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PubMed2004

Expression of active plasminogen activator inhibitor-1 reduces cell migration and invasion in breast and gynecological cancer cells.

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.

Breast Neoplasms↗
PubMed2005

Alphavbeta3 integrin and cofilin modulate K1735 melanoma cell invasion.

Cytoskeletal reorganization is partially mediated through cofilin, an actin assembly regulatory protein. Cofilin activity is modulated by reversible phosphorylation at Ser3. In this study, using K1735 murine melanoma cells, we examined the relationship between beta3-integrin expression, phosphorylation of cofilin, and metalloproteinase production. The levels of phosphorylated cofilin were 10-fold higher in cells expressing alphavbeta3 than in alphavbeta3-negative cells when plated on vitronectin for 30 min. However, by 60 min, phosphorylation of cofilin was greater in the beta3-negative cells. Expression of the wild type (WT) or non-phosphorylatable cofilin (A3 mutant) increased melanoma cell migration on vitronectin and invasion through a reconstituted basement membrane. Expression of a pseudophosphorylated, poorly active cofilin (E3 mutant) reduced cell motility. Expression of active cofilin accelerated the phosphorylation of FAK at Y397 and at Y576, strongly implicating cofilin as a mediator of cell signaling. The expression of MT1-MMP and MMP2 was also increased by expression of wild type or A3 cofilin. A 50% reduction of both enzymes was observed by the expression of the E3 cofilin. Overexpression of non-phosphorylatable cofilin was sufficient to induce the expression of MT1-MMP and MMP2 in the beta3-negative M2Tbeta3 cells. Interestingly, the invasion of M2Tbeta3 cells could be sustained by overexpression of cofilin A3. These results suggest that the integrin alphavbeta3 and cofilin together regulate K1735 melanoma cell invasion.

Actin Depolymerizing Factors↗
PubMed1999

Effect of exogenous gonadotropins on endometrial maturation in oocyte donors.

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.

Adult↗
PubMed2002

Metalloproteinase inhibitor attenuates neointima formation and constrictive remodeling after angioplasty in rats: augmentative effect of alpha(v)beta(3) receptor blockade.

Release of matrix metalloproteinases (MMP) from smooth muscle and foam cells following arterial injury facilitates cell migration, neointimal hyperplasia, and vessel wall remodeling. Inhibition of MMP activity using the hydroxamate, zinc-chelating mimicers of collagen, Batimastat and Marimastat, has shown efficacy in reducing constrictive vascular remodeling 6 weeks after experimental angioplasty but not intimal hyperplasia. Vitronectin receptor (alpha(v)beta(3)) blockade interferes with binding of this integrin to MMP-2 and proteolyzed collagen, thereby reducing cell invasion. This study tests the effect of MMP inhibition, with and without vitronectin receptor (alpha(v)beta(3)) blockade, on neointima formation and arterial remodeling in a long-term model (up to 212 months) of balloon injury in vivo. Male Sabra rats were treated with Batimastat (BB-94, British Biotech Pharmaceuticals Ltd., 30 mg/kg, intraperitoneally) and/or the alpha(v)beta(3) receptor inhibiting RGD peptide, G-Pen-GRGDSPCA (GIBCO BRL, 0.1 micromol), administered as a perivascular gel to the common carotid artery after balloon injury. Animals were sacrificed 3, 14, 25, and 75 days (n=21, 23, 22, and 21) after injury. Animals treated with BB-94, peptide, or both had markedly increased absolute luminal area with markedly reduced luminal cross-sectional-area narrowing by neointima and intima-to-media area ratio at all time points except for 3 days after balloon injury versus non-treated, ballooned animals. Combined treatment was significantly more effective than either one alone. Constrictive remodeling, most marked 212 months after balloon injury, was prevented at this time point in all treated animals. The pattern of reduction in luminal narrowing, neointimal formation, and constrictive remodeling across treatment groups correlated very significantly with the reduction in tissue MMP activity as determined by zymography at 3 days. Confirmation of the efficacy of this strategy in larger animals should be the next step toward testing the applicability of this novel approach to the interventional setting.

Angioplasty, Balloon↗
PubMed2001

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

Animals↗
PubMed1995

Differential sensitivity of thoracic malignant tumors to adenovirus-mediated drug sensitization gene therapy.

Malignant mesothelioma may prove to be an attractive candidate for somatic gene therapy with replication-deficient recombinant adenovirus transfer of a toxic, or drug sensitization gene. Transfer of the herpes simplex thymidine kinase type I gene (HSVtk), followed by exposure to the acyclic nucleoside drug ganciclovir, has been shown to be an effective tumor cell killing system. To study generalized applicability, we tested a number of thoracic malignant cell lines for their sensitivity to gancyclovir after infection with an adenoviral vector containing the HSVtk gene (Ad.RSVtk). Using the concentration of gancyclovir required to kill 50% of the cells (IC50) as a measure of sensitivity, we detected variable sensitivity among cell lines, with mesothelioma most sensitive (IC50 = 0.075 to 2.8 mumol/L gancyclovir), and non-small-cell carcinoma lines having an intermediate sensitivity (IC50 = 1.5 to 100 mumol/L). In contrast, an ovarian carcinoma line was extremely resistant (IC50 > 2000 mumol/L). To study the possible mechanisms for these differences, we studied cell lines with regard to their ability to be infected with an adenoviral vector containing a marker gene (Ad.CMVlacZ) and expression of the vitronectin receptor alpha v (an integrin cell adhesion molecule shown to be required for adenovirus internalization after initial binding). We found that the degree of lacZ transduction correlated with HSVtk sensitivity, whereas vitronectin receptor expression did not, suggesting that differences in initial viral binding ability, rather than internalization, may explain the sensitivity differences seen in vitro.

Adenoviridae↗
PubMed1999

Recombinant adenovirus vectors for cytokine gene therapy in mice.

BACKGROUND: Adenoviruses have several specific features useful for gene therapy. They infect various lineages of cells irrespective of cell cycle status. However, the exact mechanism of their infection and in vivo kinetics as a gene expression vector have not been elucidated. OBJECTIVE: Using adenovirus vectors expressing marker genes, we examined the infectivity of these vectors (including cellular and tissue tropism), the duration and intensity of transgene expression, and the side effects. METHODS: Various cells were infected with adenovirus expressing LacZ gene at various doses, and beta-galactosidase activity was measured and compared in relation with dose, time course, and cellular vitronectin receptor. Mice were injected with adenoviruses expressing LacZ, luciferase and GM-CSF, and in vivo gene expression was examined. RESULTS: Adenovirus infection induced viral dose-dependent transgene expression that persisted for 2 weeks. Adherent cells were infected much more efficiently than nonadherent cells, probably because the former expressed much higher levels of the vitronectin receptor, one of the main receptors for adenovirus. Studies performed in mice with luciferase-expressing adenovirus revealed that the liver was the main target organ after intravenous injection and showed that the intravenous route was superior to other routes with regard to transgene expression. After intravenous injection of adenovirus expressing human GM-CSF, there was a transient and dose-dependent increase in the serum level of this cytokine. Administration of adenovirus expressing mouse GM-CSF enhanced hematopoiesis in the spleen and bone marrow. CONCLUSION: These results indicated that adenoviruses can be used for in vivo cytokine gene therapy but suggested the necessity of taking into consideration the route of administration, the duration of transgene expression, the toxic dose, and host immune reactions.

Adenoviridae↗
PubMed2002

Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling.

3T3 Cells↗
PubMed2000

The importance of helix F in plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold decreased binding to vitronectin. The obtained results clearly show the importance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.

Epitopes↗
PubMed1999

Extracellular matrix-modulated differential invasion of human meningioma cell lines in vitro.

The in vitro invasive behaviour of six meningioma cell lines of various histological sub-type and grade was assessed using Boyden chemotaxis chambers ('Transwell' units) precoated with various extracellular matrix proteins. The cell lines included a benign meningothelial (IPGS), two benign transitional (IPCBR and IPGC), one atypical (IPIH) and two malignant (IPSE and IPIR) meningiomas. IPGC was a recurrent tumour. The results showed that IPCBR was most invasive through laminin and vitronectin. IPIH was moderately invasive through collagen type IV, laminin, vitronectin and fibronectin. However, both IPSE and IPIR were less invasive than IPIH whereas, IPGS was least invasive of all. Moreover, laminin was the most permissive extracellular matrix protein for most cell lines and collagen type IV, the least permissive. These results show that there is a differential in vitro invasive behaviour of cell lines derived from different histological types of meningiomas according to extracellular matrix substrate and suggests that invasion and migration of meningiomas in situ might be modulated by various extracellular components.

Chemotaxis↗
PubMed2000

[Cortex-wall connections in the apical cell of Sphacelaria].

The apical cell of Sphacelaria (Fucophyceae) exhibits a permanent polarized organization throughout asymmetric divisions. The apex organization was studied by immunolocalization of tubulin, vitronectin, alpha-actinin and beta 1 integrin. Microfilaments were stained directly by fluorescein phalloidin. The apex was highly organized around a patch of microfilaments densely packed at the tip, where vitronectin-like and alpha-actinin-like proteins colocalized. In the same area, an actin-dependent targeted secretion of sulfated polysaccharides was shown. The permanent localization of these components throughout cell elongation suggests that a cortical site involving transmembrane connections between the cytoskeleton and the extracellular matrix is required for cell polarity. A model of the organization of the tip is proposed.

Actin Cytoskeleton↗
PubMed1999

Depletion of alphaV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis.

Integrin heterodimers sharing the common alphaV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alphaV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alphaVbeta3 and alphaVbeta5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alphaV integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor alphaV integrin subunit was not affected. However, the maturation of alphaV integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of alphaV integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, alphaV integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation.

Alkaline Phosphatase↗
PubMed1999

The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed. RESULTS: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins. CONCLUSIONS: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.

Binding Sites↗
PubMed2002

Macrophage colony-stimulating factor and receptor activator NF-kappaB ligand fail to rescue osteoclast-poor human malignant infantile osteopetrosis in vitro.

Malignant infantile osteopetrosis (MIOP) is a disease characterized by failure in bone resorption, leading to dense fragile bones with a severely reduced bone marrow cavity. Normal or increased numbers of osteoclasts are present in the common variant of this disease; in such cases, the defect is likely to be inherent to the mature osteoclast and can be cured by bone marrow transplantation. However, MIOP also results from failure of osteoclast formation (osteoclast-poor MIOP). We report on two infants diagnosed with osteoclast-poor MIOP and utilize modern cell culture techniques to investigate the pathogenesis of disease. Peripheral blood mononuclear cells (PBMNCs) from these children were cultured in the presence of recombinant macrophage colony-stimulating factor and receptor activator NF-kappaB ligand for up to 3 weeks. Control cultures included PBMNCs from age-matched children, one of whom had an osteoclast-rich form of MIOP. Formation of osteoclasts (cells coexpressing vitronectin receptor and F-actin rings) occurred in all the control cultures. Significant bone resorption occurred in cultures from PBMNCs of the healthy individuals, whereas almost no bone resorption occurred in the osteoclast-rich MIOP cultures. In contrast, PBMNC cultures from the osteoclast-poor MIOP child formed only very occasional small F-actin ring-positive osteoclasts, which coexpressed vitronectin receptor and cathepsin K, and extremely rare foci of resorption. Because neither macrophage colony-stimulating factor nor receptor activator NF-kappaB ligand rescued the defect in osteoclast differentiation in the two cases of osteoclast-poor MIOP in vitro, there would be little benefit in treating these children with either of these recombinant proteins. Finally, these results demonstrate that this experimental culture model replicates the human osteopetrosis phenotype observed in vivo and should prove useful in analyzing the pathogenesis of the various forms of MIOP.

Actins↗
PubMed1998

Macrophage colony-stimulating factor increases bone resorption by osteoclasts disaggregated from human fetal long bones.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in human and murine osteoclast formation. Although M-CSF has been shown to inhibit isolated neonatal rat osteoclasts from resorbing bone, its action on the mature human osteoclast has not been described. We now report that M-CSF increases osteoclastic bone resorption in a dose-responsive manner. Bone resorption by mature human fetal osteoclasts, including pit area, depth, and volume, was increased in the presence of M-CSF compared with vehicle. The number of osteoclasts in the cultures was similar after 2 and 18 h in the presence of M-CSF, whereas there was a significant reduction in osteoclast number, whether assessed as the number of tartrate-resistant acid phosphatase (TRAP)-positive or vitronectin receptor-positive cells after 18 h in M-CSF-free cultures. The number of nuclei per osteoclast after 2 or 18 h in M-CSF was also similar and there was no difference in the number of vitronectin receptor-positive mononucleate cells at 2 and 18 h. This suggests that the increased bone resorption is likely to be accounted for by enhanced osteoclast survival in M-CSF compared with controls rather than by formation of new osteoclasts.

Acid Phosphatase↗
PubMed1995

Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries.

The integrin alpha v beta 3 binds promiscuously to cell-adhesive proteins: vitronectin, fibronectin, and several others containing the RGD motif. We have explored molecular recognition by alpha v beta 3 through selection of ligands from large random libraries of peptides displayed on phage. Ligands bound by alpha beta 3 consisted primarily of RGD peptides; however, these peptides showed considerable heterogeneity with respect to the identities of amino acids flanking RGD. The tolerance of alpha v beta 3 for RGD peptides of diverse composition is consistent with its role in vivo as a versatile receptor for RGD-containing extracellular matrix proteins. Peptide ligands for alpha v beta 3 also included a novel binding sequence, identical to a tetrapeptide found in vitronectin, which is a candidate for a synergistic site in this adhesive protein that may act in concert with RGD to promote molecular recognition.

Amino Acid Sequence↗
PubMed1999

Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.

Binding Sites↗
PubMed1996

Synthesis and solution conformation of cyclo[RGDRGD]: a cyclic peptide with selectivity for the alpha V beta 3 receptor.

Three peptides, cyclo[RGDRGD], cyclo[RGDRGd] (d = D-Asp), and the linear sequence RGDRGD, were prepared via solid phase syntheses. These were tested in binding assays based upon the alpha IIb/beta 3-fibrinogen and the alpha V beta 3-vitronectin interactions and found to be selective for the alpha V beta 3 integrin. The alpha V beta 3-vitronectin is important in bone regeneration, hence the compounds were also tested in an osteoclast regeneration assay; all three compounds, cyclo-[RGDRGD], cyclo[RGDRGd], and RGDRGD, showed modest activities. Molecular modeling, NMR, and CD studies were undertaken to elucidate the conformational preferences of cyclo-[RGDRGD], in aqueous solutions. Results from these studies strongly suggest that the molecule tends to adopt a type I beta-turn conformation with a relatively short distance between the Asp and Arg side chains. These observations are in harmony with the first correlations made between alpha V beta 3 selectivity and solution conformation for a peptide ligand (Pfaff, M.; et al. J. Biol. Chem. 1994, 269, 20233).

Cell Division↗