Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

BackgroundThymosin beta 4 (Tβ4) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.MethodsIn 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 Tβ4, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.ResultsThe 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 Tβ4 (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 (> 1 μM) the Tβ4-potentiating effect was diminished to near control levels. Tβ4 did interact with fibrinogen with an estimated KD of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.ConclusionThese results suggest that Tβ4 could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ4 could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.General significanceThis work suggests that Tβ4 might have a dual role in platelet function.