title: The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays.
abstract: Patients on P2Y12inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12inhibitors. The effect of in vitro and in vivo P2Y12inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12inhibitors was compared to VerifyNow results. P2Y12inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.|b Department of Vascular Surgery , University Medical Center Utrecht , The Netherlands.
authors.forename: Tesse C
authors.initials: TC
authors.lastname: Leunissen
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.|b Department of Vascular Surgery , University Medical Center Utrecht , The Netherlands.
authors.forename: Peter Paul
authors.initials: PP
authors.lastname: Wisman
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Thijs C
authors.initials: TC
authors.lastname: van Holten
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Philip G
authors.initials: PG
authors.lastname: de Groot
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Suzanne J
authors.identifier: http://orcid.org/0000-0002-3868-4271
authors.initials: SJ
authors.lastname: Korporaal
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Arnold C
authors.initials: AC
authors.lastname: Koekman
authors.affiliation: b Department of Vascular Surgery , University Medical Center Utrecht , The Netherlands.
authors.forename: Frans L
authors.initials: FL
authors.lastname: Moll
authors.affiliation: b Department of Vascular Surgery , University Medical Center Utrecht , The Netherlands.|c Department of Nephrology and Hypertension , University Medical Center Utrecht , The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Teraa
authors.affiliation: c Department of Nephrology and Hypertension , University Medical Center Utrecht , The Netherlands.
authors.forename: Marianne C
authors.initials: MC
authors.lastname: Verhaar
authors.affiliation: b Department of Vascular Surgery , University Medical Center Utrecht , The Netherlands.
authors.forename: Gert Jan
authors.initials: GJ
authors.lastname: de Borst
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Rolf T
authors.initials: RT
authors.lastname: Urbanus
authors.affiliation: a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Roest
mesh_terms: D001792:Blood Platelets / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D010974:Platelet Aggregation / Q000187:drug effects*; D010979:Platelet Function Tests; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism*
chemical_list: C548576:P2RY12 protein, human; D058921:Purinergic P2Y Receptor Antagonists; D058925:Receptors, Purinergic P2Y12
keywords: P-selectin; Platelet aggregation; platelet Membrane Glycoprotein IIb; platelet aggregation inhibitor; platelet function test