The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.     

肝癌细胞-胞外基质粘附性与粘附识别序列的相关性

以微管吸吮技术研究了人肝癌细胞在ＩＶ型胶原／层粘连蛋白（ＬＮ）／纤维连结蛋白（ＦＮ）裱衬表面的粘附性。进一步,用四种人工合成肽精－甘－天冬－丝（ＲＧＤＳ）、甘－精－甘－天冬－苏－脯ＧＲＧＤＴＰ）、酪－异亮－甘－丝－精（ＹＩＧＳＲ０和半胱－天冬－脯－甘－酪－异亮－甘－丝－精（ＣＤＰＧＹＩＧＳＲ）研究了肝癌细胞粘附性对两种粘附识别序列ＲＧＤ和ＹＩＧＳＲ的依赖性。为了归纳和整理实验结果,根据竞争性抑制的     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptide cyclo[-d-Val-Arg-Gly-Asp-Glu(-Ahx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugate BSA–peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated that covalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

Summary A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptidecyclo[-d-Val-Arg-Gly-Asp-Glu(εAhx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugated BSA-peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated thatcovalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Studies of peptide binding to allyl amine and vinyl acetic acid-modified polymers using matrix-assisted laser desorption/ionization mass spectrometry.

Previous studies have shown that increases in surface-peptide binding affinity result in decreases in peptide matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) ion signals. The present work demonstrates that, with appropriate corrections for peptide ionization efficiency under MALDI conditions, relative surface-peptide binding affinities can be assayed using the MALDI MS methodology. Peptides with a range of pI values are allowed to interact with amine-modified and carboxylic acid-modified polymer surfaces (produced by pulsed radio-frequency plasma polymerization of allyl amine and vinyl acetic acid) in buffered solutions of neutral pH. Because of the net positive and negative charges associated with the peptides and surfaces in solution, both electrostatic and hydrophilic interactions play a role in the surface-peptide interaction. Consistent with expectations, the peptide MALDI ion signals for peptides with net negative charges in solution are smaller than those for peptides with net positive charges in solution when the peptides are allowed to interact with positively charged surfaces. A reversal of the relative peptide MALDI ion signal intensities is observed when the same peptides are allowed to interact with negatively charged surfaces. Cumulatively, the results demonstrate that even modest changes in surface-peptide interactions can be comparatively probed by MALDI mass spectrometry.     

Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide

Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.     

Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands

In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry. Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization. These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins. These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands. Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment. These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function. Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process.     

Hydrogen-bond energetics drive helix formation in membrane interfaces

The free energy cost ΔG of partitioning many unfolded peptides into membrane interfaces is unfavorable due to the cost of partitioning backbone peptide bonds. The partitioning cost is dramatically reduced if the peptide bonds participate in hydrogen bonds. The reduced cost underlies secondary structure formation by amphiphilic peptides partitioned into membrane interfaces through a process referred to as partitioning-folding coupling. This coupling is characterized by the free energy reduction per residue, ?G(res) that drives folding. There is some debate about the correct value of ?G(res) and its dependence on the hydrophobic moment (μ(H)) of amphiphilic α-helical peptides. We show how to compute ?G(res) correctly. Using published data for two families of peptides with different hydrophobic moments and charges, we find that ?G(res) does not depend upon μ(H). The best estimate of ?G(res) is -0.37 ± 0.02 kcal mol(-1). This article is part of a Special Issue entitled: Membrane protein structure and function.     

Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined cell-adhesive substrates

The synthetic peptides Gly-Arg-Gly-Asp-Tyr and Gly-Tyr-Ile-Gly-Ser-Arg-Tyr, which contain Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR), the ligands for two important classes of cell adhesion receptors, were covalently coupled to a nonadhesive modified glass surface by the N-terminal Gly. The N-terminal Gly served as a spacer, and the C-terminal Y served as a site for radioiodination. These modified substrates supported the adhesion and spreading of cultured human foreskin fibroblasts (HFFs) independently of adsorbed proteins and, it was demonstrated that a covalently immobilized YIGSR-containing peptide has biological activity. The surface concentration of grafted peptide on the glass was measured by 125I radio-labeling and was 12.1 pmol/cm2. HFFs spread on both immobilized peptide substrates, but at much slower rates on grafted YIGSR glass surfaces than on the RGD-containing substrates. Cells formed focal contacts on the RGD-derivatized substrates in the presence or absence of serum. Focal contacts formed on the YIGSR-grafted surfaces only when serum was present in the medium and had morphologies different from those observed on the RGD-containing substrates. Serum influenced the organization of microfilaments and the extent of spreading of adherent cells, although adsorption of adhesion proteins was minimal on all substrates. This derivatization method produced chemically stable substrates which may be useful in studying receptor-mediated cell adhesion, as the quantity of peptide available at the surface may be precisely measured and controlled.     

Inhibition of adhesion of hepatocellular carcinoma cells to basement membrane components by receptor competition with RGD- or YIGSR-containing synthetic peptides

A micropipette technique was used to investigate the effects of four synthetic peptides, YIGSR, CDPGYIGSR, RGDS and GRGDTP, on the adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen/laminin/fibronectin coated surfaces. Adhesion of HCC cells to laminin was found to be YIGSR- or CDPGYIGSR-dependent while that to fibronectin and type IV collagen was RGDS- or GRGDTP-dependent. The reduction in adhesion strengths of HCC cells was slight to moderate (up to 55%), and was dependent on the peptide concentration. The decrease in adhesion strengths was reversed by an increase in ligand coating concentration and was compromised by prolonged interaction of the cells with the surfaces. These results suggested that the inhibition was due to competitive retardation rather than to a blockade of adhesion strengthening. A simple asymptotic function was adopted to fit the correlation between the mean of cell adhesion strengths and peptide concentration within defined concentration ranges. Regression analysis showed that cell adhesion strengths appeared to approach a plateau with increasing concentration of the inhibitory peptides, which was not always uniform over the entire concentration range tested. Further reduction in adhesion strengths was observed at higher peptide concentrations. It is suggested that the constants obtained by fitting over a low peptide concentration range might be kinetically representative of the inhibition during early events of adhesion or attachment.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

The Inhibitory Effect of an RGD-Human Chitin-Binding Domain Fusion Protein on the Adhesion of Fibroblasts to Reacetylated Chitosan Films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.     

Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III_8-11) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III_8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials.     

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.     

A method for probing the affinity of peptides for amphiphilic surfaces

We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.     

Synthesis, characterization and cytocompatibility of polyurethaneurea elastomers with designed elastase sensitivity

In designing a synthetic scaffold for engineering soft, mechanically active tissues, desirable properties include elasticity, support of cell adhesion and growth, ease of processability, and responsiveness to in vivo remodeling. To achieve these properties, we have developed a family of thermoplastic elastomers, polyurethaneureas (PUs), that possess enzymatic remodeling capabilities in addition to simple hydrolytic lability. PUs were synthesized using either polycaprolactone or triblock copolymer polycaprolactone-b-poly(ethylene glycol)-b-polycaprolactone as the soft segment, 1,4-butanediisocyanate as the hard segment, and the peptide Ala-Ala-Lys as a chain extender. The synthesized PUs had high molecular weights, low glass transition temperatures (< -54 degrees C), and were flexible with breaking strains of 670-890% and tensile strengths of 15-28 MPa. Incubation in buffered saline without elastase for 8 weeks resulted in mass loss from 12% to 18% depending on soft segment composition. The degradation significantly increased (p < 0.05) in the presence of elastase, ranging from 19% to 34% with degradation products showing no cytotoxicity. To encourage cell adhesion, PUs were surface-modified with radio frequency glow discharge followed by coupling of Arg-Gly-Asp-Ser (RGDS). Endothelial cell adhesion was >140% of tissue culture polystyrene on PU surfaces and >200% on RGDS-modified surfaces. The synthesized PUs thus combine mechanical, chemical, and bioresponsive properties that might be employed in soft-tissue engineering applications.     

Characterizing the role of cell-wall β-1,3-exoglucanase Xog1p in Candida albicans adhesion by the human antimicrobial peptide LL-37

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37-cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p(90-115), and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion.     

Adhesion of cells to polystyrene surfaces

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.     

The dualistic mechanisms of platelet adhesion to von Willebrand factor substrates

von Willebrand factor (vWf) which serves as a necessary factor for platelet adhesion to damaged vascular subendothelium can bind to the platelet surface via two distinct receptors. Ristocetin promotes the binding of vWf to platelet membrane glycoprotein lb, whereas platelet activation by thrombin supports binding to the glycoprotein IIb/IIIa complex. Platelet adhesion to vWf substrates mediated by these two mechanisms has been compared. Both mechanisms supported similar rates of adhesion to the substrates. Whereas adhesion via the ristocetin-dependent mechanism did not require divalent cations, adhesion mediated by the thrombin-dependent mechanism required the presence of divalent cations. Modification of vWf amino groups markedly impaired the ability of the protein to support ristocetin-dependent adhesion but did not alter its ability to support thrombin-enhanced adhesion. Reduction and carboxymethylation nearly abolished the ability of vWf to support adhesion via the ristocetin-dependent mechanism, but did not substantially impair its ability to support thrombin-enhanced adhesion. Short synthetic peptides containing the sequence Arg-Gly-Asp-Ser effectively inhibited thrombin-dependent platelet adhesion to vWf substrates but had no effect on ristocetin-dependent adhesion. Substrates composed of synthetic peptides containing the Arg-Gly-Asp-Ser sequence supported thrombin-dependent adhesion but did not support ristocetin-dependent adhesion. Scanning electron microscopic examination revealed that platelets adherent via the ristocetin-dependent mechanism almost uniformly adopted a flattened and fully spread appearance. In contrast, the thrombin-enhanced mechanism of adhesion supported only a limited degree of platelet spreading on the vWf substrate.     

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-l-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer ofl-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 g/cm² of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 g/cm² also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.