Biochemical properties of conjugates of urokinase‐type plasminogen activator with a monoclonal antibody specific for cross‐linked fibrin

Open Access
Publisher Website
Google Scholar
Abstract
Equimolar mixtures of recombinant single chain urokinase‐type plasminogen activator (rscu‐PA) and a murine monoclonal antibody (MA‐15C5) directed against fragment‐D dimer of human cross‐linked fibrin were conjugated, using the cross‐linking agent N‐succinimidyl 3‐(2‐pyridyldithio)propionate (PySSProSu). The conjugate (rscu‐PA/MA‐15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment‐D dimer with a yield of 42 ± 15% (mean ± SD, n= 3), contained an average of 1.2 ± 0.3 IgG molecules/rscu‐PA molecule. On non‐reduced SDS/PAGE it migrated as a main band with apparent Mr of 200000. Specific amidolytic activities expressed/mass of u‐PA were < 250 IU/mg for rscu‐PA/MA‐15C5 and rscu‐PA, 140000 ± 13000 IU/mg and 100000 ± 17000 IU/mg for their plasmin‐generated two chain derivatives rtcu‐PA/MA‐15C5 and rtcu‐PA respectively. Specific activities on fibrin plates were 100000 ± 24000 IU/mg and 130000 ± 49000 IU/mg for rscu‐PA/MA‐15C5 and rtcu‐PA/MA‐15C5 respectively, as compared to 180000 ± 15000 IU/mg for both rscu‐PA and rtcu‐PA. Activation of plasminogen with rscu‐PA/MA‐15C5 (Km= 0.37 ± 0.16 μM, k2= 0.0063 ± 0.0030 s−1) or rtcu‐PA/MA‐15C5 (Km= 19 ± 3.0 μM, k2= 2.0 ± 0.10 s−1) in purified systems followed Michaelis‐Menten kinetics with Km and k2 values comparable to those of rscu‐PA and rtcu‐PA. In an in vitro system composed of a 125I‐fibrin‐labeled whole human plasma clot immersed in citrated human plasma, dose‐ and time‐dependent lysis was obtained; 50% lysis in 2 h required 1.4 μg/ml of rscu‐PA or 0.33 μg/ml of rtcu‐PA, but only 0.22 μg u‐PA/ml of rscu‐PA/MA‐15C5 or 0.15 μg u‐PA/ml of rtcu‐PA/MA‐15C5. Addition of purified fragment‐D dimer reversed the increased fibrinolytic potency of rscu‐PA/MA‐15C5 in a concentration‐dependent way (50% inhibition at 7.2 μg fragment‐D dimer/ml). Thus, conjugation of u‐PA moieties with the fibrin‐specific antibody MA‐15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4‐fold for rscu‐PA and 2.2‐fold for rtcu‐PA.

This publication has 29 references indexed in Scilit: