Properties Of Tissue Plasminogen Activator Research Articles

Modulation by the Noble Gas Helium of Tissue Plasminogen Activator: Effects in a Rat Model of Thromboembolic Stroke.

Helium has been shown to provide neuroprotection in mechanical model of acute ischemic stroke by inducing hypothermia, a condition shown by itself to reduce the thrombolytic and proteolytic properties of tissue plasminogen activator. However, whether or not helium interacts with the thrombolytic drug tissue plasminogen activator, the only approved therapy of acute ischemic stroke still remains unknown. This point is not trivial since previous data have shown the critical importance of the time at which the neuroprotective noble gases xenon and argon should be administered, during or after ischemia, in order not to block tissue plasminogen activator-induced thrombolysis and to obtain neuroprotection and inhibition of tissue plasminogen activator-induced brain hemorrhages. We show that helium of 25-75 vol% inhibits in a concentration-dependent fashion the catalytic and thrombolytic activity of tissue plasminogen activator in vitro and ex vivo. In vivo, in rats subjected to thromboembolic brain ischemia, we found that intraischemic helium at 75 vol% inhibits tissue plasminogen activator-induced thrombolysis and subsequent reduction of ischemic brain damage and that postischemic helium at 75 vol% reduces ischemic brain damage and brain hemorrhages. In a clinical perspective for the treatment of acute ischemic stroke, these data suggest that helium 1) should not be administered before or together with tissue plasminogen activator therapy due to the risk of inhibiting the benefit of tissue plasminogen activator-induced thrombolysis; and 2) could be an efficient neuroprotective agent if given after tissue plasminogen activator-induced reperfusion.

Thrombolytic properties of tissue plasminogen activator (t-PA) and single chain urokinase plasminogen activator (scu-PA) in vitro: Absence of synergy as demonstrated by two quantitative analytical methods

The thrombolytic effects of tissue-type plasminogen activator (t-PA), of single chain urokinase-type plasminogen activator (scu-PA) and of combinations of t-PA and scu-PA were measured in an in vitro system composed of a 125I-fibrin labelled human plasma clot immersed in citrated human plasma. Lysis of the plasma clot was concentration and time dependent; 50% lysis in 4 h was obtained with 1.2 nM t-PA, in 8 h with 0.5 nM t-PA and in 12 h with 0.4 nM t-PA. For scu-PA corresponding values were 7, 4 and 2 nM. Combinations of t-PA and scu-PA in molar ratios of 1:2 to 1:9, were equieffective at algebraic fractions which were not significantly smaller than 1 and using the geometric isobole method coincided with the straight line connecting the equieffective concentrations of the individual plasminogen activators. Thus, both analytical methods indicated that the effects of t-PA and scu-PA on in vitro clot lysis were purely additive and not synergistic, notwithstanding the fact that the thrombolytic potency of combinations was consistently higher than the sum of the effects of the individual agents at the same concentrations as used in the combination. Our results do not support the existence of in vitro synergism between t-PA and scu-PA.

277 Change of the enzymatic properties of tissue plasminogen activator by limited proteolysis

277 Change of the enzymatic properties of tissue plasminogen activator by limited proteolysis

Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations.

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.

Comparison of the Relative Fibrinogenolytic, Fibrinolytic and Thrombolytic Properties of Tissue Plasminogen Activator and Urokinase in Vitro

The relative fibrinogenolytic, fibrinolytic and thrombolytic properties of human tissue plasminogen activator and human urokinase were compared in purified systems, in whole human plasma and in a system composed of a radioactive human blood clot (125I-fibrinogen) hanging in circulating human plasma. The human tissue plasminogen activator was highly purified from the culture fluid of a human melanoma cell line. In purified systems composed of fibrinogen of fibrin, plasminogen and alpha 2-antiplasmin as well as in whole plasma, tissue plasminogen activator digested fibrin without degrading fibrinogen significantly. Urokinase did not have this specific fibrinolytic effect. In the circulating plasma system, the degree of fibrinolysis was proportional to the amount of activator added, tissue plasminogen activator being about 10 times more efficient than urokinase. In addition, tissue plasminogen activator appeared to cause negligible fibrinogen degradation. Tissue plasminogen activator still induced significant thrombolysis at a concentration of 10 IU per ml whereas no effect of urokinase was observed at 20 IU per ml. Infusion of 100 IU (1 microgram) of tissue plasminogen activator per ml resulted in moderate activation of the fibrinolytic system as judged from a decrease of plasminogen and alpha 2-antiplasmin to 40-50 percent. Nevertheless, extensive fibrinolysis (50 to 80 percent of radioactivity released after 12 hrs) and only very limited fibrinogenolysis were observed. An equivalent amount of urokinase (100 IU per ml) only induced approximately 15 percent lysis in 12 hrs. At higher concentrations of urokinase (260 IU per ml or more) extensive activation of the fibrinolytic system was obtained as evidenced by a depletion of plasminogen, alpha 2-antiplasmin and fibrinogen. This was associated with extensive fibrinolysis (approximately 60 percent after 12 hrs). It is concluded that human tissue plasminogen activator is a more specific and effective fibrinolytic-thrombolytic agent than human urokinase.