AUTHOR=Palarasah Yaseelan , Pham Stephanie Thuy Duong , Gram Jørgen Brodersen , Graversen Jonas Heilskov , Pilely Katrine , Sidelmann Johannes Jakobsen 

TITLE=Plasma Kallikrein Cleaved H-kininogen: An End-Point Marker for Contact Activation in vitro and ex vivo

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2022.873975

DOI=10.3389/fcvm.2022.873975

ISSN=2297-055X

ABSTRACT=<sec><title>Objectives</title><p>The contact system consists of coagulation factor XII (FXII), prekallikrein, and H-kininogen (HK) and plays important roles in many diseases. Plasma kallikrein (PKa) cleaved HK (cHK) is a marker of contact activation. Presently, we developed a specific and precise enzyme-linked immunosorbent assay (ELISA) for determination of cHK <italic>in vitro</italic> and <italic>ex vivo.</italic></p></sec><sec><title>Methods</title><p>Cleaved HK specific mouse monoclonal antibodies (mAbs) were generated using a peptide corresponding to the PKa cleavage site on HK as immunogen. ELISA, surface plasmon resonance analysis, and immunoprecipitation established the specificity of the antibody, which subsequently was used in a sandwich ELISA. The analytical imprecision and the concentration of cHK in a reference population and in women receiving oral contraceptives (OC) were determined. cHK was assessed <italic>in vitro</italic> in plasma exposed to polytetrafluoroethylene, silicone, and glass tubes.</p></sec><sec><title>Results</title><p>The selected mAb showed excellent specificity towards cHK. The intra-assay and inter-assay CV of the ELISA were 3.6 and 6.0%, respectively. The reference population (60 women, 60 men) displayed a median cHK plasma concentration of 1.38 μg/mL and a reference interval of 0.82 – 2.56 μg/mL. Women receiving OC had significantly higher concentrations, <italic>p</italic> &lt; 0.001. cHK was significantly elevated in plasma exposed to polytetrafluoroethylene, <italic>p</italic> = 0.001, and glass, <italic>p</italic> &lt; 0.0001.</p></sec><sec><title>Conclusion</title><p>The ELISA showed excellent precision and specificity. cHK assessment <italic>ex vivo</italic> demonstrated ongoing contact activation in healthy individuals, augmented by OC. The cHK antibody and the ELISA could be promising tools in contact activation related diseases and <italic>in vitro</italic> investigations of the plasma compatibility of blood contacting biomaterials.</p></sec>