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Most fatalities from trauma occur due to severe blood loss. There is a need for improved hemostatic biomaterials that can address this problem. The aim of this study was to determine the in vivo efficacy of nanofibrous microspheres (NFM) loaded with hemostatic peptides, specifically ideal amphipathic peptides (IAP) that have been demonstrated to possess both procoagulant and antifibrinolyic activities. We demonstrate that IAP can be coupled to NFM (IAP-NFM) to form matrices that exhibit substantial hemostatic activity. IAP-NFM matrices were compared to a commercial zeolitic hemostatic biomaterial (QuikClot) and have superior efficacy in reducing bleeding in vivo. In both a murine tail transection and a murine hepatic injury model, bleeding times were significantly reduced (P<0.05) with the use of IAP-NFM as compared with equal masses of either QuikClot or NFM alone, or no treatment. Importantly, histological examination revealed no tissue injury when IAP-NFM or NFM were applied to hepatic lacerations. In contrast, QuikClot caused widespread hepatocyte degeneration and necrosis, with higher average injury zone thickness as determined by semiquantitative analysis. In summary, NFM was able to maintain the pro-coagulant properties of IAP in our preclinical model, caused no observable tissue damage at the site of application and had better performance than QuikClot controls., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: The leading causes of death for trauma patients in civilian and combat settings are traumatic brain injury and uncontrolled hemorrhage, respectively. This study examines the hemostatic activity of an ideal amphipathic peptide (IAP) attached to a biocompatible surface., Methods: Procoagulant properties of IAP attached to a surface were first tested, in vitro, using factor Xa and thrombin generation assays and thromboelastography. Rabbits and swine were used for in vivo studies. Injuries were performed using scalpel blade 11, and free bleeding was allowed for five seconds. While bleeding, IAP coupled to a hydrogel or QuikClot were applied to the wound and the time was recorded until bleeding stopped., Results: Results show that when IAP is attached to a surface, both factor IXa and factor Xa activities are promoted. Thromboelastography shows that surface-attached IAP results in earlier onset and stronger clot formation. In rabbits, the incorporation of IAP onto a biocompatible hydrogel reduces bleeding times by 40% (p < 0.03). In pigs, bleeding times are reduced 30% to 50% (p < 0.02) by surface-coupled IAP. Finally, using a rabbit liver laceration model, the properties of surface-coupled IAP are less damaging when compared with QuikClot, a currently used material in external hemorrhagic injuries., Conclusions: This study provides a relevant proof of concept for the development of IAP coupled to a biocompatible surface as a hemostatic agent, that is potentially safer than the commercially available QuikClot.

An ideal amphipathic peptide (IAP), composed of simply lysine and leucine residues in a 1:2 ratio (K(7)L(15)), specifically prolongs in vitro coagulation assays that use phospholipids, such as the activated partial thromboplastin time (APTT). The main hypothesis of the present work is that IAP's anticoagulant effect occurs by competing with phospholipid membranes in in vitro coagulation reactions. We verified this hypothesis by employing different phospholipid-dependent coagulation assays, such as the APTT, the dilute prothrombin time (dPT) and the dilute Russell viper venom time (dRVVT) with both low and high amounts of phospholipids. We show that coagulation times are prolonged by IAP in a concentration-dependent manner, and that this prolongation is abrogated by adding excess phospholipid, demonstrating a phospholipid dependence for this inhibition. Using an ELISA-based binding assay, we show IAP inhibits the binding of one of the vitamin K-dependent coagulation factors, factor X, to phospholipid membranes. This is further confirmed with fluorescence spectroscopy, where the interaction of IAP and factor X is inhibited by phospholipid. In summary, this work demonstrates that IAP can act as an anticoagulant by impairing the interaction of coagulation factors with phospholipid membranes and provides a paradigm for the development of novel anticoagulants., (Copyright 2009 Elsevier Inc. All rights reserved.)

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 Lys(7)Leu(15) 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 (gamma-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.