Schambeck Christian M, Grossmann Ralf, Zonnur Sarah, Berger Mario, Teuchert Kathleen, Spahn Alois, Walter Ulrich
Central Laboratory and Blood Coagulation Unit, Department of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Germany.
Thromb Haemost. 2004 Jul;92(1):42-6. doi: 10.1160/TH04-02-0063.
Theoretically, von Willebrand factor (VWF) should be capable of binding all factor VIII (FVIII), but an unbound FVIII (uFVIII) plasma fraction remains. In patients' status post deep-vein thrombosis (DVT), an altered uFVIII fraction and high FVIII levels might be indicative of dysfunctional FVIII regulation. Out of 928 consecutive DVT patients, 321 were found to have high FVIII levels. After excluding 183 patients with known causes for high FVIII levels, plasma samples with unexplainably high FVIII levels were available from 84 patients. To capture the FVIII-VWF-complex, superparamagnetic polystyrene beads with covalently attached streptavidin were coated with biotinylated anti-rabbit Ig and incubated with rabbit anti-human VWF-Ig. Slowly thawed plasma samples were added to cooled beads, which were then separated by a magnetic particle concentrator. The uFVIII fraction was calculated by dividing the FVIII activity in the supernatant of the FVIII-VWF-complex-free sample by the FVIII activity in the supernatant of the control sample. Additionally, the VWF residuum in the supernatant was determined. Compared to age- and sex-matched blood donors, thrombosis patients showed a significantly higher plasma FVIII/VWF ratio (median: 1.3 vs. 1.0, p<0.001). uFVIII fraction data were adjusted for VWF residuum. After forward stepwise logistic regression, uFVIII had an odds ratio of 0.48 (95% CI 0.34-0.65), i.e. the uFVIII fraction was reduced in thrombosis patients. Analysis of covariance confirmed these results: In thrombosis patients, the estimated mean of the uFVIII fraction was significantly lower (6.34% vs. 7.58%, p<0.001). In conclusion, thrombosis patients with high FVIII levels showed a higher FVIII/VWF ratio, similar to mice with defective FVIII clearance. The clearly reduced uFVIII fraction lends further support to the hypothesis of a modified FVIII clearance.
理论上，血管性血友病因子（VWF）应该能够结合所有的凝血因子VIII（FVIII），但仍存在未结合的FVIII（uFVIII）血浆部分。在深静脉血栓形成（DVT）后的患者状态中，uFVIII部分的改变和FVIII水平升高可能表明FVIII调节功能失调。在928例连续的DVT患者中，发现321例FVIII水平升高。在排除183例已知FVIII水平升高原因的患者后，84例患者有FVIII水平不明原因升高的血浆样本。为了捕获FVIII-VWF复合物，将共价连接有链霉亲和素的超顺磁性聚苯乙烯珠用生物素化抗兔Ig包被，并与兔抗人VWF-Ig孵育。将缓慢解冻的血浆样本加入冷却的珠子中，然后通过磁性颗粒浓缩器分离。通过将无FVIII-VWF复合物样本上清液中的FVIII活性除以对照样本上清液中的FVIII活性来计算uFVIII部分。此外，还测定了上清液中的VWF残留量。与年龄和性别匹配的献血者相比，血栓形成患者的血浆FVIII/VWF比值显著更高（中位数：1.3对1.0，p<0.001）。uFVIII部分数据针对VWF残留量进行了调整。经过向前逐步逻辑回归分析，uFVIII的优势比为0.48（95%置信区间0.34-0.65），即血栓形成患者的uFVIII部分降低。协方差分析证实了这些结果：在血栓形成患者中，uFVIII部分的估计平均值显著更低（6.34%对7.58%，p<0.001）。总之，FVIII水平升高的血栓形成患者表现出更高的FVIII/VWF比值，类似于FVIII清除缺陷的小鼠。明显降低的uFVIII部分进一步支持了FVIII清除改变的假说。
