Gert Müller-Berghaus

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells

CD40 ligand (CD40L, CD154), a transmembrane protein structurally related to the cytokine TNF-α, was originally identified on stimulated CD4+ T cells, and later on stimulated mast cells and basophils. Interaction of CD40L on T cells with CD40 on B cells is of paramount importance for the development and function of the humoral immune system. CD40 is not only constitutively present on B cells, but it is also found on monocytes, macrophages and endothelial cells, suggesting that CD40L has a broader function in vivo. We now report that platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo. Like TNF-α and interleukin-1, CD40L on platelets induces endothelial cells to secrete chemokines and to express adhesion molecules, thereby generating signals for the recruitment and extravasation of leukocytes at the site of injury. Our results indicate that platelets are not only involved in haemostasis but that they also directly initiate an inflammatory response of the vessel wall.

MONITORING OF RECOMBINANT HIRUDIN: ASSESSMENT OF A PLASMA-BASED ECARIN CLOTTING TIME ASSAY

Assay conditions of a plasma-based ecarin clotting time (ECT) were evaluated and the precision of the ECT in monitoring plasma levels of r-hirudin assessed. The snake venom enzyme ecarin converts prothrombin to meizothrombin possessing only weak coagulant but strong esterase activity. In r-hirudin containing plasma samples, meizo thrombin is rapidly neutralized by r-hirudin resulting in a dose-dependent prolongation of the clotting times. Among the different assay conditions tested, addition of 50 microliters of ecarin (4 U/ml) to 100 microliters of undiluted citrate-anticoagulated plasma gave optimal results with respect to precision and reproducibility. The measuring range of the ECT performed in this way is about 0.02-5.0 micrograms/ml r-hirudin. In vitro studies performed on r-hirudin-spiked plasma samples of 50 healthy individuals demonstrate remarkably low interindividual differences in r-hirudin responsiveness as indicated by CV-values below 5% and 7% at r-hirudin concentrations between 0-3 micrograms/ml and 4-5 micrograms/ml, respectively. The specificity for r-hirudin of the ECT is further demonstrated by the strong correlation (r = 0.94) between the results of a chromogenic assay and the ECT-measured r-hirudin concentrations obtained on 67 ex vivo blood samples. Depending on the concentration of r-hirudin the ECT is sensitive to plasma levels of prothrombin. In the absence of r-hirudin the critical prothrombin concentration was found to be 20% but increasing to 60% in the presence of 2.0 micrograms/ml r-hirudin. A fibrinogen concentration of 50 mg/dl was found to be the minimal concentration independent of the r-hirudin concentration. The precision of the ECT in measuring plasma levels of r-hirudin is not influenced by treatment with heparin, aprotinin or oral anticoagulants. The data demonstrate that the ECT is a rapid and easily perfor mable clotting assay which allows accurate measurement of r-hirudin plasma levels. ECT-monitored hirudin treatment will help to establish optimal dose regimens that are more efficacious but still as safe as heparin.

A case report on the use of recombinant hirudin as an anticoagulant for cardiopulmonary bypass in open heart surgery

We present a patient with coronary heart disease and a heparin- induced thrombocytopenia , who was successfully treated by coronary ar- tery bypass grafting (CABG) using recombinant hirudin as an anticoagu- lant for cardiopulmonary bypass (CPB) instead of heparin. (Eur J Car- dio-thorac Surg (1996) 10: 386-388)

Heparin-related thrombosis despite normal platelet counts in vascular surgery

BACKGROUND Acute graft thrombosis is a severe complication in vascular surgery that may require limb amputation or even cause death. Because nearly all patients undergoing vascular surgery have had previous exposure to heparin, the presence of heparin-related anti-platelet antibodies typical for heparin-associated thrombocytopenia (HAT) is one underlying possible mechanism of acute graft thrombosis. Although thrombocytopenia is a typical finding of HAT, it is not clear whether the occurrence of clinically important HAT is necessarily associated with thrombocytopenia. PATIENTS AND METHODS Ten out of 246 patients undergoing vascular surgery were diagnosed with HAT because of otherwise unexplained acute graft thrombosis that required recurrent surgical interventions. RESULTS In all of the 10 patients, heparin-related anti-platelet antibodies were detected although the platelet counts were within the normal range. When HAT was diagnosed, heparin administration was stopped and, after autoantibody cross-reactivity with the heparinoid Danaparoid had been excluded, anticoagulation was continued using this anticoagulant. After heparin therapy was discontinued none of the 10 patients developed further thrombotic complications. CONCLUSION The data presented demonstrate clearly that a normal platelet count does not exclude the possibility of HAT. As a consequence of this, HAT should be suspected in patients who develop thrombotic complications during heparin treatment, regardless of the actual platelet counts.

Physicochemical, immunochemical and functional comparison of human S-protein and vitronectin evidence for the identity of both plasma proteins

The comparison of the complement inhibitor s-protein, isolated from human plasma, with vitronectin, a serum spreading factor, revealed a high degree of similarity of both proteins with respect to molecular weight, band pattern in polyacrylamide gels in the presence of sodium dodecyl sulfate and amino acid composition. While radiolabeled S-protein was precipitated by antiserum against vitronectin, both proteins exhibited precipitin lines of complete identity in double immunodiffusion analysis when tested mutually against antisera of the appropriate components. The functional property of vitronectin to promote cell spreading of fibroblasts was also documented for purified S-protein. These findings indicate a high degree of similarity with respect to structural and functional properties of S-protein and vitronectin and hence may implicate that both proteins are identical.

Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin

Summary Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N‐ and O‐linked glycans of the endothelial cell thrombin receptor. While N‐linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate‐like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin‐mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly‐cationic basic proteins, whereas the CSGAG‐deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O‐linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM. These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post‐translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell‐related anticoagulant potential in different vascular beds.

Recombinant hirudin as an anticoagulant during cardiac operations: experiments in a pig model.

OBJECTIVE The efficacy and safety of recombinant hirudin (r-hirudin) compared with heparin as an anticoagulant during open-heart surgery has been studied in a pig model. METHODS A total of 18 Göttingen minipigs were randomly divided into three treatment groups and subjected to cardiopulmonary bypass for 1 h. Heparin-treated animals received a bolus of unfractionated heparin of 400 IU/kg body weight. Recombinant hirudin was given by a bolus injection of 1 mg/kg body weight, followed by a 1 h lasting infusion of 1 mg/kg body weight per h. The heparin-anticoagulated animals and one group of the hirudin-treated animals additionally received aprotinin at a dosage of 17500 KIU/kg body weight (KIU, kallikrein inhibitory units). In the second group of r-hirudin-treated animals, the aprotinin was replaced by saline. RESULTS The extracorporeal circuit remained patent for a 1 h pump period in all of the animals studied. There was no evidence of vascular occlusion or clot formation in the r-hirudin-treated animals. The anticoagulant efficacy of the hirudin protocol used is further demonstrated by the results of electron-microscopical scans of the pump-line filters. Fibrin deposits were visible only in the heparin-treated animals and not in r-hirudin-treated pigs. Despite this strong anticoagulant effect, there was no evidence of an increased bleeding tendency in r-hirudin-treated pigs. Moreover, histological studies showed a statistically significant (P < 0.05) higher incidence of tissue bleeding in the heparin/aprotinin-treated animals compared with the r-hirudin/aprotinin-treated pigs. Studying the platelet function, a statistically significant (P < 0.01) better preserved ADP- and collagen-induced platelet aggregation was seen in the r-hirudin/aprotinin-treated animals when compared with heparin/aprotinin-treated animals. CONCLUSIONS These data demonstrate that r-hirudin can be used successfully as an alternative anticoagulant to heparin during cardiac operations including cardiopulmonary bypass. The better preservation of platelet function suggests that r-hirudin may reduce the postoperative risk of bleeding.

Post-operative course of coronary artery bypass surgery patients who pre-donate autologous blood

BACKGROUND Pre-operative autologous blood donation is used to reduce the need of allogeneic blood in patients undergoing coronary bypass surgery operations, but it is not clear what impact the blood donation has on the post-operative course of these patients. METHODS We studied the post-operative course of 210 patients who pre-donated autologous blood before their coronary bypass operation (donors) and of 67 patients who were eligible to pre-donate but did not (controls). RESULTS The clinical variables and the technical operative parameters of the patients in the two groups were similar. There was no significant difference between the duration of assisted ventilation post-operatively (756 +/- 197 vs. 802 +/- 395 min; P=0.54) or length of stay in the intensive care unit (1.8 +/- 1.1 vs. 1.7 +/- 0.9 days; P=0.52) of the two groups. The number of autologous units of packed red cells and of fresh frozen plasma (FFP) received by the donors was significantly higher than the number of units of allogeneic packed red cells (1.5 +/- 0.9 vs. 0.3 +/- 0.9; P=0.001) and the units of homologous FFP received by the controls (2.3 +/- 0.8 vs. 0.6 +/- 1; P=0.001). CONCLUSIONS We found no evidence that autologous blood donation exerted a negative influence on the post-operative course of patients undergoing coronary bypass surgery. Patients who pre-donated blood received no allogeneic blood products, but the number of autologous blood products received by donors was higher than the number of blood products received by patients who did not pre-donate.

Therapie mit Blutkomponenten und Plasmaderivaten in der Geburtshilfe

ZusammenfassungAkute Blutungen und angeborene oder erworbene Gerinnungsstörungen stellen in der Geburtshilfe die Hauptindikationen für die Therapie mit zellulären und plasmatischen Blutkomponenten dar. In der akuten Blutungssituation, in der Erythrozytenkonzentrate und Frischplasma substituiert werden müssen, ist ein regelmäßiges Gerinnungsmonitoring, das neben den Globaltesten, der Bestimmung der Thrombozytenanzahl auch die Fibrinogen- und Faktor-V-Bestimmung umfassen sollte, eine wichtige Voraussetzung um begleitende oder entstehende Gerinnungsstörungen frühzeitig zu erkennen. In Abhängigkeit von der jeweiligen klinischen Symptomatik und der bestehenden Grunderkrankung ist dann eine gezielte Substitution mit Frischplasma, Thrombozyten und gereinigten Gerinnungsfaktoren möglich. Im folgenden Kapitel werden für die häufigsten hämostaseologischen Störungen in der Geburtshilfe entsprechende Diagnose- und Therapieschemata besprochen. Die Gliederung orientiert sich dabei an der jeweiligen klinischen Situation und den laboranalytisch meßbaren Gerinnungsveränderungen.