Platelet Activation Profiles on TiO2: Effect of Ca2+ Binding to the Surface

Biointerphases (2012) 7:28 DOI 10.1007/s13758-012-0028-8 ARTICLE Platelet Activation Profiles on TiO2: Effect of Ca2+ Binding to the Surface Swati Gupta • Ilya Reviakine Received: 31 December 2011 / Accepted: 15 March 2012 / Published online: 24 April 2012 Ó The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Surface ion equilibrium is hypothesized to play an important role in defining the interactions between foreign materials and biological systems. In this study, we compare two surfaces with respect to their ability to activate adhering platelets. One is a commonly used implant material TiO2, which binds Ca2?, and the other one is glass, which does not. We show, that in the presence of Ca2?, TiO2 acts as an agonist, activating adhering platelets and causing the expression on their surface of two wellknown activation markers, CD62P (P-selectin) and CD63. On the contrary, in the absence of Ca2?, platelets adhering on TiO2 express only one of the two markers, CD63. Platelets adhering on glass, as well as platelets challenged with soluble agonists in solution, express both markers independently of whether Ca2? is present or not. The expression of CD62P and CD63 is indicative of the exocytosis of the so-called a- and dense granules, respectively. It is a normal response of platelets to activation. Differences in the expression profiles of these two markers point to differential regulation of the exocytosis of the two kinds of granules, confirming the recent notion that platelets can tune their microenvironment in a trigger-specific fashion. Electronic supplementary material The online version of this article (doi:10.1007/s13758-012-0028-8) contains supplementary material, which is available to authorized users. S. Gupta
I. Reviakine (&) Biosurfaces Unit, CIC BiomaGUNE (Centro de Investigación Cooperativa en Biomateriales), Paseo Miramón 182, 20009 Donostia, San Sebastián, Spain e-mail: S. Gupta
I. Reviakine Department of Biochemistry and Molecular Biology, University of the Basque Country, 48940 Leioa, Spain 1 Introduction The interplay between surface physico-chemical properties of materials and the biological response they elicit is an active area of research [1]. The role of interfacial ion equilibrium in this process is an aspect that has not received sufficient attention despite the recognition of its importance [1]. In this context, titania–Ca2? interactions represent an interesting example. Titania (TiO2) is a relatively inert and stable material responsible for the favorable, if poorly understood, biocompatibility properties of titanium (Ti), which is used in the production of various implants—stents, heart valve housings, dental prostheses and other osseoimplants [2–4]. Titania interactions with Ca2? are manifested in terms of changes of several surface properties, such as the isoelectric point (IEP, the pH at which proton dissociation from the surface is balanced by adsorption and the surface charge is neutralized) and f-potential: the IEP of TiO2 is shifted in the presence of calcium to higher pH [5–8], and its f-potential is inverted at a near-physiological Ca2? concentration of *3 mM at physiological pH [8]. These changes can be interpreted in terms of adsorption of Ca2? ions at the oxide surface; this is the so-called ‘‘chemical’’ interpretation. For a detailed discussion of the chemical versus physical interpretation of the charge reversal phenomena, the reader is referred to Lyklema et al. [9], while IEP shifts are discussed in detail in Hunter et al. [10]. In simplest terms, Ca2? adsorption to the surface will change the balance of interactions between the surface and the material (proteins, lipids, cells) adsorbing or adhering to it. Indeed, there is some evidence suggesting that Ca2? adsorption to titania affects TiO2-protein interactions [11]. Furthermore, on TiO2, Ca2? elicits a clear response in terms of lipid behavior in phosphatidyl serine (PS)-containing liposomes and supported lipid bilayers—response 123 Page 2 of 12 that is absent on substrates such as silica or glass [12–15]. Interactions of Ca2? with silica are not manifested by the f-potential reversal [16], and at neutral pH the fraction of adsorbed Ca2? ions is quite small [17]. For more details, see Figure S1 in the supporting information. In view of these observations, we hypothesized that the behavior of more complex biological systems on TiO2 would also be affected by the TiO2–Ca2? interactions. In order to demonstrate that these interactions have an effect on a biological system, we chose to investigate platelets, because of a clearly identifiable response in terms of activation, and their relevance to material biocompatibility. Platelets are anuclear 3–4 lm cell fragments that circulate in the blood, scouring the vascular bed for sites of injury, where they become activated. Activated platelets adhere to each other and to the injured tissue, forming a platelet plug, catalyzing coagulation cascade reactions that culminate in fibrin production and clot formation, and coordinate the subsequent inflammatory response and wound healing processes [18–22]. Platelets achieve these functions by releasing and expressing on their surface a variety of soluble and membrane factors—lipids, proteins, and small molecules—that are stored internally in the quiescent platelets. Examples of membrane factors include the phospholipid phosphatidyl serine (PS), which catalyzes the assembly of the coagulation cascade proteins [18, 23, 24], transmembrane protein markers such as CD62P (P-selectin) and CD63 that are characteristic of the exocytosis of the a- and dense storage granules, respectively [25], and integrin GPIIb/IIIa, the active form of which is responsible for the adhesion events [20, 26–31]. Expression of these factors is a useful measure of platelet activation [20, 32, 33]. Contact with artificial surfaces also activates platelets, leading to thrombosis [34, 35], which can be successfully managed with anticoagulants to some extent. However, complications still arise despite the anticoagulant therapy [34]. This is a well-known problem for the development of blood-compatible biomaterials and considerable research efforts are focused on the discovery and development of materials with minimal procoagulatory properties [34, 35]. On the other hand, blood coagulation at surfaces of osseoimplants is beneficial for implant integration. Because of its importance, interactions between whole blood, or its components, and various biomaterials, has been extensively studied [34–40]. Quite a few of these studies focused specifically on TiO2 [30, 41–51]. There is an overall understanding of the sequence of events, viz., protein adsorption—platelet activation—thrombosis, and it is established that surface protein adsorption and the dynamics of the protein-adsorbed films play a major role in the process of platelet activation by foreign materials [34, 42, 52] (...truncated)