Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen

Platelet thrombospondin interacts with fibrinogen in a specific and saturable manner. Thrombospondin was found to specifically bind to the A alpha- and B beta-chains of fibrinogen; binding was independent of divalent ions. Binding could be blocked either by preincubation of thrombospondin with 9.4 microM fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. Thrombospondin bound only to the beta-chain component of the D and DD plasmin fragment of fibrinogen. Thrombospondin interaction with fibrinogen was not blocked by preincubation with synthetic peptides which have previously been identified as either the fibrinogen receptor (alpha 572-575, the synthetic tetrapeptide arginyl-glycyl-aspartyl-serine) or cell attachment (gamma 400-411) domains. Fibrinogen, therefore, possesses at least two unique and distinct sites, within the A alpha- and B beta-chains, for its interaction with thrombospondin.     

Amino acid sequence of the beta-chain of the tetrameric haemoglobin of the bivalve mollusc, Anadara trapezia

The amino acid sequence of the beta-chain of the principal haemoglobin from A. trapezia has been determined. The sequence was deduced from the sequences of tryptic peptides, which were fractionated using highperformance liquid chromatography and peptide mapping. Additional sequence data, particularly for the large tryptic peptides, was obtained from enzyme digests of both cyanogen bromide fragments and large citraconyltryptic peptides. The beta-chain has 151 residues which is longer than all the other sequenced haemoglobin chains except the alpha-chain of A. trapezia, which is 153 residues in length. The residues corresponding to those normally in the D helix are absent in this beta-chain. The additional residues are contributed by an extension of the N-terminal region, which was also found to be acetylated. Comparison of the beta-chain amino acid sequence with that of the alpha-chain of A. trapezia, the dimeric chain of A. trapezia, and the dimeric chain of A. broughtonii showed 53% identity in each case. In the E and F helices, the homology is particularly noticeable. There is 100% homology in the F helix of all four chains. The dimeric globin of A. trapezia also shows 100% homology with the beta-chain in the E helix, while the alpha-chain shows 75%. If the tertiary structure of the alpha- and beta-chains of A. trapezia haemoglobin is the same as that of horse haemoglobin, then there are many changes in the alpha 1 and beta 2 contact site residues.     

Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding

Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Amino-acid sequence of gayal hemoglobin (Bos gaurus frontalis, Bovidae)

The amino-acid sequences of the alpha- and beta-chains of gayal hemoglobin have been determined and compared with those of bovine and yak hemoglobins. The gayal alpha-chain differs from the alpha-chains of bovine by 3 amino-acid residues and from yak I alpha- and II alpha-hemoglobins by 4 and 2 residues, respectively. The gayal beta-chain differs from bovine beta A- and beta B-chains by 3 and 4 residues, respectively and from yak beta-chains by 2 residues.     

Mapping of the properdin-binding site in the third component of complement.

The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.     

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.     

Haemoglobin binding with haptoglobin. Unequivocal demonstration that the beta-chains of human haemoglobin bind to haptoglobin.

Haptoglobin binding to haemoglobin and its isolated alpha- and beta-chains was studied by use of a highly sensitive solid-phase radiometric assay. As expected, adsorbents of haemoglobin bound 125I-labelled haptoglobin more efficiently than did adsorbents of the alpha-chain. However, unexpectedly, adsorbents of the beta-chain were found to be essentially identical with those of the alpha-chain in their ability to bind haptoglobin. These results demonstrate, unequivocally, the ability of beta-chains to bind to haptoglobin, and indicate that this assay is particularly convenient and useful for studying haptoglobin interactions with haemoglobin and its alpha- and beta-chains.     

Fibrin(ogen) peptide B beta 15-42 inhibits platelet aggregation and fibrinogen binding to activated platelets

The binding of fibrinogen to activated platelets leads to platelet aggregation. Fibrinogen has multiple binding sites to platelet membrane glycoprotein IIb-IIIa complex. At least two well-defined sequences in fibrinogen, Arg-Gly-Asp sequence of A alpha 95-97 and A alpha 572-574 and gamma 400-411, have been shown to interact with glycoprotein IIb-IIIa. A possible binding site on the amino-terminal end of fibrinogen to platelet glycoprotein IIb-IIIa has also been reported. In this paper the effect of synthetic peptides derived from the amino-terminal end of the B beta chain on platelet aggregation and fibrinogen binding has been examined. B beta 15-42 peptide inhibits platelet aggregation and 125I-fibrinogen binding to activated platelets in a dose-dependent manner. Since B beta 15-42 contains a previously identified fibrinogen binding site, B beta 15-18, exposed by thrombin cleavage of native fibrinogen, we also examined the effect of B beta 15-18, B beta 19-42, and B beta 1-14 (fibrinopeptide B) on platelet aggregation and fibrinogen binding. Synthetic fibrinopeptide B and B beta 15-18 had no effect on platelet aggregation and fibrinogen binding while B beta 19-42 retained the inhibitory effect. When fibrinogen is chromatographed on a column of agarose-bound B beta 15-42, a cation-dependent retention of fibrinogen on the peptide column was observed, and fibrinogen was eluted from the column by B beta 15-42 but not by B beta 1-14. Under the same conditions, platelet glycoprotein IIb-IIIa was not retained in the column. Thus, the observed inhibitory effect is due to its interaction with fibrinogen rather than to platelet glycoprotein IIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of fibrinogen binding to GP IIb-IIIa by a GP IIIa peptide.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) mediates platelet aggregation and is a member of the cytoadhesin family of receptors that bind adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor. Despite the wide range of cell-substrate interactions mediated by these receptors, ligand binding domains have not yet been identified on any of the integrins. The present study was designed to determine potential fibrinogen binding domain(s) on the GP IIb-IIIa complex. Synthetic peptides derived from residues 1-288 of the amino-terminal portion of GP IIIa were tested for their abilities to block the binding of fibrinogen to purified GP IIb-IIIa in a solid-phase microtiter assay. Two overlapping peptides encompassing residues 204-229 of GP IIIa were identified which blocked fibrinogen binding in this assay. Polyclonal antibodies to these peptides blocked fibrinogen binding to purified GP IIb-IIIa as well as platelet aggregation. The overlapping residues of these two peptides GP IIIa (211-222), SVSRNRDAPEGG-NH2, blocked the binding of fibronectin, von Willebrand factor, and vitronectin to purified GP IIb-IIIa. Finally, direct binding of GP IIIa (204-229) to fibrinogen and fibronectin was demonstrated by enzyme-linked immunosorbent assay. We conclude from these studies that the amino acid sequence 211-222 of GP IIIa is critically involved in adhesive protein binding, and may represent an important portion of the GP IIb-IIIa ligand binding domain.     

Structural and functional analyses of glycosylation on the distinct molecules of human GM-CSF receptors.

We have previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors are composed of at least two molecules of 80 and 135 kDa, which were denoted alpha- and beta-chains, respectively [Chiba, S., Shibuya, K., Piao, Y.-F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K. & Takaku, F. (1990) Cell Regul. 1, 327-335]. In this paper, we describe an investigation of the biochemical disparity noted between the alpha- and beta-chains of GM-CSF receptors using proteolytic and deglycosidic enzymes, and further demonstrate the potential importance of carbohydrate structures of the GM-CSF receptors using different lectins and glycoprotein synthesis inhibitors. Cross-linked alpha- and beta-chains with 125I-GM-CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the alpha-chain was reduced by 25 kDa by the removal of the N-linked oligosaccharides with peptidase: N-glycosidase F treatment, whereas that of the beta-chain remained unmodified by the enzyme. These results suggest that the alpha-chain of GM-CSF receptors agrees with the recently cloned low-affinity GM-CSF receptor [Gearing, D.P., King, J.A., Gough, N. M. & Nicola, N.A. (1989) EMBO J. 8, 3667-3676] having approximately 30% N-linked oligosaccharides and is biochemically different from the alpha beta-chain. By analyses using lectins, some of the oligosaccharides in the alpha-chain seem to be the complex-type and/or hybrid-type, because wheat germ agglutinin and leukoagglutinating phytohemagglutinin inhibited both GM-CSF-induced proliferation and GM-CSF binding to its receptors. Further analyses using glycoprotein synthesis inhibitors showed that N-linked processing of the alpha-chain, especially glucose removal by glucosidase I and II (whose activities are inhibited by deoxynojirimycin), appeared to be required for the expression onto the cell surface although the beta-chain expression was little affected by their inhibitors. Thus the beta-chain, probably located near the alpha-chain on the cell surface, was associated with a high-affinity class of GM-CSF receptors.     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.     

The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets

Fibrinogen binding to receptors on activated platelets is a prerequisite for platelet aggregation. However, the regions of fibrinogen interacting with these receptors have not been completely characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover, the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin, is located near the carboxyl-terminal region of the alpha-chain of fibrinogen. We have examined the ability of this tetrapeptide to inhibit platelet aggregation and fibrinogen binding to activated platelets. Arg-Gly-Asp-Ser, but not the peptide Arg-Gly-Tyr-Ser-Leu-Gly, inhibited platelet aggregation stimulated by ADP, collagen, and gamma-thrombin without inhibiting platelet shape change or secretion. At a concentration of 60-80 microM, Arg-Gly-Asp-Ser inhibited the aggregation of ADP-stimulated gel-filtered platelets approximately equal to 50%. Arg-Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding to ADP-stimulated platelets. This inhibition was competitive with a Ki of approximately equal to 25 microM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gly-Asp-Ser is a partial competitive inhibitor of fibrinogen binding. These data suggest that a region near the carboxyl-terminus of the alpha-chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes.     

Characterization of carbohydrates in the alpha 2-macroglobulin receptor.

Carbohydrates were characterized in the human placental alpha 2-macroglobulin receptor and its associated protein. Carbohydrates, largely N-linked, contributed to about 18% of the size of the receptor alpha-chain and to about 25% of the beta-chain. The 40 kDa receptor-associated protein also contained carbohydrate. The alpha- and beta-chains contained a wide variety of carbohydrates as judged by binding of lectins. Monosaccharide-competing inhibition of alpha 2M-methylamine binding by WGA suggested a functional significance of sugars in binding of ligands to the alpha-chain.     

Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Cell surface T3 expression requires the presence of both alpha- and beta-chains of the T cell receptor

We have identified a surface T3- Jurkat variant which has a defective alpha-chain but which possesses an intact beta-chain. The transfection of a functional mouse alpha-chain into this human T cell induces the expression of surface T3 molecules associated with mouse alpha-human beta-heterodimers detected by anticlonotypic antibodies. Treatment of the transfectant with anti-T3, anti-mouse Ti-alpha, or anti-human Ti-beta antibodies clears all Ti-T3 complexes from the surface. These results demonstrate that functional alpha- and beta-chains are both required for expression of T3 on the cell membrane, and that the Ti heterodimers present and associated with T3 on Jurkat cells involve only alpha- and beta-chains.     

Phosphorylation of human fibrinogen in vitro with protein kinase C: characterization of the phosphorylated sites

Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin.     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-N epsilon-(4-azido-5- [125I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.     

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)