Glycocalicin is contained inside the thrombus

Glycoprotein Ibα (GPIbα) is a platelet-specific receptor that is subjected to proteolytic shedding by ADAM17, releasing its ectodomain glycocalicin. GPIbα proteolysis is induced by strong platelet activation in a process too slow to be crucial in primary hemostasis. The spatiotemporal fate of glycocalicin was assessed during thrombosis using human and murine samples, integrating in vitro approaches and an inferior vena cava (IVC) ligation model. We found that the glycocalicin concentration inside human thrombi was 4-fold higher than in initial plasma (4.0 ± 0.8 µg/mL vs 0.9 ± 0.0 µg/mL, respectively). This was confirmed in vitro starting from human platelet-rich plasma or murine whole blood, with 6-fold and 3-fold increases, respectively. In vivo, containment of glycocalicin inside thrombi following IVC ligation was even more pronounced with glycocalicin levels reaching 135.1 ± 44.3 µg/mL in thrombi compared to 8.2 ± 1.0 µg/mL in plasma. In the presence of ADAM17 inhibitors, the glycocalicin concentration inside human thrombi was 3-fold lower than in thrombi with competent ADAM17. In contrast, inhibition of ADAM17 in mice, either pharmacologically or genetically, had no limiting effect on intrathrombic GPIbα shedding. In conclusion, GPIbα shedding occurs inside thrombi and results in maximal retention of glycocalicin. Intrathrombic GPIbα shedding is conserved between species, but is regulated differently.KEY POINTS Glycocalicin is contained within the thrombus microenvironmentRegulation of GPIbα ectodomain shedding is species-dependent and not universally mediated by ADAM17