Abstract：
OBJECTIVE: 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 (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios.
CONCLUSIONS: rTM-mediated TF-triggered CWA might be useful for screening 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 (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios.
CONCLUSIONS: rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.
摘要：
目的：绝对或相对蛋白（P）C通路异常（PC缺乏，PS缺陷,抗磷脂综合征（APS），因子(F)V异常，和高FVIII水平）引起血栓形成。尽管这些血栓性患者的筛查方法是可用的，一个利用凝块波形分析（CWA）仍然未知。我们旨在建立一种基于CWA的筛查方法，以区分PC通路异常相关的血栓形成。
方法：样品与组织因子（TF）/磷脂和重组血栓调节蛋白（rTM；最佳20nM）反应，其次是CWA测量。计算凝块波形的一阶导数曲线的峰比(有/没有rTM)。
结果：健康血浆中的峰比（n=35）为0.36±0.13；因此，截止值设置为0.49。PC缺乏的等离子体中的峰比，PS缺陷,高FVIII（峰值300IU/dl），和APS均高于临界值（分别为0.79/0.97/0.50/0.93）。PC缺乏血浆或PS缺乏血浆与正常血浆混合（25％/50％/75％/100％PC或PS水平）显示出峰比的剂量依赖性降低（PC缺乏：0.85/0.64/0.44/0.28；PS缺乏：0.69/0.53/0.40/0.25），表明PC或PS水平≤50%时的峰值比率超过了临界值。FV≤25％的FV缺乏症的峰值比率高于临界值。掺入40IU/dlrFV-R506Q(FVLeiden)或rFV-W1920R(FVNara)的缺乏FV的血浆显示>90%的峰比。
结论：rTM介导的TF触发的CWA可能用于筛查PC通路异常相关的血栓形成。
方法：样品与组织因子（TF）/磷脂和重组血栓调节蛋白（rTM；最佳20nM）反应，其次是CWA测量。计算凝块波形的一阶导数曲线的峰比(有/没有rTM)。
结果：健康血浆中的峰比（n=35）为0.36±0.13；因此，截止值设置为0.49。PC缺乏的等离子体中的峰比，PS缺陷,高FVIII（峰值300IU/dl），和APS均高于临界值（分别为0.79/0.97/0.50/0.93）。PC缺乏血浆或PS缺乏血浆与正常血浆混合（25％/50％/75％/100％PC或PS水平）显示出峰比的剂量依赖性降低（PC缺乏：0.85/0.64/0.44/0.28；PS缺乏：0.69/0.53/0.40/0.25），表明PC或PS水平≤50%时的峰值比率超过了临界值。FV≤25％的FV缺乏症的峰值比率高于临界值。掺入40IU/dlrFV-R506Q(FVLeiden)或rFV-W1920R(FVNara)的缺乏FV的血浆显示>90%的峰比。
结论：rTM介导的TF触发的CWA可能用于筛查PC通路异常相关的血栓形成。
