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PubMed1994

The somatomedin B domain of vitronectin. Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor.

We recently localized the high affinity binding site for activated type 1 plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e. amino acid (aa) 1-51) of vitronectin (Vn). In this study to further define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity. Vn polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding activity and stabilized PAI-1 to a similar extent as intact Vn, but polypeptides containing aa 1-30 did not bind to PAI-1 nor stabilize its activity. The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 binding was also determined. One mAb bound to Vn and blocked its ability to bind to PAI-1. It also dissociated pre-existing PAI-1.Vn complexes, and prevented the incorporation of PAI-1 into extracellular matrix of HT 1080 cells. This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30. A second randomly chosen mAb with similar affinity for Vn was inactive in these assays and bound to the region between aa 52 and 239. These results indicate that the high affinity binding site for active PAI-1 in Vn is between aa 1 and 40, and that this domain may also stabilize active PAI-1.

Amino Acid Sequence↗
PubMed1994

Vitronectin, fibronectin, and gp120 antibody enhance macrophage release of TNF-alpha in response to Pneumocystis carinii.

Alveolar macrophages (AMS) initiate inflammation during Pneumocystis carinii pneumonia by releasing cytokines including TNF-alpha. Recent studies suggest that macrophage responses to P. carinii are enhanced by serum opsonization, but the mechanisms of enhancement are not well defined. To determine whether macrophage release of TNF-alpha in response to P. carinii was augmented by immune opsonization, alveolar macrophages obtained from rabbits were cultured with P. carinii that had been opsonized with either nonimmune rabbit serum, immune serum generated against P. carinii, or an affinity-purified polyclonal Ab recognizing the major P. carinii surface Ag gp120. Each experiment also included organisms maintained in media alone (nonopsonized P. carinii). Opsonization of P. carinii with immune serum or gp120 Ab significantly enhanced macrophage TNF-alpha release. Interestingly, however, opsonization with nonimmune serum also increased TNF-alpha response to the organism. Because P. carinii is known to interact with the adhesive glycoproteins, vitronectin (VN) and fibronectin (FN), we hypothesized that they might also augment TNF-alpha release. Opsonization of P. carinii with VN or FN resulted in significant potentiation of macrophage TNF-alpha liberation. We further determined that VN and FN were present in increased quantities in the lower respiratory tract of patients with P. carinii pneumonia compared with normal volunteers. Additionally, VN and FN were demonstrated on the surface of freshly isolated P. carinii organisms by immunoblot analysis. Our study suggests that immune and nonimmune opsonins contribute to host defenses during P. carinii pneumonia by enhancing regional TNF-alpha release in response to the organism.

Animals↗
PubMed1994

The role of fibronectin, laminin, vitronectin and their receptors on cellular adhesion in proliferative vitreoretinopathy.

PURPOSE: To examine the possible role of some adhesion multifunctional glycoproteins of the extracellular matrix, such as fibronectin (FN), laminin (LN), vitronectin (VN) and their receptors (beta 1-subunit complex and alpha v beta 3 integrins) in events of cell migration and adhesion in proliferative vitreoretinopathy (PVR). METHODS: Optical and electron-immunocytochemical techniques were carried out on epiretinal membranes. Electrophoretic immunoblotting methods and densitometric analysis of normal and PVR vitreous were also undertaken. Chi-square (chi 2) and unbalanced analysis of variance were employed for statistical analysis. RESULTS: FN was detected as a major component in the extracellular matrix in both fibrillar and pericellular arrangement. A change in pericellular distribution to more fibrillous organization was related to the time of intraocular proliferative tissue development (P < 0.001). LN and VN were observed as minor components in extracellular matrix. A colocalized pattern between VN and FN in collagenic bundles of the matrix was often observed. Beta-1 subunit and alpha v beta 3 receptors were usually localized in a position that could mediate the interaction of FN, VN, and/or LN to the cell plasma membrane. Increased levels of FN concentration were observed in both subretinal fluid and pathologic vitreous; intravitreal FN concentration tends to increase with clinical stages of the evolution of PVR, whereas intravitreal VN levels tend to decrease. CONCLUSIONS: Results suggest that FN could mediate the initial events involved in epiretinal membrane formation, and VN could modulate the adhesion mechanisms in established membranes.

Blotting, Western↗
PubMed1994

Clustering of vitronectin and RGD peptides on microspheres leads to engagement of integrins on the luminal aspect of endothelial cell membrane.

In previous work (Conforti et al, Blood 80:437, 1992), we have shown that integrins in endothelial cells (EC) are not polarized to the basal cell membrane, but are also exposed on the apical cell surface, in contact with blood. Therefore, endothelial integrins might be available for binding circulating plasma proteins. However soluble plasma vitronectin (vn) bound very poorly to EC apical surface and this interaction was unaffected by Arg-Gly-Asp (RGD) peptides or an anti-alpha v beta 3 serum. In contrast, beads (diameter, 4.5 microns) coupled with plasma vn associated to EC apical surface in a time- and concentration-dependent way. Addition of antibodies directed to vn, alpha v beta 3, and RGD-containing peptides blocked the interaction of vn beads with EC. In contrast, heparin and antibodies directed to alpha v beta 5 and beta 1 integrin chain had no effect. Beads coupled with Gly-Arg-Gly-Asp-Ser-Pro bound to the EC surface, but not those coupled with Gly-Arg-Gly-Glu-Ser-Pro. This interaction was blocked by alpha v beta 3 antibodies and RGD peptides, but not by alpha v beta 5 antibody. Overall, these results indicate that luminal alpha v beta 3 retains its binding capacity for surface-linked vn and RGD-containing ligands, but binding is observed only when the ligand is offered in a clustered, multivalent form. We propose that when vn or RGD-containing proteins are bound to circulating cells, they can act as bridging molecules by promoting adhesion of the cells to the endothelium via apical integrins.

Amino Acid Sequence↗
PubMed1994

Vitronectin gene expression in vivo. Evidence for extrahepatic synthesis and acute phase regulation.

A competitive polymerase chain reaction (PCR) assay was developed to quantitate vitronectin (Vn) mRNA in murine tissues using a synthetic RNA as an external standard. Although the liver contained the highest concentration of Vn mRNA, significant levels were also detected in the brain (25-fold less) and in adipose tissue, heart, and skeletal muscle (100-fold less than liver). Lower concentrations also were detected in the lung, uterus, testis, and thymus, and little or no Vn mRNA could be detected in kidney, spleen, and blood. These results indicate that significant amounts of Vn mRNA are produced in extrahepatic organs. The regulation of Vn gene expression in vivo was studied in a murine model system in which acute systemic inflammation was induced by endotoxin administration. Plasma Vn levels increased 2- to 3-fold within 16 h after endotoxin administration and remained elevated for up to 72 h. This increase appeared to result from increased synthesis in the liver since the steady-state level of hepatic Vn mRNA increased 4-fold after endotoxin administration. Moreover, Vn mRNA levels in heart, lung, and brain were not significantly increased by endotoxin. These results suggest that Vn gene expression in vivo is regulated in a tissue-specific manner and identify Vn as a novel acute phase reactant.

Acute-Phase Reaction↗
PubMed1994

Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin.

The fate of neutrophils (PMNs) at sites of inflammation is important to our understanding of many disease processes. Previously, it had been widely assumed that extravasated PMNs inevitably disintegrated before their fragments were removed by local phagocytes, but we have recently described an alternative process whereby senescent PMNs undergo apoptosis (programmed cell death). This process leads to macrophage (Mphi) ingestion of the intact cell by a novel phagocytic recognition process. In this study, we show that monolayers of fibroblasts also can selectively phagocytose apoptotic PMNs and that the recognition of apoptotic PMNs by fibroblasts involves two distinct mechanisms: one uses the vitronectin receptor, as in Mphi ingestion of PMNs; the other uses a mannose/fucose-specific lectin, which plays no part in Mphi phagocytosis of apoptotic PMNs. The direct interactions between PMNs and fibroblasts demonstrated herein may have implications for our understanding of the relationship between inflammation and scarring in many diseases.

Amino Acid Sequence↗
PubMed1995

Engagement of the vitronectin receptor (alpha V beta 3) on murine T cells stimulates tyrosine phosphorylation of a 115-kDa protein.

The murine vitronectin receptor (VNR, alpha V beta 3) is expressed on T cell hybridomas expressing both the alpha beta and the gamma delta TCR as well as on TCR-alpha beta cells activated for prolonged periods of time by mitogens or alloantigens. The VNR functions as a costimulatory molecule for the activation of a subset of gamma delta T cells that express the V gamma 1.1 C gamma 4 V delta 6 TCR and that may recognize a ubiquitously expressed autoantigen. To characterize further some of the signal transduction parameters observed after engagement of the VNR in stimulated T lymphocytes, we have examined the effect of ligation of the VNR by RGDS-containing proteins on the pattern of protein phosphorylation. We demonstrate the appearance of a 115-kDa, tyrosine-phosphorylated protein (pp115) after engagement of the VNR with its ligand, RGDS. pp115 was shown to be immunologically distinct from focal adhesion kinase, did not possess inherent kinase activity, and may represent an as yet unidentified substrate in the integrin signal transduction pathway. Although induction of pp115 was independent of TCR expression, because it was seen in the TG40 hybridoma, which expresses neither the alpha beta nor the gamma delta TCR, pp115 could also be induced by cross-linking of the TCR in a murine TCR gamma delta hybridoma in the absence of any extracellular matrix proteins. This result raises the possibility that induction of pp115 is a common biochemical step in signal transduction by both the TCR and the VNR.

3T3 Cells↗
PubMed1995

Vessel wall-dependent metabolic pathways of the adhesive proteins, von-Willebrand-factor and vitronectin.

The integrity of the vessel wall under quiescent conditions as well as its appropriate responsiveness under conditions of stimulation, inflammation or vascular injury is controlled by a number of adhesive interactions involving distinct cellular receptor systems and various multifunctional adhesive ligands. While a number of these extracellular matrix components of the vessel wall are endogenously produced, secreted and deposited, exogenous adhesion proteins may become translocated from the intra- to the extravascular space by virtue of endothelial cell-specific transport systems. Two prominent examples for each metabolic pathway are discussed. Endothelial cell-specific biosynthesis and secretion as well as deposition of multimeric von-Willebrand-factor within intracellular granules (Weibel-Palade bodies) relates to the first possibility of processing, whereas binding of reactive forms of circulating vitronectin to diverse cellular receptors with subsequent extravasation and deposition into the extracellular matrix appears to be characteristic for the second case. In this review the known features of the metabolic routes of both adhesion proteins are discussed and set in perspective to their functional properties. Their localized mode of action in the vessel wall appears to be crucial for balanced haemostasis and immune systems, two major defence mechanisms of the organism.

Animals↗
PubMed1995

[Migration and adhesiveness of malignant glioma cells to fibronectin or vitronectin and their expression of integrin subunits].

In order to better understand the cellular mechanism of glioma invasion, we investigated migratory responses and adhesiveness of human malignant glioma cells to fibronectin (FN) or vitronectin (VN). In addition, an expression of integrin subunits for FN and VN was analyzed by flow cytometry. All glioma cells tested migrated to both FN and VN to a various degree. Glioma cells which strongly migrated to FN or VN showed an intense expression of alpha 5 or alpha v, respectively, while there was no correlation between cell adhesiveness to FN or VN and intensity of the integrin expression. Studies using primary tumor cells from surgical specimen revealed that only an intensity of cell adhesiveness to FN was negatively correlated well with the degree of clinical invasion of gliomas. That is, the more glioma cells adhered to FN, the less the original tumor tissues showed tumor invasion.

Brain Neoplasms↗
PubMed1995

IL-6 stimulates vitronectin gene expression in vivo.

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.

Animals↗
PubMed1993

Ternary vitronectin-thrombin-antithrombin III complexes in human plasma. Detection and mode of association.

Radiolabeled antithrombin III (ATIII) was incubated at 37 degrees C with purified vitronectin (VN) or fibrinogen-deficient plasma before thrombin was added to initiate complex formation. Incorporation of radiolabeled ATIII was detected using polyacrylamide gel electrophoresis (PAGE) and autoradiography. The PAGE conditions appeared to be crucial for the detection of VN.TAT complexes. In the absence of SDS, ternary complexes formed instantaneously, whereas in the presence of SDS, only 50% of the TAT was associated with VN after a 60-min incubation. Formation of ternary complexes could be confirmed by gel filtration of the plasma to which thrombin was added. Furthermore, TAT in patient plasmas (disseminated intravascular coagulation and sepsis) was found to bind to heparin-Sepharose, indicating that this endogenously formed TAT was also associated with VN. The amino-terminal region of VN and the thrombin moiety of the TAT complex were found to be responsible for their interaction, which was stabilized by disulfide bridges. These results indicate that in normal plasma all TAT is complexed with VN. This association alters the conformational state of plasma VN, which appears to be responsible for the clearance of thrombin complexes from the circulation.

Antibodies, Monoclonal↗
PubMed1993

Expression of vitronectin and fibronectin binding by Candida albicans yeast cells.

Expression of binding to vitronectin (Vn or S-protein) and fibronectin (Fn) was common among clinical isolates of Candida albicans. Growth at 37 degrees C enhanced expression of both Vn and Fn binding. Some strains expressed higher binding after growth in liquid media and others after growth on solid media. Most strains expressed higher cell surface hydrophobicity after growth on agar media. Vn binding was less influenced by expression of cell surface hydrophobicity than Fn binding. Vn binding to yeast cells was optimal around pH 4 and Fn binding around pH 6. Binding to soluble Vn was inhibited by unlabelled Vn and to a lesser extent by Fn. Fn binding to the same C. albicans strain was inhibited by unlabelled Fn, Vn, fibrinogen and to some extent collagens. C. albicans strain 3248 expressed specific high binding of Vn, and high binding of Fn. Binding of both proteins was sensitive to heat and protease treatment, but in different ways. Vn binding differed significantly from the earlier reported Fn binding and may represent a novel type of tissue adherence.

Blood Proteins↗
PubMed1993

Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein.

The adhesive glycoprotein vitronectin (VN) shows a high degree of conformational flexibility implicating that different molecular forms of the molecular may exist. Conformation-dependent monoclonal antibodies 13H1 or 16A7 that, per se, did not react with plasma VN bound to VN treated with heparin, chaotropes, detergents, pH below 6, or by heating at 56 degrees C. Dependent on the stimulus, recognition of VN by these antibodies varied and preceded heparin binding and self-association of VN resulting in the formation of noncovalently linked multimeric species of the protein. Both monoclonal antibodies also reacted with VN in serum or in platelet releasates as well as with VN in extracellular matrices of endothelial cells and inhibited cell adhesion on immobilized VN. Critical VN levels were needed for concentration-dependent multimerization indicating a nonlinear type of polymerization process. The nature of VN multimers was judged by nondenaturing gel electrophoresis, gel filtration, and sucrose gradient ultracentrifugation and revealed the formation of 3- to 16-mer multimeric species within an M(r) range of 200-1200 kDa representing a mean sedimentation coefficient of 9.6 S. In electron microscopy, multimeric VN occurred as globular specimens with an average diameter of 15-28 nm (monomeric plasma VN, 6-8 nm). In contrast to plasma VN, VN multimers were efficiently stabilized by covalent inter-molecular bonds following chemical or transglutaminase-induced cross-linking. A synthetic peptide comprising the central heparin binding region of VN (residues 348-361) not only bound to plasma VN but induced its multimerization also in plasma. During plasmin proteolysis of VN, fragments were generated that lacked the heparin binding region and that lost the ability to multimerize following urea or detergent treatment, implicating that the highly basic region is essential for multimer formation. These data suggest that non-plasma forms of VN, which are abundant in platelets and subendothelium, represent the prototype conformer of the reactive heparin binding form of VN. Our findings implicate that conformationally altered forms of VN enable the adhesive protein to multimerize in a characteristic fashion and thereby endow extracellular matrix sites with unique multivalent properties.

Amino Acid Sequence↗
PubMed1996

Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line.

Type 1 plasminogen activator inhibitor (PAI-1) and its cofactor vitronectin (Vn) are stored within the alpha-granules of platelets. The two possible sources for their biosynthetic origin are endogenous synthesis in megakaryocytes or endocytosis from plasma. Using ultrastructural and confocal laser scanning microscopic (CLSM) image analysis, we observed that treatment of Dami cells, a human megakaryocytic cell line, with phorbol myristate acetate (PMA) induces the accumulation of PAI-1 and Vn in intracellular storage vacuoles that contain other alpha-granule proteins such as von Willebrand factor. To examine evidence for biosynthesis of PAI-1 and Vn by Dami cells, we immunoprecipitated PAI-1 and Vn from the conditioned media of cells biosynthetically radiolabeled with 35S-methionine in the presence or absence of PMA. In contrast to Hep G2 cells, which synthesize both PAI-1 and Vn, only 35S-PAI-1 was recovered from PMA-treated Dami cells. Reverse transcription-PCR analysis of RNA extracted from resting and PMA-treated Dami cells confirmed that PAI-1 mRNA expression was detectable at low levels in resting cells and induced by PMA treatment. In contrast, Vn mRNA was not detected. We examined binding and internalization (endocytosis) of PAI-1 and Vn by Dami cells using biotinylated analogs (b-PAI-1 and b-Vn). Flow cytometry analysis indicated that the binding of b-Vn to Dami cells was dose-dependent, saturable, and specific for multimeric forms of Vn. Cells were incubated at 4 degrees C or 37 degrees C and endocytosis of b-Vn was shown by probing electrophoretically fractionated cell lysates with 125I-labeled streptavidin. Only cells incubated at 37 degrees C internalized b-Vn. CLSM image analysis confirmed that the b-Vn was internalized and that it colocalized with PAI-1 in storage granules. The binding of b-Vn to cells was inhibited by the presence of PAI-1, and there was no evidence of specific b-PAI-1 binding or uptake to resting or PMA-treated cells. These data suggest that accumulation of PAI-1 in Dami cell storage granules is due to endogenous synthesis and that the accumulation of Vn is due to endocytosis of serum-derived Vn.

Base Sequence↗
PubMed1996

Two functionally distinct pools of vitronectin (Vn) in the blood circulation: identification of a heparin-binding competent population of Vn within platelet alpha-granules.

The biological functions of vitronectin (Vn) are dependent on its conformation. Whereas plasma Vn is present in a conformation that does not bind to heparin, platelet Vn has been recognized to be in a multimeric, conformationally altered form. To further understand the characteristics of platelet Vn, the molecules present in plasma and total and size-fractionated platelet releasates were compared (1) immunologically using three conformationally sensitive epitope-defined monoclonal antibodies, (2) functionally for their ability to interact with heparin, and (3) structurally using denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Our data indicate that Vn is present in platelet releasates in two molecular weight (M(r) forms. The high M(r) fractions contain conformationally and structurally altered Vn capable of interacting with heparin, and this form is distinct from plasma Vn and purified denatured Vn. In contrast, the lower M(r) forms of Vn are similar to plasma Vn. To determine if the presence of multimeric Vn requires platelet activation, platelets were disintegrated by sonication and fractionated by density gradients. Combined sodium dodecyl sulfate-PAGE (SDS-PAGE) and immunoblotting analysis showed a codistribution of multimeric Vn and type 1 plasminogen activator inhibitor in alpha-granule-rich fractions. Thus, platelet Vn is stored in a structurally and functionally distinct form from the molecule in plasma, raising the possibility that platelet-derived heparin-binding competent Vn will accumulate in areas of vascular injury.

Antibodies, Monoclonal↗
PubMed1996

Modification of vitronectin by advanced glycation alters functional properties in vitro and in the diabetic retina.

Early diabetic retinopathy is characterized by areas of acellular capillaries and the accumulation of advanced glycation end products (AGE). Endothelial cell maintenance, growth, and migration is regulated by a complex system involving the partitioning of growth factors between extracellular matrix-bound proteoglycans and cells, as well as by the activation and control of pericellular proteolysis. Using specific antibodies, we compared the immunolocalization of the vascular heparan sulfate proteoglycan (HSPG)-binding protein vitronectin (VN), HSPG, basic fibroblast growth factor, and collagen type 1 in retinae prepared from 11-month-old diabetic and nondiabetic Wistar rats. In normal rats, VN immunostaining was prominent in vascularized parts of the inner retina and colocalized with HSPG, which itself showed costaining with basic fibroblast growth factor in the extracellular matrix, vascular walls, and the inner limiting membrane. In contrast, the recognition of VN, HSPG, and basic fibroblast growth factor in diabetic retinae was greatly reduced in the inner limiting membrane and the extracellular matrix, where increased immunoreactive AGE colocalizing with VN were detectable. Capillary labeling of retinal vessel walls for plasminogen activator inhibitor-1 was moderate in both normal and diabetic retinae. AGE-VN was demonstrated in tissue extracts from retina by immunoprecipitation and Western blotting and presented a similar increase of AGE-related immunoreactivity compared with AGE-VN generated in vitro. AGE-VN in vitro had lost its native conformation, yielded high Mr SDS-resistant products, and was resistant to proteolysis because of modification of about 30% of lysines. Because binding of glycosaminoglycans, as well as interaction of type I collagen and the morphoregulatory proteins osteonectin and tenascin with AGE-VN, were reduced to at least 50% of control, alteration of basic residues in the heparin-binding domain of VN is plausible. In comparison to nonmodified VN, AGE-VN exhibited reduced cell attachment-promoting activity. Together, these in vitro results suggest that AGE-VN found in vivo is related to morphologic and functional changes in the diabetic retina and may contribute to the genesis of acellular capillaries in early diabetic retinopathy.

Animals↗
PubMed1997

The composition of complexes between plasminogen activator inhibitor 1, vitronectin and either thrombin or tissue-type plasminogen activator.

Vitronectin (VN) is an obligatory cofactor for the inhibition of thrombin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the rate of association between thrombin and PAI-1 more than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we reported that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 fully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we show that this area is also required for the interaction between thrombin and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural requirements for the interaction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, the intermediate reaction products are clearly distinct. By employing surface plasmon resonance (SPR), using the BIAcore equipment, and by immunoprecipitation we demonstrate that, in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombin, PAI-1 and VN generate equimolar, binary thrombin/PAI-1 complexes and in addition equimolar, ternary complexes and multimers.

Antibodies, Monoclonal↗
PubMed1997

Human glomerular mesangial cell phagocytosis of apoptotic neutrophils: mediation by a novel CD36-independent vitronectin receptor/thrombospondin recognition mechanism that is uncoupled from chemokine secretion.

In this study we examined the mechanisms by which glomerular mesangial cells ingest apoptotic cells and the mesangial cell response to this event, since there is in vivo evidence that such semiprofessional phagocytes participate in phagocytic clearance of both apoptotic leukocytes and apoptotic resident cells from inflamed glomeruli, thereby promoting resolution of glomerulonephritis. Mesangial cell phagocytosis of apoptotic neutrophils in vitro was not affected by inhibitors of lectin-like receptors, phosphatidylserine receptors, the 61D3 Ag, and beta1 and beta2 integrins, receptors which have been implicated in phagocytosis of apoptotic cells by particular populations of semiprofessional and professional phagocytes. However, the specific inhibitory effects of cationic aminosugars, Arg-Gly-Asp-Ser (RGDS) peptide, and mAbs to phagocyte alpha(v)beta3 vitronectin receptor integrin and "bridging" thrombospondin 1 (TSP1) indicated that mesangial cell phagocytosis of apoptotic cells involved an alpha(v)beta3/TSP mechanism akin to that described for human monocyte-derived macrophages (Mphi) in which Mphi CD36 plays an important role in binding "bridging" TSP1. However, mesangial cells did not express CD36 and there was no evidence for involvement of alternative phagocyte receptors for TSP1, heparan sulfate proteoglycan and sulfatides. Nevertheless, phagocytosis of apoptotic neutrophils by either mesangial cells or Mphi failed to elicit secretion of IL-8 and MCP-1, representatives of each major class of proinflammatory chemotactic cytokines. We conclude that mesangial cell phagocytosis of apoptotic neutrophils involves a novel CD36-independent, alpha(v)beta3/TSP-mediated mechanism that is uncoupled from chemokine secretion, emphasizing the injury-limiting potential of mesangial cell phagocytosis of apoptotic cells.

Apoptosis↗