STRUCTURE FUNCTION ANALYSIS OF HUMAN FACTOR-XII USING RECOMBINANT DELETION MUTANTS - EVIDENCE FOR AN ADDITIONAL REGION INVOLVED IN THE BINDING TO NEGATIVELY CHARGED SURFACES/

The binding site of human factor XII (FXII) for negatively charged sur faces has been proposed to be localized in the N-terminal region of fa ctor XII. We have generated two recombinant factor XII proteins that l ack this region: one protein consisting of the second growth-factor-li ke domain, the kringle domain, the proline-rich region and the catalyt ic domain of FXII (rFXII-U like), and another consisting of only 16 am ino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-lpc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 c ells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied, Radiolabeled rFXII-U-like and, to a lesser extent, rFXII-lpc bound to glass in a concentration-d ependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of n on-labeled native factor XII. On native polypacrylamide gel electropho resis, bath truncated proteins appeared to bind also to dextran sulfat e, a soluble negatively charged compound. Glass-bound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but le ss efficiently than rFXII, rFXII-U-like and rFXII-lpc exhibited coagul ant activity, but this activity was significantly lower than that of r FXII. These data confirm that the N-terminal part of the heavy-chain r egion of factor XII contains a binding sit for negatively charged acti vating surfaces, and indicate that other sequences, possibly located o n tile second epidermal-growth-factor-like domain and/or the kringle d omain, contribute to the binding of factor XII to these surfaces.