Supplementary Material for: Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

皮肤损伤可使受损及濒死细胞的细胞表面暴露磷脂酰丝氨酸（phosphatidylserine, PS），以激活凝血与修复进程。膜联蛋白（Annexins）可结合PS，并可能调控愈合反应。本研究利用膜联蛋白A1（AnxA1）与膜联蛋白A5（AnxA5）敲除小鼠，以及具有不同PS结合特性的重组膜联蛋白，探究膜联蛋白在皮肤伤口愈合中的相关性。
研究显示，AnxA1或AnxA5敲除小鼠的伤口炎症反应、伤口闭合及肉芽组织形成均无显著改变；在野生型小鼠体内升高血清AnxA5浓度至100 nM时，上述指标亦未出现变化。当血清AnxA5浓度升至1 µM时，则会引发大量出血，但AnxA1并不会延迟伤口止血过程。
两类膜联蛋白均可与PS结合，但仅AnxA5可在细胞表面形成二维（2D）阵列。将可结合PS但丧失二维阵列形成能力的AnxA5突变体注射后，并未引发出血现象。具备高PS亲和力的二维晶格形成型膜联蛋白A4（AnxA4）同样可引发出血，而对PS亲和力较低的AnxA8，以及兼具中等PS亲和力且丧失二维阵列形成能力的AnxA8突变体，则不会对止血过程造成影响。
体外实验中，升高AnxA4与AnxA5的浓度可延缓凝血通路的激活，该抑制效应在AnxA5突变体、AnxA1及AnxA8中均有所减弱。
综上，内源性AnxA1与AnxA5并非皮肤伤口止血与修复所必需，但药理浓度过高的AnxA4与AnxA5会抑制皮肤伤口的止血过程。