Factor seven activating protease (FSAP): does it activate factor VII?
Factor seven activating protease (FSAP): does it activate factor VII?
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DOI：
10.1111/j.1538-7836.2012.04619.x
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发表时间：
2012-05-01
影响因子：
10.4
通讯作者：
Mertens, K.
中科院分区：
文献类型：
--
作者：
Stavenuiter, F.;Dienava-Verdoold, I.;Mertens, K.
.背景资料：第七因子活化蛋白酶（FSAP）最初被报道为单链尿激酶型纤溶酶原激活物（scuPA）和第七因子（FVII）的活化剂。随后，已经确定了许多其他底物，并报告了多种其他生物学效应。由于明显缺乏特异性，FSAP的生理作用变得越来越不清楚。严格的研究受到难以从血液或重组来源获得完整FSAP的限制。目的：我们的目的是生产完整的重组人FSAP，并评估其作为凝血和纤溶触发剂的作用。结果如下：野生型FSAP在各种哺乳动物细胞中的表达总是导致降解的FSAP由于自激活和降解而积累。为了克服这个问题，我们构建了一个变体，其中Arg313在天然活化位点被替换为谷氨酰胺，创建一个裂解位点的细菌蛋白酶嗜热菌蛋白酶。HEK293细胞以其完整形式产生FSAPR313Q。嗜热菌蛋白酶激活的FSAP显示出与血浆来源的FSAP相同的对底物S-2288的反应性，并保留其激活scuPA的能力。聚磷酸盐和肝素使Vmax增加23倍，而不影响scuPA活化的Km（62 nm）。令人惊讶的是，活化的FSAP的FVII活化被证明是可忽略的，即使在钙离子、磷脂囊泡和重组可溶性组织因子的存在下。在100%心磷脂的膜上确实发生FVII裂解，但这导致瞬时活化和快速降解。结论：虽然FSAP确实激活了scuPA，但FVII似乎对激活有显著的抵抗力。因此，重新评价FSAP在止血中的假定作用似乎是适当的。
. Background: Factor seven activating protease (FSAP) was initially reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources. Objectives: Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis. Results: Expression of wild-type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg313 at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAPR313Q in its intact form. Thermolysin-activated FSAP displayed the same reactivity toward the substrate S-2288 as plasma-derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased Vmax by 23-fold, without affecting Km (62 nm) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation. Conclusions: While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.