PO090 | INCOMPLETE THROMBIN INHIBITION: BIOCHEMICAL AND FUNCTIONAL EVIDENCE OF ADP P2Y1 RECEPTOR DESENSITIZATION IN HUMAN PLATELETS
Abstract
Background and Aims. Adenosine diphosphate (ADP) is a key agonist of platelet activation and aggregation through the stimulation of purinergic receptors P2Y₁ and P2Y₁₂. Tight regulation of ADP-mediated signaling is essential during platelet plug formation. It has recently been demonstrated that the P2Y₁ receptor, upon ADP stimulation, can be downregulated through a protein kinase C (PKC)-dependent phosphorylation mechanism, leading to receptor desensitization. First we demonstrated that a prior platelet thrombin activation reduces PLC activation and Calcium mobilization induced by ADP, suggesting a capability of thrombin to densitize P2Y1 receptor. When pre-analytical procedures are not properly followed, ADP responses is lacking. The aim of this study was to investigate whether residual thrombin activity, under such conditions, induces desensitization of platelet P2Y₁ signaling pathways in a PKC-dependent manner, thereby to verify if limits ADP induced platelet response. Methods. Human platelets obtained from sodium citrate-anticoagulated blood were prepared under different agitation conditions (3 times manual inversions vs mild shaking; in presence or absence of hirudin, a thrombin inhibitor). Platelets were stimulated with ADP (0.8, 2, and 4 μM) to assess both platelet aggregation and in washed, aspirin-treated platelets, intracellular calcium mobilization was measured using FURA-2 as a probe. It was also evaluated PKC activity by analyzing the phosphorylation of PKC substrates at baseline (T=0) and after 2 hours (T=2h) by Western blot analysis. Results. Platelets analyzed 2 hours after blood collection, subjected to mild shaking and not treated with hirudin, showed reduced ADP-induced aggregation, accompanied by a marked attenuation of intracellular calcium mobilization, indicating decreased responsiveness of the P2Y₁ receptor. Normal response were observed both in the shaking hirudin treated sample and in samples correctly agitate by inversion. In contrast, PKC shows a slightly activation immediately after the blood sampling that become higher after two hours, in shaking treated platelets. Hirudin treatment showed no PKC activation either immediately or after 2 hours, as well as agitate sample and no treated with hirudin. Conclusions. Our findings demonstrate that insufficient inhibition of thrombin during platelet preparation induces desensitization of the P2Y₁ receptor, leading to reduced platelet responsiveness to ADP. This artefact can be the cause of variability in platelet aggregation results across different laboratories. A thrombin-dependent, PKC-mediated mechanism may represent an additional regulatory pathway limiting platelet activation during sustained stimulation. To the best of our knowledge, this is the first evidence that a thrombin contributing to the prevention of excessive platelet aggregate, similar to the limitation of platelet plug formation endothelial dependent.
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BibTeX
@article{schiera2026po090,
title = {{PO090 | INCOMPLETE THROMBIN INHIBITION: BIOCHEMICAL AND FUNCTIONAL EVIDENCE OF ADP P2Y1 RECEPTOR DESENSITIZATION IN HUMAN PLATELETS}},
author = {Eleonora Schiera},
journal = {Bleeding, Thrombosis and Vascular Biology},
year = {2026},
month = oct,
url = {https://www.btvb.org/btvb/article/view/906},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - PO090 | INCOMPLETE THROMBIN INHIBITION: BIOCHEMICAL AND FUNCTIONAL EVIDENCE OF ADP P2Y1 RECEPTOR DESENSITIZATION IN HUMAN PLATELETS
AU - Eleonora Schiera
PY - 2026
DA - 2026/10/26
JO - Bleeding, Thrombosis and Vascular Biology
UR - https://www.btvb.org/btvb/article/view/906
AB - Background and Aims. Adenosine diphosphate (ADP) is a key agonist of platelet activation and aggregation through the stimulation of purinergic receptors P2Y₁ and P2Y₁₂. Tight regulation of ADP-mediated signaling is essential during platelet plug formation. It has recently been demonstrated that the P2Y₁ receptor, upon ADP stimulation, can be downregulated through a protein kinase C (PKC)-dependent phosphorylation mechanism, leading to receptor desensitization. First we demonstrated that a prior platelet thrombin activation reduces PLC activation and Calcium mobilization induced by ADP, suggesting a capability of thrombin to densitize P2Y1 receptor. When pre-analytical procedures are not properly followed, ADP responses is lacking. The aim of this study was to investigate whether residual thrombin activity, under such conditions, induces desensitization of platelet P2Y₁ signaling pathways in a PKC-dependent manner, thereby to verify if limits ADP induced platelet response. Methods. Human platelets obtained from sodium citrate-anticoagulated blood were prepared under different agitation conditions (3 times manual inversions vs mild shaking; in presence or absence of hirudin, a thrombin inhibitor). Platelets were stimulated with ADP (0.8, 2, and 4 μM) to assess both platelet aggregation and in washed, aspirin-treated platelets, intracellular calcium mobilization was measured using FURA-2 as a probe. It was also evaluated PKC activity by analyzing the phosphorylation of PKC substrates at baseline (T=0) and after 2 hours (T=2h) by Western blot analysis. Results. Platelets analyzed 2 hours after blood collection, subjected to mild shaking and not treated with hirudin, showed reduced ADP-induced aggregation, accompanied by a marked attenuation of intracellular calcium mobilization, indicating decreased responsiveness of the P2Y₁ receptor. Normal response were observed both in the shaking hirudin treated sample and in samples correctly agitate by inversion. In contrast, PKC shows a slightly activation immediately after the blood sampling that become higher after two hours, in shaking treated platelets. Hirudin treatment showed no PKC activation either immediately or after 2 hours, as well as agitate sample and no treated with hirudin. Conclusions. Our findings demonstrate that insufficient inhibition of thrombin during platelet preparation induces desensitization of the P2Y₁ receptor, leading to reduced platelet responsiveness to ADP. This artefact can be the cause of variability in platelet aggregation results across different laboratories. A thrombin-dependent, PKC-mediated mechanism may represent an additional regulatory pathway limiting platelet activation during sustained stimulation. To the best of our knowledge, this is the first evidence that a thrombin contributing to the prevention of excessive platelet aggregate, similar to the limitation of platelet plug formation endothelial dependent.
SN - 2785-5309
ER -