Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin. Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22 degrees C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr approximately 90,000) into a previously described, major membrane-bound fragment with Mr 60,000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr approximately 120,000), which was converted into a faster migrating species with Mr approximately 115,000). This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37 degrees C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37 degrees C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.     

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation

The Arg-Gly-Asp (RGD)-binding domain of GPIIb-IIIa has been localized in a fragment of the GPIIIa subunit that includes the sequence between amino acids 109 and 171. To examine, in a platelet membrane environment, the activated versus nonactivated status of this domain, we have produced a monoclonal antibody against a synthetic peptide (residues 109-128) located within the RGD-binding region on GPIIIa. This kappa-IgM, named AC7, was specific for GPIIIa peptide 109-128 and interacted only with activated platelets. Fibrinogen, RGDF peptide, and the fibrinogen phi chain decapeptide LGGAKQAGDV inhibited the binding of AC7 to ADP-stimulated platelets. AC7 IgM and "small fragments" inhibited fibrinogen binding and platelet aggregation in a dose-dependent fashion. Induction of AC7 binding by D33C, a monoclonal antibody recognizing the GPIIb 426-437 sequence and stimulating fibrinogen binding, indicated that the GPIIb 426-437 and the GPIIIa 109-128 sequences were both involved in a stimulation-dependent conformational modification of the receptor. AC7 was able to recognize beta subunits other than GPIIIa on leucocyte surfaces but only after cell fixation with glutaraldehyde. The results are consistent with the implication of the RGD-binding domain in receptor ligand interaction on the platelet surface and its conformational modification and exposure upon receptor induction.     

Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.     

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Study of the endoproteolytic cleavage of platelet glycoprotein IIb using oligonucleotide-mediated mutagenesis.

The precursor of platelet membrane glycoprotein IIb (GPIIb) undergoes endoproteolytic cleavage into heavy and light chains post-translation. Endoproteolysis occurs within a 17-amino acid stretch of the precursor that contains 4 arginine residues, 3 in dibasic sequences [Lys-Arg (855-856) and Arg-Arg (858-859)] and a single arginine at 871. To determine the site of GPIIb cleavage and its role in the function of the glycoprotein IIb/IIIa heterodimer, we mutated arginine 856, the di-arginine sequence 858-859, and arginine 871 and coexpressed the mutants with glycoprotein IIIa (GPIIIa) in COS-1 cells. Each GPIIb mutant formed recombinant GPIIb-IIIa heterodimers, but mutants lacking arginine at 856 or 858-859 failed to undergo cleavage. Nevertheless, heterodimers containing the uncleaved GPIIb were expressed on the cell surface. Because endoproteolysis most often occurs after arginines in dibasic sequences, we next expressed GPIIb mutants containing lysine at 856 or aspartic acid at 855 with GPIIIa. Both mutants were cleaved and surface-expressed, indicating that the dibasic sequence at 858-859, but not at 855-856, is required for GPIIb cleavage. Lastly, we tested the function of GPIIb-IIIa containing uncleaved GPIIb by measuring adhesion of transfected cells to immobilized fibrinogen. We found no difference in the adhesion of cells expressing either wild-type or mutant GPIIb, indicating GPIIb-IIIa heterodimers containing uncleaved GPIIb maintain their ability to interact with fibrinogen.     

Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3

Summary The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-pb GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.     

Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane

As reported previously, homologous plasma lipoproteins specifically bind to the plasma membrane of human blood platelets. The two major lipoprotein-binding membrane glycoproteins were purified to apparent homogeneity and identified by their mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both in the nonreduced and reduced state, by specific antibodies against glycoproteins IIb (GPIIb) and IIIa (GPIIIa), respectively, including the alloantibody anti-PlA1 and monoclonal antibodies. Furthermore, lipoprotein binding to intact platelets is also inhibited in a dose-dependent fashion by preincubation of the platelets with antibodies against these glycoproteins. From these experiments it can be concluded that lipoproteins bind to both components of the glycoprotein IIb-IIIa complex in isolated membranes and intact platelets. High density lipoprotein and low density lipoprotein bind to GPIIIa blotted to nitrocellulose in a way that binding of one species interferes with the binding of the other. Addition of fibrinogen significantly inhibits this binding. The specific binding of fibrinogen to GPIIIa is strongly inhibited in the presence of either of the two lipoproteins. LDL and HDL are specifically bound by isolated GPIIb, too. In our blotting experiments fibrinogen shows no binding to this membrane glycoprotein. On the other hand, fibrinogen significantly interferes with the interaction between GPIIb and the lipoproteins.     

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.     

Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets

A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa.     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

Synthesis by cultured human umbilical vein endothelial cells of two proteins structurally and immunologically related to platelet membrane glycoproteins IIb and IIIa

Human platelets participate in a number of adhesive interactions, including binding to exposed subendothelium after vascular injury, and platelet-platelet cohesion to form large aggregates. Platelet membrane glycoproteins (GP) IIb and IIIa constitute a receptor for fibrinogen that, together with fibrinogen and calcium, is largely responsible for mediating the formation of the primary hemostatic plug. Using highly specific polyclonal and monoclonal antibodies as probes, we could detect the presence of both of these glycoproteins in cultured human umbilical vein endothelial cells. Western-blot analysis showed that the endothelial cell analogues were similar in size to their platelet counterparts, and were present in cells that had been in culture for over 2 mo. Metabolic labeling of endothelium with [35S]methionine demonstrated that both GPIIb and GPIIIa were actively synthesized in culture. Using the technique of crossed immunoelectrophoresis, evidence was obtained that the endothelial cell forms of GPIIb and GPIIIa may exist complexed to one another after solubilization in Triton X-100. The presence of GPIIb-IIIa analogues in cultured endothelial cells may provide an opportunity to examine the structure, function, and synthesis of these two membrane glycoproteins, as well as provide a source of genetic material with which to begin detailed molecular genetic studies.     

C-terminal amino acid determination of the transmembrane subunits of the human platelet fibrinogen receptor, the GPIIb/IIIa complex

Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.     

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.     

The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit

The extreme carboxyl-terminal amino acid sequence of the gamma chain of fibrinogen is involved in the binding of this adhesive protein to the platelet integrin glycoprotein (GP) IIb-IIIa, and synthetic peptides corresponding to this region inhibit fibrinogen as well as fibronectin and von Willebrand factor binding to platelets. A chemical cross-linking approach was used to characterize the interaction of a 16-amino acid fibrinogen gamma chain peptide with platelets and to localize the site of its binding to GPIIb-IIIa. This peptide became specifically cross-linked to GPIIb, and platelet stimulation selectively enhanced its cross-linking to this alpha subunit. The cross-linking reaction was specifically inhibited by fibrinogen and an Arg-Gly-Asp peptide but not by an unrelated protein or a substituted peptide. Utilizing a combination of immunochemical mapping, enzymatic and chemical digestions, and amino acid sequencing, the cross-linking site of the gamma chain peptide in GPIIb was localized to a stretch of 21 amino acids. The identified region, GPIIb 294-314, contains the second putative calcium binding domain within GPIIb. The primary structure of this region is highly conserved among alpha subunits of other integrin adhesion receptors. These results identify a discrete region of GPIIb that resides in close proximity to a ligand binding site within GPIIb-IIIa. The homologous region may be involved in the functions of other integrin receptors.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.