Abstract
Spontaneous fibrinolysis of plasma clots was studied by following the lysis of the clots formed in 125I-fibrinogen-supplemented citrated plasma. Lysis of the clots invariably follows sigmoidal kinetics with S50 (the time required for 50% clot lysis) ranging from 3.5 to 4.7 days in 8 samples of pooled blood bank plasma and in the majority of apparently healthy donor plasmas. The spontaneous lysis of factor XII-deficient and prekallikrein-deficient plasmas was found to be similar to that of normal plasma. Addition of ellagic acid or antibodies against kallikrein or urokinase to normal pooled plasma did not alter significantly its rate of spontaneous lysis. On the other hand the addition of antibody against tissue activator (t-PA) inhibited over 80% of the spontaneous fibrinolysis in a 7-day incubation period at 37 degrees C, and the clot visually persisted for more than a month. Therefore, the factor XII-dependent components and prourokinase/urokinase system do not contribute significantly in whole plasma fibrinolysis in vitro, while the t-PA-related protein appears to be the major plasminogen activator responsible for initiating spontaneous fibrinolysis in whole plasma. Exogenous addition of increasing amounts of purified HeLa cell t-PA to normal pooled plasma in the ng/ml range cause progressively faster clot lysis. By extrapolation, normal pooled plasma is found to contain endogenous tissue activator in an amount functionally equivalent to 2 ng/ml of purified 60-kDa t-PA. The molecular nature of the t-PA-related proteins in plasma was studied by zymographic and immunological methods. The major t-PA-related protein in plasma was found to have a molecular mass of 100 kDa as determined by zymography. By incubating purified HeLa 60-kDa t-PA with a t-PA-depleted plasma, the 100-kDa component can be generated in plasma, suggesting that the latter is formed as a result of the binding of 60-kDa t-PA to a binding protein in plasma.
Highlights
Spontaneous fibrinolysis of plasma clots was studied degrading enzyme-plasmin from its precursor plasminogen by following the lysis of the clots formed in ‘261-fibrin- [1].as reviewedin many articles [2,3,4], the exact ogen-supplemented citrated plasma
Lysis of the clots identity of the plasminogen activators involved in the develinvariably followssigmoidal kinetics with5’50 ranging from 3.5 to 4.7 itively defined
On the other hand the additioofnantibody against tissue activator (t-PA) inhibited over 80% of from uterine [6], heart ( 7 ), and several cell lines such as melanoma [8] and HeLa [9]; 2) a plasma urokinase/prourokinase has been identified and purified and found to be the same as u-PA by many biochemical and immunological criteria [10,11,12]; 3) factor XII-dependent components are thought to be involved in plasma fibrinolysis since glass, the spontaneous fibrinolysis in a 7-day incubation pe- kaolin, or similar compounds enhance fibrinolysis in normal riod at 37 “C, and the clot visually persisted for more plasma but not in factor XII-deficient plasma [13]
Summary
IDENTIFICATION OF TISSUE ACTIVATOR-RELATED PROTEIN AS THE MAJOR PLASMINOGEN ACTIVATOR CAUSING SPONTANEOUS ACTIVITY IN VITRO*. Thefactor XII-dependent more, purified factor XI1 and kallikrein have been shown to components and prourokinase/urokinasesystem do not be capable of plasminogen activation [14] Despite these excontribute significantly in whole plasma fibrinolysis tensive studies, there is neither direct proof that these proi n uitro, while the t-PA-related protein appears tobe teins initiate fibrinolysis in whole plasma in vitro nor firm the major plasminogen activator responsible for initi- evidence that these proteins arephysiologically relevant plasating spontaneous fibrinolysis in whole plasma. Enous addition of increasing amountsof purified HeLa In an attempt to delineate the mechanism of the developcell t-PA to normal pooled plasma in the ng/ml range ment of fibrinolytic activity in whole human plasma, we have cause progressively faster clot lysis.
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