Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser-->Cys substitution associated with thrombosis.

A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.

Basics and practice in evaluating plasminogen.

There exist different ways of assays of plasminogen which give information about different properties of this proenzyme. The concentration of plasminogen can be determined by its antigenicity. Since the normal concentration of plasminogen in plasma is between 15 and 25 mg/dl the test can be carried out by simple methods such as radial immunodiffusion on Partigen plates. The possibility of errors is small and there is no need of special apparatus. The disadvantages are the lapse of 24 h until the result is available and the fact that the knowledge of the concentration does not give any information about the activity. The activity can be measured by different coagulation tests. A typical assay would involve activation of plasminogen to plasmin, addition of plasminogen-free thrombin and measuring of the lysis time. The result is however, dependent on more than one variable. Plasmin is rapidly inhibited by alpha-2-antiplasmin (APL) and there is also a dependence of the lysis time on the amount of clottable fibrinogen in the test system. Better results can be obtained by the use of diluted test plasma and addition of a constant amount of plasminogen-free fibrinogen. A different way would be the use of the euglobulin fraction instead of plasma. This has however, the possible disadvantage of incomplete precipitation of plasminogen. Instead of coagulation tests the activity can also be determined when diluted activated plasma is placed on plasminogen-free fibrin plates and the amount of lysis in the plate is recorded. All assays of this group also depend on the method of activation of plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Standardization of plasminogen assays.

Like a number of the components of the fibrinolytic and coagulation systems, plasminogen (plgn) is a multifunctional molecule. As a proenzyme, a number of its activities such as its binding to fibrin, histidine-rich glycoprotein (HRGP) and alpha 2-antiplasmin (AP) are expressed while its major enzymatic activity remains unexpressed. This latter activity has been used as a yardstick of plasminogen potency, despite the fact that no such activity resides in the native plasminogen molecule. Assay procedures usually involve the activation of the plasminogen to plasmin using an activator such as streptokinase (SK) or urokinase (UK) and a major problem involves the establishment of a properly-timed plasminogen-activator ratio to fully express the plasminogen as the active enzyme plasmin (Gaffney, P.J. et al. Activation of plasminogen as a feature of its assay. Haemostasis 1977, 6, 72-78). Substrates such as casein, fibrinogen and fibrin have been used to assess the plasmin activity developed while more recently the tripeptide chromogenic substrate S-2251 has been successfully used. These assays have been standardised using a reference preparation of the active enzyme, plasmin, and both a 1st and 2nd International Reference Preparation (IRP) have been established. These IRP's differed in that the fibrin binding kringle-structures were missing in the 1st IRP yielding differing fibrinolytic and chromogenic activities (Philo, R.D. and Gaffney, P.J. Plasmin potency estimates. Influence of substrate used in assay. Thrombosis and Haemostasis 1981, 45, 107-109). Activation procedures of plasminogen and subsequent assays of plasmin using a variety of substrates have been recently superseded by an assay which involves the formation of a plgn-SK complex which complex has an active site which hydrolyses the chromogenic substrate S-2251. This avoids the problems highlighted above involved in measuring plasminogen activity at the optimum stage during activation. While plasmin standards have been suitable for the standardisation of plasminogen when it is measured by activation-based procedures, a British Standard for glutamic acid-plasminogen has now been established in order to standardise the plgn-SK assay (Gaffney, P.J. and Curtis, A.D. The establishment of a standard for plasminogen (glu-type). Thrombosis and Haemostasis 1984, 51, 376-378). The calibration of this standard using the 2nd IRP for plasmin and the value of this standard in the measurement of plasminogen in plasma is discussed.

Characterization of commercially available plasminogen preparations in vitro: purity and reactivity.

Three different commercially available plasminogen preparations (Immuno-, Kabi-, and Behring-plasminogens) were examined regarding purity and reactivity to different activators (high molecular weight [HMW] or low molecular weight [LMW] two-chain urokinase type plasminogen activator [tcu-PA], single chain urokinase type plasminogen activator [scu-PA], tissue type plasminogen activator [t-PA], and streptokinase [SK]). The Immuno-preparation was a Lys-plasminogen, commercially available for therapeutical use, whereas the research reagents for KabiVitrum and Behringwerke were Glu-plasminogen. Activity data provided by the manufacturers correlated well with our findings. Also a good correlation of reactivity to activators measured with a chromogenic substrate and on fibrin plates could be observed. The Immuno-plasminogen showed a minimum contamination with plasmin which has to be taken into consideration for the interpretation for its apparently higher activation by plasminogen activators compared to the plasmin-free plasminogens. Further in vitro and in vivo research has to be performed to find out criteria for a practicable scheme of administration of fibrinolytic agents for therapeutical thrombolysis.

Production and quality assurance of Lys-plasminogen steam treated.

A highly purified plasminogen concentrate, LYS-PLASMINOGEN Steam Treated, has been developed for thrombolytic therapy of arterial and venous occlusions in combination with fibrinolytic agents. In search of a highly efficient drug covering this indication, we decided to select the lys-form of plasminogen because of its higher affinity to fibrin in contrast to the glu-form. This property of lys-plasminogen also led us to expect an improved thrombolytic activity as opposed to other forms of the proenzyme. The intermediate product is manufactured from pooled human citrated plasma by ethanol fractionation after separation of coagulation factor proteins. Further processing includes specific transformation and purification steps. The final product is a freeze-dried preparation characterized by a high specific activity greater than or equal to 18.0 CU/mg protein and a content of lys-plasminogen of greater than or equal to 95%. To reduce the risk of viral infections, the plasma pool includes only plasma donations which are ALT tested and negative for HBsAg and anti-HIV. In addition the intermediate freeze-dried bulk powder is subjected to a virus inactivation procedure based on steam treatment for 10 hours under standardized product specific conditions without using special protein stabilizers. Physical parameters of steam treatment provide for a maximum virus killing effect without impairing the biological plasminogen activity or changing the molecular integrity of the product. In a preclinical test HIV was inactivated by 6 log 10 after 3 hours of steam treatment leaving a 7 hour safety margin for inactivation of more heat resistant viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Reference values and variability of plasminogen in healthy blood donors and its relation to parameters of the fibrinolytic system.

During a survey of four month's duration the following parameters were determined in 43 healthy blood donors (22 males, 21 females; mean age 29 years/20-49/): plasminogen activity, plasminogen concentration, alpha 2-antiplasmin (alpha 2-AP) activity, alpha 2-AP concentration, tissue type plasminogen activator (t-PA) activity, t-PA concentration, plasminogen activator inhibitor--I (PAI-I) activity, AT III activity, AT III concentration and heparin cofactor II (HC II) activity. Normal values including standard deviation (x +/- 2s) were: plasminogen activity: 96.3% (65.9-126.8), plasminogen concentration: 12.2 mg/dl (7.7-16.8), alpha 2-AP activity: 99.9% (83.8-116), alpha 2-AP concentration: 108.1% (84.5-131.8), t-PA activity: 0.85 IU/l (0.0-1.92), t-PA concentration: 10.3 ng/ml (2.5-18.1), PAI-I activity: 15.2 AU/ml, AT III activity: 111.4% (87.8-134.9), AT III concentration: 31.6 mg/dl (24.2-39.1) and HC II activity: 110.7% (81.4-140.0). Concerning plasminogen values no sex related difference could be stated. Women who were smokers and used oral contraceptives tended to present elevated t-PA activity levels due to a lower activity of PAI-I, although this tendency was not significant. Determining concentration and activity of components in the fibrinolytic system plays an important part in the diagnosis, therapy and prognosis of thrombophilic disorders.

The plasminogen content of commercial preparations and of normal donor plasma in relation to the plasmin content of the 1st international plasmin reference preparation.

Kabi human plasminogen and plasmin and two Behringwerke preparations of human plasminogen were examined for antigen content, purity and specific activity with the 1st International W.H.O. human plasmin reference preparation and with the plasminogen content of an 8 donor normal plasma pool. In relation to the plasminogen content of the preparation with the highest specific activity, the 8 donor plasma pool contained 0.186 mg/ml of plasminogen. This plasminogen on complete conversion to plasmin by streptokinase or urokinase corresponded to 4.35 International units/ml of plasmin as defined by the International reference preparation. Protein adsorption from highly purified plasminogens of low protein content induced variable underestimates of antigen and of biological activity. To prevent this it is recommended to issue these purified preparations in an inert carrier medium or alternatively to release these preparations with data pertaining to salt content and optical measurement prior to lyophilisation. When standards of high purity and low protein content are being examined for antigen and enzyme, it is recommended likewise that an inert protein carrier should be present in the diluent. Measurement of proactivator was considered to be unsuitable in reference to proactivator content of highly purified plasmin and plasminogen.