The alpha 2 beta 1 integrin binds to the CB4 peptide of the alpha 2(I) collagen chain.

The adhesion of platelets and other cells to type I collagen is mediated by the alpha 2 beta 1 integrin. A binding site for the alpha 2 beta 1 integrin within the alpha 1(I) collagen chain has previously been localized to the cyanogen bromide fragment alpha 1(I)-CB3. We noe show by use of inhibitory monoclonal antibodies against the alpha 2 beta 1 integrin, that platelets also adhere to purified alpha 2(I) collagen chains by a mechanism mediated by the alpha 2 beta 1 integrin. Moreover, following isolation of cyanogen bromide fragments of the alpha 2(I) collagen chain by HPLC, we demonstrate that alpha 2 beta 1 integrin-mediated adhesion is restricted to the CB4 fragment of the alpha 2(I) collagen polypeptide. These findings indicate the presence of at least two spatially distinct binding sites for the alpha 2 beta 1 integrin on the native type I collagen triple helix.

Regulation of alpha 2 integrin gene expression in cells with megakaryocytic features: a common theme of three necessary elements.

The alpha 2 beta 1 integrin mediates interactions between cells and the extracellular matrix molecules, collagen and/or laminin. The alpha 2 beta 1 integrin is expressed in a variety of cell types, but in cells of hematopoietic lineage, expression is restricted to megakaryocytes and platelets. Increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of transcriptional activation of the alpha 2 gene. We have begun to characterize the role of the 5' flanking region of the alpha 2 integrin gene in regulating expression during megakaryocyte differentiation. A 5-kb fragment of the 5' region directs both cell type and differentiation-dependent expression of a reporter gene in the pluripotent hematopoietic K562 cells upon megakaryocytic differentiation and in the megakaryocytic cell line, Dami. Analysis of a series of 5' deletion mutants indicates that expression of the alpha 2 integrin gene in cells with megakaryocytic features requires a core promoter region, a silencer region, and megakaryocytic enhancers in the distal 5' end. The organization of these three distinct regulatory regions of the alpha 2 promoter/enhancer suggests a common theme for megakaryocytic gene regulation shared with other megakaryocyte-specific proteins, including alpha IIb integrin subunit and platelet factor 4.

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells.

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.

Preparation of soluble peptide libraries: application to studies of platelet adhesion sequences.

We describe chemical synthesis of peptide mixtures that equally express many sequence combinations. Using 65 couplings of single amino acids, five mixtures were prepared with the sequences Tyr-Gly-Arg-Gly-Yyy-Xxx-Xxx, where Yyy is Ser, Asp, Arg, Asn, or Glu, and Xxx is any amino acids. Compositional and sequence analyses supported full representation of all amino acids, except isoleucine was deficient in the sixth position. The data suggest formation of a repertoire of 1,900 sequence combinations (5 x 19 x 20). The mixture with Asp as the fifth residue inhibited platelet adhesion to fibronectin more effectively than the other mixtures. Peptide libraries offer a new tool for investigating bioactive peptides.

Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes.

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen.

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.

Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex.

Recent studies have shown that the platelet membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg(++)-dependent adhesion of platelets to collagen and that this adhesion is inhibited by Ca++ in a simple, linear, noncompetitive manner. These findings suggested that separate binding sites for Mg++ and Ca++ stabilize different divalent cation-dependent structures within the receptor complex. To provide evidence for the existence of such structures purified platelet Ia-IIa complex was subjected to limited proteolytic digestion in the presence of Mg++, Ca++, Mg++ and Ca++, or EDTA and the resulting peptides mapped by SDS-PAGE using both one and two-dimensional techniques. Unique patterns of tryptic peptides were produced under each of the conditions. The results indicate that Mg++ and Ca++ stabilize different structures within the Ia-IIa (VLA-2) complex and that these structures influence both the collagen binding activity and proteolytic susceptibility of the complex.

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen.

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.

The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++-dependent adhesion of platelets to collagen.

We have purified the platelet membrane glycoprotein Ia-IIa complex by detergent solubilization and sequential affinity chromatography on Concanavalin A-Sepharose and collagen-Sepharose. The complex, which is identical to the VLA-2 complex of lymphocytes and other cells and contains subunits of 160 and 130 kD on SDS-PAGE, was labeled with 125I and incorporated into phosphatidyl choline liposomes. The liposomes, like intact platelets, adhered to collagenous substrates in an Mg++-dependent manner with a K'a(Mg++) of 3.5 mM. Little adhesion of the liposomes to collagen occurred when Mg++ was replaced by Ca++ or EDTA. Calcium ions inhibited the Mg++-dependent adhesion with a K'i(Ca++) of 5.5 mM. Liposomes containing the Ia-IIa complex adhered to substrates composed of types I, II, III, and IV collagen, but did not effectively adhere to substrates composed of type V collagen or gelatin. Adhesion to collagen was specific. The liposomes did not adhere to fibronectin, vitronectin, laminin, thrombospondin, fibrinogen, or von Willebrand factor substrates. The monoclonal antibody P1H5, which specifically immunoprecipitated the Ia-IIa complex, also specifically inhibited the Mg++-dependent adhesion of both platelets and Ia-IIa-containing liposomes to collagen substrates. These findings provide additional evidence that the platelet membrane Ia-IIa complex is the mediator of Mg++-dependent platelet adhesion to collagen and suggest that the VLA-2 complex may also function as an Mg++-dependent collagen receptor in other cells.

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2.

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.

Effects of timolol on bovine corneal endothelial cultures.

The chronic use of timolol (Timoptic) maleate to control glaucoma may produce cytotoxic complications in the cornea. We have therefore compared the relative toxic effects of the commercial ophthalmic preparation with that of the pure compound. Commercial vehicle, either with or without 16 mM timolol maleate, killed cultures within the first five minutes of exposure. Pure timolol maleate, however, caused rapid but reversible cellular contractions, and cells remained viable in it for over 24 hours. Dilution with culture medium reduced both the cytotoxicity and the speed of the contractions. Incubation in 1:100 dilutions of vehicle or commercial drug preparations or in 0.16 mM pure timolol maleate did not alter cellular morphology. The results indicate that while undiluted vehicle is toxic, timolol maleate is not.

Effects of ionophores X537a and A23187 and calcium-free medium on corneal endothelial morphology.

Past studies have shown that apical junctional complexes (AJCs) of corneal endothelial cells break down in the presence of a Ca++-free medium. The purpose of this study was to examine the ability of Ca++ ionophores to maintain the AJCs in the Ca++-free media in both isolated perfused corneas and cultured endothelial cells. In addition, the ability of disintegrated AJCs to re-form when the endothelium is returned to a medium containing calcium ws also examined. Rabbit corneas were mounted in an in vitro specular microscope and perfused with a Ca++-free medium, or a Ca++-free medium containing 10(-5)M X537A or A23187 calcium ionophore. Also, confluent monolayer cultures of bovine corneal endothelial cells were placed in a Ca++-free medium or a Ca++-free medium containing 10(-5)M X537A or A23187 Ca++ ionophore and incubated for selected time periods. When junctional breakdown occurred, one cornea or culture plate was fixed for scanning and transmission electron microscopy (SEM and TEM), and the other was returned to a medium containing Ca++ and subsequently fixed for SEM and TEM. Both isolated perfused and cultured corneal endothelial cell AJCs exhibited marked disintegration in the presence of Ca++-free medium. The presence of an ionophore in the medium cultured cells. When returned to a medium containing Ca++, the corneas that had been perfused with Ca++-free medium containing an ionophore re-formed the junctions sooner than did those that had been perfused with a Ca++-free medium alone. These results suggests that the ionophores may be capable of mobilizing intracellular calcium to protect the AJCs.

The effects of aging and inflammation on corneal endothelial wound healing in rabbits.

After transcorneal freezing, the rates and patterns of corneal endothelial wound healing were compared in mature and young rabbits by autoradiographic analysis of 3H-thymidine incorporation and scanning electron microscopy. Healing was slower and less extensive in mature corneas than in young ones. Regardless of animal age, however, healing occurred by cell division and the migration of newly divided cells onto the wound surface. Spontaneously occurring severe inflammation appeared to reduce the ability of corneal endothelial cells to replicate their DNA.

Cytotoxicity of pivalylphenylephrine and pivalic acid to corneal endothelium.

We examined the effects of topically applied pivalylphenylephrine (PPE) and pivalic acid (PA) on the corneal endothelium of rabbits and the direct effects of PPE and PA on monolayer cultures of bovine corneal endothelium. The PPE-treated corneas without epithelium significantly increased in thickness, whereas no change in thickness was observed in corneas with epithelium intact. The PA did not alter the thickness of corneas with or without epithelium. Although 0.001% PE had no noticeable effect in two hours, 0.01% PPE caused breakdown of intercellular junctions in cultured cells in five minutes. Higher concentrations of PPE caused the cells to detach from the culture dishes within 30 minutes of treatment. Only 1% PA caused cell elongation and loss of intercellular contact after 60 to 90 minutes of exposure; lower concentrations did not effect cultured cells.