The Effector Roles of Kringle 1 and Kringle 2 in the Enzymatic Properties of Recombinant Tissue-Type Plasminogen Activator as Revealed by Generation of Recombinant Molecules Containing Each Kringle Linked to the Protease Domain

Recombinant DNA technology has been employed to construct and express in human kidney 293 cells cDNAs encoding deletion-mutant recombinant (r) tissue-type plasminogen activators (tPA) retaining only the kringle 1 ([K1tPA]) and serine protease (P) functional domains (r-[K1tPA]P), and the kringle 2 ([K2tPA]) and P domains (r-[K2tPA]P), along with a variant of r-tPA containing a W253S mutation (r-tPA/W253S). Of these mutants, only r-[K2tPA]P retained its ability to interact with ω-amino acid effector molecules. The Km for single-chain wild-type (wt) r-tPA toward the synthetic substrate H-D-Ile-LPro-L-Arg-p-nitroanilide (S2288) was decreased by approximately 3-fold in the presence of a saturating concentration of human fibrinogen (Fg), along with a small (1.14-fold) increase in the kcat for this same reaction. The kinetic activation (dissociation) constant (KA) for Fg was 2.4 μM. Fg did not influence the steady-state amidolytic properties of two-chain wtr-tPA. Similar effects of Fg on the Km for hydrolysis of 52288 were displayed for single-chain forms of r-[K1tPA]P, r-[K2tpA]P, and r-tPA/W253S, with additional small effects of Fg on the kcat of this reaction. The KA for Fg toward these proteins ranged from 2.4 μM for wtr-tPA to 5.2 μM for r-[K1tPA]P. The amidolytic properties of the two-chain forms of these variants were also unaffected by Fg. The activation rate of [Glu1]-plasminogen ([Glu1]Pg) by wtr-tPA was stimulated approximately 7-fold by Fg and approximately 139-fold by the same concentration (in Fg equivalents) of human fibrin (Fn) (Fn/Fg stimulatory ratio = 19.9). The Fn/Fg ratio was 10.6,20.9, and 18.0 for r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, respectively. Quantitative [Glu1]Pg-enriched clot lysis assays revealed that r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S were approximately 18, 72, and 54%, respectively, as effective as wtr-tPA in catalyzing the plasminogen activation event leading to lysis. The antifibrinolytic agent ϵ-aminocaproic acid, inhibited clot lysis with approximately equal effectiveness in [Glu1]Pg-enriched clots when wtr-tPA, r-[K1tPA]P, r-[K2tPA]P, or r-tPA/W253S were employed as the activators. These studies demonstrated that Fg- and Fa-based stimulatory effects on the enzymatic properties of r-tPA and its variants were generally not proportional to the macroscopic binding abilities of these proteins with ω-amino acids or with Fg and Fn. This suggests that these effector molecules exert their effects with tPA via kinetically relevant sites and mechanisms not entirely reflected by their macroscopic binding properties to tPA.