Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg). t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model. The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.
DSPA (Desmodus salivary plasminogen activator) is a new thrombolytic agent corresponding to a natural plasminogen activator discovered in the saliva of the vampire bat Desmodus rotundus. Compared with tissue plasminogen activator (TPA), DSPA, produced in a recombinant cell line, is more fibrin cofactor dependent than TPA.The thrombolytic properties of DSPA and TPA were compared in a canine model of copper coil-induced coronary thrombosis. All dogs received heparin 200 IU/kg IV and SC. Whereas controls did not reperfuse within 180 minutes (none of six), intravenous bolus administration of DSPA at 25, 50, and 100 micrograms/kg resulted in a 100% incidence (6 of 6) of recanalization within 37, 23, and 18 minutes, respectively. TPA at 63 and 125 micrograms/kg reopened the coronaries in 33% (two of six) and 50% (three of six) of cases within 40 minutes. Eighty-three percent (5 of 6) of the arteries were still patent 3 hours after 50 and 100 micrograms/kg DSPA, whereas only 20% (one of five) of all coronaries originally recanalized with both doses of TPA were still open at 3 hours. Plasma levels of alpha 2-antiplasmin decreased significantly only with 125 micrograms/kg TPA. The clearance of DSPA (2.3 to 3.5 mL.min-1.kg-1) was lower compared with TPA (11.4 to 20 mL.min-1.kg-1) due to a prolonged terminal half-life.In a canine coronary thrombosis model, DSPA exhibited higher potency and recanalized coronary arteries faster and with a lower incidence of reocclusion than TPA. Its properties may translate into a higher efficacy in patients compared with available thrombolytic agents. The long half-life of DSPA may allow for single bolus administration in the treatment of acute myocardial infarction.
The saliva of D. rotundus contains at least four plasminogen activators (PAs) which all require fibrin as a cofactor. D. rotundus salivary PAs (DSPAs) exhibit a sequential array of structural motifs such as "Finger" (F), "EGF" (E), "Kringle" (K) and "Protease" (P) which was elucidated by cDNA cloning and sequencing. The respective domain organizations are: FEKP (DSPA alpha 1 and DSPA alpha 2), EKP (DSPA beta) and KP (DSPA gamma). In all four forms the plasmin-sensitive site of tPA is obliterated, indicating that they function as single-chain enzymes. DSPA alpha 1 differs from alpha 2 by amino acid substitutions found mainly in the F, E and K domain, 11% of the total sequence. DSPA beta and gamma, while being closely related to alpha 2, still exhibit 2 and 13 amino acid exchanges, respectively. These sequence heterogeneities, together with results of Southern blot hybridization experiments, strongly suggest that the four DSPA mRNA species originate from different genes. All four forms of DSPA have been expressed in animal cell culture and DSPA alpha 1 was chosen for a detailed pharmacological characterization. In vitro DSPA alpha 1 activity is enhanced 50,000-fold in the presence of fibrin, whereas the activity of single chain tPA is only enhanced 100-fold. At equally effective thrombolytic doses DSPA causes lower bleeding incidence in a rat mesenteric vein model and exhibits high potency, clot selectivity, and speed in the dissolution of fibrin embolized into the lung of anesthetized rats. In the copper coil-induced dog coronary heart infarction model, at doses that achieve patency at equal rates, reocclusion is significantly less frequent than with tPA. These results indicate that DSPA alpha 1 may be a safer and more efficacious thrombolytic agent than the PAs currently in clinical use.
