Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
J Thromb Haemost. 2017 Mar;15(3):507-512. doi: 10.1111/jth.13607. Epub 2017 Feb 14.
Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo.
Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics.
要点 在小鼠中，因子（F）VIIa-内皮蛋白 C 受体（EPCR）结合的缺失尚未得到解决。在小鼠 FVIIa（mFVIIa）中，将亮氨酸 4 替换为苯丙氨酸可使其与 EPCR 相互作用。与 mFVIIa 相比，具有苯丙氨酸 4 的 mFVIIa 显示出 EPCR 结合依赖性增强的体内止血功能。定义小鼠中的 FVIIa-EPCR 相互作用可以进一步研究其体内生物学。
背景 人激活的因子 VII（hFVIIa）用于血友病治疗，与内皮蛋白 C（PC）受体（EPCR）结合，但不清楚其止血后果。有趣的是，小鼠缺乏激活的 FVII（FVIIa）-EPCR 相互作用。因此，为了研究这种相互作用在血友病中的止血后果，我们之前设计了一种小鼠 FVIIa（mFVIIa）分子，该分子通过来自小鼠 PC（mPC）的三个取代基结合小鼠 EPCR（mEPCR），即亮氨酸 4→苯丙氨酸，亮氨酸 8→蛋氨酸和色氨酸 9→精氨酸。所得分子 mFVIIa-FMR 模拟了 hFVIIa 的 EPCR 结合特性，并显示出与 mFVIIa 相比，在血友病小鼠中具有增强的止血能力。这些数据暗示了 EPCR 在血友病治疗中 hFVIIa 作用中的作用。然而，mFVIIa-FMR 中的取代仅广泛定义了其 mEPCR 相互作用和体内增强功能的序列决定因素。目的 确定 mPC 苯丙氨酸 4、蛋氨酸 8 和精氨酸 9 对 mFVIIa-FMR 的体外/体内特性的单独贡献。方法 研究了 mFVIIa 或 mPC 位置 4、8 或 9 处的单个氨基酸变异体与 mEPCR 的相互作用特性。结果与结论 mFVIIa 或 mPC 中的苯丙氨酸 4 是与 mEPCR 相互作用所必需的。在血友病小鼠中，与 EPCR 结合依赖性相比，mFVIIa 中存在苯丙氨酸 4 的给药导致止血能力增加 1.9-2.5 倍。这与之前用三重突变 mFVIIa-FMR 所做的观察结果一致。由于亮氨酸 8 对 hFVIIa-EPCR 结合至关重要，因此我们描述了在小鼠中该相互作用的序列差异，现在可以进一步在体内对其进行表征。我们还说明了调节 EPCR-FVIIa 相互作用可能会导致更好的 FVIIa 治疗。
