Design of a DNA-Programmed Plasminogen Activator.

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.

[Thrombolysis with plasminogen activators: use and research of serine proteinases, promise and actuality].

Physiological plasminogen activators of tissue and urokinase type (serine proteinases) are effective thrombolytic agents. Research and development of their novel forms contributed to progress of thrombolytic therapy, advanced treatment of acute coronary syndrome and a marked decrease in the lethal index.

Synthesis and characterization of positively charged tPA as a prodrug using heparin/protamine-based drug delivery system.

Positively charged peptides [(Arg)7 Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7 Cys (termed modified tPA, mtPA) was 3900 IU/microg as compared to 5800 IU/microg of the parent tPA. Both activation of plasminogen with mtPA (Km= 2.7 mM(-1)) and tPA (Km= 1.1 mM(-1)) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7 Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and alpha2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.

[The thrombolytic activity of acylated activator complexes of plasmin-streptokinase with different rates of reactivation]. / Tromboliticheskaia aktivnost' atsilirovannykh aktivatornykh kompleksov plazmin--streptokinaza s razlichnymi skorostiami reaktivatsii.

In the experiments on guinea-pigs with venous thrombosis there were studied the fibrin- and thrombolytic effects of streptokinase, the plasmin-streptokinase complex and the acylated derivatives of the complex with various rates of reactivation. It was established that the acylated derivatives of the plasmin-streptokinase complex possess greater stability in the blood flow and lead to more prolonged stimulation of fibrinolysis at less magnitude of its systemic activation. Due to this the acylated derivatives of the plasmin-streptokinase complex produce less pronounced fibrinogenolysis. In connection with a high affinity to fibrin their thrombolytic action does not depend on the systemic activation of fibrinolysis.

Challenges and opportunities in biotechnology.

The biotechnology industry is thriving, and many predicted accomplishments have actually occurred during the last decade. Cloning and expression of genetic information is now simple and routine. Initial commercial products have been realized, but there is much yet to be accomplished in evaluating the clinical significance of many other gene products made available by biotechnology resources. During the next decade, human health care and the pharmaceutical industry should be affected substantially by first- and second-generation recombinant DNA products. Recombinant vaccines, blood coagulation factors, and known biological modulators produced by rDNA technologies should be widely used. Further opportunities will be realized with increasing discoveries of new bioactive molecules and identification of NANB hepatitis and AIDS infectious agents. Full exploitation of health care products will depend on innovative new delivery systems or the ability to reconstruct mammalian and plant genes, providing for in-situ delivery of the necessary gene products.