On the separation of fibrinogen degradation products D and E.

Separation of fibrinogen degradation products D and E by means of gel chromatography cannot be achieved at neutral pH even in the presence of high ionic strength of the elution buffer. It is assumed that fragments D and E are linked together in a complex preventing the separation despite different molecular weights of both components. By means of addition of chaotropic substances like 1 M Kl to the elution buffer clear separation of degradation products D and E on Sephadex G-200 columns can be achieved.

Adsorption of fibrinogen and fragment D of fibrinogen onto the insolubilized alpha-chain of fibrin.

After thrombin treatment insolubilized fibrinmonomer, which is obtained from insolubilized fibrinogen covalently bound to agarose, adsorbs soluble fibrin and its derivatives from solutions. The immobilized proteins are attached to the agarose by the 'A' alpha-chain. After reduction of the disulfide bridges the beta- and gamma-chains can be removed from the agarose. After thrombin treatment the immobilized alpha-chain adsorbs fibrinogen and fragment D. To some extent the beta- and gamma-chain do not seem necessary for the adsorption. The amount adsorbed increases, when thrombin treatment of the insolubilized protein follows the reduction process. This may indicate that the fibrinopeptides 'A' of the insolubilized alpha-chain are better accessible after the removal of the beta- and gamma-chains.

Separation of fibrinogen degradation products (FDP) by means of adsorption to insolubilized fibrinmonomer (FM-ag).

The phenomenon of complex formation between fibrinmonomer and fibrinogen degradation products was investigated by means of adsorption of FDP to insolubilized thrombin-modified fibrinogen (FM-ag). Since it could be demonstrated that there are different adsorption characteristics for early FDP and late FDP, the possibility of separation of FDP by means of affinity chromatography on FM-ag columns was evaluated using plasmic digests of 3H-Ac-labelled fibrinogen. The identification of FDP was performed by disc-electrophoresis. The results indicate that the adsorption of early FDP is comparable to the behaviour of fibrinogen, whereas late FDP show essential difference in the affinity towards FM-ag, evident by the result that fragment E adsorbs only to a minimal extents. Fragments D and E derived from fibrinogen as well as from non-crosslinked fibrin, revealed identical adsorption characteristics. Under specified conditions the procedure is suitable as a preparative method for the separation of fragments D and E.

Studies on chemically modified fibrinogen.

Treatment of fibrinogen with maleic acid anhydride renders fibrinogen unclottable depending on the degree of modification of the molecule. According to radioactive studies the release of fibrinopeptides by thrombin or reptilase is undisturbed. The incoagulability is due to inhibition of the polymerization process of fibrinmonomers derived from modified fibronogen, mainly caused by the increase of electronegative charges upon the fibrogen molecule. According to discelectrophoretic analysis modified fibrinogen fails to produce fragments D and E following plasmic digestion, however, may be degraded to high molecular weight products. Modified fibrinogen reveals some similarities to abnormal fibrinogens in congenital dysfibrinogenemia with regard to its functional properties.

Hemostatic abnormalities and the severity of illness in patients at the onset of clinically defined sepsis. Possible indication of the degree of endothelial cell activation?

OBJECTIVE: To find out whether changes within the hemostatic system are related to the severity of illness and organ failure in patients at the onset of clinically defined sepsis and to find some indications for the contribution of endothelial cell activation or perturbation to the patient's status. The following measurements were undertaken: Acute Physiology and Chronic Health Evaluation (APACHE) II score, multiple organ failure (MOF) score, plasma levels of thrombin-antithrombin III complexes (TAT), antithrombin III (AT III), protein C antigen, factor XII, and plasminogen activator inhibitor type 1 antigen (PAI-1), neopterin, and interleukin 6 (IL-6). DESIGN: A prospective case series study. SETTING: Intensive care unit (ICU) of the Department of Internal Medicine, Justus Liebig University, Giessen, Germany. PATIENTS: 28 consecutive patients (11 females, 17 males; mean age 58 years) with clinically defined sepsis. Eleven patients were admitted from the surgical ICU (9 after elective surgery, 2 after trauma surgery). The operations were done 1-26 days (mean 14 days) prior to the onset of sepsis. MAIN RESULTS: At the onset of sepsis we found elevated plasma levels of TAT, PAI-1, neopterin, and IL-6, and lowered plasma levels of AT III, factor XII, and protein Cantigen. Neopterin, PAI-1, IL-6, and factor XII showed a statistically significant correlation with the APACHE II score. The MOF score is significantly correlated with IL-6 and neopterin. The extent of hemostatic abnormalities was related to increasing levels of IL-6. CONCLUSIONS: Clinical evidence of a septic process is most likely to be preceded by activation of the hemostatic system, the vascular endothelium, and the monocyte/macrophage system. IL-6 may have a regulatory function for hemostasis in inflammation. Laboratory monitoring could be helpful in deciding whether to start early intensive therapy in patients at risk for sepsis.

Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled, double-blind multicenter trial plus a meta-analysis on all randomized, placebo-controlled, double-blind trials with antithrombin III in severe sepsis.

OBJECTIVES: To evaluate the safety and potential efficacy of antithrombin III (AT III) in reducing mortality in patients with severe sepsis. DESIGN: Prospective, randomized, placebo-controlled, double-blind, phase II, multicenter, multinational clinical trial. SETTING: Seven academic medical center intensive care units (ICU) in Belgium, Denmark, the Netherlands, Norway and Sweden. PATIENTS: 42 patients with severe sepsis who received standard supportive care and antimicrobial therapy, in addition to the administration of AT III or placebo. INTERVENTIONS: Patients received either an intravenous loading dose of 3000 IU AT III followed by a maintenance dose of 1500 IU every 12 h for 5 days or equivalent amounts of placebo. MEASUREMENTS AND RESULTS: All patients were evaluated for safety and for 30-day all-cause mortality. CONCLUSIONS: The administration of AT III was safe and well-tolerated. It was followed by a 39 % reduction in 30-day all-cause mortality (NS). The reduction in mortality was accompanied by a considerably shorter stay in the ICU. Patients treated with AT III exhibited a better performance in overall severity of illness and organ failure scores (Acute Physiology and Chronic Health Evaluation II, multiple organ failure, organ system failure), which was noticeable soon after initiation of treatment. Patients treated with AT III demonstrated a better resolution of pre-existing organ failures and a lower incidence of new organ failures during the observation period. A meta-analysis comprising this and two other double-blind, placebo-controlled trials with AT III with a total of 122 patients suffering from severe sepsis confirms the positive trend. The results of the meta-analysis demonstrate a 22.9 % reduction in 30-day all-cause mortality in patients treated with AT III. Although still too small to be confirmative, the meta-analysis clearly points to the fact that a sufficiently powered phase III trial is warranted to prove whether AT III has a beneficial role in the treatment of severe sepsis.

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.

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.

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.

Effects of prostacyclin during cardiopulmonary bypass in men on plasma levels of beta-thromboglobulin, platelet factor 4, thromboxane B2, 6-keto-prostaglandin F1 alpha and heparin.

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 (PGI2), beginning two minutes before extracorporeal circulation (ECC) and ending together with ECC. Compared to the placebo-treated patient group (n = 20), PGI2-treatment significantly reduced the ECC-induced release of platelet alpha-granule proteins, beta-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 PGI2-treated patients. Application of PGI2 had no effect on the increase in thromboxane B2 (TxB2) plasma levels, which amounted to 0.6 ng/ml at the end of ECC. PGI2-treatment resulted in significantly elevated plasma concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (2.1 ng/ml) throughout the infusion off prostacyclin. 6-keto-PGF1 alpha plasma levels increased up to 1.2 ng/ml in the control group patients, indicating a stimulation of endogenous PGI2 formation during ECC.

[Microcirculation and hemostasis in inflammatory processes. Modulation by administration of physiologic protease inhibitors as a therapeutic approach]. / Mikrozirkulation und Hämostase bei entzündlichen Prozessen. Modulation durch den Einsatz physiologischer Protease-Inhibitoren als therapeutischer Ansatz.

BACKGROUND: The course of an inflammatory process is based upon complex interactions between the vessel wall and the humoral or cellular compounds of the vascular content as a consequence of a defense reaction. There are no substantial differences between the pathophysiology of local or of whole body inflammation on the molecular and cellular level. Sepsis, which is clinically regarded as a systemic inflammatory process requires the broad therapeutical spectrum of nowaday intensive care medicine, but still has a high mortality. The pathophysiology and clinical examples for both systemic and local inflammatory reactions are presented in this paper. Thereby, similar interactions between the vascular endothelium, the mediator systems to which the hemostatic system has to be considered as a part of, and the microcirculation remain in the foreground at first. Based on that, the use of polyvalent protease inhibitors in the therapy of local or systemic inflammatory reactions of different origin will be discussed. The spotlight falls on physiological inhibitors of the hemostatic and complement system, antithrombin III and C1-esterase inhibitor, which may have a regulatory function within these systems because of their multiple targets. CONCLUSION: The possibility of an adjuvant therapy of local or generalized inflammatory processes with physiologic protease inhibitors seems to be very promising. Nevertheless, at yet the substantial mechanisms of action are not fully understood.

[Coronary risk factors in chronic hemodialysis patients]. / Koronare Risikofaktoren bei chronisch hämodialysierten Patienten.

BACKGROUND: In contrast to persons with normal renal function, coronary risk factors or indicators until yet could not clearly be defined in renal insufficiency. PATIENTS AND METHODS: 30 patients under chronic hemodialysis therapy were investigated; 15 patients with severe coronary artery disease and 15 patients with normal coronary angiogram were compared. Numerous factors of the manner of living (diet, smoking behaviour etc.) were registered and glucose and lipid metabolism, hemostatic and fibrinolytic system as well as blood pressure level were investigated. RESULTS: Besides higher HDL-cholesterol and tissue plasminogen activator (TPA) levels in patients without coronary heart disease, no significant difference could be found between both groups. The higher HDL levels were mainly due to the higher percentage of women in the coronary healthy group. There was no evidence of insulin resistance as a major pathogenic factor in the group with coronary heart disease. The blood pressure levels were not significantly different in both groups. CONCLUSION: Our quantitative examination of accepted or suspected coronary risk factors revealed no entity which turned out to be a reliable risk indicator for practical purposes.

[Disseminated intravascular coagulation and circulatory shock]. / Disseminirovannoe vnutrisosudistoe svertyvanie i tsirkuliatornyi shok.

The authors describe the pathophysiology of disseminated intravascular coagulation and the coagulopathy of utilization. The clinical and laboratory data on different types of shock are described. The efficacy of different antithrombotic agents in shock with disseminated intravascular coagulation is shown.