Summary
Inactivation by slow acting inhibitors in plasma is of little consequence for thrombolysis with wild type t-PA, since it is rapidly cleared from the blood stream and constantly replenished through infusion. However, it becomes increasingly important as the clearance rate of t-PA is reduced, through mutagenesis, to enable the molecule to be long acting and administered by a single bolus injection. The substitution of serine for alanine at position 473 substantially reduced the slow inactivation that occurs at pharmacological levels of t-PA in plasma. Approximately 70% of the activity of A473S remained after 4 h incubation in human plasma compared to approximately 25% for wild type t-PA. Wild type t-PA and A473S showed the same stability in alpha-2-antiplasmin depleted plasma, indicating that the resistance of A473S to inactivation is a result of reduced reactivity towards alpha-2-antiplasmin, the primary slow acting inhibitor of t-PA. The second order rate constant for the inactivation of A473S by purified alpha-2-antiplasmin was approximately 4 fold less than that of wild type t-PA, which is consistent with the results obtained in plasma. Substitution of threonine at position 473 also produced inhibitor resistance, but glycine did not. The substitution of charged or bulky residues at position 473 destroyed enzymatic activity. The mechanism of inhibitor resistance for A473S and A473T appears to be a reduced reactivity towards substrates with arginine at the PI position. The A473S mutation adds well to T103N, a mutation that causes an approximate 9 fold reduction in the clearance rate of t-PA. The double mutation variant, T103N, A473S had normal plasma clot lysis activity, and was stable in plasma over a 4 h incubation period at 37° C in vitro.
