A REGION OF TISSUE PLASMINOGEN-ACTIVATOR THAT AFFECTS PLASMINOGEN ACTIVATION DIFFERENTIALLY WITH VARIOUS FIBRIN(OGEN)-RELATED STIMULATORS

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (K(M)) of the reaction, whereas the catalytic rate constant (k(cat)) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the K(M) values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The k(cat) however, was lower with "KHRR" t-PA than with wild-type t-PA. This led to a decrease in the rate of plasminogen activation with "KHRR" t-PA in the presence of these stimulators. In the presence of the more potent stimulators (fibrin or degraded fibrin), the k(cat) of the reaction with "KHRR" t-PA was actually more efficient than that observed with Activase alteplase. The K(M) was also higher, indicating that the variant and plasminogen may interact less well than wild-type t-PA and plasminogen in the presence of fibrin-related stimulators. However, the kinetic constants predict that at concentrations of plasminogen above 1-mu-M, the variant would display a more rapid activation of plasminogen in the presence of these stimulators. These properties would suggest that "KHRR" t-PA may be a superior thrombolytic agent to Activase alteplase by virtue of its decreased ability to be stimulated by fibrinogen-related molecules found systemically (i.e., fibrinogen and fibrinogen degradation products) but have increased activity in the presence of components found in a clot (i.e., fibrin and slightly degraded fibrin).