Biomedical subjects

K T Preissner

Publications and source records attributed to K T Preissner.

At least 19 recordsLinked to original sources

PubMed2002

Kinetics of integrin expression in the mouse model of proliferative retinopathy and success of secondary intervention with cyclic RGD peptides.

AIMS/HYPOTHESIS: Vitronectin-receptor-type integrins (alpha(v) beta(3) and alpha(v) beta(5)) are thought to be involved in the selective ablation of tumorigenic and other pathologic angiogenesis. Specifically, it has been shown that ligation inhibition of the alpha(v)-type integrins with cyclic penta-peptid peptide inhibits proliferative retinopathy by almost 80 % in a hypoxia-induced mouse model. METHODS: On the basis of growth factor and integrin expression dynamics in this model, secondary intervention approaches with cyclic RGDfV peptide were investigated. RESULTS: alpha(v)-integrin expression started immediately after induction of hypoxia (at postnatal day 12, p12) and persisted only during the initial period of neovascularization (until day p14). Vascular endothelial growth factor (VEGF) expression started at high values immediately after return of the mice into room air, and dropped rapidly to low values beyond day 13. In contrast, basic fibroblast growth factor (bFGF) was predominantly expressed during the phase of maximum angiogenesis which was noted between day p17 and 19. Based on these findings, cyclic penta peptide was administered subcutaneously at varying doses (2-20 microg/kg/day) for 5 days beginning either at day p14 (early intervention) or at day p17 (late intervention). Early secondary intervention showed a dose-dependent reduction of new vessels with maximum inhibition of 57 % (control 68.08 +/- 3.21 nuclei/section compared with RGDfV-treated 29.35 +/- 2.39 nuclei/section; p < 0.0001), whereas late secondary intervention had no effect. CONCLUSION/HYPOTHESIS: These data indicate that angiogenesis-related alpha(v)-integrin expression is VEGF- rather than bFGF-dependent, and the efficacy of cyclic penta-peptid (RGDfV)-treatment in proliferative retinopathy is only effective as long as the alpha(v)-integrin target is prominently expressed.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed2002

Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix.

Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.

Ammonia↗

PubMed2001

Ligand binding regions in the receptor for urokinase-type plasminogen activator.

The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion. In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages. Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains. This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule. Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III. All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR. Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA. The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction. These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.

Amino Acid Sequence↗

PubMed2001

Factor VII and single-chain plasminogen activator-activating protease: activation and autoactivation of the proenzyme.

Structural and biological characteristics of a recently described plasma serine protease, which displayed factor VII as well as pro-urokinase-activating properties in vitro, indicated a dual role for this factor VII-activating protease (FSAP) in hemostasis. Only the active protease (two-chain FSAP) has been isolated from plasma and from a prothrombin complex concentrate, whereas activators of the proenzyme have not been identified so far. After purification of the FSAP proenzyme from cryo-poor plasma by adsorption to an immobilized mAb and subsequent ion-exchange chromatography, activation to generate two-chain FSAP was followed by a direct chromogenic assay as well as by the ability of two-chain FSAP to activate pro-urokinase. Purified single-chain FSAP underwent autoactivation leading to the typical protease two-chain pattern and subsequent degradation products, as demonstrated by Western-blotting analysis using a site-specific mAb. This autoactivation was significantly enhanced in the presence of heparin, whereas Ca2+ ions stabilized single-chain FSAP (the proenzyme) resulting in slower autoactivation kinetics. Correspondingly, the heparin-augmented reaction, which was associated with autodegradation particularly of the protease domain, was slowed down by co-incubation with Ca2+. Of the other proteases and cofactors tested, only urokinase (uPA) was able to generate the typical two-chain FSAP pattern. Studies with different forms of uPA suggest that the catalytic activity of pro-urokinase/uPA is needed to activate single-chain FSAP, indicating that it is the only hemostatic protease that can act as a physiological activator of FSAP.

Blood Coagulation↗

PubMed2001

Topical application of integrin antagonists inhibits proliferative retinopathy.

The expression of alphav-integrins is highly selective for angiogenic endothelial cells; ligation inhibition by cyclic RGD peptides prevents pathological neovascularization in tumor or retinopathy models to a large extent. We have previously demonstrated that proliferative retinopathy in a mouse model of retinopathy of prematurity (ROP model) can be reduced by more than 70%. To minimize systemic side effects and unwanted interference with responsive angiogenesis, we investigated topical application of cyclic RGD-peptides. In preliminary experiments, we could exclude any inhibiting effects of the carrier solution containing EDTA, Na2S, mannitol, hydroxyethyl starch, and benzalconium chloride on the inhibitory effect of cyclic RGD peptides. Retinal presence of small molecular-mass integrin antagonists after topical application was confirmed using fluorescein-labeled cyclic RGD peptide. Topical application of the peptide to the eye inhibited proliferative retinopathy in a dose-dependent fashion with a maximum of almost 50%. These results suggest that small molecular-mass peptide antagonists of alphav-type integrins are efficient in inhibiting proliferative retinopathy by topical application.

Adhesiveness↗

PubMed2001

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

Biological Transport↗

PubMed2001

Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen.

Proteolytic cleavage of single-chain, high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind a two-chain, high molecular weight kininogen (HKa) reported to bind to the beta2-integrin Mac-1 (CR3, CD11b/CD18, alphaMbeta2) on neutrophils and exert antiadhesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of beta2-integrin-mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM-1 to immobilized Mac-1, but not the binding of ICAM-1 to immobilized LFA-1 (CD11a/CD18, alphaLbeta2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for binding to Mac-1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac-1 but not LFA-1-transfected K562 human erythroleukemic cells to ICAM-1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475-G497 (and to a lesser extent, G440-H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycollate-provoked peritonitis, decreased the recruitment of neutrophils by approximately 70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac-1 but not with LFA-1, thereby blocking Mac-1-dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leukocyte recruitment into the inflamed tissue.

Amino Acid Sequence↗

PubMed2001

Release of soluble urokinase receptor from vascular cells.

Urokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1beta (IL-1beta), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1beta stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall.

Arteriosclerosis↗

PubMed2000

VLA-4 (alpha(4)beta(1)) engagement defines a novel activation pathway for beta(2) integrin-dependent leukocyte adhesion involving the urokinase receptor.

During acute inflammatory processes, beta(2) and beta(1) integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (alpha(4)beta(1)) engagement on beta(2) integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)-Fc or by 2 anti-CD29 (beta(1) chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via beta(2) integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of beta(2) integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1-transfected Chinese hamster ovary cells, but not to ligands of beta(1) integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1-Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1-Fc was required for beta(2) integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-beta(1) integrin mAb were ineffective. Activation of beta(2) integrins by alpha(4)beta(1) integrin ligation (VCAM-1-Fc or anti-beta(1) mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1-Fc or anti-beta(1) integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of beta(2) integrin-dependent cell-to-cell adhesion that requires alpha(4)beta(1) integrin ligation for initiation and uPAR as activation transducer. (Blood. 2000;96:506-513)

Antibodies, Monoclonal↗

PubMed2000

Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions.

Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent alphavbeta(3) integrin-mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with alphavbeta(3) integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn(2+)-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN ("somatomedin B region"). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn(2+)-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys-rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions. (Blood. 2000;96:514-522)

Arteriosclerosis↗

PubMed2000

Vascular protease receptors: integrating haemostasis and endothelial cell functions.

The endothelium plays a crucial dynamic role as a protective interface between blood and the underlying tissues during the haemostatic process, which maintains blood flow in the circulation and prevents life-threatening blood loss. Following vessel wall injury with initial platelet adhesion and aggregation to exposed subendothelial extracellular matrix, the initiation, amplification, and control of haemostasis depend on structurally unrelated membrane-associated receptors for blood coagulation proteases including tissue factor, G-protein-coupled protease-activatable receptors, thrombomodulin, and protein C receptor, respectively. In addition to their regulatory role in haemostasis, the respective (pro-)enzyme ligands such as Factors VIIa and Xa, thrombin or protein C mediate specific signalling pathways in vascular cells related to migration, proliferation or adhesion. The functional importance of these receptors beyond haemostasis has been manifested by various lethal and pathological phenotypes in knock-out mice. These protease receptors thereby provide important molecular links in the vascular system and serve to integrate haemostasis with endothelial cell functions which are relevant for the (patho-)physiological responses to injury or inflammatory challenges.

Animals↗

PubMed2000

Release of the angiogenesis inhibitor angiostatin in patients with proliferative diabetic retinopathy: association with retinal photocoagulation.

AIMS/HYPOTHESIS: Proliferative diabetic retinopathy is a major debilitating disease causing most cases of blindness in humans in the Western world. Photocoagulation is the established therapy of proliferative diabetic retinopathy, although the molecular mechanisms of its effects are still not known. Recently angiostatin has been characterized as a potent inhibitor of neovascularization. Apart from a possible down-regulation of angiogenic cytokines, release of angiostatin could initiate the anti-angiogenic effects of retinal photocoagulation. METHODS: We investigated the regulation of angiostatin and the angiogenic cytokines vascular endothelial growth factor and basic fibroblast growth factor in vivo by comparing vitreal concentrations of 18 control patients and 34 patients with proliferative diabetic retinopathy with and without previous photocoagulation. Concentrations of basic fibroblast growth factor and angiostatin were additionally measured in serum, while vascular endothelial growth factor is known to be regulated locally in the eye. Cytokines were measured by immunological methods. RESULTS: Angiostatin could be detected in 2 out of 18 control patients and in 25 out of 34 diabetic patients (p < 0.00001). Most importantly, production of angiostatin in human vitreous correlated significantly with previous retinal photocoagulation (p < 0.0001) in patients with proliferative diabetic retinopathy. Only two patients (one control and one diabetic) had detectable serum concentrations of angiostatin. Additionally patients with proliferative diabetic retinopathy and with previous photocoagulation had significantly lower concentrations of vascular endothelial growth factor (0.9 +/- 0.1 ng/ml; p < 0.0001) than diabetic patients without previous photocoagulation (4.0 +/- 0.8 ng/ml). The investigation of vitreal and serum basic fibroblast growth factor concentrations yielded no significant differences between the groups. CONCLUSION/INTERPRETATION: Angiostatin is not a regularly expressed angiogenesis inhibitor in human vitreous. The alterations we observed suggest that local release of angiostatin and down-regulation of vascular endothelial growth factor mediate the therapeutic effects of retinal photocoagulation in proliferative diabetic retinopathy.

Aged↗

PubMed2000

Urokinase receptor: a molecular organizer in cellular communication.

In a variety of cell types, the glycolipid-anchored urokinase receptor (uPAR) is colocalized pericellularly with components of the plasminogen activation system and endocytosis receptors. uPAR is also coexpressed with caveolin and members of the integrin adhesion receptor superfamily. The formation of functional units with these various proteins allows the uPAR to mediate the focused proteolysis required for cell migration and invasion and to contribute both directly and indirectly to cell adhesive processes in a non-proteolytic fashion. This dual activity, together with the initiation of signal transduction pathways by uPAR, is believed to influence cellular behaviour in angiogenesis, inflammation, wound repair and tumor progression/metastasis and open up the way for uPAR-based therapeutic approaches.

Animals↗

PubMed2000

Hemostatic protease receptors and endothelial cell function: insights from gene targeting in mice.

Hemostasis constitutes a major defense system of our body that maintains blood flow in the circulation and prevents life-threatening blood loss. The endothelium plays a crucial dynamic role as a protective interface between blood and the underlying tissues during this process. Following vessel wall injury with initial platelet adhesion and aggregation to exposed subendothelial extracellular matrix, the initiation, amplification, and control of hemostasis depend on structurally unrelated membrane-associated receptors for blood coagulation proteases including tissue factor, G protein-coupled protease-activatable receptors, thrombomodulin, or protein C receptor. In addition to their regulatory role in hemostasis, the respective (pro-)enzyme ligands such as factors VIIa and Xa, thrombin, and protein C mediate specific signaling pathways in vascular cells related to migration, proliferation, or adhesion. The functional importance of these receptors beyond hemostasis has been manifested by various lethal or pathological phenotypes in knock-out mice. These protease receptors thereby provide important molecular links in the vascular system and serve to integrate hemostasis with endothelial cell functions that are relevant for the (patho-) physiological responses to inflammatory challenges, injury, or infection.

Animals↗

PubMed2000

Vitronectin and substitution of a beta-strand 5A lysine residue potentiate activity-neutralization of PA inhibitor-1 by monoclonal antibodies against alpha-helix F.

Some monoclonal antibodies against plasminogen activator inhibitor-1 (PAI-1) are able to inhibit its reaction with its target proteinases. We have characterized the effect on PAI-1 of two monoclonal antibodies, Mab-2 and Mab-6, with overlapping epitopes in a sequence encompassing beta-strand 1A, alpha-helix F, and the loop connecting alpha-helix F and beta-strand 3A (the hF/s3A loop). Mab-2 reduced the inhibitory activity of wild type PAI-1 and almost totally abolished the inhibitory activity of a PAI-1 variant harboring an Ala substitution of Lys 325 (335 in the alpha1-proteinase inhibitor template residue numbering) in beta-strand 5A. In both cases, the neutralizing effect of the antibody was strongly potentiated by vitronectin. Mab-6 had no effect on wild type PAI-1, but reduced the inhibitory activity of the K325A variant. The effect of Mab-6 was not potentiated by vitronectin. With both Mab-2 and Mab-6, the neutralization of PAI-1 activity was associated with PAI-1 substrate behaviour. Mab-2, but not Mab-6, prevented vitronectin from rescuing PAI-1 from cold-induced substrate behaviour. We propose that the antibodies act by weakening the anchoring of alpha-helix F to the adjacent structures, resulting in an increased flexibility of beta-strand 5A and the hF/s3A loop and a changed conformational response to the binding of vitronectin in the alpha-helix E region. The potentiating effect of vitronectin on neutralization of PAI-1 by antibodies is a novel concept in the development of compounds for neutralizing PAI-1 in vivo.

Amino Acid Motifs↗

PubMed1999

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells.

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.

Cells, Cultured↗

PubMed1999

Novel glycosylated forms of human plasma endostatin and circulating endostatin-related fragments of collagen XV.

Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117)HLRPAR. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII, respectively. These polypetides are found to be O-glycosylated at T(125) (residue 9) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate. These polypeptides exhibit the N-terminal sequences P(66)HLLPPP. and Y(81)EKPALH. of the collagen XV NC-1 domain. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments.

Amino Acid Sequence↗