Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway.

Interleukin-11 (IL-11) reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum- and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dose-dependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours. Survivin protein expression was maximal at 6 hours of treatment and remained elevated through 24 hours. Survivin induction may be mediated by activation of protein kinase B/Akt, but IL-11 failed to activate this pathway in HUVEC. IL-11 did activate signal transducer and activator of transcription (STAT)-3 and IL-11 failed to induce survivin expression in HUVEC transduced with a dominant-negative STAT3 mutant, whereas control-transduced HUVEC responded normally. An IL-11 transgene caused increased survivin mRNA expression in mice compared with control littermates. Intradermal injection of IL-11 (500 ng) into human skin xenografts on immunodeficient mice up-regulated survivin protein in microvascular endothelium and epithelial keratinocytes. We conclude that IL-11 induces expression of survivin, an antiapoptotic protein, in vitro and in vivo, and identify STAT3 as a critical mediator of this response.

Urine detection of survivin and diagnosis of bladder cancer.

CONTEXT: Dysregulation of apoptosis may favor onset and progression of cancer and influence response to therapy. Survivin is an inhibitor of apoptosis that is selectively overexpressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. OBJECTIVE: To investigate the potential suitability of survivin detection in urine as a novel predictive/prognostic molecular marker of bladder cancer. DESIGN, SETTING, AND PATIENTS: Survey of urine specimens from 5 groups: healthy volunteers (n = 17) and patients with nonneoplastic urinary tract disease (n = 30), genitourinary cancer (n = 30), new-onset or recurrent bladder cancer (n = 46), or treated bladder cancer (n = 35), recruited from 2 New England urology clinics. MAIN OUTCOME MEASURES: Detectable survivin levels, analyzed by a novel detection system and confirmed by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR), in urine samples of the 5 participant groups. RESULTS: Survivin was detected in the urine samples of all 46 patients with new or recurrent bladder cancer using a novel detection system (31 of 31) and RT-PCR (15 of 15) methods. Survivin was not detected in the urine samples of 32 of 35 patients treated for bladder cancer and having negative cystoscopy results. None of the healthy volunteers or patients with prostate, kidney, vaginal, or cervical cancer had detectable survivin in urine samples. Of the 30 patients with nonneoplastic urinary tract disease, survivin was detected in 3 patients who had bladder abnormalities noted using cystoscopy and in 1 patient with an increased prostate-specific antigen level. Patients with low-grade bladder cancer had significantly lower urine survivin levels than patients with carcinoma in situ (P =.002). CONCLUSIONS: Highly sensitive and specific determination of urine survivin appears to provide a simple, noninvasive diagnostic test to identify patients with new or recurrent bladder cancer.

Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin.

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34(cdc2) on the mitotic apparatus, and is phosphorylated on Thr(34) by p34(cdc2)-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr(34) resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34(cdc2)-cyclin B1 and suggest that survivin phosphorylation on Thr(34) may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.

AlphaMbeta2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to alphaM I domain that is divalent cation-sensitive.

The beta2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the alphaM subunit, which directly induces adhesion of alphaMbeta2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the alphaM I domain. The alphaM I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of alphaMbeta2 ligand-binding affinity. Thus, these studies have identified a novel alphaM I domain activation epitope of alphaMbeta2 and support the idea that the I domain modulates the activational state of the beta2 integrins.

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Control of apoptosis and mitotic spindle checkpoint by survivin.

Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin-microtubule interactions results in loss of survivin's anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.

Molecular identification of the cross-reacting epitope on alphaM beta2 integrin I domain recognized by anti-alphaIIb beta3 monoclonal antibody 7E3 and its involvement in leukocyte adherence.

The monoclonal antibody (mAb) 7E3 directed to the platelet integrin alphaIIb beta3 was tested for its cross-reactivity with the homologous leukocyte integrin alphaM beta2. Nested recombinant fragments of alphaM I domain were expressed as glutathione S-transferase fusion proteins and analyzed for antibody recognition. In enzyme-linked immunosorbent assay, mAb 7E3 bound alphaM I domain fragments containing the amino-terminal sequence Cys128-Ser172, whereas the carboxyl-terminal region Leu173-Pro291 was ineffective. A synthetic peptide designated R1.1 and duplicating the alphaM sequence G127CPQEDSDIAFLIDGSGSIIPHDF150 bound mAb 7E3. In contrast, the adjacent alphaM region F150RRMKEFVSTVMEQLKKSKTLFS172 or a control peptide with a scrambled R1.1 sequence was not recognized by mAb 7E3. Binding of mAb 7E3 to alphaM I domain blocked monocyte and neutrophil adhesion to immobilized fibrinogen and fibrinogen-dependent leukocyte-endothelium bridging, indistinguishably from bona fide anti-beta2 mAb IB4. In contrast, leukocyte binding to stable transfectants expressing intercellular adhesion molecule-1 was not affected by mAb 7E3. Balloon-mediated injury of iliofemoral arteries in rabbits resulted in prominent deposition of fibrinogen and increased monocyte adhesion to the injured vessel, in a reaction inhibited by mAb 7E3, but unaffected by control mAb 14E11. Through its cross-reactivity between alphaIIb beta3 and alphaM beta2, mAb 7E3 may initiate a new class of integrin antagonists, capable of simultaneously targeting platelet and leukocyte adhesion mechanisms in vascular injury.

Dual regulation of ligand binding by CD11b I domain. Inhibition of intercellular adhesion and monocyte procoagulant activity by a factor X-derived peptide.

The role of coagulation factor X as a ligand for CD11b/CD18 (Mac-1, alphaMbeta2) in leukocyte adhesion was investigated. A factor X peptide, (G)L238YQAKRFKV246(G), blocked ligand binding to CD11b/CD18 and prevented monocyte procoagulant activity. This peptide also inhibited monocytic THP-1 cell adhesion to tumor necrosis factor alpha-stimulated endothelium and blocked neutrophil migration through tumor necrosis factor alpha-activated endothelial cell monolayers. In contrast, other factor X-derived peptides were ineffective. Radiolabeled peptide (G)LYQAKRFKV(G) bound specifically and saturably to isolated recombinant CD11b I domain. Functionally, the factor X sequence (G)LYQAKRFKV(G) dose-dependently inhibited THP-1 cell attachment to intercellular adhesion molecule 1 (ICAM-1) transfectants (IC50 = approximately 50 microg/ml), indistinguishably from anti-CD18 monoclonal antibodies 60.3 and IB4. In contrast, peptide (G)LYQAKRFKV(G) failed to reduce binding of 125I-fibrinogen to immobilized CD11b I domain, which was abolished by the fibrinogen-derived peptide KYG190WTVFQKRLDGSV202. By Lineweaver-Burke analysis, peptide (G)LYQAKRFKV(G) inhibited factor X binding to CD11b/CD18 in a noncompetitive fashion, and intact factor X did not reduce monocyte-endothelial cell interaction. These data suggest that the factor X sequence (G)LYQAKRFKV(G) defines an ICAM-1-binding site on CD11b I domain physically distinct from and nonoverlapping with the fibrinogen interacting region(s). Engagement of this site induces a conformational change in the holoreceptor, which disrupts a distant factor X-binding site required for monocyte procoagulant activity. These observations demonstrate a dual regulatory role of CD11b I domain in ligand binding and provide a molecular basis for the recently reported anti-inflammatory properties of factor X homologous sequences in vivo.

Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1.

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.

Molecular identification of a novel fibrinogen binding site on the first domain of ICAM-1 regulating leukocyte-endothelium bridging.

Binding of fibrinogen to intercellular adhesion molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the structure-function requirements of this recognition. All four mAbs bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted ICAM-1 from detergent-solubilized JY lymphocyte extracts. Functionally, mAbs 1G12 and 2D5 inhibited binding of 125I-fibrinogen to ICAM-1-transfectants and abrogated the enhancing effect of fibrinogen on mononuclear cell adhesion to endothelium and transendothelial migration. In contrast, mAbs 3D6 and 6E6 did not affect ICAM-1 recognition of fibrinogen. With respect to other ligands, mAbs 1G12 and 2D5 completely inhibited attachment of Plasmodium falciparum-infected erythrocytes to immobilized recombinant ICAM-1-Fc, whereas they had no effect on LFA-1-dependent T cell binding to ICAM-1-Fc. Conversely, mAbs 3D6 and 6E6 completely abolished LFA-1 binding to ICAM-1-Fc. Epitope assignment using ICAM-1 chimeras and receptor mutants revealed that the fibrinogen-blocking mAbs 1G12 and 2D5 reacted with domain 1 of ICAM-1, and their binding was disrupted by 97 and 70% by mutations of D26 and P70, respectively, whereas mAbs 3D6 and 6E6 bound to domain 2 of ICAM-1. By recognizing a site distinct from that of beta2 integrins Mac-1 or LFA-1, fibrinogen binding to ICAM-1 may provide an alternative pathway of intercellular adhesion and/or modulate integrin-dependent adherence during inflammation and vascular injury.

Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation.

Leucocyte initiation of coagulation preserves the haemostatic balance and may aberrantly contribute to vascular injury. In addition to the extrinsic activation mediated by tissue factor: factor VIIa, monocytes express an alternative procoagulant response after binding of the zymogen factor X to the integrin Mac-1 (CD11b/CD18). Here, factor X-activating activity was found in purified monocyte granules, and coincided with size-chromatographed fractions containing cathepsin G. In contrast, elastase-containing granule fractions did not activate factor X. In the presence of Ca2+ ions, purified cathepsin G, but not elastase, cleaved factor X to a approximately 54 kDa catalytically active derivative, structurally indistinguishable from the procoagulant product generated on monocytes after binding to Mac-1. Factor X activation by purified cathepsin G involved limited proteolysis of a novel Leu177-Leu178 peptide bond in the zymogen's activation peptide. Cathepsin G activation of factor X was completely inhibited by alpha 1 antichymotrypsin, or soybean trypsin inhibitor, or by a neutralizing antiserum to cathepsin G, while eglin, or an anti-elastase antibody, were ineffective. Affinity chromatography on active-site-dependent inhibitors Glu-Gly-Arg-chloromethyl ketone or benzamidine completely abolished factor Xa activity generated by cathepsin G. Cathepsin G was not constitutively detected on the monocyte surface by flow cytometry. However, inflammatory stimuli, including formyl peptide or phorbol ester, or Mac-1 engagement with its ligands fibrinogen, factor X or serum-opsonized zymosan, triggered monocyte degranulation and cathepsin G activation of factor X. These findings demonstrate that monocytes can alternatively initiate coagulation in a sequential three-step cascade, including (i) binding of factor X to Mac-1, (ii) discharge of azurophil granules, and (iii) limited proteolytic activation of membranebound factor X by cathepsin G. By rapidly forming thrombin and factor Xa in a protected membrane microenvironment, this pathway may contribute a "priming' signal for clotting, anticoagulation and vascular cell signal transduction, in vivo.

Protease receptors in Hodgkin's disease: expression of the factor Xa receptor, effector cell protease receptor-1, in Reed-Sternberg cells.

The expression of a cellular receptor for the blood-clotting protease factor Xa, designated effector cell protease receptor-1 (EPR-1), was investigated in lymphoma. Immunohistochemical analysis demonstrated prominent reactivity of monoclonal antibodies to EPR-1 with Reed-Sternberg cells in 30 of 35 cases of nodular-sclerosis, lymphocyte-depletion, and mixed-cellularity Hodgkin's disease (HD). In contrast, several non-Hodgkin's lymphomas, or the nonneoplastic cellular components of HD, did not react with anti-EPR-1 monoclonal antibodies. A single molecular species of approximately 62 kD, consistent with the size and structural organization of EPR-1, was immunoblotted by an anti-EPR-1 monoclonal antibody from tissue samples of HD, but not from normal lymph nodes. Expression of EPR-1 transcripts in Reed-Sternberg cells was demonstrated by in situ hybridization with an antisense EPR-1 riboprobe, and by amplification of reverse-transcribed HD RNA with EPR-1-specific primers. These findings identify the factor Xa receptor, EPR-1, as a novel marker of Reed-Sternberg cells, and suggest its potential role in the histopathogenesis of HD.

Structural recognition of a novel fibrinogen gamma chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction.

In addition to its role in hemostasis, fibrinogen is obligatorily required to mount competent inflammatory responses in vivo. A molecular prerequisite of fibrinogen-dependent inflammation may reside in its ability to associate with intercellular adhesion molecule-1 (ICAM-1), and enhance monocyte adhesion to endothelium by bridging the two cell types. Structure-function characterization of the novel ICAM-1 recognition of fibrinogen was carried out by synthetic peptidyl mimicry of the fibrinogen gamma chain. A novel peptide sequence, N117NQ-KIVNLKEKVAQLEA133, designated gamma 3, dose-dependently inhibited (IC50 approximately 20-40 micrograms/ml) binding of 125I-fibrinogen to endothelial cells or ICAM-1-expressing B lymphoblastoid Daudi cells. In contrast, none of the previously identified vascular cell fibrinogen interacting sequences was effective. Increasing concentrations of gamma 3 completely inhibited fibrinogen-mediated adhesion of peripheral blood mononuclear cells or vitamin D3-differentiated monocytic HL-60 cells to endothelium, but did not affect leukocyte-endothelium interaction in the absence of fibrinogen. 125I-Labeled gamma 3 bound specifically and saturably to genetically engineered ICAM-1 transfectants, but not to control non-transfected cells, and associated with ICAM-1 on cytokine-activated endothelium with a Kd of 34 microM. Consistent with functional recognition of ICAM-1, immobilized gamma 3 supported adhesion of JY lymphoblasts in a dose-dependent reaction inhibited by monoclonal antibodies to ICAM-1. We conclude that a novel fibrinogen gamma 3 sequence N117NQKIVNLKEKVAQLEA133 binds to ICAM-1 and modulates ICAM-1-dependent adhesion. These findings define the structural basis of fibrinogen:ICAM-1 recognition and provide a potential selective target for inhibiting fibrinogen-dependent inflammatory responses.

Differential ligand binding specificities of recombinant CD11b/CD18 integrin I-domain.

The alpha subunits of leukocyte CD11/CD18 integrins contain an approximately 200-amino acid "inserted" domain (I-domain) that may be important for multivalent adhesive recognitions. A recombinant form of the I-domain of CD11b/CD18 was generated and analyzed directly for interaction with complementary integrin ligands. CD11b I-domain bound the activation-dependent monoclonal antibody 7E3, and the functionally blocking anti-CD11b monoclonal antibodies OKM9, 60.1, and LM2/1, but not OKM1 or M1/70. Fibrinogen or soluble intercellular adhesion molecule-1 associated with CD11b I-domain in a concentration-dependent manner. Binding of 125I-fibrinogen to recombinant CD11b I-domain was saturable, governed by a Kd of approximately 0.22 +/- 0.06 microM, and fully inhibited by molar excess of unlabeled fibrinogen, or by the P1 peptide (KY)GWTVFQKRLDGSV (IC50 approximately 2.5-5 microM), duplicating the fibrinogen gamma chain sequence Gly190-Val202. In contrast, 125I-factor X binding to CD11b I-domain was only partially inhibited (50-60%) by a molar excess of unlabeled factor X, and entirely unaffected by functionally blocking anti-CD11b monoclonal antibodies or by factor X-derived synthetic peptidyl antagonists. We conclude that the I-domain of CD11b participates in qualitative mechanisms of receptor activation and contains the binding site(s) for the CD11b/CD18 ligands fibrinogen and intercellular adhesion molecule-1, while it is only minimally implicated in the recognition of factor X.

Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway.

Leukocyte traffic in immune-inflammatory responses requires regulated adhesion of leukocyte subsets to vascular endothelium. We show that fibrinogen or normal human plasma enhances by 2- to 5-fold the adhesion of cells of myeloid and lymphoid lineage to endothelium. This mechanism is mediated by fibrinogen binding to complementary membrane receptors on leukocytes and endothelial cells. Using an affinity chromatography purification strategy, genetically engineered transfectants, and direct binding studies to the isolated recombinant protein, we identified a novel hematopoietic fibrinogen receptor participating in this adhesion pathway as intercellular adhesion molecule 1 (ICAM-1). Accordingly, a new model can be proposed, in which fibrinogen binding to a variety of vascular cell receptors mediates a specific pathway of cell to cell adhesion by bridging together leukocytes and endothelial cells.

The structural motif glycine 190-valine 202 of the fibrinogen gamma chain interacts with CD11b/CD18 integrin (alpha M beta 2, Mac-1) and promotes leukocyte adhesion.

The leukocyte-restricted integrin CD11b/CD18 (alpha M beta 2, Mac-1) is a receptor for fibrinogen on stimulated monocytes and neutrophils. At variance with platelet alpha IIb beta 3 or endothelial cell alpha v beta 3 integrins, CD11b/CD18 interacts with a approximately 30-kDa plasmic fragment D (D30) of fibrinogen that lacks the Arg-Gly-Asp sequences in the A alpha chain and the carboxyl terminus of the gamma chain. Using epitope-mapped antibodies and synthetic peptidyl mimicry, we have now identified a unique linear sequence in fibrinogen that mediates ligand binding to CD11b/CD18. Anti-fibrinogen antibodies directed to the gamma chain region 95-264 inhibited 125I-fibrinogen or 125I-D30 binding to chemoattractant-stimulated neutrophils or monocytic THP-1 cells in a dose-dependent fashion. Partially overlapping synthetic peptides reproducing this gamma chain region were tested for their ability to inhibit fibrinogen binding to leukocytes. A synthetic peptide designated P1, duplicating gamma chain Gly190-Val202, inhibited 125I-fibrinogen binding to stimulated neutrophils or THP-1 cells and blocked adhesion of these cells to immobilized fibrinogen in a dose-dependent fashion. Increasing concentrations of P1 inhibited 125I-fibrinogen binding to isolated CD11b/CD18 in a cell-free system. Consistent with genuine peptidyl mimicry, 125I-P1 bound saturably to THP-1 cells in a reaction inhibited by molar excess of unlabeled peptide, fibrinogen, or D30. Finally, immobilized P1 effectively supported adhesion of THP-1 cells in a CD11b/CD18-dependent manner. These data suggest that the fibrinogen gamma chain region Gly190-Val202 functions as a minimal recognition sequence for the leukocyte integrin CD11b/CD18. Given the participation of fibrinogen:leukocyte interaction in inflammation and atherogenesis, antagonists based on this unique structural motif would effectively interfere with aberrant leukocyte adhesion mechanisms without affecting Arg-Gly-Asp-directed vascular integrins.

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.

Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines.

The capacity of human monocytoid cell lines and peripheral blood monocytes to modulate their expression of plasminogen receptors has been assessed. After PMA stimulation, THP-1 or U937 monocytoid cells were separated into adherent and nonadherent populations. Plasminogen bound to adherent cells with similar capacity and affinity as to nonstimulated cells. In contrast, the nonadherent cells bound plasminogen with 5-17-fold higher capacity (without a change in affinity). This increase was selective as urokinase bound with similar affinity and capacity to the adherent and nonadherent populations. Upregulation of plasminogen receptors on the nonadherent monocytoid cells was rapid, detectable within 30 min, and reversible, adhesion of the nonadherent cells resulted in a sixfold decrease in plasminogen binding within 90 min. The increase in plasminogen binding to the nonadherent cells was associated with a marked increase in their capacity to generate plasmin activity from cell-bound plasminogen. PMA stimulation of human peripheral blood monocytes increased their expression of plasminogen receptors by two- to fourfold. This increase was observed in both adherent and nonadherent monocytes. Freshly isolated monocytes maximally bound 5.0 x 10(5) plasminogen molecules per cell, whereas monocytes cultured for 18 h or more maximally bound 1.7 x 10(7) molecules per cell, a 30-fold difference in receptor number. These results indicate that both monocytes and monocytoid cell lines can rapidly and markedly regulate their expression of plasminogen binding sites. As enhanced plasminogen binding is correlated with an increased capacity to generate plasmin, an enzyme with broad substrate recognition, modulation of plasminogen receptors may have profound functional consequences.

A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac-1 (CD11b/CD18).

Mac-1 (CD11b/CD18), a leukocyte-restricted integrin receptor, mediates neutrophil/monocyte adhesion to vascular endothelium and phagocytosis of complement-opsonized particles. Recent studies have shown that Mac-1 also functions as a receptor for fibrinogen in a reaction linked to fibrin deposition on the monocyte surface. In this study, we have used extended proteolytic digestion of fibrinogen to identify the region of this molecule that interacts with Mac-1. We found that an Mr approximately 30,000 plasmic fragment D of fibrinogen (D30) produced dose-dependent inhibition (IC50 = 1.6 microM) of the interaction of intact 125I-fibrinogen with stimulated neutrophils and monocytes. 125I-D30 bound saturably to these cells with specific association of 136,200 +/- 15,000 molecules/cell in a reaction inhibited by OKM1 and M1/70, monoclonal antibodies specific for the alpha subunit of Mac-1. Direct microsequence analysis and an epitope-mapped monoclonal antibody showed that D30 lacks the COOH-terminal dodecapeptide of the gamma chain as well as the Arg-Gly-Asp sequences in the A alpha chain. We conclude that fibrinogen interacts with the leukocyte integrin Mac-1 through a novel recognition site that is not shared with other known integrins that function as fibrinogen receptors.