THE POSITION OF THE STRUCTURALLY AUTONOMOUS KRINGLE-2 DOMAIN INFLUENCES THE FUNCTIONAL FEATURES OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains, From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned, The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation, To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA, The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography, All molecules were expressed in their single-chain form and could be converted to their two-chain form, With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied, All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose, Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose. All variants bound to a forming fibrin clot, Reversal of the K domains (FGK1K2P versus FGK2K1P) reduced fibrin binding, In the presence of the lysine analogue epsilon-amino caproic acid, only FGK1K2P bound to fibrin, All variants activated plasminogen, In the absence of fibrinogen CNBr fragments (mimic of fibrin), the reversal of the K domain (FGK2K1P) resulted in a 2-fold improved plasminogen activation, In the presence of a fibrin mimic, the plasminogen activations of the F domain deletion analogues GK1K2P and GK2K1P were found to be decreased 2- to 4-fold, From these results we concluded that the function of t-PA in lysine binding, fibrin binding and fibrin-dependent plasminogen activation is dependent on the correct spatial orientation of the K2 domain within the t-PA molecule.