Occhipinti Rossana, Zhao Pan, Moss Fraser J, Boron Walter F
bioRxiv. 2025 May 19:2025.03.05.639964. doi: 10.1101/2025.03.05.639964.
In this third of three papers, we develop a reaction-diffusion model for O offloading from a red blood cell (RBC), treated as a sphere with diameter approximating RBC thickness. Stopped-flow (SF) analysis (paper #1) of hemoglobin/oxyhemoglobin (Hb/HbO ) absorbance spectra during O efflux from intact murine RBCs show that membrane-impermeant inhibitor p-chloromercuribenzenesulfonate (pCMBS) reduces the HbO -deoxygenation rate constant (k ) by ~61%. SF experiments show that k falls by (1) 9% for aquaporin-1 knockouts (AQP1-KOs), (2) 17% for Rhesus A-glycoprotein knockouts (RhAG-KOs), (3) 30% for double knockouts (dKOs), and (4) ~78% in dKOs/pCMBS. Here, we simulate HbO dissociation (rate constant, k → Hb); HbO , Hb, and O diffusion through RBC cytosol; transmembrane O diffusion; and O diffusion through extracellular unconvected fluid (EUF) to bulk extracellular fluid. Informed by automated-hematology data (paper #1) and imaging-flow-cytometry data (paper #2), simulations predict that observed k decreases cannot reflect changes in RBC size/shape or [Hb/HbO ]. Instead, membrane O permeability ( ) must fall by (1) 22% to account for AQP1-KO data, (2) 36% for RhAG-KOs, (3) 55% for dKOs, and (4) 91% for dKOs/pCMBS. Exploring predicted k sensitivities to eight key parameters (e.g., [Hb/HbO ], diffusion constants, k → Hb, thickness , diameter ) shows that no reasonable changes explain the k data. We introduce a linear-combination approach to accommodate for the presence of poikilocytes. Finally, contrary to common beliefs, the model predicts that, in the absence of inhibitors, the RBC membrane represents >30% of total diffusive "resistance" to O offloading, even for a WT mouse.
在这三篇系列论文的第三篇中，我们构建了一个反应扩散模型，用于研究氧气从红细胞（RBC）卸载的过程，该红细胞被视为直径近似于红细胞厚度的球体。在完整的小鼠红细胞释放氧气期间，对血红蛋白/氧合血红蛋白（Hb/HbO₂）吸收光谱进行的停流（SF）分析（第一篇论文）表明，膜不通透性抑制剂对氯汞苯磺酸盐（pCMBS）使HbO₂的脱氧速率常数（k₂）降低了约61%。SF实验表明，k₂下降的情况如下：（1）水通道蛋白-1基因敲除小鼠（AQP1-KOs）下降9%；（2）恒河猴A糖蛋白基因敲除小鼠（RhAG-KOs）下降17%；（3）双基因敲除小鼠（dKOs）下降30%；（4）dKOs/pCMBS下降约78%。在此，我们模拟了HbO₂的解离（速率常数，k₂→Hb）；HbO₂、Hb和O₂在红细胞胞质溶胶中的扩散；跨膜O₂扩散；以及O₂通过细胞外非对流液（EUF）扩散到细胞外总体液中。根据自动血液学数据（第一篇论文）和成像流式细胞术数据（第二篇论文），模拟结果预测，观察到的k₂下降不能反映红细胞大小/形状或[Hb/HbO₂]的变化。相反，膜对O₂的通透性（Pm）必须下降：（1）22%以解释AQP1-KO的数据；（2）36%以解释RhAG-KO的数据；（3）55%以解释dKO的数据；（4）91%以解释dKO/pCMBS的数据。研究预测的k₂对八个关键参数（例如，[Hb/HbO₂]、扩散常数、k₂→Hb、厚度δ、直径D）的敏感性表明，没有合理的变化能够解释k₂的数据。我们引入了一种线性组合方法来适应异形红细胞的存在。最后，与普遍看法相反，该模型预测，在没有抑制剂的情况下，即使对于野生型小鼠，红细胞膜对氧气卸载的总扩散“阻力”也超过30%。
