The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins Megan S. Lord a, * , Bill Cheng a , Simon J. McCarthy b , MoonSun Jung a , John M. Whitelock a a Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, New South Wales 2052, Australia b HemCon Medical Technologies Inc, Portland, USA article info Article history: Received 9 May 2011 Accepted 20 May 2011 Available online 14 June 2011 Keywords: Chitosan Platelets Integrins Protein adsorption abstract Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by a IIb b 3 integrins, while a 2 b 1 integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of a IIb b 3 integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Chitosan is derived from the deacetylation of chitin, which is a naturally occurring poly-glucosamine. Chitin is found in the exoskeleton of crustaceans, cuticle of insects and cell wall of fungi. Chitosan is a generic term used to describe linear polysaccharides which are composed of glucosamine and N-acetyl glucosamine residues linked by b(1e4) glycosidic bonds and have been deac- teylated by more than 50% [1]. Chitosan has many properties that make it well suited for wound healing applications including biocompatibility and antimicrobial properties. Various forms of chitosan have been shown to promote wound closure and healing in various animal models [1,2] as well as blood coagulation in vitro [3,4]. The extents of platelet adhesion and activation, induced by contact with biomaterials, are considered early indicators of the thrombogenic potential and hemocompatability of materials. Platelet adhesion and activation are also crucial initial events, after traumatic bleeding, to promote thrombus formation that prevents further blood loss. These events have been analysed in vitro by various groups and reported to be in part due to the ability of chitosan to promote erythrocyte aggregation [5] as well as platelet adhesion and activation [6,7]. Chitosan has been found to promote coagulation in vitro with whole blood clotting time reduced by 40% compared to whole blood alone [8]. These events were found to be independent of the classical coagulation pathway, which involves the formation of fibrin, suggesting that coagulation in the presence of chitosan was mediated by physical interactions with cells through the polycationic nature of chitosan [8,9]. Platelet adhesion and activation occur in vivo upon exposure of circulating platelets to the endothelial basement membrane. These events are primarily mediated by integrins, leading to platelet aggregation and thrombus formation. The endothelial basement membrane is rich in collagen, with platelet adhesion to collagen mediated through the integrin a 2 b 1 [10e12]. Platelets have recently been found to bind to another basement membrane molecule, perlecan when it is devoid of its heparan sulfate (HS) chains [13]. The integrin a 2 b 1 acts in concert with glycoprotein (GP) VI to promote the activation of platelets through conformational changes in a IIb b 3 integrins on platelets that bind to fibrinogen and von Willebrand factor thus promoting platelet aggregation [14e17]. Platelets have been found to express five integrins, a IIb b 3 , a 2 b 1 , a 5 b 1 , a 6 b 1 and a V b 3 , with a IIb b 3 being the most abundant [18e20], thus allowing interactions with many extracellular matrix and plasma proteins including collagen, fibrinogen, fibronectin, von Willebrand factor, vitronectin and thrombospondin [21]. Few studies have investigated the role of adsorbed plasma proteins in promoting platelet adhesion to chitosan. When a bioma- terial is placed in contact with biological fluids it becomes exposed to * Corresponding author. Tel.: þ61 2 9385 3910; fax: þ61 2 9663 2108. E-mail address: m.lord@unsw.edu.au (M.S. Lord). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.05.062 Biomaterials 32 (2011) 6655e6662