The extrinsic pathway of blood coagulation is initiated when factor VIIa (FVIIa) 1 circulating in plasma binds to the integral membrane protein, tissue factor (TF), exposed to the blood upon injury of the vessel wall. TF consists of a 219-residue (1-219) extracellular domain, a 23-residue (220 -242) transmembrane domain, and a 21-residue (243-263) cytoplasmic domain. The extracellular part of TF is structured in two fibronectin type III-like domains, which shows structural and sequence homology to the cytokine receptor superfamily. The important role of TF in hemostasis and thrombotic disorders such as atherogenesis is well established (see (1-5)). Recent findings suggest that TF may participate in biological processes other than hemostasis such as angiogenesis (6), embryo vascularization (7), and tumor metastasis (8, 9). Furthermore, it has been reported that binding of FVIIa to cell surface TF-induced intracellular Ca 2ϩ oscillations in a number of TFexpressing cells (10, 11), transient phosphorylation of tyrosine in monocytes (12), alteration in gene expression in fibroblasts (13), and enhanced expression of urokinase receptor in pancreatic cancer cells (14). Additional information about FVIIa/TFinduced signal transduction comes from our previous report showing that binding of FVIIa to cell surface TF resulted in phosphorylation of p44/42 MAPK (15).A potential role for the TF cytoplasmic domain in signal transduction was indicated by studies showing that TF expression markedly increased the metastatic potential of melanoma cells (16) and that the pro-metastatic property was critically dependent on this domain (8, 9). This supposition was further substantiated by experiments showing that the cytoplasmic domain of TF can be phosphorylated by a protein kinase C-dependent mechanism (17) and that a synthetic peptide based on the cytoplasmic domain can work as a substrate for cell lysate protein kinase activity (18). Furthermore, cysteine 245 in the TF cytoplasmic domain was shown to be acylated with long chain fatty acids (19).Although a number of studies, as referred above, suggest that TF is involved in induction of an intracellular activity, it is not clear how signal transduction is mediated across the membrane as a result of binding of FVIIa to TF, just as the role of the cytoplasmic domain of TF in this process is still unclear. The present study explores the effect of removing the TF cytoplasmic domain on p44/42 MAPK signaling and further examines the importance of FVIIa catalytic activity in mediating the activation of the p44/42 MAPK pathway. Our data show that the cytoplasmic TF domain with its putative sites for regulatory modifications is not required for FVIIa-induced p44/42 MAPK phosphorylation. Furthermore, the data provide evidence that specific FVIIa catalytic activity is required and that other serine proteases fail to mimic the response, suggesting that TF/FVIIa may be activating a novel receptor on the cell surface. EXPERIMENTAL PROCEDURESCell Culture-The baby hamster kidney cell line BHK-21 tk...

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.

Factor VIIa is a vitamin K-dependent enzyme whose ~-carboxyglutamic acid (Gla)-containing domain is important for calcium ion-dependent binding to the cofactor tissue factor and membrane surfaces. This domain contains 10 Gla residues, the individual roles and importance of which are not known. Comparisons with the homologous protein C, factor IX and prothrombin may provide functional information on the first nine Gla residues, whereas no data can be extrapolated to Gla-35 in factor VIIa. Therefore, the effects of posttranslational ycarboxylation and site-directed mutagenesis of Glu-35 were investigated. Mutations to Asp, Gin or Val all lead to a lower affinity for tissue factor by decreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Gin or Gla side chains at position 35 appear to fulfil the functional roles equally well.

Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in ␥-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca 2؉ binding site, the function of which is largely unknown. Sitedirected mutagenesis of two Ca 2؉ -liganding Asp residues in this domain abolished Ca 2؉ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca 2؉ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower k cat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50 -60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca 2؉ binding site in the first epidermal growth factorlike domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.

The protease domain of coagulation factor VIIa~FVIIa! is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor~TF!. To further elucidate the mechanisms behind these transformations, the effects of Zn to FVIIa, which was influenced by the presence of Ca 2ϩ , resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn 2ϩ inhibition. A search for putative Zn 2ϩ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn 2ϩ binding sites in the Glu210-Glu220 Ca 2ϩ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn 2ϩ -mediated allosteric down-regulation of FVIIa's activity.

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Plasminogen contains a unique disulphide bond, Cys558 Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558 Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558 Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.Keywords : disulphide bond; plasminogen mutant; urokinase-type plasminogen activator ; tissue-type plasminogen activator ; streptokinase.Plasminogen is the zymogen form of the serine protease, necting kringles 2 and 3, may also serve to stabilise the protein in a conformation important for its presentation of fibrinplasmin, consisting of 791 amino acids with a molecular mass of 92 000 Da. The protein contains an N-terminal Glu1-Lys77 binding sites. Plasminogen is activated by cleavage of a peptide bond at Arg561-Val562 located in a short disulphide linked peptide, five homologous kringle domains and a C-terminal serine protease domain. Native Glu1-plasminogen is converted to (Cys558 Cys566) peptide loop, CPGRVVGGC. Activation is Lys78-plasminogen by plasmin which cleaves at the Lys77-mediated by urokinase (uPA), tissue-type plasminogen activator Lys78 peptide bond. Kringle 1 and kringle 4 each contain a ly-(tPA) or a complex of streptokinase and plasminogen/plasmin. sine-binding site and kringle 5 contains an aminohexyl-binding After activation, the heavy and light chains are linked by two site. Several studies have indicated that kringle 1 and kringle 5 interdomain Cys548 Cys670 and Cys558 Cys566 disulphide are involved in fibrin binding via their lysine-binding sites, bonds. Plasminogen activators are remarkably specific proteinwhereas this may not be the case for kringle 4 although it con-ases since the activation loop in plasminogen is their only known tains such a site ([1] and [2] for reviews). Specific functions physiologically relevant subs...