Delayed Plasma Clot Lysis in Adult Patients with Primary Immune Thrombocytopenia (ITP)
Authors: Schramm Theresa , Machacek Jennifer , Fillitz Michael , Dixer Barbara , Quehenberger Peter , Mehic Dino , Ay Cihan , Pabinger Ingrid , Gebhart Johanna 
Published: November 2021

Abstract
Introduction: Primary immune thrombocytopenia (ITP) is an orphan disease characterized by very low platelet counts. Patients have heterogeneous bleeding phenotypes, which are not only determined by platelet counts, also a paradoxically increased thrombotic risk has been observed.
Aim: To investigate, whether the fibrinolysis inhibitors plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin are associated with impaired plasma clot lysis in primary ITP patients in comparison to non-immunologic thrombocytopenic controls (TPC) and healthy controls (HC). Furthermore, associations with bleeding severity and previous thrombotic events were investigated.
Methods: Patients from the Vienna ITP biobank (EC 1843/2016), a multi-centric study including adult patients with primary ITP were investigated and compared to age- and sex-matched control groups: TPC with thrombocytopenia after chemotherapy and HC. Informed consent was obtained from all individuals before study inclusion. A clot formation and lysis assay (CLA) was performed according to the recommendations of the ISTH SSC. Platelet poor plasma samples were measured in duplicates for each patient and control. PAI-1 (PAI-1 Actibind ELISA, Technoclone, Vienna, Austria) and α2-antiplasmin by the chromogenic STA Stachrom antiplasmin assay (Diagnostic Stago, Asnieres, France) were measured. Bleeding severity was measured using the ITP-specific ITP-ISTH BAT (Rodeghiero et al. 2013).
Results: In total, 37 primary ITP patients, 18 TPC and 156 healthy controls were analyzed (Table 1). Primary ITP patients had a higher BMI than HC. Bleeding severity was higher and more ITP patients had a thrombosis history compared to HC, whereas there was no difference in comparison to TPC. PAI-1 activity was highest in ITP patients, with a statistically significant difference in comparison to HC. α2-antiplasmin activity was higher in ITP patients than in TPC, whereas there was no difference in comparison to HC.
After adjustment for sex, age, BMI and fibrinogen, primary ITP patients had a reduced clot formation rate (V max) and significantly delayed plasma clot lysis compared to TPC and HC (Table 2). Also, the lag phase and time to peak absorbance (TTP) were prolonged with a significant difference in comparison to HC.
To investigate outliers of PAI-1 and α2-antiplasmin, we calculated cut-offs at the 75 th percentile of healthy controls (PAI-1: ≥ 3.1 U/mL, α2-antiplasmin: ≥ 107.0 %). 14 (37.8 %) ITP patients had PAI-1 levels and 10 (27.0 %) ITP patients had α2-antiplasmin activity above the cut-off. ITP patients with high PAI-1 levels had mildly delayed clot lysis in comparison to those below with a significantly lower maximal lysis rate (mLR). ITP patients with α2-antiplasmin activity above the cut-off had a significantly shorter lag phase, faster V max and shorter TTP than patients below the cut-off, whereas there was no difference in clot lysis. No differences between ITP patients above or below the respective cut-offs of PAI-1 and α2-antiplasmin regarding their bleeding severity and thrombosis incidence were observed (Table 3).
Conclusion: Primary ITP patients have a tendency towards increased PAI-1 activity, which is associated with considerably delayed plasma clot lysis. Albeit an association with the bleeding score could not be identified, this impaired lysis could be seen as a counter-regulation and at least contribute to the relatively mild bleeding tendency in patients with ITP.
Figure 1 Figure 1.

Disclosures
Pabinger: CSL Behring: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Daiichi Sanchyo: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.