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The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF·VIIa complex but did not reduce macromolecular substrate affinity for TF·VIIa. Similarly, mutations of the S and S′ subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.

The tissue factor (TF)-initiated blood coagulation protease cascade can be greatly inhibited in vivoby a potent anti-human-TF monoclonal antibody, 5G9. This antibody binds the carboxyl module of the extracellular domain of TF with a nanomolar binding constant and inhibits the formation of the TF·VIIa·X ternary initiation complex. We have determined the crystal structures of the extra-cellular modules of human TF, Fab 5G9, and their complex (TF·5G9) to 2.4 Å, 2.5 Å, and 3.0 Å, respectively, and measured the apparent inhibition constants of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7° change in the relative orientation between the D1 and D2 modules was observed when compared with other published TF structures. Comparison of the free and bound Fab 5G9 indicates that small segmental and side chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recognition involves 18 TF antigen residues and 19 Fab residues from six CDR with one of the largest buried surface areas seen to date. A combination of structural and mutagenesis data indicate that Tyr156, Lys169, Arg200, and Lys201 play the major role in the antibody recognition. The TF·5G9 structure provides insights into the mechanism by which the antibody 5G9 inhibits formation of the TF·VIIa·X ternary complex.

To study the signaling role or CD11a/CD18 in the early events of T cell activation we have examined the induction of transcription of two important cytokines, namely TNFα and IL-2. Human peripheral blood T cells were stimulated with PMA/ionophore or immobilized anti-CD3 mAb (OKT3) with or without CD11a/CD18 engagement. Induced cytokine production by immobilized OKT3 was enhanced (3- to 10-fold) in cells adhering to OKT3 and ICAM-1 coimmobilized surfaces and anti-CD11a mAb abolished this enhancement effect. Similarly, inhibition of the PMA/ionophore-induced CD11a/CD18-mediated homotypic aggregations of T cells by mAbs specific for either CD11a or ICAM-1 reduced the induced cytokine production by more than 70%. We have also observed that greatly enhanced cytokine production resulted from cellular interactions between activated T cells and monolayers of endothelial cells. This enhancement was inhibited by a combination of CD11a-, CD18-, and ICAM-1-specific mAbs implicating a role of CD11a/CD18 in leukocyte adhesion to endothelium and diapedesis as part of the inflammatory process. By Northern analyses induced TNFα mRNA expression was significantly enhanced by the engagement of CD11a/CD18 in all the conditions mentioned above. These results, together with our previous studies on monocytes, lead to the conclusion that engagement of the CD11/CD18 family of receptor results in the transduction of cellular signals that quantitatively enhance the expression of important leukocyte-mediated immune and inflammatory responses. Copyright 1993, 1999 Academic Press

The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196→ Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.

Thromboembolic disorders are commonly associated with cardiovascular, infectious, and neoplastic disease. A major link in the pathophysiology of thrombosis is the excessive triggering of the coagulation pathways by the initiating cofactor molecule termed tissue factor, an integral membrane glycoprotein. The tissue factor extracellular ligand binding domain is predicted to fold in an architecture similar to the cytokine receptor homology module. Functional sites in tissue factor have been defined by a combination of antibody, chemical cross‐linking, and mutational analyses providing a model for cofactor function that involves discrete interactions with both enzyme and substrate. The understanding of the structural basis of tissue factor function promises to facilitate rational design of inhibitor molecules for defined functional sites, eventually leading to effective in vivo therapeutics.—Ruf, W., Edgington, T. S. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB J.8: 385‐390; 1994.

We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureusV-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multim-eric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin-induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino-terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165→Ala, Lys166→Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158→Arg, Ser160→Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TFR158G160. In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TFA165A166 as the cofactor, but only 3.5-fold with TFR158G160. The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TFR158G160 and 7Ő8-fold with TFA165A166. This suggests that the molecular recognition of VII in complex with TF by the enzymes TFŐVIIa and factor Xa are similar. Generation of factor IXa by TFR158G160ŐVIIa was unaltered, but reduced 2-fold with TFA165A166. In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TFŐVIIa complex.

The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa. Enhancement of VIIa amidolytic function by TF Ala44 was reduced by 20–25% relative to wild-type and support of proteolytic function was diminished 6-fold indicating that this cofactor residue is significantly enhancing proteolysis of the macromolecular substrate by VIIa. Replacement of Asp44 by Glu, Thr and Arg exhibited a less severe phenotype with an approx. 4-fold reduced affinity for VIIa and a 2–3-fold diminished activation of Factor X. The improved activity of these mutants as compared with the Ala replacement is consistent with functional importance of an extended side chain at this position. The specific influence of the Asp44 exchange on catalytic function of the TF·VIIa complex indicates fine specificity of the TF ligand interface in mediating receptor and cofactor function.

Vascular endothelial cells derived from rabbit vena cava and maintained in continuous culture exhibited properties characteristic of the intact endothelium. These cells were used as a model for characterizing the fibrinolytic components specified by the endothelium. Endothelial cells in culture digested radiolabeled fibrinogen. Digestion resulted from the synthesis and secretion of a plasminogen activator. Fibrinolysis was not detected when cells were grown in medium lacking plasminogen, indicating the absence of plasminogen-independent fibrinolytic enzymes. Phorbol-myristate-acetate increased extracellular plasminogen activator activity dramatically. This increase was prevented when actinomycin D or cycloheximide was included in the growth medium, indicating that new gene expression was required for it. Intracellular plasminogen activator could not be detected unless the cell extracts were exposed briefly to mildly acidic conditions. Mixing experiments between acid-treated and untreated extracts suggested that the cells contained a potent, acid-labile inhibitor of fibrinolysis. As little as 10 μg of protein from whole cell extracts inhibited both cell and urokinase-mediated fibrinolysis by more than 70%. Cell fractionation studies localized the inhibitor to the cytosol whereas plasminogen activator activity was restricted to the membrane-rich fraction. This membrane fraction did not require acidification for activity, suggesting that the inhibitor had been removed and that acidification did not activate a plasminogen proactivator. These observations demonstrate that regulation of endothelial fibrinolytic activity is far more complex than had been anticipated and raise several uncertainties in regard to detecting the presence of plasminogen activators in cells and tissues.

In order to examine the control of human factor X biosyn thesis we have molecularly cloned the cDNA and investi gated the expression of the Factor X gene. A recombinant clone of approximately 1100 base pairs in length containing the sequence of factor X was identified in a Xgt11 human liver cDNA library by screening with polyclonal antibodies. One plaque was selected and confirmed for specificity with a mixture of five factor X specific monoclonal antibodies (MoAbs). A partial nucleic acid sequence of the 5' end of the cDNA corresponded to the described amino acid sequence between residues 41 and 56 of the light chain of factor X. Northern blot analysis of RNA from human liver and the hepatoma cell line. Hep G2, identified the factor X mRNA as a single molecular species of approximately 1700 bases. Cell lines which do not secrete factor X did not contain factor X mRNA indicating restriction of tran scription to hepatocytes. Slot-blot hybridization analysis of factor X and actin mRNA demonstrated no change in the levels of total or specific factor X mRNA in Hep G2 cells following treatment with warfarin or vitamin K. We con clude that modulation of factor X production by these drugs, known to influence 7-carboxylation and total factor X secretion by these cells, is mediated by changes in posttranscriptional events rather than by effects on the steady state levels of factor X mRNA.

Cells of monocytic lineage (Mo) persistently infected with human immunodeficiency virus (HIV) have been suspected to be a major reservoir for in vivo transmission of virus to susceptible target cells. Cellular events and mechanisms that upregulate viral gene expression in such cells are important issues. Because the traffic of such cells is central to biodistribution of HIV, we have explored the impact of interaction of endothelium with HIV-1–infected U1 promonocytic cells. Cocufturing of U1 with human umbilical endothelial cells (HUVEC) for 24 to 72 hours in the absence of stimulation induced HIV-1 p24 biosynthesis significantly. Antibody-blocking experiments indicated that CD11/CD18 integrins play a role in upregulation of HIV expression elicited by interaction with HUVEC. Engagement of CD11b/ CD18 by adherence of U1 to surfaces coated with either the cognate ligand fibrinogen or monoclonal antibody specific for CD11b/CD18 also enhanced p24 biosynthesis. Furthermore, endothelial cells were found to constitutively synthesize and secrete soluble factors that enhanced HIV-1 synthesis. The enhancing factors, of estimated size 10 to 45 kD, were induced in HUVEC to high levels by monokines or by lipopolysaccharide, resulting in markedly enhanced HIV-1 expression by U1. These endothelial cell-derived HIV-1–enhancing factors consist of, among others, interleukin-6 (IL-6), IL-1β, and granulocyte-macrophage CSF (GM-CSF). Our results suggest that activation of HIV biosynthesis in infected Mo via interaction with endothelium may impact significantly on the tissue distribution and pathogenesis of HIV infections.

The capacity of normal human T lymphocytes to form rosettes with sheep red blood cells can be inhibited by drugs or agents which induce elevations in intracellular levels of cyclic AMP. The effect is early in the presence of agents which elicit rapid elevations in intracellular cyclic AMP (isoproterenol, aminophylline) and occurs later in the presence of cholera toxin which induces a dalayed increase in endogenous cyclic AMP. Dibutyryl cyclic AMP is inhibitory, and the effects of dibutyryl cyclic AMP and the adenyl cyclase stimulators are potentiated by inhibition of phosphodiesterase. These data provide substantial evidence that elevation of intracellular cyclic AMP diminishes E rosette function of lymphocytes.

Biosynthesis of tumor necrosis factor-α (TNF-α) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-α promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-α promoter localized enhancer elements in a region (−182 to −37 base pairs (bp)) that were required for optimal transcription of the TNF-α gene in response to LPS. Two regions were identified: region I (−182 to −162 bp) contained an overlapping Sp1/Egr-1 site, and region II (−119 to −88) contained CRE and NF-κB (designated κB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and κB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and κB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the κB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-α promoter is mediated by concerted participation of at least two separatecis-acting regulatory elements.

The regional structure of fibrinogen was considered in reference to the terminal plasmin-cleavage fragments, D and E. By two independent approaches, a yield of two D and two E fragments was determined and this established the presence of two D and two E regions in each fibrinogen molecule. Immunochemically, it was shown that the expression of native fibrinogen determinants by the D:E complex was fully reconstituted by an equimolar mixture of D and E fragments, while other recombinant ratios failed to yield optimal reconstitution. Utilizing the cleavage-associated neoantigen of fibrinogen, fg-Dneo, as a quantitative immunochemical marker, it was determined that complete digestion of fibrinogen by plasmin yielded two D fragments. Since the D:E complex appeared to consist of an equal number of D and E fragments, the presence of two E regions and two D regions in fibrinogen was indicated. In polyacrylamide gel electrophoresis, the linear relationship between the concentration of the D or E fragment and its densitometric area in the gel was utilized to quantitate the yield of D and E fragments in terminal digests. The yield of two D and two E fragments was demonstrated and it was also shown that the yield of fragments was independent of the plasmin concentration and of the length of exposure of the D and E fragments to the enzyme. Thus, it appears that fibrinogen is a highly symmetrical molecule consisting of two E regions as well as two D regions.

Discrimination between the physiological cleavage fragments of fibrinogen and fibrin offers an approach to differentiation between fibrinogenolytic processes and fibrinolysis after coagulation. By use of the cleavage-associated neoantigen of fibrinogen (fg-Dneo) as a molecular marker, characteristic differences between the D regions of fibrinogen derivatives and fibrin derivatives can be demonstrated. The expression of fg-Dneoby X, Y, D:E complex, and D-fragments of fibrinogen or fibrin is shown to be quantitative and unitary. Characteristic differences between fg-Dneosites present on fibrinogen cleavage fragments, as contrasted to fibrin cleavage fragments, are indicated by different competitive inhibition slopes, and appear to reflect differential binding affinity of selected anti-fg-Dneoantibodies for the specific molecular site. There is a linear relationship between the slope of quantitative competitive inhibition and the relative molar ratio of fibrinogen and fibrin derivatives. Identical immunochemical expressions are observed in vitroand in vivo, and support the thesis that cleavage in vivois produced by plasmin. The differential immunochemical features of fg-Dneoexpression may be the result of stable conformational and/or subtle structural differences between the D region of fibrinogen and fibrin cleavage fragments and suggest that precise changes in the D region are associated with the fibrin transition. These molecular features not only provide additional insight into the molecular immunology and structure of fibrinogen, but also appear to offer a new molecular approach to discrimination between fibrinogenolytic mechanisms as contrasted to fibrinolysis secondary to coagulation.

The comparative reactivities of the non-fibrous and fibrous forms of desoxy-ribonucleoprotein were studied in the complement fixation test with LE sera. On the basis of “antigen” dilution experiments the non-fibrous DNP was shown to be between 10 and 100 times more reactive than fibrous DNP.

Molecular events responsible for modulation of neoantigenic expressions of a defined molecule have been explored in relation to three hypothetical molecular models (see below). Fibrinogen and its cleavage-associated neoantigen have been used as a prototype system. Physicochemical and enzymatic factors influencing neoantigenic expression were evaluated. The cleavage-associated neoantigen was not only exposed by plasmin and enzymes of similar specificity, but also in a qualitatively and quantitatively deficient fashion by enzymes of differing specificities. Denaturation of fibrinogen via reduction or pH alteration did not induce the neoantigen, but oxidation of the native fibrinogen molecule did elicit this neoantigenic expression. The neoantigen, once exposed on the D-fragment, was relatively stable to physical and chemical denaturation. These results are inconsistent with proposed cleavage site specificand neoconformationaldeterminant models and are consistent with a stericmodel, which postulates that the cleavage-associated neoantigenic determinant is buried in native fibrinogen but is exposed in certain altered molecular species. The importance of molecular conformation in the exposure of antigenic expressions of a molecule and in modulation of the binding affinity of a neoantigen for specific antibody is demonstrated.

Factor VIII coagulant activity (VIIIC) and the von Willebrand's disease antigen (Factor VIII-like antigen, vW-Ag) are biologically linked, and it has been suggested that they reside on the same molecule. However, insolubilized human isoantibody to VIIIC and rabbit antiserum containing antibodies to VIIIC and vW-Ag differentially bind and remove these entities from plasma, thus physically segregating one from the other. These findings indicate that Factor VIII coagulant activity resides on a molecule distinct from that expressing the von Willebrand's antigen.

The pathogenetic mechanism involved in a form of experimental allergic glomerulonephritis induced by immunization of rats with renal tubular antigen has been investigated. A single immunization with less than a milligram of a crude renal tubular preparation, probably containing less than 25 µg of the specific nephritogenic antigen, is effective in the induction of this form of chronic membranous glomerulonephritis. In the nephritic kidney autologous nephritogenic tubular antigen is found in the glomerular deposits along with γ-globulin and complement. When large amounts of antigen are injected during induction of the disease the exogenous immunizing antigen can also be detected in the glomerular deposits. It appears that this disease results from the formation of circulating antibodies capable of reacting with autologous renal tubular antigen(s) and the deposition of these antibodies and antigen(s) plus complement apparently as immune complexes in the glomeruli. This pathogenetic system has been termed an autologous immune complex disease and the resultant glomerulonephritis has been similarly designated.

The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-α5, is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 µg N of RTE-α5 in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-α5 is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of γ-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.

SH virus-associated antigens have been identified in the liver and blood by immunofluorescence. This antigenic expression of the hepatitis agent or its genome is found in the cytoplasm of hepatocytes and other cells of the liver. Acute hepatitis is associated with hepatocellular localization of SH antigen, whereas alternative or additional SH antigen in the cytoplasm of nonhepatocytes is observed in chronic or recurrent hepatitis. Persistent intrahepatic SH antigen has also been observed in conversion from acute to chronic hepatitis, and the persistence of this virus-associated antigen in the liver may serve a significant pathogenetic role in mediating direct anti-tissue responses as well as local immune complex reactions. Competitive inhibition studies of SH antigen suggest at least two antigenically and sterically independent determinants on the SH particle.

A renal tubular epithelial antigen is deposited in association with gamma globulin and complement in glomeruli from rats with experimental allergic glomerulonephritis induced by immunization with renal tubular antigens. Apparently, in normal kidneys this antigen is concentrated in the distal segment of the proximal convoluted tubular epithelium, and the principal source of this antigen in the glomerular deposits is autologous. This form of glomerulonephritis provides an experimental prototype for what may be termed "autologous immune-complex" diseases.

Alpha2-macroglobulin (α2M) is a broad-spectrum proteinase inhibitor (M~718,000) which is unique in its ability to inhibit proteinases from all four mechanistic classes. α2M inhibits coagulation proteinases, such as thrombin and factor Xa, and fibrinolytic proteinases, such as plasmin. α2M is also a major carrier of growth factors and may regulate growth factor activity. The primary receptors for α2M are the low density lipoprotein receptor related protein (LRP, KD~5 nM) and a higher affinity receptor recently identified as the 78 kDa glucose regulated protein (Grp78, KD~50 pM). In its native conformation α2M is present in plasma at very high concentrations (up to 5 μM). Upon binding to serum proteinases or small primary amines α2M is converted to the receptor-recognized form (α2M*) and then binds to LRP. LRP-associated α2M-proteinase complexes are rapidly internalized and degraded by phagocytic cells such as macrophages. Previous work in our laboratory has demonstrated that Grp78 binds to tissue factor (TF) and inhibits its functional activity. We now demonstrate that α2M* enhances TF-dependent procoagulant activity and factor Xa generation and this function is dependent on α2M* binding to LRP, but not Grp78, since receptor-associated protein (RAP) blocks the response. The effects of α2M on TF-dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but also observed in THP-1 monocytic cells. We are performing additional studies, using both α2M mutants and LRP-deficient cells, to further elucidate this novel mechanism by which α2M* may regulate hemostasis.

Alpha2-macroglobulin (α2M) is a broad-spectrum proteinase inhibitor (M~718,000) which is unique in its ability to inhibit proteinases from all four mechanistic classes. α2M inhibits coagulation proteinases, such as thrombin and factor Xa, and fibrinolytic proteinases, such as plasmin. α2M is also a major carrier of growth factors and may regulate growth factor activity. The primary receptors for α2M are the low density lipoprotein receptor related protein (LRP, KD~5 nM) and a higher affinity receptor recently identified as the 78 kDa glucose regulated protein (Grp78, KD~50 pM). In its native conformation α2M is present in plasma at very high concentrations (up to 5 μM). Upon binding to serum proteinases or small primary amines α2M is converted to the receptor-recognized form (α2M*) and then binds to LRP. LRP-associated α2M-proteinase complexes are rapidly internalized and degraded by phagocytic cells such as macrophages. Previous work in our laboratory has demonstrated that Grp78 binds to tissue factor (TF) and inhibits its functional activity. We now demonstrate that α2M* enhances TF-dependent procoagulant activity and factor Xa generation and this function is dependent on α2M* binding to LRP, but not Grp78, since receptor-associated protein (RAP) blocks the response. The effects of α2M on TF-dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but also observed in THP-1 monocytic cells. We are performing additional studies, using both α2M mutants and LRP-deficient cells, to further elucidate this novel mechanism by which α2M* may regulate hemostasis.

In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

Recently, a new class of RNAs has been identified that, like mRNAs, are usually transcribed by RNA polymerase II, but lack significant translated open reading frames. They are non-coding RNAs (ncRNAs) that exert roles directly as RNAs, and function as genetic regulators, or riboregulators. In the present study, we discovered a novel very large ncRNA. From differential gene profiling of CD31 positive tumor microvascular endothelial cells from a murine colon carcinoma, we amplified an ~380 bp nucleotide sequence, DP100. This RNA was present in tumor cells as well as tumor vascular endothelium. The full-length DP100 transcript is an ~7 Kb RNA lacking an open reading frame set for more than 66 amino acids. Also the hypothetically predicted proteins lacked significant similarity to known proteins, consistent with identity of DP100 as an unusually large non-coding RNA transcript. Further bioinformatic search demonstrated that the full-length DP100 sequence is highly homologous to the human alpha gene, which encodes an 8.5 Kb, non-coding RNA transcript, and is located in a region implicated in chromosomal abnormalities of human tumors. The full-length DP100, here designated the mouse alpha gene, is evolutionally highly conserved among vertebrates, suggesting its functional significance. The role for this ncRNA in cellular behavior is under investigation.

Recently, a new class of RNAs has been identified that, like mRNAs, are usually transcribed by RNA polymerase II, but lack significant translated open reading frames. They are non-coding RNAs (ncRNAs) that exert roles directly as RNAs, and function as genetic regulators, or riboregulators. In the present study, we discovered a novel very large ncRNA. From differential gene profiling of CD31 positive tumor microvascular endothelial cells from a murine colon carcinoma, we amplified an ~380 bp nucleotide sequence, DP100. This RNA was present in tumor cells as well as tumor vascular endothelium. The full-length DP100 transcript is an ~7 Kb RNA lacking an open reading frame set for more than 66 amino acids. Also the hypothetically predicted proteins lacked significant similarity to known proteins, consistent with identity of DP100 as an unusually large non-coding RNA transcript. Further bioinformatic search demonstrated that the full-length DP100 sequence is highly homologous to the human alpha gene, which encodes an 8.5 Kb, non-coding RNA transcript, and is located in a region implicated in chromosomal abnormalities of human tumors. The full-length DP100, here designated the mouse alpha gene, is evolutionally highly conserved among vertebrates, suggesting its functional significance. The role for this ncRNA in cellular behavior is under investigation.