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Covalent Prourokinase-dendrimer Complexes with Increased Plasma Stability and High Thrombolytic Activity in vitro

1Lomonosov Moscow State University, Chemistry Faculty, Moscow, Russian Federation, 2Bauman Moscow State Technical University, Moscow, Russian Federation, 3Emanuel Institute of Biochemical Physics of RAS, Moscow, Russian Federation

Abstract Number: PB0768

Meeting: ISTH 2020 Congress

Theme: Fibrinolysis and Proteolysis » Thrombolytic Therapy

Background: Single-chain prourokinase (scuPA) is an effective plasminogen activator used as thrombolytic agent. However, it is quickly cleared from the bloodstream. In addition, with the formation of plasmin (Pm), scuPA loses its fibrin specificity owing to rapid conversion to two-chain urokinase (tcuPA). Nanoscale polyamidoamine (PAMAM) dendrimers are widely used as drug carriers due to their unique structure and polyvalent surface.

Aims: Increasing the scuPA stability in human plasma by its modification with anionic PAMAM dendrimer G2.5.

Methods: COOH-groups of PAMAM dendrimer G2.5 was activated the mixture of EDC and NHS (pH 2.0) and purified. ScuPA was conjugated with activated dendrimer in ratios of protein:polymer 1:1, 1:5, 1:10 and 1:20 (М:М) during 2h at pH 8.5. Methods for studying the plasma stability of fibrinolytic activity, plasminogen-activating, and thrombolytic activities of scuPA and conjugates of scuPA-dendrimer were based on measuring the fibrin plate lysis area, Glu-plasminogen activation rate and plasma clot lysis rate in vitro, respectively.

Results: The conversion rate of scuPA-dendrimer to tcuPA-dendrimer by plasmin slowed somewhat with increasing the modification degree of the scuPA positively charged groups by PAMAM-COOH dendrimer (from 1:1 to 1:20), and the resulting tcuPA-dendrimer conjugates retained 92-85% of amidase activity. Plasminogen-activating activity of scuPA-dendrimer conjugates was comparable or slightly higher than free scuPA. Thrombolytic activity of scuPA-dendrimer conjugates was 90-95% of the scuPA activity measured by clot lysis in 4h. Conjugation with dendrimer increased the plasma stability of scuPA fibrinolytic activity (τ1/2 48 min): the τ1/2 of conjugates increased from 60 to 130 min with at increasing the ratio of scuPA: dendrimer from 1:1 to 1:20.

Conclusions: Supposedly, due to the modification of the PAI-1-binding Arg-Arg-Arg-Arg-cluster in scuPA by anionic dendrimer, the scuPA-dendrimer conjugates are significantly more stable in plasma than scuPA, but retain high plasminogen-activating and thrombolytic activities.

To cite this abstract in AMA style:

Ivanova E, Mukhametova L, Aisina R, Gershkovich K. Covalent Prourokinase-dendrimer Complexes with Increased Plasma Stability and High Thrombolytic Activity in vitro [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/covalent-prourokinase-dendrimer-complexes-with-increased-plasma-stability-and-high-thrombolytic-activity-in-vitro/. Accessed September 29, 2023.

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