Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion Harmanpreet Kaur a , Rebecca Heeney a , Rupp Carriveau b , Gabriel Sosne c , Bulent Mutus a, ⁎ a Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada b Department of Civil and Environmental Engineering, University of Windsor, Windsor, Ontario, Canada c Department of Opthalmology and Anatomy/Cell Biology, Wayne State University School of Medicine, Detroit, MI, USA abstract article info Article history: Received 7 April 2010 Received in revised form 22 July 2010 Accepted 18 August 2010 Available online 24 August 2010 Keywords: Platelet deposition Thymosin β-4 Flow-chamber studies Collagen Fibrinogen ADP Ca 2+ Kinetics Background: Thymosin beta 4 (Tβ 4 ) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing. Methods: In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ 4 , on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers. Results: The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s -1 , corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ 4 (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (N 1 μM) the Tβ 4 -potentiating effect was diminished to near control levels. Tβ 4 did interact with fibrinogen with an estimated K D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively. Conclusion: These results suggest that Tβ 4 could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ 4 could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition. General significance: This work suggests that Tβ 4 might have a dual role in platelet function. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Tβ 4 is a water-soluble, ubiquitous, highly conserved across species, 43-amino acid acidic polypeptide (pI 5.1) with a molecular weight of 4.9 kDa [1–5]. It is found at concentrations of ~5 nM to ~80 nM in plasma to ~500 μM in platelets [6–10]. The major intracellular function of Tβ 4 in mammalian cells is to form a 1:1 complex with G-actin, preventing its polymerization [2,7,11]. Apart from its interactions with actin, Tβ 4 is capable of modulating a wide array of biological functions including tissue [12] and wound repair [13] as well as anti-inflammatory [14] antiapoptotic and neurotrophic [15] properties. Tβ 4 has been also shown to play a relevant role in angiogenesis [16] and neural development [17]. The ability of Tβ 4 to affect these diverse biological functions has been ascribed to an array of short, bioactive, peptide sequences within the Tβ 4 -primary sequence [18]. The largest concentrations of Tβ 4 are found in leukocytes and platelets [1–9]. In resting platelets, it has been estimated that ~ 60% of the actin is prevented from polymerizing by complexing with Tβ 4 [19]. Upon platelet activation, Tβ 4 is secreted and cross-linked enzymatically by factor XIIIa (a transglutaminase) to fibrin in a time- and Ca 2+ -dependent manner [20,21] thus increasing the local concentration of Tβ 4 near sites of clots and tissue damage, where it is postulated to contribute to wound healing, angiogenesis and inflammatory responses. Platelets carry and release large amounts of Tβ 4 . Yet the role of Tβ 4 on platelet thrombus formation has yet to be fully investigated. The current study represents a small step towards this goal. Here, we have tested the effects of Tβ 4 extraneously added to whole blood, on deposition of ADP activated platelets onto fibrinogen under condi- tions of continuous flow and shear. Biochimica et Biophysica Acta 1800 (2010) 1256–1261 Abbreviations: PDMS, Polydimethylsiloxane; ADP, Adenosine diphosphate; Tβ 4 , Thymosin beta 4; Ca 2+ , Calcium; APTMES, aminopropyltrimethoxysilane; DSS, disuccinimidyl suberate; EITC, eosin-isothiocyanate ⁎ Corresponding author. Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4. Tel.: + 1 519 253 3000x3533. E-mail address: mutusb@uwindsor.ca (B. Mutus). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.08.003 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen