"Plasminogen steal" and clot lysis.

Although initially developed to reduce the risk of bleeding, second-generation (clot-selective) thrombolytic agents have been found to induce more prompt and frequent recanalization than do nonselective, first-generation agents. To determine whether they do so in part by preserving clot-associated plasminogen, human whole blood clots formed in Chandler tubes were studied. Addition of suprapharmacologic concentrations of recombinant tissue-type plasminogen activator (rt-PA) to the media bathing mature clots led to a paradoxic impairment of clot lysis and a concomitant concentration-dependent depletion of clot-associated plasminogen (Western blot analysis). In contrast, supplementation of the plasma with plasminogen (0.27 mg/ml) led to significant conservation of both plasma and clot-associated plasminogen (p less than or equal to 0.05, n = 4), and prevented the diminution of clot lysis (p less than or equal to 0.05; n = 4). Fibrinogen degradation products did not account for the attenuation of lysis with the highest concentrations of rt-PA. In concentrations equivalent to those that were induced by the highest concentrations of rt-PA evaluated, fibrinogen degradation products potentiated rather than inhibited lysis (p less than or equal to 0.05, n = 4), probably by stimulating rt-PA activity directly. When preformed clots were incubated with plasminogen-depleted plasma plus 1,000 ng/ml rt-PA, the plasminogen content in residual clot declined (9.36 +/- 0.46 versus 12.39 +/- 0.69 ng/mg clot found in nondepleted plasma; p less than or equal to 0.05; n = 6). Furthermore, clot lysis was attenuated completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented and sustained plasma concentrations after intramuscular injections of molecular variants and deglycosylated forms of tissue-type plasminogen activators.

We have previously explored induction of coronary thrombolysis with tissue-type plasminogen activator (t-PA) administered intramuscularly. Absorption-enhancing agents that rendered the approach feasible were identified, but large amounts of activator were required and initial elevations of concentrations in plasma could not be sustained. The present study was designed to determine whether more therapeutically favorable plasma concentrations could be induced by genetically engineering or chemically modifying t-PA to prolong its half-life based on the hypothesis that the ratio of absorption to clearance would be increased. Each of four genetically engineered variants (one variant with growth factor and kringle 1 domains deleted and kringle 2 duplicated, a second variant with a cysteine for Arg substitution in the growth factor domain, a third variant with an additional urokinase kringle inserted, and a fourth variant with the growth factor domain deleted) and enzymatically deglycosylated t-PA exhibited prolonged half-life after bolus intravenous injection in rabbits. Each elicited substantially higher and more sustained elevations in plasma after intramuscular injection in rabbits or dogs with absorption-enhancing agents as compared with wild-type t-PA that were not accompanied by a systemic lytic state. Thus, use of molecular variants of t-PA with prolonged half-lives in the circulation permits induction of augmented and sustained elevations of plasma concentrations after intramuscular injection with absorption-enhancing agents as compared with wild-type t-PA, rendering potentially therapeutic blood levels more attainable with relatively modest amounts of material.

Biochemical determinants of clearance of tissue-type plasminogen activator from the circulation.

Biochemical modification of tissue-type plasminogen activator (t-PA) designed to alter pharmacokinetics and pharmacodynamics offers promise for development of pharmaceuticals particularly suitable for treatment of specific disorders and for induction of coronary thrombolysis by intramuscular as well as intravenous administration. Accordingly, to identify biochemical determinants of clearance of t-PA from the circulation, we injected rabbits intravenously with three different preparations of t-PA synthesized from the same human gene and expressed in Chinese hamster ovary cells cultured under disparate conditions. Influences of glycosylation on clearance were defined by experiments with enzymatically treated t-PA in which clearance was assessed with concomitant administration of selected neoglycoproteins that compete with t-PA for specific glycoprotein receptors. The role of an intact active catalytic site, as reflected by differences in clearance with and without prior treatment of t-PA with the protease inhibitor PPACK, was defined also. Results indicate that clearance is altered by inhibition of the active site and that the nature and extent of glycosylation--not evident simply by analysis of peptide structure--influence clearance as well. These findings suggest that mannose/N-acetylglucosamine-specific glycoprotein receptors expressed on hepatic reticuloendothelial cells participate in clearance of t-PA from the circulation but that galactose-specific glycoprotein receptors probably do not. The observations may explain differences in clearance seen with different preparations of t-PA that have been seen in clinical pilot studies and may identify biochemical determinants of clearance amenable to modification for development of agents with potentially desirable, specific biological properties.