Localization of the human tissue factor recognition determinant of human factor VIIa

Tissue factor is an integral membrane glycoprotein that serves as an essential cofactor for the blood coagulation factor VIIa. Recent studies have attempted to localize the tissue factor recognition determinant of human factor VIIa. While several regions of factor VIIa have been implicated as important for tissue factor binding, the high affinity tissue factor recognition determinant of human factor VIIa is unknown. In order to define the determinant, we constructed a set of six chimeric proteins composed of portions of factor VII and factor IX. We then utilized the chimeras in competition experiments with 125I-labeled factor VIIa for recombinant tissue factor bound to an Immobilon-P membrane. The data indicate that the high affinity tissue factor recognition determinant of human factor VIIa is within the epidermal growth factor domains.     

The factor V activation paradox

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.     

The tissue factor region that interacts with factor Xa in the activation of factor VII

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.     

Biological activity of vegetalizing and neuralizing inducing factors after binding to BAC-cellulose and CNBr-sepharose

Summary Covalent binding to bromoacetyl-cellulose inactivates the vegetalizing factor. The bound factor is however still able to form a complex with an inhibitor for the factor. Covalent binding to CNBr-Sepharose likewise inactivates the vegetalizing factor. The neuralizing factor on the other hand is not inactivated when covalently bound to CNBr-Sepharose. When a crude fraction which contains the neuralizng factor as well as the vegetalizing factor is bound to CNBr-Sepharose the vegetalizing activity is greatly decreased whereas the neuralizing activity is not reduced. This suggests that the mechanisms of action of the two factors are quite different. Whereas the vegetalizing factor must be incorporated into the cells, the neuralizing factor interacts with the plasma membrane of competent ectoderm cells.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.     

Mapping of the factor Xa binding site on factor Va by site-directed mutagenesis

Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on the introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells, and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and 501A/510A/511D/513A/577A/578A exhibited markedly reduced factor Xa-cofactor activity tested in the prothrombinase assay, and reduced binding affinity as judged by the direct binding assay. These factor Va variants were normally cleaved at Arg-506 by activated protein C, and the interaction between the factor Xa-factor Va complex and prothrombin was unaffected by the introduced mutations. Based on the integration of all available data, we propose a key factor Xa binding surface to be centered on Arg-501, Arg-510, Ala-511, Asp-513, Asp-577, and Asp-578 in the factor Va A2 domain. These residues form an elongated charged factor Xa binding cluster on the factor Va surface.     

Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X

The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.     

A serum factor capable of stimulating hyaluronic acid synthesis in cultured rat fibroblasts.

Calf serum as well as rat and mouse sera has a factor that stimulates hyaluronic acid synthesis in cultured rat fibroblasts. Such a factor was partially purified from calf serum and characterized. It has a molecular weight of approximately 150,000. The activity of the factor is lost by treatment with pronase and by periodate oxidation. It is suggested, therefore, that the factor is a glycoprotein. Its susceptibility to alpha-mannosidase and affinity for Con A-Sepharose may suggest that the factor contains a mannose residue(s) which is essential for the activity to induce hyaluronic acid synthesis.     

The emerging neuropoietic cytokine family: first CDF/LIF, CNTF and IL-6; next ONC, MGF, GCSF?

CDF/LIF is a polyfunctional cytokine that shares a remarkable overlap with ciliary neurotrophic factor in its actions on neurons, and with interleukin-6 in its actions on other tissues. Moreover, the receptors for this cytokine, as well as those for ciliary neurotrophic factor, share homology with the subunits of the interleukin-6 receptor. The predicted structural similarity of these proteins with oncostatin M, myelomonocytic growth factor and granulocyte colony-stimulating factor, as well as at least a partial overlap in biological activities, is now prompting further examination of their roles in neuronal gene expression.     

Effects of mutations of a phosphorylation site in an exposed loop in acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) contains a phosphorylation site recognized by protein kinase C. A non-mitogenic mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to high affinity receptors as well as to heparin. We have also studied the ability of the mutants to stimulate DNA synthesis and cell proliferation as well as phosphorylation of mitogen-activated protein kinase and the ability of the growth factor mutants to be transported to the nucleus. The results indicate that while the mutations strongly affect the ability of the growth factor to bind to heparin, they do not affect much the binding to the specific FGF receptors, activation of mitogen-activated protein kinase or transport of the growth factor to the nucleus. The mutations affect to various extents the ability of the growth factor to stimulate DNA synthesis and to induce cell multiplication. We find that phosphorylation of aFGF is not required for mitogenic activity. The data suggest that altered interaction of the growth factor with a cellular component different from the receptor, possibly a component in the nucleus, is the reason for the different mitogenicity of the different growth factor mutants.     

Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.     

Peptide mimics of epidermal growth factor (EGF) with antagonistic activity

Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.     

Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.     

Stem cell factor presentation to c-Kit. Identification of a basolateral targeting domain

Stem cell factor (also known as mast cell growth factor and kit-ligand) is a transmembrane growth factor with a highly conserved cytoplasmic domain. Basolateral membrane expression in epithelia and persistent cell surface exposure of stem cell factor are required for complete biological activity in pigmentation, fertility, learning, and hematopoiesis. Here we show by site-directed mutagenesis that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal to this motif, a cluster of acidic amino acids serves to increase the efficiency of basolateral sorting mediated by the leucine residue. Hence, basolateral targeting of stem cell factor requires a mono-leucine determinant assisted by a cluster of acidic amino acids. This mono-leucine determinant is functionally conserved in colony-stimulating factor-1, a transmembrane growth factor related to stem cell factor. Furthermore, this leucine motif is not capable of inducing endocytosis, allowing for persistent cell surface expression of stem cell factor. In contrast, the mutated cytoplasmic tail found in the stem cell factor mutant Mgf(Sl17H) induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore present mono-leucines as a novel type of protein sorting motif for transmembrane growth factors.     

Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer.

Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.