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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.

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.

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.

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.