Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma: evidence for a rapid plasminogen activator inhibitor

• Published in: Biochimica et biophysica acta Vol. 802; no. 1; pp. 111 - 118
• Main Authors: THORSEN, S, PHILIPS, M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 06.11.1984; North-Holland
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Glycoproteins - isolation & purification; Humans; Molecular and cellular biology; Molecular Weight; Plasminogen Activators - antagonists & inhibitors; Plasminogen Inactivators; Sodium Dodecyl Sulfate; Human; Cell culture; Endothelial cell; Purification; Activator; Gel electrophoresis; Gel filtration; Inhibitor; Immunosorption; Plasminogen
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/0304-4165(84)90040-0

Plasminogen activator-inhibitor complexes were analyzed by SDS-polyacrylamide gel electrophoresis and enzymography. The complexes appeared as fibrinolytically active bands in the fibrin-indicator gel. A high-molecular-weight t-PA form comigrating with a t-PA-inhibitor complex (Mr 95 000-135 000) from cultured human endothelial cells was purified from plasma by immunoadsorption on anti-t-PA-Sepharose followed by gel filtration on Sephadex G-150. The high-molecular-weight t-PA form was fibrinolytically inactive when assayed by the fibrin-plate method. It was converted to a form with the same electrophoretic mobility as t-PA (Mr 72 000) when treated with 1.5 M NH4OH/39 mM SDS. These observations suggested that the plasma high-molecular-weight t-PA form was an enzyme-inhibitor complex. The complex did not show immunological cross-reactivity with a number of known plasma serine proteinase inhibitors. Both t-PA and u-PA rapidly formed complexes with an inhibitor which was present in plasma in pmolar concentrations. p-Aminobenzamidine blocked the reaction, indicating that the active center of the activator was indeed implicated in complex formation. The complex between the plasma inhibitor and t-PA and the high-molecular-weight t-PA had the same electrophoretic mobilities. The rapid plasminogen activator inhibitor in plasma showed remarkable similarity to a plasminogen activator inhibitor from cultured human endothelial cells. In addition to the high-molecular-weight t-PA form described above, three other t-PA forms were isolated from plasma. Our results indicated that they represented free t-PA and t-PA in complex with respectively C1-esterase inhibitor and alpha 2-antiplasmin.