Platelet sensitivity to GPVI stimulation decreases during platelet recovery after apheresis

Introduction Platelet turnover is elevated in conditions such as sepsis, immune thrombocytopenia, and following acute blood loss. During platelet count recovery, a surge of young, hyperreactive platelets is released, which potentially increase the risk of arterial and venous thrombosis [ 1 ]. While upregulation of GPVI-surface expression of these platelets is well documented [ 2 ], less is known about how the expression of other key platelet receptors changes and how this translates into functional platelet responses. We studied blood from healthy platelet apheresis donors as a model of enhanced platelet turnover to investigate mechanisms underlying platelet hyperreactivity during platelet count recovery. Method Washed platelets were prepared from>8 platelet apheresis donors before (day 0) and after apheresis (day 3 and 10) and analysed by flow cytometry. Young platelets were identified via high residual RNA content (SYTO13 stain) or high HLA-I expression [ 3 ]. Surface expression of the platelet receptors GPVI, P2Y12 and PAR-1 was quantified. Functional assays measured intracellular calcium fluxes (FLUO-4) following stimulation with TRAP-6 or CRP as well as platelet activation (PAC-1) and degranulation (CD62P) after stimulation of GPVI with CRP (1.25 µg/ml – 5 µg/ml), PAR-1 with TRAP-6 and P2Y12 with ADP (both 2.5 µM – 10 µM). Results Receptor surface expression increased on day 3 compared to day 0 among young platelets (GPVI:+26%, p=0.0013; PAR-1:+34%, p=0.066; P2Y12:+44%, p=0.0063; data refer to SYTO13-stained platelets). However, receptor levels among the total platelet population remained equal after platelet apheresis. Stimulation with low-dose TRAP-6 elicited a stronger platelet response on days 3 (79% increase) and 10 (51% increase) compared to day 0, which was accompanied by a 1.5 fold increase in maximum intracellular calcium mobilization on day 10. In contrast, stimulation of GPVI with low-dose CRP resulted in 88% reduced platelet activation on day 3 (p=0.0063) with no full recovery by day 10 (58% reduced activation, p=0.63). This was supported by a delay of intracellular calcium mobilization after CRP stimulation three days post-apheresis (maximum calcium mobilization on day 0 after 300 s and on day 3 after 500 s). Surprisingly, maximum calcium fluxes on day 10 were two times higher compared to baseline. Platelet activation after stimulation with medium to high doses of CRP, TRAP-6 and ADP was not significantly altered after apheresis. Conclusion Platelets undergo functional changes during recovery after apheresis, predominantly driven by young platelets. Platelet apheresis increases the number of receptors in young platelets. Notably, platelet sensitivity to CRP stimulation (calcium fluxes, activation) was decreased during platelet count recovery after apheresis. Our in vitro findings may translate into in vivo effects in platelet apheresis donors or patients with increased turnover. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany