Brodin Ellen, Appelbom Hege, Osterud Bjarne, Hilden Ida, Petersen Lars C, Hansen John-Bjarne
Center for Atherothrombotic Research (CART), Department of Medicine, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway.
Transl Res. 2009 Mar;153(3):124-31. doi: 10.1016/j.trsl.2008.12.004. Epub 2009 Jan 7.
The purpose of this study was to investigate the impact of recombinant glycosylated TFPI (rg-TFPI) from BHK cells, nonglycosylated TFPI (r-TFPI) from Escherichia coli, and truncated TFPI (1-161) on thrombin generation (TG) in plasma treated with and without heparin in vitro and ex vivo. Fasting plasma samples were collected from 6 healthy persons. TG was assessed by the calibrated automated thrombography (CAT) method. The addition of increasing concentrations (0-200 ng/mL) of different TFPI caused a 5% to 30% prolongation of lag time for TF (3.0 pM) induced TG, with the most pronounced effect for rg-TFPI and the least pronounced effect for truncated TFPI, but without affecting endogenous thrombin potential (ETP) in TF-induced coagulation. Removal of native TFPI from plasma by anti-TFPI IgG treatment shortened lag time by 35 +/- 4% without affecting ETP. Increasing concentrations (0-200 ng/mL) of various TFPI in the presence of low heparin concentrations (0.1 IU/mL) prolonged lag time and decreased ETP by 25% to 75% with the most prominent effect promoted by glycosylated full-length TFPI. The effect of neutralizing antibodies against TFPI and antithrombin (AT) was studied in plasma in the presence of heparin administered in vitro or ex vivo. The results revealed that TFPI and AT acted in synergy as inhibitors of coagulation in terms of the effect on both initiation (lag time) and propagation (ETP). Our data demonstrated that the CAT assay appropriately assessed the impact of TFPI on initiation and propagation of TG in a physiological plasma milieu with and without heparin. TFPI contributed significantly to regulation of coagulation initiation (lag time). The C-terminal region and, to a lesser extent, glycosylation of the TFPI molecule were essential for its anticoagulant function in the absence and presence of heparin.
本研究的目的是调查来自BHK细胞的重组糖基化TFPI（rg-TFPI）、来自大肠杆菌的非糖基化TFPI（r-TFPI）和截短的TFPI（1-161）在体外和体内对经肝素处理和未经肝素处理的血浆中凝血酶生成（TG）的影响。从6名健康人身上采集空腹血浆样本。通过校准自动血栓形成测定法（CAT）评估TG。添加不同浓度（0-200 ng/mL）递增的不同TFPI导致由TF（3.0 pM）诱导的TG的滞后时间延长5%至30%，其中rg-TFPI的作用最明显，截短的TFPI作用最不明显，但不影响TF诱导的凝血中的内源性凝血酶潜力（ETP）。用抗TFPI IgG处理从血浆中去除天然TFPI可使滞后时间缩短35±4%，而不影响ETP。在低肝素浓度（0.1 IU/mL）存在的情况下，添加不同浓度（0-200 ng/mL）递增的各种TFPI可延长滞后时间，并使ETP降低25%至75%，糖基化全长TFPI的促进作用最为显著。在体外或体内给予肝素的情况下，研究了针对TFPI和抗凝血酶（AT）的中和抗体在血浆中的作用。结果显示，就对起始（滞后时间）和传播（ETP）的影响而言，TFPI和AT作为凝血抑制剂发挥协同作用。我们的数据表明，CAT测定法适当地评估了TFPI在有肝素和无肝素的生理血浆环境中对TG起始和传播的影响。TFPI对凝血起始（滞后时间）的调节有显著贡献。TFPI分子的C末端区域以及较小程度上的糖基化对于其在无肝素和有肝素情况下的抗凝功能至关重要。
