Biochem. J. (2008) 412
(545–551) (Printed in Great Britain)
Characterization of an ideal amphipathic peptide as a procoagulant agent
Jorge G. GANOPOLSKY* 1, Sophie CHARBONNEAU* 1, Henry T. PENG†, Pang N. SHEK† and Mark D. BLOSTEIN*2
*Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Room E-156, SMDB-Jewish General Hospital, 3755 Côte Sainte Catherine Road, Montreal, Quebec, Canada H3T 1E2, and †Defence Research and Development Canada– Toronto, 1133 Sheppard Avenue West, P.O. Box 2000, Toronto, Ontario, Canada M3M 3B9
On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000–12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys7Leu15 composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (γ-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.
Key words: coagulation, fibrinolysis, haemostatic peptide, ideal amphipathic peptide (IAP), prothrombinase, tenase.
Abbreviations used: FRET, fluorescence resonance energy transfer; FVIII2303–2323, Factor VIII residues 2303–2323; Gla, γ-carboxyglutamic acid; IAP, ideal amphipathic peptide; PPACM, Phe-Pro-Arg-chloromethane; tPA, tissue plasminogen activator.
1These authors contributed equally to this work and should be considered primary authors.
2To whom correspondence should be addressed (email mark.blostein@mcgill.ca).
Received 22 October 2007/22 February 2008; accepted 12 March 2008
Published as BJ Immediate Publication 12 March 2008, doi:10.1042/BJ20071448
© The Authors Journal compilation © 2008 Biochemical Society
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