Tissue Plasminogen Activator Binding to the Annexin II Tail Domain
DIRECT MODULATION BY HOMOCYSTEINE*
- Katherine A. Hajjar‡§,
- Laura Mauri‡,
- Andrew T. Jacovina‡¶,
- Fengming Zhong‡,
- Urooj A. Mirza‖,
- Julio Cesar Padovan‖ and
- Brian T. Chait‖
- From the ‡Divisions of Hematology-Oncology, Departments of Pediatrics and Medicine, Cornell University Medical College, New York, New York 10021 and the ‖Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York 10021
Abstract
Tissue plasminogen activator binds to endothelial cells via the calcium-regulated phospholipid-binding protein annexin II, an interaction that is inhibited by the prothrombotic amino acid homocysteine. We sought to identify the tissue plasminogen activator binding domain of annexin II and to determine the mechanism of its modulation by homocysteine. Tissue plasminogen activator binding to immobilized annexin II was inhibited by intact fluid phase annexin II but not by its “core” fragment (residues 25–339). Two overlapping “tail” peptides specifically blocked 65–75% of binding. Localization of the tissue plasminogen activator binding domain was confirmed upon specific inhibition by the hexapeptide LCKLSL (residues 7–12). Expressed C9G annexin II protein failed to support tissue plasminogen activator binding, while binding to C133G, C262G, and C335G was equivalent to that of wild type annexin II. Upon exposure to homocysteine, annexin II underwent a 135 ± 4-Da increase in mass localizing specifically to Cys9 and a 60–66% loss in tissue plasminogen activator-binding capacity (I50 = 11 μm). Upon treatment of cultured endothelial cells with [35S]homocysteine, the dithiothreitol-sensitive label was recovered by immunoprecipitation with anti-annexin II IgG. These data provide a potential mechanism for the prothrombotic effect of homocysteine by demonstrating direct blockade of the tissue plasminogen activator binding domain of annexin II.
Footnotes
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↵* This work was supported by National Institutes of Health (NIH) Grants HL 42493, HL 46403, and HL 58981 (to K. A. H.), Grant RR 00862 (to B. T. C.), and a FAPESP Fellowship (to J. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Cornell University Medical College, 1300 York Ave., Box 45, New York, NY 10021. Tel.: 212-746-2034; Fax: 212-746-8809; E-mail: khajjar{at}cornell.mail.med.edu.
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↵¶ Supported by NIH Training Grant HL 07423.
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↵1 The abbreviations used are: t-PA, tissue plasminogen activator; Ann-II, annexin II; ESI, electrospray ionization, nAnn-II, native annexin II; PLG, plasminogen; rAnn-II recombinant annexin II; HC, homocysteine; HPLC, high pressure liquid chromatography; HUVEC, human umbilical vein endothelial cell(s).
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↵2 K. A. Hajjar, unpublished observations.
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- Received November 24, 1997.
- Revision received January 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
