Substitution of Serine or Threonine at Position 473 of Tissue-type Plasminogen Activator Increases its Stability in Plasma

Abstract

SummaryInactivation 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.

Effective lysis of model thrombi by a t‐PA mutant (A473S) that is resistant to α2‐antiplasmin

“…Lysis of plasma clots and Chandler model thrombi by A473S was found to be unaffected by inclusion of antibodies to a 2 -AP. This, taken together with the fact that this t-PA mutant was only poorly inhibited by a 2 -AP (Alibhai et al, 1996; this study), suggests that t-PA rather than plasmin is an important target for a 2 -AP in fibrin clot lysis. These data are in agreement with the protection of fibrin-bound plasmin from the action of a 2 -AP (Collen, 1976;Moroi & Aoki, 1976;Mu È llertz & Clemmensen, 1976;Wiman & Collen, 1977).…”

“…Both u-PA and plasminogen have a serine in this position; the other residues in this region are remarkably well-conserved. The mutant was also found to be more stable in plasma than wild-type t-PA as a result of poorer complex formation with plasma inhibitors, including a 2 -AP (Alibhai et al, 1996). Here, we extend its characterization and show that it was totally resistant to complex formation with a 2 -AP.…”

“…Previous studies have suggested that the A473S mutant is resistant to plasma inhibitors (Alibhai et al, 1996). We examined complex formation between t-PA A473S with a 2 -AP (both 40 nmol/l) by SDS±PAGE (Fig 3).…”

“…Lysis was achieved with wild-type t-PA and with two mutants, one of which is resistant to PAI-1 by virtue of the KHRR296±299AAAA mutation (Madison et al, 1989(Madison et al, , 1990. The second mutant is A473S, which has increased stability in plasma (Alibhai et al, 1996). Here, we show that the t-PA mutant A473S was more efficient than wild-type t-PA in the lysis of model thrombi by virtue of its resistance to a 2 -AP.…”

“…The variants of t-PA A473S and KHRR296±299AAAA (Genentech) were produced as described previously (Madison et al, 1989(Madison et al, , 1990Alibhai et al, 1996).…”