MAPPING OF THE DISCONTINUOUS KININOGEN BINDING-SITE OF PREKALLIKREIN - A DISTAL BINDING SEGMENT IS LOCATED IN THE HEAVY-CHAIN DOMAIN A4

Prekallikrein, the precursor to the serine proteinase kallikrein, circ ulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting o f four ''apple'' domains, A1-A4, which attaches to H-kininogen with hi gh specificity and affinity (K-D = 83 nM). At least two distinct porti ons of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy c hain encompassing A1, and distal segment(s) located in COOH-terminal f ragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the dist al binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-pre kallikrein complex formation. Monoclonal antibody 13G11 binds to recom binant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epi tope of 13G11 to the center portion of domain A4, positions 284-331. D irect binding studies of H-kininogen to various domain A4 constructs r evealed that the distal H-kininogen binding portion is located on a se gment of 48 residues, which overlaps the 13G11 epitope. Hence the tigh t interaction of H-kininogen add prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respecti vely, of the prekallikrein heavy chain. The isolated distal binding se gment significantly prolongs the partial thromboplastin time of recons tituted Williams plasma thus stressing the critical role of the prekal likrein-H-kininogen complex formation in the initiation of the endogen ous blood coagulation cascade.