Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active siteinhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.The in vivo initiation of the coagulation cascade is triggered by the binding of plasma factor VIIa (FVIIa) 1 to the cell surface receptor tissue factor (TF) (1). TF is normally expressed in adventitial cells and pericytes surrounding blood vessels but not in cells that come in contact with blood, such as monocytes and endothelial cells (2, 3). Tissue injury disrupting the endothelial cell barrier is normally required for FVIIa to come in contact with TF. However, under pathological conditions monocytes and endothelial cells could be perturbed to induce TF (4 -7).Factor VII circulates as a single chain zymogen, and the binding to TF markedly increases the susceptibility of factor VII to cleavage at Arg 152 resulting in formation of a two-chain serine protease, FVIIa. TF is a glycoprotein consisting of 263 amino acids with a 219-amino acid extracellular part. The extracellular part is structured in two fibronectin type III-like domains (8, 9). The crystal structure of D-Phe-L-Phe-L-Arg (FFR)-FVIIa-soluble TF (sTF) complex has recently been determined (10). In this complex, FVIIa has been shown to adopt an extended confirmation and wrap around TF with the Gla domain near the cell membrane and the catalytic domain distal to it.Administration of inactivated FVIIa was shown to reduce angiographic restenosis and decrease neointimal hyperplasia in a rabbit atherosclerotic injury model (11) and also to reduce thrombus formation at sites of vascular injury in a baboon femoral balloon artery angioplasty model (12). In a preliminary study, it was shown that infusion of a low concentration of inac...
Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on cell surfaces trigger the intracellular signaling events. At present, it is unclear how these signals influence phenotype. To elucidate this, we have used cDNA microarray technology to examine changes in transcriptional program in human fibroblasts in response to exposure to FVIIa. cDNA microarrays revealed that FVIIa binding to TF up-regulated the expression of Cyr61 and CTGF (connective tissue growth factor), the genes that encode extracellular matrix signaling proteins Cyr61 and CTGF, respectively. Northern blot analysis confirmed that FVIIa binding to TF markedly increased the expression of Cyr61 and CTGF in a time- and dose-dependent manner. FVIIa catalytic activity is required for the gene induction. In addition to FVIIa, thrombin also induced the expression of Cyr61 and CTGF. Hirudin abolished the thrombin-induced expression of these mRNAs but not the FVIIa-induced expression. FVIIa-induced expression of Cyr61 appears not to involve the currently known protease-activated receptors (PARs), whereas thrombin-induced expression involves the activation of PAR1 and possibly an additional PAR. Various intracellular signaling pathway inhibitors exhibited different inhibitory pattern on FVIIa and thrombin-induced up-regulation of Cyr61. Cyr61 and CTGF could act as downstream mediators of FVIIa x TF in affecting various biological processes.
Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K m for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...
The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.
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