Sedzro Josepha C, Smith Stephanie A, Scott Alexander, Wang Yuqi, Travers Richard J, Hemp Rachel, Morse Chase N, Morrissey James H
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Res Pract Thromb Haemost. 2024 Aug 16;8(6):102550. doi: 10.1016/j.rpth.2024.102550. eCollection 2024 Aug.
Inorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP has been shown to reduce thrombosis.
To identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents.
Hybridomas were prepared from spleen cells from autoimmune NZBWF1/J female mice and screened for anti-polyP antibodies. Antibodies that bound polyP using enzyme-linked immunosorbent assay and pull-down assays were further characterized with plate binding, surface plasmon resonance, and plasma-based clotting assays. Antithrombotic potential was evaluated in a murine ferric chloride-induced carotid artery thrombosis model.
Of 4 antibodies that bound polyP in our pull-down assay, 2 (PP2069 and PP2099) were available for further characterization. While analyzing these anti-polyP antibodies, we found secretory leukocyte peptidase inhibitor (SLPI) to be a common contaminant of these antibodies and that SLPI binds polyP. We removed SLPI quantitatively from our purified immunoglobulin G. Both PP2069 and PP2099 immunoglobulin G displayed high affinity for polyP but also bound to other polyanions such as DNA, heparin, and certain other glycosaminoglycans, indicating limited specificity. Both antibodies inhibited polyP-initiated plasma clotting . When tested in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect.
Autoimmune mice spontaneously produce antibodies against polyP. The 2 examples of anti-polyP monoclonal antibodies studied here not only bound to polyP with high affinity but also cross-reacted with DNA and heparin. Neither antibody protected against thrombosis in a mouse model, but they might have some utility for studies of polyP.
无机多聚磷酸盐（多聚磷酸）是加速血液凝固的磷酸盐线性链。靶向多聚磷酸已被证明可减少血栓形成。
鉴定和表征可作为分析工具和抗血栓药物的抗多聚磷酸单克隆抗体。
从自身免疫性NZBWF1/J雌性小鼠的脾细胞制备杂交瘤，并筛选抗多聚磷酸抗体。使用酶联免疫吸附测定和下拉测定法结合多聚磷酸的抗体，通过平板结合、表面等离子体共振和基于血浆的凝血测定法进一步表征。在小鼠氯化铁诱导的颈动脉血栓形成模型中评估抗血栓潜力。
在我们的下拉测定中结合多聚磷酸的4种抗体中，有2种（PP2069和PP2099）可用于进一步表征。在分析这些抗多聚磷酸抗体时，我们发现分泌型白细胞蛋白酶抑制剂（SLPI）是这些抗体的常见污染物，并且SLPI结合多聚磷酸。我们从纯化的免疫球蛋白G中定量去除了SLPI。PP2069和PP2099免疫球蛋白G对多聚磷酸均显示出高亲和力，但也与其他多阴离子如DNA、肝素和某些其他糖胺聚糖结合，表明特异性有限。两种抗体均抑制多聚磷酸引发的血浆凝固。然而，在小鼠血栓形成模型中进行测试时，PP2069和PP2099均未表现出显著的抗血栓作用。
自身免疫小鼠自发产生抗多聚磷酸抗体。此处研究的2例抗多聚磷酸单克隆抗体不仅与多聚磷酸具有高亲和力结合，而且与DNA和肝素发生交叉反应。在小鼠模型中，这两种抗体均不能预防血栓形成，但它们可能对多聚磷酸的研究有一定用途。
