{
  "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.",
  "abstract_shown": true,
  "affiliations": [],
  "article_type": null,
  "arxiv": null,
  "author_total": 1,
  "authors": [
    {
      "name": "Eleonora Schiera"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/cy6ahv3ytqhmr.bib",
  "cited_by": 0,
  "doi": null,
  "fulltext_url": "https://www.btvb.org/btvb/article/view/906",
  "id": "cy6ahv3ytqhmr",
  "issns": [
    "2785-5309"
  ],
  "landing_url": "https://www.btvb.org/btvb/article/view/906",
  "language": "en",
  "licence": "CC-BY-NC",
  "licence_label": "CC BY-NC",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "RC666-701"
    ]
  ],
  "openalex": null,
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-26",
  "publisher": "PAGEPress Publications",
  "ris": "https://research.draconitic.com/paper/cy6ahv3ytqhmr.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:47:32Z",
      "native_id": "b81fd52e4c534ea3be155ed0610f507d",
      "source": "doaj",
      "url": "https://www.btvb.org/btvb/article/view/906"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "lcc:RC666-701",
      "subfield": "cardiovascular"
    }
  ],
  "title": "PO090 | INCOMPLETE THROMBIN INHIBITION: BIOCHEMICAL AND FUNCTIONAL EVIDENCE OF ADP P2Y1 RECEPTOR DESENSITIZATION IN HUMAN PLATELETS",
  "title_text": "PO090 | INCOMPLETE THROMBIN INHIBITION: BIOCHEMICAL AND FUNCTIONAL EVIDENCE OF ADP P2Y1 RECEPTOR DESENSITIZATION IN HUMAN PLATELETS",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:47:32Z",
  "url": "https://research.draconitic.com/paper/cy6ahv3ytqhmr",
  "venue": "Bleeding, Thrombosis and Vascular Biology",
  "versions": []
}