A-123 Nascent ADAM17 synthesis potentiates GPIba cleavage in resting and stimulated stored platelets

Abstract

Background Platelet concentrates stored at room temperature have a shelf life of five days. During storage, platelets undergo glycoprotein cleavage by metalloproteases, notably ADAM17 cleavage of GPIba, which leads to decreased post-transfusion reactivity and recovery. Aim: To investigate putative ADAM17 synthesis and its contributions to GPIba proteolysis in stored platelets. Methods Human platelets maintained in autologous plasma were treated on day 1 of storage with naked endonuclease-resistant ADAM17 siRNA and monitored for molecular and cellular effects. Platelet-specific Adam17-deleted mice were generated and dynamics of GpIba cleavage were assessed. Results Platelets translated nascent ADAM17 during storage as evidenced by increased expression blocked by puromycin, and by metabolic labeling with azidohomoalanine, coinciding with progressive GPIba ectodomain cleavage. SiRNA treatment suppressed ADAM17 translation, and rescued total but not surface levels of full-length GPIba in resting platelets during storage. Flow cytometry and confocal microscopy in permeabilized platelets confirmed the existence of an internal pool of GPIba as suggested by prior EM studies. Stimulation of washed platelets with thrombin or calcium ionophore led to 27.3 ± 4.7% and 47.5 ± 0.96% decrease in surface GPIba, respectively. Platelet pre-treatment with cell-permeable ADAM17 inhibitor KP-457 protected GPIba from ectodomain cleavage following platelet stimulation. However, cell-impermeable 5G6 antibody against the scissile domain of GPIba failed to protect GPIba upon platelet stimulation. ADAM17 siRNA did not alter thrombin-induced decrease in surface GPIba across 5 days in storage. However, surface GpIba was increased in resting and thrombin-stimulated Adam17-deleted murine platelets. Adam17-deficient platelets showed similar lifespan to wild type platelets as assessed by in vivo pulse chase labeling. Conclusions: An internal pool of GPIba, possibly in the open canalicular system (OCS), is exposed upon platelet stimulation but subject to rapid cleavage by ADAM17. Chemical inhibition or genetic deletion of ADAM17 allows the internal reservoir of GPIba to reach the surface in stimulated platelets. However, the pool of existing, siRNA-resistant ADAM17 is sufficient to cleave GPIba upon stimulation in stored platelets.