From the Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler (V.K., U.R.P., L.V.M.R.).
Department of Molecular Medicine, Scripps Research Institute, La Jolla, CA (X.X., J.H.G.).
Arterioscler Thromb Vasc Biol. 2020 May;40(5):1275-1288. doi: 10.1161/ATVBAHA.120.314244. Epub 2020 Mar 26.
Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1.
Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.
最近的研究表明，VIIa 因子（因子 VIIa）与内皮细胞蛋白 C 受体（endothelial cell protein C receptor，EPCR）结合后，通过激活 PAR（蛋白酶激活受体）-1 介导的信号通路，引起内皮屏障稳定和抗炎作用。目前尚不清楚 VIIa 是否通过在 canonical 位点或类似于 APC（活化蛋白 C）的非 canonical 位点切割 PAR1 来诱导 PAR1 依赖性细胞保护信号。
使用携带 PAR1 中 R41Q（canonical 位点）或 R46Q（非 canonical 位点）点突变的纯合子小鼠（QQ41-PAR1 和 QQ46-PAR1 小鼠）来研究 VIIa 诱导的 PAR1 依赖性药理学有益作用的体内机制。在野生型（WT）和 QQ46-PAR1 小鼠中，VIIa 可降低脂多糖诱导的炎症、屏障通透性和 VEGF（血管内皮生长因子）诱导的屏障破坏，但在 QQ41-PAR1 小鼠中则不然。用从 WT、QQ41-PAR1 和 QQ46-PAR1 小鼠分离的脑内皮细胞进行的体外信号研究表明，VIIa 在血管内皮细胞中激活 Akt（蛋白激酶 B）需要 PAR1 中的 R41 切割位点。我们的研究表明，VIIa 可在内皮细胞中切割内源性 PAR1，而不像 APC 切割的 PAR1 那样仍保留在细胞表面，VIIa 切割的 PAR1 很容易被内化。进一步的研究表明，内皮细胞预先用 VIIa 处理可减少随后凝血酶诱导的信号。该过程依赖于β-arrestin1。
我们的结果表明，在体内，VIIa 在小鼠中的药理学益处源于 PAR1 依赖性偏向信号，这是在 canonical R41 位点切割 PAR1 后的信号。VIIa 诱导细胞保护信号的机制与 APC 明显不同。我们的数据为 PAR1 的偏向激动和信号多样性提供了另一层复杂性。
