Abstract Number: PB2229
Meeting: ISTH 2020 Congress
Background: Protein C deficiency (PC-def), protein S deficiency (PS-def), anti-phospholipid syndrome (APS), and high level of factor VIII (high-FVIII) cause absolute or relative dysfunction of PC pathway. Although a few screening assays for these thrombophilia are available, such as thrombin-generation-based assay (Thrombopath®) and APTT-based assay (ProC® Global), clot waveform analysis (CWA) which is a simple and quantitative global assay for monitoring bleeding disorders such as hemophilia has little been used for this purpose.
Aims: To establish a screening assay using a modified-PT-based CWA for detecting dysfunctional PC pathway.
Methods: CWA was performed on ACL-TOP® (IL-Japan). Sample plasma was incubated with recombinant tissue factor (rTF), synthesized phospholipid (PL) in the presence or absence of recombinant thrombomodulin (rTM). Clot formation was initiated with CaCl2 and absorbance change was monitored. The peak of the first derivative curves (maximum coagulation velocity) was measured.
Results: Optimal concentrations of reagents (rTF 1 pM, PL 12 µM, rTM 20 nM, CaCl2 10 mM) were determined in pooled normal plasma (PNP) so that the peak ratio with or without rTM reached ~0.3, which mean that added rTM reduced the peak down to 30% [Fig1]. The peak ratio in 35 healthy plasmas was 0.36±0.13 (mean±SD), hence the cut-off value was set as 0.49. Plasma of PC-def, PS-def, high-FVIII (300 IU/dl), and APS showed higher peak ratio than cut-off value (0.79, 0.97, 0.52, 0.93, respectively) [Fig2A]. PC-def, PS-def, high-FVIII showed higher peak values reflecting hypercoagulation (182, 207, 141 mAbs/sec, respectively) than healthy plasmas (109±22). PC-def or PS-def plasma mixed with PNP (25, 50, 75, 100%) showed dose-dependent reduction in peak ratio (PC-def; 0.85, 0.64, 0.44, 0.28 or PS-def; 0.69, 0.53, 0.40, 0.25, respectively). Therefore, peak ratio exceeded the cut-off value at 50% or less concentration of PC/PS [Fig2B].
Conclusions: Thrombomodulin-mediated CWA successfully distinguished PC pathway-associated thrombophilia from normal individuals.
To cite this abstract in AMA style:Hashimoto N, Ogiwara K, Nogami K, Shima M. A New Global Assay for Detection of Protein C Pathway-Associated Thrombophilia Using Thrombomodulin-Mediated Clot Waveform Analysis [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-new-global-assay-for-detection-of-protein-c-pathway-associated-thrombophilia-using-thrombomodulin-mediated-clot-waveform-analysis/. Accessed May 18, 2021.
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