File S1 - A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

Figures S1–S5 and Table S1. Figure S1. Mass spectra of 6 kDa LC N-terminus peptide fragment of FVIII variants after thrombin digestion and LC-MS (TOF) analysis. (a) rFVIIIFc (b) BDD rFVIII (c) SC rFVIIIFc (d) SC rFVIIIFc R1645A/R1648A. Major digestion products are indicated. Figure S2. Deconvoluted spectra of A2 fragment of FVIII variants after thrombin digestion and LC-MS (TOF) analysis. (a) rFVIIIFc (b) BDD rFVIII (c) SC rFVIIIFc (d) SC rFVIIIFc R1645A/R1648A. Major digestion products are indicated. Figure S3. Representative SPR sensorgrams of interactions between FVIII variants and VWF. One representative graph is displayed for (A) rFVIIIFc and (B) SC rFVIIIFc, respectively. Black indicates the binding curve and red indicates the best fit to a 1∶1 interaction model. Figure S4. Thrombin-mediated release of activated FVIII variants from VWF at 25°C. (A) Single reference subtracted sensorgrams. (B) Double reference subtracted sensorgrams for the phase corresponding to thrombin application. (C) Thrombin-mediated release rate as a function of time. (D) Peak thrombin-mediated release rate as a function of thrombin concentration. Figure S5. Thrombin-mediated release of activated FVIII variants from VWF at 37°C. (A) Single reference subtracted sensorgrams. (B) Double reference subtracted sensorgrams for the phase corresponding to thrombin application. (C) Thrombin-mediated release rate as a function of time. (D) Peak thrombin-mediated release rate as a function of thrombin concentration. Table S1. Summary of results from thrombin map by LC-MS (TOF) analysis. For SC rFVIIIFc R1645A/R1648A, mass analysis confirmed the presence of the R1645A/R1648A mutations. (DOCX)补充图S1至S5及补充表S1。 补充图S1：经凝血酶酶切及液相色谱-质谱（飞行时间，TOF）分析后，第八凝血因子（Factor VIII, FVIII）变体6 kDa轻链（Light Chain, LC）N端肽段的质谱图。(a) 重组FVIII-Fc融合蛋白（recombinant Factor VIII-Fc fusion protein, rFVIIIFc）；(b) B结构域缺失重组FVIII（B-domain deleted recombinant Factor VIII, BDD rFVIII）；(c) 单链重组FVIII-Fc融合蛋白（single-chain recombinant Factor VIII-Fc fusion protein, SC rFVIIIFc）；(d) SC rFVIIIFc R1645A/R1648A。已标注主要酶切产物。 补充图S2：经凝血酶酶切及LC-MS (TOF)分析后，FVIII变体A2片段的解卷积质谱图。(a) rFVIIIFc；(b) BDD rFVIII；(c) SC rFVIIIFc；(d) SC rFVIIIFc R1645A/R1648A。已标注主要酶切产物。 补充图S3：FVIII变体与血管性血友病因子（von Willebrand Factor, VWF）相互作用的代表性表面等离子体共振（Surface Plasmon Resonance, SPR）传感图。分别展示(a) rFVIIIFc与(b) SC rFVIIIFc的代表性结果。黑色曲线为结合曲线，红色曲线为1:1相互作用模型的最优拟合曲线。 补充图S4：25℃条件下凝血酶介导的活化FVIII变体从VWF上的释放过程。(A) 单参比扣除传感图；(B) 对应凝血酶施加阶段的双参比扣除传感图；(C) 凝血酶介导的释放速率随时间的变化趋势；(D) 峰值凝血酶介导的释放速率随凝血酶浓度的变化趋势。 补充图S5：37℃条件下凝血酶介导的活化FVIII变体从VWF上的释放过程。(A) 单参比扣除传感图；(B) 对应凝血酶施加阶段的双参比扣除传感图；(C) 凝血酶介导的释放速率随时间的变化趋势；(D) 峰值凝血酶介导的释放速率随凝血酶浓度的变化趋势。 补充表S1：LC-MS (TOF)分析的凝血酶酶切图谱结果汇总。针对SC rFVIIIFc R1645A/R1648A，质谱分析证实了R1645A/R1648A突变的存在。(DOCX)