The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa

Margaret L. Rand; Marian A. Packham; Desirée M. Taylor; Erik L. Yeo; Cynthia H. Gemmell; Sonali Patil; Stephen C.-T. Lam

doi:10.1055/s-0037-1614899

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1999; 82(06): 1680-1686
DOI: 10.1055/s-0037-1614899

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The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa

Margaret L. Rand

¹From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada

³Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Canada

,

Marian A. Packham

¹From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada

,

Desirée M. Taylor

¹From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada

,

Erik L. Yeo

²Medicine, University of Toronto and The Hospital for Sick Children, Toronto, Canada

,

Cynthia H. Gemmell

²Medicine, University of Toronto and The Hospital for Sick Children, Toronto, Canada

,

Sonali Patil

⁴Department of Pharmacology, University of Illinois, Chicago, IL, USA

,

Stephen C.-T. Lam

⁴Department of Pharmacology, University of Illinois, Chicago, IL, USA

› Author Affiliations

Abstract

Summary

The HHLGGAKQAGDV (H₁₂) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H₁₂ or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H₁₂ inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H₁₂ inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H₁₂ with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble H₁₂, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H₁₂ inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H₁₂.

Keywords

Rabbit platelets - fibrinogen - GPIIb-IIIa - gamma chain

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References

References
1 Mustard JF, Packham MA, Kinlough-Rathbone RL, Perry DW, Regoeczi E. Fibrinogen and ADP-induced platelet aggregation. Blood 1978; 52: 453-66.
2 Marguerie GA, Edgington TS, Plow EF. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 1980; 255: 154-61.
3 Plow EF, Ginsberg MH. Cellular adhesion: GPIIb-IIIa as a prototypic adhesion receptor. Prog Hemost Thromb 1989; 9: 117-56.
4 Phillips DR, Charo IF, Scarborough RM. GPIIb-IIIa: The responsive integrin. Cell 1991; 65: 359-62.
5 Hawiger J. Mechanisms involved in platelet vessel wall interaction. Thromb Haemost 1995; 74: 369-72.
6 Andrieux A, Hudry-Clergeon G, Ryckewaert J-J, Chapel A, Ginsberg MH, Plow EF, Marguerie G. Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa. J Biol Chem 1989; 264: 9258-65.
7 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: 11891-4.
8 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: 946-52.
9 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: 8057-61.
10 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: 1767-74.
11 Plow EF, Srouji AH, Meyer D, Marguerie G, Ginsberg MH. Evidence that three adhesive proteins interact with a common recognition site on activated platelets. J Biol Chem 1984; 259: 5388-91.
12 Lam SC-T, Plow EF, Smith MA, Andrieux A, Ryckwaert J-J, Marguerie G, Ginsberg MH. Evidence that arginyl-glycyl-aspartate peptides and fibrinogen γ chain peptides share a common binding site on platelets. J Biol Chem 1987; 262: 947-50.
13 D'Souza SE, Ginsberg MH, Lam SC-T, Plow EF. Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets. J Biol Chem 1988; 263: 3943-51.
14 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: 867-73.
15 Harfenist EJ, Packham MA, Mustard JF. Effects of variant γ chains and sialic acid content of fibrinogen upon its interactions with ADP-stimulated human and rabbit platelets. Blood 1984; 64: 1163-8.
16 Kirschbaum NE, Mosesson MW, Amrani DL. Characterization of the γ chain platelet binding site on fibrinogen fragment D. Blood 1992; 79: 2643-8.
17 Farrell DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogen α and γ chain sites in platelet aggregation. Proc Natl Acad Sci USA 1992; 89: 10729-32.
18 Farrell DH, Thiagarajan P. Binding of recombinant fibrinogen mutants to platelets. J Biol Chem 1994; 269: 226-31.
19 Rooney MM, Parise LV, Lord ST. Dissecting clot retraction and platelet aggregation. Clot retraction does not require an intact fibrinogen γ chain C terminus. J Biol Chem 1996; 271: 8553-5.
20 Hettasch JM, Bolyard MG, Lord ST. The residues AGDV of recombinant γ chains of human fibrinogen must be carboxy-terminal to support human platelet aggregation. Thromb Haemost 1992; 68: 701-6.
21 Liu Q, Matsueda G, Brown E, Frojmovic M. The AGDV residues on the gamma chain carboxyl terminus of platelet-bound fibrinogen are needed for platelet aggregation. Biochim Biophys Acta 1997; 1343: 316-26.
22 Holmbäck K, Danton MJS, Suh TT, Daugherty CC, Degen JL. Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin α_IIbβ₃ . EMBO J 1996; 15: 5760-71.
23 Weisel JW, Nagaswami C, Vilaire G, Bennett JS. Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy. J Biol Chem 1992; 267: 16637-43.
24 Harfenist EJ, Packham MA, Mustard JF. Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats. Blood 1988; 71: 132-6.
25 Moyle M, Bullens S, Deisher T, Gantzos R, Bunting S, Napier M. Evolutionary divergence of the platelet GP IIbIIIa complex. Blood 1989; 74: 92a.
26 Verhallen PFJ, Barth M. Species comparison of anti-aggregatory properties of three fibrinogen receptor-antagonists: RGDS, echistatin and the carboxyterminal duodecapeptide of the fibrinogen gamma-chain. Thromb Haemost 1991; 65: 1144.
27 Hawiger J. Adhesive interactions of platelets and their blockade. Ann NY Acad Sci 1991; 614: 270-8.
28 Kloczewiak M, Timmons S, Bednarek MA, Sakon M, Hawiger J. Platelet receptor recognition domain on the γ chain of human fibrinogen and its synthetic peptide analogues. Biochemistry 1989; 28: 2915-9.
29 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-63.
30 Kinlough-Rathbone RL, Packham MA, Mustard JF. Platelet Aggregation. In: Methods in Hematology: Measurements of Platelet Function. Harker LA, Zimmerman TS. eds. Edinburgh: Churchill-Livingstone; 1983: 64-91.
31 IUPAC-IUB Commisson on Biochemical Nomenclature. A one-letter notation for amino acid sequences. Tentative rules. J Biol Chem 1968; 243: 3557-9.
32 Frelinger III AL, Cohen I, Plow EF, Smith MA, Roberts J, Lam SC-T, Ginsberg MH. Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem 1990; 265: 6346-52.
33 Aster RH, Jandl JH. Platelet sequestration in man, I: Methods. J Clin Invest 1964; 43: 843-69.
34 Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphate-induced platelet aggregation in suspensions of washed rabbit platelets. Br J Haematol 1970; 19: 7-17.
35 Ardlie NG, Perry DW, Packham MA, Mustard JF. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc Soc Exp Biol Med 1971; 136: 1021-3.
36 Rand ML, Gross PL, Jakowec DM, Packham MA, Mustard JF. In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin. Thromb Haemost 1989; 61: 254-8.
37 Hatton MWC, Moar SL, Richardson M. Deendothelialization in vivo initiates a thrombogenic reaction at the rabbit aorta surface. Correlation of uptake of fibrinogen and antithrombin III with thrombin generation by the exposed subendothelium. Am J Pathol 1989; 135: 499-508.
38 Straughn III W, Wagner RH. A simple method for preparing fibrinogen. Thrombos Diathes Haemorrh 1967; 16: 198-206.
39 Lawrie JS, Ross J, Kemp GD. Purification of fibrinogen and the separation of its degradation products in the presence of calcium ions. Biochem Soc Trans 1979; 7: 693-4.
40 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-15.
41 The TH, Feltkamp TEW. Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology 1970; 18: 865-73.
42 Faraday N, Goldschmidt-Clermont P, Dise K, Bray PF. Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry. J Lab Clin Med 1994; 123: 728-40.
43 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4.
44 Packham MA, Rand ML, Kinlough-Rathbone RL. Similarities and differences between rabbit and human platelet characteristics and functions. Comp Biochem Physiol 1992; 103A: 35-54.
45 Harfenist EJ, Packham MA, Mustard JF. Expression of fibrinogen on the surface of ADP-stimulated platelets: Comparison of human and rabbit platelets. Thromb Haemost 1988; 59: 319-22.
46 Wencel-Drake JD. Plasma membrane GPIIb/IIIa. Evidence for a cycling receptor pool. Am J Pathol 1990; 136: 61-70.
47 Nurden P, Humbert M, Piotrowicz RS, Bihour C, Poujol C, Nurden AT, Kunicki TJ. Distribution of ligand-occupied α_IIbβ₃ in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6. Blood 1996; 88: 887-99.
48 D'Souza SE, Ginsberg MH, Matsueda GR, Plow EF. A discrete sequence in a platelet integrin is involved in ligand recognition. Nature 1991; 350: 66-8.
49 Taylor DB, Gartner TK. A peptide corresponding to GPIIb_α 300-312, a presumptive fibrinogen γ-chain binding site on the platelet integrin GPIIb/IIIa, inhibits the adhesion of platelets to at least four adhesive ligands. J Biol Chem 1992; 267: 11729-33.
50 Alemany M, Concord E, Garin J, Vinçon M, Giles A, Marguerie G, Gulino D. Sequence 274-368 in the β3-subunit of the integrin αIIbβ3 provides a ligand recognition and binding domain for the γ-chain of fibrinogen that is independent of platelet activation. Blood 1996; 87: 592-601.
51 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: 91-3.
52 Loftus JC, O’Toole TE, Plow EF, Glass A, Frelinger III AL, Ginsberg MH. A β₃ integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 1990; 249: 915-8.
53 Jennings LK, White MM, Mandrell TD. Interspecies comparison of platelet aggregation, LIBS expression and clot retraction: Observed differences in GPIIb-IIIa functional activity. Thromb Haemost 1995; 74: 1551-6.
54 Lam SC-T, Plow EF, D’Souza SE, Cheresh DA, Frelinger III AL, Ginsberg MH. Isolation and characterization of a platelet membrane protein related to the vitronectin receptor. J Biol Chem 1989; 264: 3742-9.
55 Smith JW, Ruggeri ZM, Kunicki TJ, Cheresh DA. Interaction of integrins α_vβ₃ and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites. J Biol Chem 1990; 265: 12267-71.
56 Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 945-53.