Current treatment with tissue painogen activator (tPA) requires an Intravenous infusion (1.5-3 h) became the clearance of tPA from the circulation is rapid (t1/2 6 min).

SummarySite directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.

A Cl(-)-HCO3- exchanger in the brush-border membrane mediates active Cl- absorption and regulates intracellular pH in rabbit ileum. The molecular identity of the ileal Cl(-)-HCO3- exchanger has not been established. The best-characterized plasma membrane Cl(-)-HCO3- exchanger is erythroid band 3. Structurally related proteins in nonerythroid tissues comprise an anion exchanger (AE) family. We used the polymerase chain reaction to amplify and clone a cDNA encoding an ileal band 3-related protein (B3RP) from rabbit ileal enterocytes. The composite sequence is 3,909 bp and is predicted to encode a protein of 136 kDa. The deduced amino acid sequence is 95% identical to murine renal AE2, indicating that ileal B3RP is rabbit AE2. Antisera generated against a cytoplasmic fragment of ileal B3RP recognized a 160- to 170-kDa polypeptide in the brush-border membrane, but not the basolateral membrane, of ileal crypt and villus enterocytes. This correlates with previous studies indicating that a Cl(-)-HCO3- exchange is present in brush-border but not basolateral membrane vesicles from rabbit ileal enterocytes. We conclude that ileal B3RP is a product of the AE gene family, and is present in the brush-border of ileal enterocytes, where it may mediate Cl(-)-HCO3- exchange.

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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.