High shear induced platelet rolling velocity and adhesion onto von Willebrand factor are mediated by GpIb alpha clustering as demonstrated by FRET/FLIM and can be inhibited by alpha linolenic acid

S. Stivala1,2, D. van den Heuvel3, S. De Meyer4, S. Gobbato1, N. Bonetti1, H. Deckmyn4, G. G. Camici1, T. F. Lüscher1, R. Urbanus3, H. Gerritsen3, J. H. Beer1,2 (1Zurich, Switzerland, 2Baden, Switzerland, 3Utrecht, The Netherlands, 4 Kortrijk, Belgium)

Date: 18.02.2017,
Time: 10:00 - 11:15

Objective: We studied the shear-dependent GpIb alpha clustering and the structural and functional interference with this process by the C18 omega-3 fatty acid alpha linolenic acid (ALA).

Methods: Citrated- and EDTA-anticoagulated blood was incubated with vehicle (ethanol 0,1% v/v) or ALA at 7.5/15/30 μM final concentrations. Calcein-stained platelets were perfused on flow channels coated with human vWF at a shear flow of 100 dyne/cm2 (shear force of 2500 s-1). The platelet-covered area, platelet rolling velocity and distance travelled were calculated with the Bioflux software. GpIb clustering was analysed by Förster resonance energy transfer/ fluorescence lifetime imaging (FRET/FLIM). Platelet-rich plasma was pre-incubated with vehicle or ALA 30 uM for 1h, then subjected to 10’000 s-1 shear in a cone and plate viscometer, and GpIb stained with the 6B4 Fab fragment conjugated with the donor and acceptor fluorophores 488 and 594, respectively. Fluorescence lifetime was analyzed on a CLSM C1 microscope equipped with a lifetime module with an objective of 60x/1.49 numerical aperture.

Results: Shear-induced platelet aggregation to vWF was dose-dependently reduced by ALA (platelet-positive area: 64,112 μm2 in the vehicle versus 21,102 μm2 ALA 30 uM, n=6, p=0.018). A reduction in GPIb-mediated platelet adhesion was also observed in EDTA-anticoagulated blood (platelet-positive area: 99,781 μm2 vehicle versus 70,405 μm2 ALA, n=6, p=0.016). Platelet velocity was double in ALA-treated samples compared to vehicle (velocity: 0.75 μm/sec vehicle versus 1.56 μm/sec ALA, n=10, p=0.023). Incubation with ALA did not change GPIb surface expression, as shown by flow cytometry (MFI: 3217 vehicle versus 3477 ALA, p=0.3). GpIb clustering (as measured by fluorescence lifetime) was increased by shear duration and partially inhibited by ALA as indicated by an increase in the donor fluorophore lifetime after shear in preliminary experiments (lifetime: 2.59±0.09 nsec vehicle versus 2.72±0.1 nsec ALA, n=3).

Conclusion: a) platelet adhesion and aggregation to vWF under high-shear flow can be inhibited by ALA, an n-3 fatty acid abundantly found in the Mediterranean diet; b) clustering of GPIb alpha on the platelet surface mediates the adhesion process under flow and ALA interferes with this process; c) this mechanism may contribute to explain the protective effects of the Mediterranean diet on platelet mediated vascular events.