Patients with immune thrombocytopenia (ITP) remain a challenge to diagnose, manage, and predict bleeding risk. A comprehensive assessment of platelet function may aid clinical management. This study assessed platelet parameters to predict bleeding in ITP. Blood from 103 clinically-annotated cases with isolated thrombocytopenia or 123 healthy donors was evaluated. In the ITP cohort, 75/110 encounters (68%) had platelet counts below 50 x 10⁹/L. Platelet surface proteins, reticulated platelets, and activation were quantified using flow cytometry. Soluble receptor fragments, citrullinated histone-DNA (CitH3-DNA) complexes, and thrombopoietin (TPO) were quantified by ELISA. Whole blood clotting and platelet contribution to clot formation were evaluated using viscoelastography. Elevated levels of glycoprotein (GP) VI (p=0.0012), Trem-like transcript-1 (TLT-1) (p=0.0248), platelet-bound immunoglobulin (Ig) G (p<0.0029), CitH3-DNA complexes (p=0.0022), TPO (p<0.0001), and reduced platelet contribution to clot formation (p<0.0001), were observed in primary ITP patients with bleeding and bruising symptoms. A multivariable analysis revealed that measuring platelet indices strengthened a predictive bleeding model over platelet count alone (78.1% vs. 70.48%). Symptomatic ITP patients have measurable platelet dysfunction and quantifiable differences on platelet surface, increased evidence of NETosis and elevated TPO levels. Identifying biomarkers for ITP outcomes can define subsets of disease with clinical relevance.

Key biomarkers and assays evaluated in this study. The top left panel depicts flow cytometry-based approaches performed in whole blood samples for studying platelet surface proteins, including integrins, glycoproteins, and activation markers, as well as the assessment of pathways regulating integrin αIIbβ3 activation. The right panel highlights ELISA-based detection of soluble fragments (sTLT-1, sGPVI) released from activated platelets, along with serum TPO and NET formation in plasma and serum samples. The bottom panel illustrates ROTEM analysis in normal clot formation, ITP patients with thrombocytopenia and its ability to detect platelet dysfunction.

PYIC received honoraria, speaking fees and travel assistance from Sobi, Novartis and Amgen; advisory boards for Janssen, Sanofi and Sobi; and research grants from Janssen and Novartis. EEG received speaking fees from Sobi. The other authors have no conflicts of interest to disclose.

This work was supported by the National Health and Medical Research Council of Australia, the National Blood Authority of Australia, and ACT Health. SAA and SMH were recipients of scholarships from the Australian Government Research Training Program, and SAA received an ISTH Reach the World fellowship.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study received Human Research Ethics Committee approval from ANU (2022/372 and 2017/924) and ACT Health (ETHLR.18.058). All patients and healthy donors provided informed consent prior to participation, and the study was conducted as per the Declaration of Helsinki under the National Platelet Research and Referral Centre protocol at The Canberra Hospital.

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All data produced in the present study are available upon reasonable request to the authors.