Tenecteplase (TNK) and other TNK formulations lose PAI-1 resistance in their two-chain form: Comparison with alteplase

Alteplase is synthesized as a single-chain protease that requires plasmin-mediated conversion to its fully active two-chain form for maximal thrombolysis. Tenecteplase (TNK), an alteplase variant, is widely regarded as being more fibrin-selective and more resistant to plasminogen activator inhibitor-1 (PAI-1) than alteplase. However, the PAI-1 sensitivity of TNK after two-chain conversion, and that of newly available TNK formulations, has not been evaluated. Alteplase and four TNK formulations: Metalyse (Boehringer Ingelheim), Tenectase (Gennova, India), GenetPA (BioApower, China) and Mingfule (CSPC, China) were compared in their native state and after two-chain conversion. Proteolytic activity, PAI-1 resistance and binding were evaluated using amidolytic assay, fibrinolysis assays and Western blotting. Alteplase, but not Metalyse was converted into its two-chain form in plasma in a fibrinogen-dependent manner, resulting in off-target fibrinogenolysis. When directly reconstituted from the manufacturers vial (native form), TNK was ~4-fold more resistant to PAI-1 than alteplase; however, two-chain conversion significantly reduced PAI-1 resistance by ~40%, concomitant with increased PAI-1 binding. Native Metalyse contained ~3-fold more pre-existing two-chain species than the other TNK formulations, explaining its higher amidolytic activity. However, after two-chain conversion, all TNK formulations showed similar fibrinolytic activity albeit with substantially reduced PAI-1 resistance. Metalyse and all new TNK formulations display similar fibrinolytic activity in their fully active two-chain states, but this is associated with substantial loss of PAI-1 resistance, challenging the view that the fully active two-chain form of TNK maintains PAI-1 resistance during thrombolysis.