Characterization of an Ideal Amphipathic Peptide as a Potential Hemostatic Agent.

Abstract

We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.