Thrombocytopenia under ECMO and Shear-induced shedding of platelet receptor Glycoprotein-(GP)Ialpha

Abstract Background Several mechanisms are suspected to thrombocytopenia under Extracorporeal Membrane Oxygenation (ECMO) such as platelet-consumption or sepsis. Shedding of glycoprotein-(GP)Ibα is a recently identified mechanism of platelet clearance. ECMO generates high shear stress forces that could impact GPIbα-shedding. We hypothesized that ECMO continuous-flow devices could directly induce thrombocytopenia through shear-induced GPIbα-shedding. Aims Determine if ECMO induce GpIb-shedding in vitro and in vivo and determinates the kinetic evolution of platelet-count and GpIb-shedding after patient's implantation. Methods Platelet GPIbα-shedding was first investigated in vitro using a high-shear pump loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma obtained from healthy donors in fresh-frozen-plasma. Samples were collected before and after (5, 30, 60 and 180 min) perfusion at 37°C of plasma-NPC at intermediate and high speed (2.6 and 3.6 L min–1 respectively, n=4 each). Platelet count and GPIbα-shedding were next investigated in 20 ECMO patients before/after implantation (WITECMO trial) and in 20 healthy volunteers. The geometric mean-fluorescence-intensity (gMFI) of platelet GPIbα (PE-staining) and GPIX (FITC-staining) was measured with a Navios flow cytometer (Beckman Coulter, Miami, FL). Results are expressed as GPIbα/GPIX gMFI-ratio. Results A significant time-dependent loss of GPIbα/GPIX gMFI-ratio was already apparent after 30 min in vitro and was significantly more pronounced at high-speed compared to intermediate-speed (pANOVA<0.001 and p<0.01 at 180 min respectively). GPIbα/GPIX gMFI-ratio was significantly increased in ECMO patients compared to healthy subjects 1- and 24-hour after implantation (p<0.001). A significantly lower platelet count was observed 1 hour after ECMO implantation (−23% vs baseline, p<0.01) with a further significant decrease at 24-hours (−53% vs baseline, p<0.0001). Figure 1. A. Significant time-dependent loss of platelet GPIbα/GPIX gMFI-ratio (pANOVA <0.001) assessed by flow-cytometry after 30 min of perfusion at 3.6 L/min with a high-shear continuous-flow device in vitro. B. Representative experiment showing the apparition of a platelet sub-population with loss of GPIbα expression after 30 min of perfusion at 3.6 L/minwith a high-shear continuous-flow device in vitro.