properties and inhibit platelet activation at low thrombin concentration in vitro. We hypothesized that the rapid occlusive thrombosis in HK-deficient (HKd) rats following endothelial injury of the aorta results from enhanced platelet aggregation by thrombin. The effects of D3 (G235-M357) or D3-derived peptides on thrombosis in vivo were tested. D3 and its exon 7C terminal peptide (E7CP, K270-Q292), expressed as glutathione S-transferase (GST) fusion proteins (GST-D3, GST-E7CP), or GST alone, as well as cleaved HK (HKa) or synthetic peptide E7CP, were infused intravenously 10 min before endothelial injury. Blood flow was reduced down to 10% of baseline flow within 28 +/- 5.2 min by a platelet-fibrin thrombus in GST-treated HKd rats compared with >240 min in GST-treated normal HK rats (wild type). GST-D3, GST-E7CP, HKa, or E7CP infusion prolonged the flow time to 233, >240, 223, and >240 min, respectively, in HKd rats. When GST-E7CP was infused 10 min after the injury, blood flow was maintained for >240 min. Thrombin-antithrombin concentrations were elevated by injury in HKd rats receiving GST from 35 to 55 microg/l and decreased with GST-E7CP, HKa, or E7CP reconstitution to 40, 15, and 9 microg/l, respectively. We conclude that HKd rats are prothrombotic and that HKa, kininogen D3, and its fragment E7CP modulate arterial thrombosis after endothelial injury.
Assuntos
Aorta/metabolismo , Endotélio Vascular/metabolismo , Fibrinolíticos/metabolismo , Cininogênio de Alto Peso Molecular/metabolismo , Fragmentos de Peptídeos/metabolismo , Trombose/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Antitrombina III , Aorta/efeitos dos fármacos , Aorta/lesões , Aorta/patologia , Aorta/fisiopatologia , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/lesões , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fibrina/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Glutationa Transferase/genética , Cininogênio de Alto Peso Molecular/química , Cininogênio de Alto Peso Molecular/genética , Cininogênio de Alto Peso Molecular/farmacologia , Cininogênio de Alto Peso Molecular/uso terapêutico , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/sangue , Agregação Plaquetária , Estrutura Terciária de Proteína , Ratos , Ratos Endogâmicos Lew/genética , Proteínas Recombinantes de Fusão/metabolismo , Fluxo Sanguíneo Regional , Trombina/metabolismo , Trombose/patologia , Trombose/fisiopatologia , Trombose/prevenção & controleRESUMO
We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer.