Reexamination of the Extent of the Activation of Lys₇₈ Plasminogen by Tissue Plasminogen Activator in the Presence of Polymerized Fibrin

Abstract

The rate of the conversion of Lys₇₈ plasminogen (Lys₇₈-plg) to Lys₇₈ plasmin by tissue plasminogen activator (tPA) was directly quantitated by SDS-PAGE. The apparent second-order rate constant (Kapp) was (0.27 ± 0.07) × 10³/M/s for single-chain tPA and Lys₇₈-plg, and was enhanced approximately 5-fold by fibrinogen and 17-fold by polymerized fibrin. Kapp was (1.57 ± 0.46) × 10³/M/s for two-chain tPA and Lys₇₈-plg, and was enhanced approximately 2.6-fold by fibrinogen and 3.6-fold by fibrin. The factor of the enhancement by polymerized fibrin was far less than in previous reports in which chro-mogenic substrate was commonly employed.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) in plasminogen activator inhibitor‐1 knockout mice: the effect of fibrinolysis during neuroinflammation

“…For specimen collection, mice were killed with CO2 and immediately perfused intracardially with phosphate buffered saline (PBS). Samples were taken from various time points throughout CREAE to correspond to the acute, remission and relapse stages of disease [15][16][17][18][19][20]30 and 40 days post induction (dpi)]. In PAI-1 -/-mice, there was no relapse of disease; hence samples for the remission phase were taken at 30 and 40 dpi.…”

“…In order to investigate the fibrinolytic capacity of different CNS tissue extracts, and to determine whether this changed during experimental neuroinflammation, a clot lysis assay was performed as previously described [19]. Spinal cord tissue protein extracts were mixed 1:10 with dilution buffer (50 mM Tris, 0.2% Triton X-100, pH 7.4 with HCl).…”

A Role for the Plasminogen Activator System in Inflammation and Neurodegeneration in the Central Nervous System during Experimental Allergic Encephalomyelitis

“…26 Briefly, spinal cord tissue protein extracts were mixed 1:50 with a reaction buffer (50 mmol/L Tris-HCl, pH 7.4) containing 7.3 mol/L human fibrinogen (Sigma), 0.25 mol/L human lys-plasminogen (Chromogenix, Milan, Italy), 1.7 mmol/L CaCl 2 , 0.7 mmol/L MgCl 2 , and 12.5 mmol/L NaCl. Samples were added in duplicate to 96-well microtitre plates containing 20 l of human thrombin per well (100 U/ml) and incubated at 37°C.…”

Impaired fibrinolysis in multiple sclerosis: a role for tissue plasminogen activator inhibitors

“…A clot lysis assay was used to evaluate ®brinolytic activity in the CNS tissue extracts (Urano et al, 1996). The turbidity assay relies on the degradation of an in vitro formed ®brin clot in the presence of plasminogen activators and plasminogen, resulting in a decrease in absorbance.…”