F.R. Matthias

Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrinmonomer

Abstract Insolubilized fibrinogen was prepared by chemical fixation of purified fibrinogen to CNBr activated agarose. Under optimal preparative conditions a fibrinogen/agarose ratio of 1:1 was achieved on the dry weight basis. Chromatographic experiments using FG-ag columns on thrombin incubated plasma containing ethanol test positive material (fibrinmonomer) revealed adsorption of a fibrinogen derivative eluted at pH 4.1 the amount of which correlated significantly to the quantitative ethanol test most likely indicating adsorption of fibrinmonomer. Fibrinogen was considerably adsorbed on FM-ag columns under the same conditions. The results are in favour of the interaction of fibrinogen and fibrinmonomer and possible complex formation.

Affinity chromatography and quantitation of soluble fibrin from plasma.

Abstract By affinity chromatography on agarose-fixed fibrinogen (FG-ag) soluble fibrin can be isolated from plasma samples or aqueous solutions and quantified by radio-activity measurement or by the staphylococcal clumping test. Using radiolabelled fibrinogen for the insolubilization it could be shown that the FG-ag column is stable and is not degraded even after many runs of plasma samples over a long period of time. By addition of radioactive fibrinogen and fibrin to plasma samples standardized and reproducible conditions are established for clinical use of the method. The amount of soluble fibrin in normal donors was found to be about 0.6 - 0.7 mg%. An elevated fibrin concentration up to 6 mg% above the normal value was detected in plasma of patients after myocardial infarction, even when the coagulation parameters were still normal.

Behaviour of fibrinogen and fibrinogen degradation products (FDP) towards insolubilized fibrinogen and fibrinmonomer

Abstract Adsorption studies were performed between insolubilized fibrinogen and fibrinmonomer, and the isolated fibrinogen degradation products. All fragments (X, Y, D, E,) were adsorbed onto insolubilized fibrinmonomer. Fragment X and fragment Y were adsorbed in a higher yield than the isolated end products fragments D and E.

Beneficial effects of prostacyclin in a rabbit endotoxin shock model.

Thirty rabbits received an infusion of lipopolysaccharide B (75 micrograms/kg.h) over 4 hours (groups E, EI, EA; n = 10 each). Saline was given to a control group (C; n = 8). In group EI, prostacyclin (PGI2; 500 ng/kg.min) was given simultaneously to endotoxin. Into group EA animals, aspirin (20 mg/kg) was injected before the endotoxin infusion was started. PGI2 and aspirin both improved survival of animals (6/10 each vs. 2/10 in group E). The drop of platelet counts was significantly reduced by PGI2, while leukocyte depletion was similar in all endotoxin groups. PGI2 preserved the functional capacity of platelets as indicated by collagen stimulated aggregation and thromboxane formation. PGI2 but not aspirin significantly reduced renal fibrin deposition.

Effects of prostacyclin during cardiopulmonary bypass in men on plasma levels of β-thromboglobulin, platelet factor 4, thromboxane B2, 6-keto-prostaglandin F1α and heparin

Abstract A randomized, double-blind study was carried out on 40 male patients requiring aorto-coronary bypass surgery. 20 patients received a constant dose of 8 ng kg −1 min −1 of prostacyclin (PGI 2 ), beginning two minutes before extracorporeal circulation (ECC) and ending together with ECC. Compared to the placebo-treated patient group (n = 20), PGI 2 -treatment significantly reduced the ECC-induced release of platelet α-granule proteins, β-thromboglobulin (1178 ng/ ml vs. 1926 ng/ml) and platelet factor 4 (837 ng/ml vs. 1245 ng/ml) into plasma (mean of max. values). Furthermore the decrease of platelet counts during ECC was less pronounced in PGI 2 -treated patients. Application of PGI 2 had no effect on the increase in thromboxane B 2 (TxB 2 ) plasma levels, which amounted to 0.6 ng/ml at the end of ECC. PGI 2 -treatment resulted in significantly elevated plasma concentrations of 6-keto-prostaglandin F 1α (6-keto-PGF 1α ) (2.1 ng/ml) throughout the infusion of prostacyclin. 6-keto-PGF 1α plasma levels increased up to 1.2 ng/ml in the control group patients, indicating a stimulation of endogenous PGI 2 formation during ECC.

Isolation of fibrinogen from plasma by affinity chromatography on insolubilized fibrinogen (FG-ag) and insolubilized fibrin-monomer (FM-ag)

Abstract Insolubilized fibrinmonomer, which is converted from insolubilized fibrinogen by thrombin action, adsorbs soluble fibrinogen from solutions or plasma. After treatment with 6 molar urea without removal of the fibrinopeptides insolubilized fibrinogen gets a high affinity for soluble fibrinogen, too. Insolubilized fibrinmonomer and urea-treated insolubilized fibrinogen are suitable adsorbents for the purification of fibrinogen from plasma by affinity chromatography.

Increase in the degree of phosphorylation of circulating fibrinogen under thrombolytic therapy with urokinase

Human fibrinogen is phosphorylated in vivo to an equal extent at two positions, one at Ser 3 located on fibrinopeptide A, the other at Ser 345 of the A alpha-chain. As has been shown previously, the degree of phosphorylation of the circulating fibrinogen pool can be determined in vitro from the ratio between the HPLC peaks formed by phosphorylated and non-phosphorylated fibrinopeptide A which has been cleaved from plasma fibrinogen by thrombin or reptilase. Plasma samples were obtained from patients with venous thrombosis undergoing fibrinolytic therapy with urokinase (n = 8). The degree of phosphorylation increased from about 35% before treatment to values between 50% and 70% within 48 hours. It remained at these high levels as long as urokinase was administered and declined slowly thereafter. This behaviour of the degree of phosphorylation of fibrinogen is explained by a model which assumes that fibrinogen is secreted in the phosphorylated form and then dephosphorylated in the circulation by an up to now unidentified phosphatase by first order kinetics. When this system is in steady state, the degree of phosphorylation is about 25% under normal conditions. If the elimination rate of fibrinogen is greatly enhanced by fibrinogenolysis the system will approach a new steady state with a higher degree of phosphorylation, the magnitude of which will depend on the new ratio of dephosphorylation and elimination.

Reduction of insolubilized fibrinogen

Abstract Insolubilization of fibrinogen is achieved by covalent fixation onto cyanogen bromide activated agarose. When insolubilized fibrinogen (FG-ag) is reduced in order to separate the fibrinogen molecule into its chains, disc-electrophoretic patterns reveal that the (‘A’) α-chains remain with the insoluble agarose whereas the (B) β-chains and the γ-chains appear in the supernatant. It is assumed that covalent fixation of fibrinogen to agarose almost exclusively takes place at the (‘A’) α-chain and that the (‘A’) α-chain is located at the surface of the fibrinogen molecule.

In vitro and ex vivo effects of aspirin in patients on a low-dose aspirin therapy

In 19 patients on a low-dose aspirin therapy with 100 mg/d, an insufficient effect of aspirin was observed in five patients (aggregations induced by arachidonic acid and collagen, thromboxane B2-formation in serum and after collagen). Aspirin added in vitro increased the inhibition to a degree comparable to that seen in the other 14 patients, i.e. the insufficient effect could be due to a lack of compliance or to a reduced availability of the drug. In another 20 patients there was a good inhibitory effect of aspirin; additional aspirin did not increase the inhibition of arachidonic acid-induced aggregation and serum-thromboxane B2, but slightly increased collagen-induced aggregation and thromboxane B2 formation. The effect was the same, whether the aspirin was given in vivo or added in vitro.

Studies on chemically modified fibrinogen. II. Physicochemical properties of maleylated fibrinogen

Abstract Modification of up to 60% of epsilon-amino lysine residues in fibrinogen molecule with maleic acid anhydride does not change hydrodynamic properties of fibrinogen molecule. Some small differences in its optical properties could be observed. Total modification leads to the gelification of fibrinogen preparation and to increased stability to the proteolytic action of plasmin and trypsin.