Tissue-type plasminogen activator (t-PA) activates the proenzyme plasminogen to the
active protease plasmin which degrades fibrin. The unique properties of t-PA, fibrin
binding and stimulation of activity by fibrin make it an interesting molecule for
specific thrombolysis. t-PA is thought to consist of five structural regions designated
finger (F), growth factor (G), kringle 1 (Kl), kringle 2 (K2) and protease (P). Previous
studies have shown that the interaction of t-PA with fibrin is mediated by the F and
K2 regions.
Mutated t-PA cDNA molecules were expressed in Chinese hamster ovary cells and t-PA
analog proteins were purified from serum free culture media using affinity chromatography
with immobilized monoclonal antibodies. Besides FGK1K2P (native t-PA) the following
analogs were used GK1K2P, klK2P, K2P, P, FP and FGKlk2P (kl and k2 have partial deletions
of the kringle). All the molecules comprising K2P could be stimulated in plasminogen
activation activity by fibrinogen fragments comparable to normal t-PA. The activities
of FP and FGKlk2P were only slightly influenced by these fragments. It was shown that
the fibrin binding site in K2 was plasminogen dependent whereas that in F was not.
K2 was found to contain a binding site for lysine, 6-amino-hexanoic acid but also
6-amino-hexane and thus to differ from the high affinity lysine binding sites in plasminogen.
Chemical modification of lysine and arginine residues in t-PA with citraconic anhydride
and cyclohexanedione respectively, revealed no involvement of these residues in interaction
with lysine or analogs nor in stimulation of activity by fibrinogen fragments. Arginine
modification led to inhibition of plasminogen activation activity, both in the presence
and absence of fibrinogen fragments, but the amidolytic activity as measured with
a tripeptide paranitroanilide was not changed. The involvement of one or more arginine
residues in interaction of t-PA with plasminogen seems likely.
