Research and practice in thrombosis and haemostasis · 2024
Antipolyphosphate monoclonal antibodies derived from autoimmune mice.
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Sedzro Josepha C, Smith Stephanie A, Scott Alexander, Wang Yuqi, Travers Richard J, Hemp Rachel, Morse Chase N, Morrissey James H
Аннотация
BACKGROUND: Inorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP in vivo has been shown to reduce thrombosis.
OBJECTIVES: To identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents.
METHODS: 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.
RESULTS: 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 in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect.
CONCLUSION: 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 in vitro studies of polyP.
OBJECTIVES: To identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents.
METHODS: 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.
RESULTS: 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 in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect.
CONCLUSION: 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 in vitro studies of polyP.