Blood Research Institute, Blood Center of Wisconsin, 8727 Watertown Plank Road, Milwaukee, WI 53201, USA.
Biochem J. 2012 Apr 1;443(1):259-66. doi: 10.1042/BJ20111994.
TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.
组织因子途径抑制剂（TFPI）是一种抗凝蛋白，通过抑制 fXa（因子 Xa）和 TF（组织因子）-fVIIa（因子 VIIa）复合物来防止血管内凝血。TFPI 在小窝中的定位增强了其抗凝活性。为了进一步确定小窝如何促进 TFPI 的抗凝活性，CHO（中国仓鼠卵巢）细胞共转染 TF 和靶向小窝的膜相关 TFPI [TFPI-GPI（TFPI-糖基磷脂酰肌醇锚嵌合体）]或质膜 [TFPI-TM（TFPI-跨膜锚嵌合体）]。稳定的克隆具有相等的表面 TF 和 TFPI 表达。TX-114 细胞裂解证实 TFPI-GPI 定位于去污剂不溶性膜部分，而 TFPI-TM 定位于水相。TFPI-GPI 和 TFPI-TM 在酰胺酶测定中都是 fXa 的有效直接抑制剂。然而，TFPI-GPI 作为 TF-fVIIa 的抑制剂比 TFPI-TM 好得多，这在酰胺酶测定和血浆凝固测定中都得到了证实。通过从产生 TFPIα 的 EA.hy926 细胞中去除膜胆固醇来破坏小窝，不会影响转染 TFPIβ 的 CHO 细胞和人脐静脉内皮细胞（HUVEC）的 fXa 抑制，但显著降低了它们对 TF-fVIIa 的抑制。这些研究证实并量化了定位在小窝中的 TFPI 的增强抗凝活性，表明小窝增强了两种 TFPI 同工型的抑制活性，并将小窝的作用定义为特异性增强 TFPI 的抗 TF 活性。
