H.C. Godal

Thrombin generation during collection and storage of blood.

Abstract. Thrombin generation, as evidenced by radioimmunologic fibrinopeptide A (FPA) determination, was studied upon drawing and storage of blood. Blood from 5 healthy subjects was drawn into Fenwal bap (PL 146, 16‐gauge needle, 92‐cm collecting tube), and blood samples for FPA analysis were taken from the proximal and distal part of the collecting tubes. The FPA concentrations were considerably higher in the distal part of the tubes both at zero time and after 5 and 10 min, implying substantial thrombin generation to have occurred upon blood passing through the tubes. In 20 healthy blood donors (age 19–52 years) subjected to a standard collection procedure (Fenwal bags PL 146, CPDA formula‐1 anticoagulant solution, 450 ml blood), higher FPA values were observed in the bags immediately upon collection, as compared to control venipuncture values (p<0.02). Furthermore, in 6 women using oral contraceptives, even larger increases were observed, indicating thrombin generation to be more pronounced in such donors (p<0.01). No statistically significant increase in FPA concentrations was found upon storage of CPDA‐blood at 4°C for 24 h. There was no correlation between FPA and beta‐thromboglobulin (BTG) concentrations after blood collection, indicating alpha‐granule release from platelets to be non‐thrombin‐mediated.

Does Lp(a) lipoprotein inhibit the fibrinolytic system

Lp(a) lipoprotein contains a unique apolipoprotein, apolipoprotein (a), that has a striking homology with plasminogen. This homology has brought forward speculations as to an inhibitory effect of Lp(a) lipoproteins on fibrinolysis. The present investigation was undertaken to study the influence of Lp(a) lipoprotein on the fibrinolytic system. In an in vitro model, we have studied the influence of purified Lp(a) lipoprotein on plasminogen activation by tissue plasminogen activator (t-PA) in the presence of soluble fibrin. Increasing concentrations of Lp(a) lipoprotein (0-32 mg/dl) did not inhibit plasminogen activation by t-PA in the presence of thrombin or bathroxobin digested fibrinogen. When purified Lp(a) lipoprotein was added to whole blood, the degree of fibrin degradation obtained following standardized coagulation, as evaluated by the generation of D-dimer, was not reduced. D-dimer levels in plasma and in serum after standardized coagulation, as well as conventional parameters for evaluation of the fibrinolytic system, were determined in 10 individuals with high and 10 individuals with low levels of Lp(a) lipoprotein. No differences in the fibrinolytic parameters were observed between the groups. Thus, we found no evidence that Lp(a) lipoprotein interferes with the fibrinolytic process in the present experiments.

Impaired coagulation of fibrinogen due to digestion of the C-terminal end of the Aα-chain by human neutrophil elastase

Human neutrophil elastase (HNE) possesses fibrinogenolytic capacity, with a high susceptibility towards degradation of the A alpha-chain. To study the influence of HNE digestion of the A alpha-chain on the coagulation of fibrinogen by thrombin, fibrinogen was incubated with human neutrophil elastase (HNE). At intervals, thrombin clotting time (TCT) and clottability were determined and compared with the patterns obtained with SDS electrophoresis and Western blotting with subsequent immunostaining, using monoclonal antibodies against the N-terminal end and C-terminal half of the A alpha-chain. Apparently, initial HNE digestion of the fibrinogen molecule occurred from the C-terminal end of the A alpha-chain, and was associated with prolongation of the TCT. With further C-terminal degradation TCT became indefinite and the degradation products were no longer clottable. Finally, N-terminal degradation of the A alpha-chain was observed. The present observations suggest that initial HNE-digestion of fibrinogen occurs from the C-terminal end of the A alpha-chain, and that the C-terminal end is crucial for the coagulation of fibrinogen.

A novel principle for assessment of stimulated fibrinolysis

After venous occlusion (VO), D-dimer levels were measured by means of an ELISA technique, in citrated plasma clotted by thrombin and in serum from whole blood. D-dimer levels increased with duration of incubation (30 min to 24 hours). D-dimer values, both in clotted plasma and in serum (n = 12), incubated 4 hours at room temperature, correlated well with euglobulin clot lysis time (ECLT) (r = -0.85 and -0.89, respectively, p less than 0.002) and tissue plasminogen activator (t-PA) activity, (r = 0.82 and 0.83, respectively, p less than 0.002). D-dimer concentrations from plasma and serum (n = 25) were compared (r = 0.90, p less than 0.001). Healthy volunteers (n = 65) were tested to establish reference values in serum from post-occlusive whole blood samples incubated 4 hours prior to centrifugation. Finally, a patient group (n = 62) was examined. For the whole material (n = 152) such D-dimer concentrations correlated well with both ECLT (r = -0.85, p less than 0.001) and t-PA activity (r = 0.81, p less than 0.001). D-dimer levels in serum were determined by a latex agglutination test as well. These semi-quantitative values also correlated significantly with both ECLT (r = -0.86, p less than 0.001) and t-PA activity (r = 0.87, p less than 0.001). We conclude that measurement of D-dimer as described above, represents a simple and accurate method for assessment of global fibrinolytic activity following VO. The latex agglutination test is particularly suitable as a screening procedure.

The fibrin-solubilizing effect of fibrinogen as studied by light scattering

Abstract The solubility and polymerization of preformed human des-AA and des-AABB fibrin monomers (prepared from fibrinogen in urea by insolubilized reptilase or thrombin) was studied. In solutions of purified fibrinogen, des-AA fibrin to approximately 17% of the fibrinogen concentration could be kept solubilized (I=0.15, pH 7.4, temp.+20°C) If calcium ions (2.5 mM) were present, or if fibrinogen-boosted serum was used, only 3.5% were soluble. The solubility of des-AA fibrin monomers in fibrinogen-boosted, heat-defibrinogenated, citrated plasma was about 10%. Since the ionic strength of fibrinogen-boosted serum and fibrinogen-boosted, heat-defibrinogenated, citrated plasma was measured to be nearly identical, the higher fibrin solubility in the latter milieu is either due to: the presence of citrate, or the calcium-binding effect of citrate, or both. The corresponding figures for the solubility of des-AABB fibrin monomers were 13%, 3% and 8%, respectively. In the abscence of fibrinogen, identical polymerization rates for both types of fibrin monomers (as judged by light scattering at 605 nm) were observed, but the polymerization of des-AA fibrin monomers was more retarded by the presence of fibrinogen than was polymerization of des-AABB fibrin monomers, thus reflecting the higher solubility of the former. The present findings suggest that fibrinogen is capable of retarding fibrin polymerization on a molar basis.

A daily glass of red wine induces a prolonged reduction in plasma viscosity: a randomized controlled trial.

Moderate red wine consumption has been associated with decreased risk of coronary heart disease. Reduced plasma viscosity and fibrinogen levels have been launched as possible contributors to this risk reduction. The effect of moderate red wine consumption on plasma viscosity, however, has not been investigated in a prospective, randomized trial. We wanted to evaluate the effect of moderate red wine consumption on plasma viscosity, fibrinogen concentration and fibrinogen subfractions. Healthy, nonsmoking volunteers were assigned to consume one glass of red wine daily for 3 weeks in a prospective, randomized cross-over study. In the second 3-week period the volunteers abstained from alcohol use. The plasma viscosity, fibrinogen concentration and the distribution of the main fibrinogen subfractions were determined at inclusion, after wine drinking and after abstention. Plasma viscosity was reduced by 0.026 and 0.024 mPa.s in the two groups following wine intake (95% confidence interval, 0.009–0.043, P = 0.004; 95% confidence interval, 0.0083–0.039, P = 0.003). The decrease in plasma viscosity was maintained following 3 weeks of abstention. The fibrinogen concentration was reduced by 0.17 g/l following wine drinking in the group starting with abstention (95% confidence interval, 0.04–0.29, P = 0.01). The distribution of the fibrinogen subfractions remained unaltered. We conclude that a daily glass of red wine for 3 weeks significantly reduces plasma viscosity. Fibrinogen concentrations are also significantly reduced, when preceded by an abstention period. The decreased viscosity levels are maintained after 3 weeks of abstention, suggesting a sustained viscosity lowering effect of red wine.

Purification and insolubilization of reptilase for the preparation of human des-AA fibrin monomers in urea

Abstract Reptilase was extensively purified from crude Bothrops atrox venom, and insolubilized on CNBr-activated Sepharose 4 B. When human fibrinogen, I-125-labelled or unlabelled, was exposed to insolubilized highly purified reptilase in the presence of 2.5 M urea, pH 7.4, at +20°C for 1 h, complete transformation of fibrinogen to des-AA fibrin monomers (labelled or unlabelled) as judged by N-terminal amino acid analysis and FPA-RIA quantitation, occurred without gelation. Subunit chain pattern of mercaptolysed, carboxymethylated des-AA fibrin monomers in acid polyacrylamide gel electrophoresis substantiated that only fibrinopeptides A had been proteolysed, and that the B beta chain had a mobility similar to that of control fibrinogen. Agarose gel chromatography of the des-AA fibrin monomer preparation in 2.5 M urea demonstrated a symmetrical peak. When the urea concentration was reduced by dilution, the fibrin monomers had the same clottability and polymerization speed as control fibrinogen exposed to large amounts of reptilase. Storage for 4 weeks at +4°C or quick freezing in liquid nitrogen with subsequent thawing and testing, caused only slightly prolonged polymerization times. Identical results were obtained with both labelled and unlabelled des-AA fibrin monomers.

Degradation of the α-chain of fibrin by human neutrophil elastase reduces the stimulating effect of fibrin on plasminogen activation

Abstract The degradation of fibrin by human neutrophil elastase (HNE) and the interference of such degradation on the stimulating effect of fibrin on plasminogen activation by tissue plasminogen activator (t-PA) was studied. By using SDS electrophoresis and Western blotting with subsequent immunostaining with monoclonal antibodies, degradation of the fibrin molecule was monitored. This degradation was related to the stimulating effect on plasminogen activation. Degradation of the α-chain was seen to occur before degradation of the β- and γ-chains. On the α-chain it was found that C-terminal degradation occurred prior to visible degradation of the N-terminal end. This C-terminal degradation was associated with a fall in the stimulation of plasminogen activation, coinciding with a corresponding reduction in the polymerization of fibrin. With further degradation, including N-terminal proteolysis of the α-chain, the stimulating effect of fibrin was reduced to that of fibrinogen. Conclusions: Our results indicate that HNE degradation of the α-chain of fibrin occurs initially from the C-terminal end, affecting the polymerization of fibrin. This impaired polymerization may be important for the observed reduction in the t-PA mediated plasminogen activation.

D-dimers are degraded by human neutrophil elastase

Abstract To see if D-dimers were degraded by human neutrophil elastase (HNE), cross-linked fibrin was obtained by adding thrombin to purified fibrinogen in the presence of calcium ions and factor XIII, and the fibrin clot subsequently degraded by plasmin. Thereafter, the supernatant containing fibrin degradation products was removed and incubated with HNE. D-dimer levels were measured by two rapid semiquantitative tests, a latex agglutination test and the Nycocard immunofiltration test, and a quantitative ELISA-method. With increasing incubation time, D-dimer levels as measured by the latex and Nycocard tests rapidly decreased and subsequently became undetectable, while the ELISA D-dimer values remained essentially unchanged. By using SDS-electrophoresis and immunoblotting, the degradation of plasmic derivatives of cross-linked fibrin by HNE was visualised. We conclude that in a purified system, D-dimers formed during plasmin mediated lysis of cross linked fibrin are further degraded by HNE. Such HNE degradation reduces the D-dimer concentration as measured by rapid semiquantitive tests, and may be partly responsible for discrepant results when using different D-dimer assays.

Does qualitatively altered fibrinogen contribute to the increased heparin precipitable fraction (HPF) in acute myocardial infarction (AMI)

The amount and protein composition of heparin precipitable fraction (HPF), and the plasma concentrations of fibrinogen, fibronectin, alpha 1-acid glycoprotein, alpha 1-antitrypsin, haptoglobin and C-reactive protein (CRP) were determined in healthy subjects as well as during the course of acute myocardial infarction (AMI). In all samples tested, HPF consisted nearly exclusively of fibrinogen and fibronectin. In the small precipitates from healthy subjects, approximately equal amounts of these two proteins were recovered. During the first days of AMI, plasma fibrinogen increased 2-3 fold. At the same time, however, the amount of HPF increased 5-10 fold. This increase was associated with increased precipitation of fibrinogen, whereas no simultaneous increase in fibronectin was found. In fact, a slight drop in plasma as well as HPF-fibronectin was regularly observed during this period. During the following week HPF returned to normal values, whereas the plasma fibrinogen remained elevated. No satisfactory explanation for this discrepancy can be offered at present. Thus, no evidence could be provided that other acute phase proteins than fibrinogen itself were involved. Some experiments, however, indicated qualitative changes in the fibrinogen participating in the HPF precipitation. Further studies are necessary to clarify this point. Such studies are in progress.