Interaction of High Molecular Weight Kininogen with Endothelial Cell Receptors suPAR, gC1qR and Cytokeratin 1 by Surface Plasmon Resonance (BiaCore).

The physiologic activation of the plasma kallikrein kinin system (KKS) requires the assembly of these proteins on the cell membrane. High molecular weight kininogen (HK) binds to endothelial cells through an interaction with a multiprotein receptor complex that consist of: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR) and cytokeratin 1 (CK1). The affinity of HK and cleaved HK (Hka) for endothelial cells is K_D=7–52 nM but the affinity for each of the three binding proteins is unknown. We first examined the direct binding of HK and Hka to the soluble receptor form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance (BiaCore). We linked suPAR, gC1qR and CK1 (800–1100 pg/mm²) by amine coupling to a CM-5 chip and perfused HK and Hka at a concentration ranging from 50 to 400 nM in the presence or absence of 10 μM ZnCl₂. A Langmuir binding model with local fit (stoichiometry of 1:1) was used to analyse k_on (association rate constant), k_off (dissociation rate constant) and K_D (equilibrium dissociation constant) for HK and Hka.

The binding of HK and Hka to the three receptors was zinc dependent. The affinity for HK and Hka was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK and Hka and was due to a very slow k_off. The affinity for Hka compared to HK was 2.5-fold tighter for CK1 but 50-fold tighter for suPAR. A reversed immobiization was then performed. We immobilized HK, Hka and LK (800 pg/mm²) and suPAR, gC1qR and CK1 were flowed over the chip at a concentration ranging from 50 to 700 nM. Only gC1qR bound to immobilized HK and Hka, with a K_D of 6.31± 2.8 pM for HK and 2.86±1.5 pM for Hka while the other 2 receptors did not bind. Receptor integrity was shown by preincubating HK or Hka with the pure receptors and observing competition with the immobilized receptors. Among the HK/Hka receptors, complex formation was only observed between gC1qR and immobiized CK1 with or without Hka indicating that a CK1-gC1qR complex can form independently of kininogen. This study indicates that although suPAR has the weakest affinity of the three receptors it is the only one that distinguishes between HK and Hka. Only one binary complex was revealed by this technique between CK1 and gC1qR. These results lay the foundation for anlayzing the affinity of these receptors in a cellular environment.