(E-162) Development of function-blocking antibodies to plasma prekallikrein for the study of the interplay between coagulation and inflammation

Introduction:: Prekallikrein (PK) is a zymogen in the contact pathway with roles in both coagulation and inflammation. Specifically, PK is involved in the intrinsic pathway of coagulation through activation of Factor XII (FXIIa). The activated form of PK, plasma kallikrein, initiates inflammation via liberation of the vasoactive peptide bradykinin from high molecular weight kininogen. Bradykinin is a potent vasodilator and induces vascular permeability. How the bi-directional relationship between coagulation and inflammation within the contact pathway contributes to vascular physiology or vascular disease pathogenesis in select settings remains unclear. The aim of this study is to develop and validate anti-human and anti-mouse PK monoclonal antibodies for use in the study of PK in health and disease.


Materials and Methods:: PK-deficient Balb-C mice were immunized with recombinant mouse or human PK. Antibody producing plasma cells were isolated from the spleen and fused with immortalized myeloma cells to generate hybridomas. IgG antibodies were collected from the hybridomas and evaluated for strength of PK binding by ELISA. Antibodies with high affinity were screened for anticoagulant properties by activated partial thromboplastin time assay (aPTT), using PK-depleted plasma supplemented with mouse or human PK. Using a chromogenic substrate assay we evaluated the effect of the antibodies on kallikrein activity and prekallikrein activation by FXIIa.

Results, Conclusions, and Discussions:: 576 anti-human and 192 anti-mouse PK antibodies were generated. 10 anti-human and 14 anti-mouse PK antibodies were selected for further characterization based on binding and clotting time assays. Chromogenic assay looking at antibody effect on kallikrein activity showed 4H11, 5H1, 3B8, and 6B9 increase kallikrein activity. Of particular interest, 4H11 dramatically decreased PK activation by FXIIa. Structure-function studies, including FXII activation by kallikrein and bradykinin generation, are currently underway and will further characterize the antibodies. These function-blocking anti-PK antibodies will be useful tools for the study of PK in physiology as well as under thrombotic and inflammatory conditions.

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