Tissue-type Plasminogen Activator (t-PA)

t-PA is a plasminogen activator produced and released in its single chain form with an apparent molecular weight of 70kD. Single chain t-PA is converted to double chain t-PA by plasmin mediated cleavage of the Arg-278-Ile-279 peptide bond. The resulting 2-chain molecule consist of a light chain (B-chain) containing the catalytic site Ser, His, Arg and a heavy chain (A-chain) containing two kringle-structure, an EGF-domain, and a fibronectin finger domain.

The fibrin specificity of t-PA is mediated by fibrin binding via finger and/or kringle-2-domain, wherby, in latter case, lysine binding sites are involved.

t-PA displays a unique fibrin-specific plasminogen activating function. In the absence of fibrin both single-chain and double-chain t-PA are poor activators of Glu-plasminogen due to a relatively high K_M value and a low K_cat value. However, in the presence of physiological concentrations of fibrin the plasminogen activating activity of t-PA is increased 200-400 fold resulting from a decrease in K_M and an increase in K_cat values. While both single and double-chain t-PA share similar plasminogen activating properties in physiological systems, double chain t-PA is more active than single-chain t-PA in an amidolytic assay with low molecular weight paranitroanilide substrates as well as in Glu-plasminogen  activating assay systems. The two forms also differ in their rates of inactivation by physiological inhibitors: Double-chain t-PA is more easily inactivated by PAI-2 than single-chain t-PA.

t-PA circulates in plasma at a concentration of approximately 5ng/ml. However, 95% of circulating t-PA is complexed with plasminogen activator inhibitor (PAI-I) and less than 5% circulates in its free form. Upon venous occlusion the concentration of t-PA antigen increases to 10 ng/ml or higher leading to measurable fibrinolytic activity. This is probably due to a combination of increased t-PA release by endothelial cells and a reduction in the t-PA clearance from the occlusion site.

Cadaver vessel perfusate plasminogen activator purified according to the method of Binder et al. (B.R. Binder, J.Spragg, K.F. Austen: Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates. J. Biol. Chem. 1998-2003, 1979).