Heterogeneous regulation of platelet adhesive-receptor shedding in thrombus formation

C. C. F. M. J. Baaten1, F. Swieringa1, T. Misztal2, T. G. Mastenbroek1, M. A. H. Feijge1, M. M. P. C. Donners1, P. W. Collins3, P. E. J. van der Meijden1, J. W. M. Heemskerk1 (1Maastricht, The Netherlands, 2Bialystok, Poland, 3Cardiff, United Kingdom)

Date: 17.02.2017,
Time: 11:00 - 12:00

Objective: The surface expression of platelet GPIbalpha and GPVI is known to be irreversibly downregulated by members of the family of A Disintegrin And Metalloproteases, ADAM10 and ADAM17. Multiple agents can provoke ADAM-induced receptor shedding, but how these functionally integrate and coordinate is still unknown. We hypothesized that adhesion receptor shedding is induced by different activation routes that signal via elevated Ca2+, protein kinase C (PKC) or apoptosis. We aimed to determine their involvement in a formed thrombus.

Methods: Platelet receptor shedding was studied in platelets, stimulated with different agents, from healthy controls and a Scott patient. Surface expression of GPIbalpha and GPVI (GPIX as control) was assessed by flow cytometry. Receptor shedding in thrombi was investigated using the Maastricht flow chamber. Heterogeneity in GPIbalpha and GPVI expression was monitored in time, and compared to phosphatidylserine (PS) exposure and von Willebrand Factor (VWF) binding.

Results: Calcium-rising agonists rapidly induced shedding of GPIbalphaand GPVI in platelet populations showing PS exposure. Cleavage was abolished by ADAM inhibitors, but not by blockage of caspases or calpain. Scott platelets with a deficiency in PS exposure displayed normal shedding, indicating independency of phospholipid scrambling. The slow shedding of only GPIbalpha with phorbol ester was abrogated by PKC inhibition (Ro318425), but delayed by Ca2+ chelation (BAPTA). Shedding induced by the apoptotic agent ABT-737 was suppressed by caspase inhibition, and delayed with BAPTA. Residual shedding induced by the mitochondrial uncoupling agent CCCP was Ca2+-dependent. In thrombus formation, PS-exposing platelets displayed ADAM-mediated cleavage of both GPIbalpha and GPVI. Secondary elevation of Ca2+ stimulated cleavage, and abolished the binding of VWF (reversed by ADAM10/17 inhibition).

Conclusion: We conclude that Ca2+ elevation is the main physiological pathway of adhesive receptor shedding, displayed by a platelet subpopulation and regulated independently of PS exposure.