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Immobilized asparaginase was prepared by embedding asparaginase (which is effective for remission children with leukemia) into fibrin polymer formed by fibrinogen-fibrin conversion in the presence of thrombin. The immobilized asparaginase film did not dissolve in 6M urea, suggesting that blood coagulation factor XIII participates in the cross-linking between fibrins and between fibrin and asparaginase.

Fibrin polymers formed from fibrinogen with thrombin in the presence of EDTA were suspended in a medium containing glucose, arabic gum and imidazole-HCl buffer and were sonicated at 20kHz for 20min to make a suspension containing fibrin particles of small size. The fibrin suspension was used as a substrate for determining the enzymic activity of plasmin and plasminogen activated with urokinase. The kinetic study on the reaction of the fibrin particles with plasmin in the presence and the absence of fibrinogen revealed that the Km value of fibrin for plasmin is 4.2×10-7M and the Ki value of fibrinogen is 1.2×10-5M.

An antifibrinolytic activity was found in the supernatant of washed platelet suspension in the process of platelet aggregation induced by thrombin, ADP and 5-hydroxytryptamine. The antifibrinolytic activity was closely associated with inhibitors in platelets, which specifically inhibited plasmin but not inhibited other proteases such as urokinase, thrombin and trypsin. One casein unit of plasmin activity was inhibited by the inhibitors released from approximately 108 platelets during the aggregation with thrombin. By the activity staining analysis, it was found that there are two kinds of plasmin inhibitors with the molecular weights of ≅25, 000 and ≅17, 000. These two inhibitors were stable after the treatment of the inhibitors with acid or denaturants, though the inhibitor with higher molecular weight lost the activity by dithiothreitol- and heat-treatments and two inhibitors lost also the activity by alkali-treatment. These inhibitors have been purified from the supernatant of aggregated platelets by ammonium sulfate precipitation, ion exchange chromatography, gel filtration and affinity adsorption to plasmin.

The endothelial cell is a rich source of plasminogen activator that is associated with fibrinolytic activity in blood vessel. Addition of fucosterol or sitosterol to the culture medium of endothelial cells from bovine carotid artery gave rise to a marked increment in the intracellular and extracellular activities of plasminogen activator. Removal of fucosterol or sitosterol from the culture medium resulted in a decrease of plasminogen activator activity back to normal levels. No enhancement of plasminogen activator activity in cultured endothelial cells was observed by cholesterol, 5-androsten-3β-ol and other steroids and sex hormones including androsterone, testosterone, estrone and estradiol. Synthesis of plasminogen activator in cells induced with fucosterol or sitosterol was inhibited by protein synthesis inhibitor, cycloheximide. The plasminogen activators produced in cells were classified into urokinase-type activators with molecular weights of 31, 000 and 55, 000 and tissue-type ones with molecular weights of 81, 000 and 130, 000, which were identified with respective antibodies. The synthesis of each type of plasminogen activator in endothelial cells was stimulated by fucosterol or by sitosterol.

A hitherto unknown synergism exerted by vitamin A (retinol, retinal, retinoic acid) and vitamin C (L-ascorbic acid) was discovered using endothelial cells. Vitamin A alone stimulated the extracellular and intracellular activities of plasminogen activator up to 8- and 4-fold from control values, respectively. Vitamin C alone enhanced the extracellular and intracellular activities up to approximately 1.5-fold. It was, however, demonstrated that their effects on extracellular activity were synergistic: simultaneous administration of these two vitamins enhanced the extracellular activity up to a 20- to 50-fold. Increase of plasminogen activator activity induced with these vitamins was inhibited by a protein synthesis inhibitor, cycloheximide.

The transglutaminase activity was found in cytosol and membrane fractions of endothelial cells from bovine carotid artery. The molecular weight was estimated to be approximately 58, 000 by gel permeation chromatography. Addition of retinoids (retinol, retinal and retinoic acid) to the endothelial cell culture medium caused a marked enhancement of the activity, and of the specific uptake of low density lipoprotein (LDL) into endothelial cells. It may be suggested that transglutaminase is involved in the receptor-mediated LDL uptake in bovine endothelial cells.