Screening of the protein C pathway abnormality‐related thrombophilia by using thrombomodulin‐mediated tissue factor‐triggered clot waveform analysis

Abstract Objectives Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V‐abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA‐based screening assay to distinguish PC pathway abnormality‐related thrombophilia. Methods Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated. Results The peak ratio in healthy plasmas ( n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high‐FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC‐deficient plasma or PS‐deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose‐dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV‐deficient plasma spiked with 40 IU/dl rFV‐R506Q (FV Leiden ) or rFV‐W1920R (FV Nara ) showed >90% peak ratios. Conclusions rTM‐mediated TF‐triggered CWA might be useful for screening PC pathway abnormality‐related thrombophilia.