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Thromb Haemost 1993; 69(05): 490-495
DOI: 10.1055/s-0038-1651639
Original Article
Platelets
Schattauer GmbH Stuttgart

Effects of Hybrid Peptide Analogs to Receptor Recognition Domains on α- and γ-Chains of Human Fibrinogen on Fibrinogen Binding to Platelets

Hiroshi Mohri
The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama, Japan
,
Takao Ohkubo
The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama, Japan
› Author Affiliations
Further Information

Publication History

Received 07 September 1992

Accepted after revision 21 December 1992

Publication Date:
25 July 2018 (online)

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Summary

We synthesized a series of hybrid peptides that correspond to the γ-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAK-QAGDV(G) n RGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 ± 1.6 μM, 15 ± 2.1 μM, and 87 ± 6.8 μM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 ± 0.14 μM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 ± 0.6 μM. When the peptide Y-HHLGGAK-QAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (K d = 3.1 ± 0.5 × 10-7 M) to stimulated platelets and 12,500 molecules/platelet (K d = 1.4 ± 0.2 × 10-7 M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or γ-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa. These domains may be the source of hybrid peptide, expanding a new class of platelet inhibitors that act at membrane receptors for adhesive proteins.

 

  • References

  • 1 Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 1979; 64: 1393-1400
    CrossrefPubMedGoogle Scholar
  • 2 Parise LV, Phillips DR. Reconstitution of the purified platelet fibrinogen receptor.Fibrinogen binding properties of the glycoprotein IIb-IIIa complex. J Biol Chem 1985; 260: 10698-10707
    PubMedGoogle Scholar
  • 3 Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-5363
    PubMedGoogle Scholar
  • 4 Gardner JM, Hynes RO. Interaction of fibronectin with its receptor on platelets. Cell 1985; 42: 439-448
    CrossrefPubMedGoogle Scholar
  • 5 Plow EF, Ginsberg MH. Specific and saturable binding of plasma fibronectin to thrombin-stimulated human platelets. J Biol Chem 1981; 256: 9477-9482
    PubMedGoogle Scholar
  • 6 Ruggeri ZM, Bader R, De Marco L. Glanzmann thrombasthenia: Deficient binding of von Willebrand factor to thrombin-stimulated platelets. Proc Natl Acad Sci USA 1982; 79: 6038-6041
    CrossrefPubMedGoogle Scholar
  • 7 Berlinger S, Niiya K, Roberts JR, Hougten RA, Ruggeri ZM. Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. J Biol Chem 1988; 263: 7500-7505
    PubMedGoogle Scholar
  • 8 Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF, Ruoslahti E. Platelet membrane glycoprotein IIb/IIIa; Member of a family of Arg-Gly-Asp-specific adhesion receptors. Science 1986; 231: 1559-1562
    CrossrefPubMedGoogle Scholar
  • 9 Thiagarajan P, Kelly KL. Exposure of binding sites for vitronectin on platelets following stimulation. J Biol Chem 1988; 263: 3035-3038
    PubMedGoogle Scholar
  • 10 Mohri H, Hashimoto Y, Ohba M, Kumagai H, Ohkubo T. The novel effect of cyclicization of Arg-Gly-Asp-containing peptide on vitronectin binding to platelets. Am J Hematol 1991; 37: 14-19
    CrossrefPubMedGoogle Scholar
  • 11 Kloczewiak M, Timmons S, Lukas TJ, Hawiger J. Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the γ-chain. Biochemistry 1984; 23: 1767-1774
    CrossrefPubMedGoogle Scholar
  • 12 Hawiger J, Timmons S, Koczewiak M, Strong DD, Doolittle RF. γ-and α-chains of human fibrinogen possess sites reactive with human platelet receptors. Proc Natl Acad Sci USA 1982; 2068-2071
    PubMedGoogle Scholar
  • 13 Plow EF, Srouji AH, Meyer D, Marguerie GA, Ginsberg MH. Evidence that three adhesive proteins interact with a common recognition site on activated platelets. J Biol Chem 1984; 259: 5388-5391
    PubMedGoogle Scholar
  • 14 Haverstick DM, Cowan JF, Yamada KM, Santoro SA. Inhibition of platelet adhesion to fibronectin, fibrinogen and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin. Blood 1985; 66: 946-952
    PubMedGoogle Scholar
  • 15 Gartner TK, Bennett JS. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. J Biol Chem 1985; 260: 11891-11894
    PubMedGoogle Scholar
  • 16 Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci USA 1985; 82: 8057-8061
    CrossrefPubMedGoogle Scholar
  • 17 Doolittle RE. Fibrinogen and fibrin. Annu Rev Biochem 1984; 53: 195-229
    CrossrefPubMedGoogle Scholar
  • 18 Lam SC-T, Plow EF, Smith MA, Andrieux A, Ryckwaert JJ, Marguerie GA, Ginsberg MH. Evidence that Arginyl-Glycyl-Aspartate peptides and fibrinogen γ-chain peptides share a common binding site on platelets. J Biol Chem 1987; 62: 947-950
    PubMedGoogle Scholar
  • 19 Andrieux A, Hudry-Cllergeon G, Rockwaert JJ, Chapel A, Ginsberg MH, Plow EF, Marguerie G. Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIb/IIIa. J Biol Chem 1989; 264: 9258-9265
    PubMedGoogle Scholar
  • 20 Bennett JS, Shattil SJ, Power JW, Gartner TK. Interaction of fibrinogen with its platelet receptor.Differential effects of α- and γ-chain fibrinogen peptides on the glycoprotein IIb-IIIa complex. J Biol Chem 1988; 263: 12948-12953
    PubMedGoogle Scholar
  • 21 Santoro SA, Lawing Jr WJ. Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins. Cell 1987; 48: 867-873
    CrossrefPubMedGoogle Scholar
  • 22 D’Souza SE, Ginsberg MH, Lam SC-T, Plow EF. Chemical crosslinking of Arginyl-Glycyl-Aspartic acid peptides to an adhesion receptor on platelets. J Biol Chem 1988; 263: 3943-3948
    PubMedGoogle Scholar
  • 23 D’Souza SE, Ginsberg MH, Burke TA, Lam SC-T, Plow EF. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 1988; 242: 91-93
    CrossrefPubMedGoogle Scholar
  • 24 Smith JW, Cheresh DA. The Arg-Gly-Asp binding domain of the vitronectin receptor. J Biol Chem 1988; 263: 18726-18731
    PubMedGoogle Scholar
  • 25 Charo If, Nannizzi L, Phillips DR, Hsu MA, Scarborough RM. Inhibition of fibrinogen binding to GPIIb-IIIa by a GPIIIa peptide. J Biol Chem 1991; 266: 1415-1421
    PubMedGoogle Scholar
  • 26 D’Souza SE, Ginsberg MH, Burke TA, Plow EF. The ligand binding site on the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its c subunit. J Biol Chem 1990; 265: 3440-3446
    PubMedGoogle Scholar
  • 27 Timmons S, Bednarek MA, Kloczewiak M, Hawiger J. Antiplatelet “hybrid” peptides analogous to receptor recognition domains on γ-and c-chains of human fibrinogen. Biochemistry 1989; 28: 2919-2923
    CrossrefPubMedGoogle Scholar
  • 28 Beer JH, Springer KT, Coller BS. Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor. Blood 1992; 79: 117-128
    PubMedGoogle Scholar
  • 29 Fraker DJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloramide 1, 3, 4, 6 tetrachloro 3a, 6a diphenylglycoluril. Biochem Biophys Res Commun 1978; 80: 849-856
    CrossrefPubMedGoogle Scholar
  • 30 Kazal LA, Amsel S, Miller OP, Tocantins LM. The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc Soc Exp Biol Med 1963; 113: 989-994
    CrossrefPubMedGoogle Scholar
  • 31 Niiya K, Hodson E, Bader R, Ward K, Koziol JA, Plow EF, Ruggeri ZM. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship of the binding of fibrinogen and platelet aggregation. Blood 1987; 70: 475-483
    PubMedGoogle Scholar
  • 32 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12479-12487
    PubMedGoogle Scholar
  • 33 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 1978; 15: 429-436
    CrossrefPubMedGoogle Scholar
  • 34 Walsh PN, Mills DCB, White JG. Metabolism and function of human platelets washed by albumin density gradient separation. Br J Haematol 1977; 36: 281-288
    CrossrefPubMedGoogle Scholar
  • 35 Lombardo VT, Hodson E, Roberts JR, Kunicki TJ, Zimmerman TS, Ruggeri ZM. Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. J Clin Invest 1985; 76: 1950-1958
    CrossrefPubMedGoogle Scholar
  • 36 Ruggeri ZM, Hougten RA, Russell SR, Zimmerman TS. Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets. Proc Natl Acad Sci USA 1986; 83: 5708-5712
    CrossrefPubMedGoogle Scholar
  • 37 Plow EF, Marguerie GA. Induction of fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP. J Biol Chem 1980; 255: 10971-10977
    PubMedGoogle Scholar