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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
1   From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada
3   Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Canada
,
Marian A. Packham
1   From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada
,
Desirée M. Taylor
1   From the Departments of Biochemistry and The Hospital for Sick Children, Toronto, Canada
,
Erik L. Yeo
2   Medicine, University of Toronto and The Hospital for Sick Children, Toronto, Canada
,
Cynthia H. Gemmell
2   Medicine, University of Toronto and The Hospital for Sick Children, Toronto, Canada
,
Sonali Patil
4   Department of Pharmacology, University of Illinois, Chicago, IL, USA
,
Stephen C.-T. Lam
4   Department of Pharmacology, University of Illinois, Chicago, IL, USA
› Author AffiliationsThis work was supported in part by grants-in-aid T2651 (M.L.R.) and NA2924 (M.A.P.) from the Heart and Stroke Foundation of Ontario, and by NIH Grant HL-41793 (S.C.-T.L.) and was done during the tenure of an Established Investigator Award from the American Heart Association and Genentech to S.C.-T.L. We thank T.M. Leisner, J.A. Russell, E.K. Latta,and D.K. Fort for expert technical assistance and contributions to various aspects of the study, and Dr. M.W.C. Hatton for advice in the purification of rabbit fibrinogen.
Further Information

Publication History

Received 23 October 1998

Accepted after resubmission 12 July 1999

Publication Date:
10 December 2017 (online)

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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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