Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.

Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.

Endogenously Increased Fibrinolytic Activity Induced by Liver-Bypass in Pigs and its in Vitro Synergism with Synthetic Fibrinolytic Compounds

The marked increase of fibrinolytic activity during the unhepatic phase during liver transplantation in animals and man is well known. Based on these observations the liver-bypass in the pig was used for producing endogenous increase of fibrinolytic activity of several hours duration thus providing fibrinolytic blood specimens suitable for in vitro studies of the synergism of endogenously induced fibrinolytic activity with the fibrinolytic activity induced by synthetic compounds. Liver-bypass in the pig results within a few minutes in a marked drop of the euglobulin lysis time from an average of 16 hrs. to one hour or less. Whereas the antiplasmin activity does not show any clear-cut change during the bypass, the antiactivator activity had dropped after about 2 hrs. to half of the original values. The fibrinolytic activity of the lover-bypass plasma is markedly enhanced by synthetic fibrinolytic compounds such as flufenamic acid applied at concentrations which themselves do not induce fibrinolytic activity. This can be demonstrated in various ways such as the use of thrombelastograms or lysis of clots formed by the bypass plasma. Also, as the bypass goes on, the clots start to dissolve spontaneously and the lysis time is considerably shortened by addition of synthetic compounds. Seen from the point of view of a potential clinical use, the most pertinent observation is that plasma obtained during an early bypass phase which does not dissolve preformed human clots, does so after addition of synthetic compounds.

Mechanism of Action of Synthetic Fibrinolytic Compounds

SummaryThe mechanism of dissolution of a preformed plasma clot was explored.Our experiments showed clearly that the purified fibrin clot, made by extensive washing of a plasma clot, was resistant to lysis and that the fibrinolytic potential of the active fibrinolytic compounds was related to the presence of other plasma proteins in addition to fibrinogen.The activation of the fibrinolytic precursors was reversible inasmuch as removal of the fibrinolytic compounds negated the fibrinolytic activity of the protein-fibrinolytic compound mixture.Antifibrinolytic compounds which had been shown to interfer with fibrinolysis by streptokinase-activated plasminogen inhibited dissolution of the preformed plasma clot by fibrinolytically active compounds.The fibrinolytic potential of fibrinolytic compounds was additive; however, no apparent synergism was observed.The implication of these results to the mechanism of synthetic fibrinolysis was discussed.

On the Effect on Human Thrombocytes of Synthetic Organic Fibrinolytic Compounds

SummaryThe effect of various synthetic organic fibrinolysis-inducing compounds was investigated on platelets in plasma and on washed platelets. To a varying degree the com pounds induced a concentration-dependent platelet aggregation. Some at higher molarities cause lysis of platelets. The same molecular modifications which reduce the fibrinolytic activity also seem to reduce the effects of the compounds on platelets. Platelet aggregation by the compounds was reduced by adenosine, adenosine monophosphate, and some other ADP-inhibitors, but there was no proof that the compounds act by releasing ADP from the platelets. With fluorescent compounds their attachment to platelets was demonstrated.