Günter Huhle

Matrix-metalloproteinases and their inhibitors are elevated in severe sepsis: prognostic value of TIMP-1 in severe sepsis.

The enzyme group of matrix metalloproteinases (MMPs) and their inhibitors, so-called tissue inhibitors of matrix-metalloproteinases (TIMPs), are crucial mediators responsible for wound repair after parenchymal damage. Little is known about the role of MMPs and TIMPs in severe sepsis. The aim of the present study was therefore to investigate their levels in patients with severe sepsis and to examine their association with prognosis. MMP-2, MMP-9, TIMP-1, TIMP-2 and interleukin-6 (IL-6) plasma levels were measured by ELISA methods in 37 patients on day 1 of severe sepsis. 37 healthy volunteers served as controls. Levels of MMP-9, TIMP-1, TIMP-2 and IL-6 in septic patients were significantly higher compared to healthy controls (p<0.001), whereas MMP-2 levels were not different in patients and controls. TIMP-1 levels were significantly higher in non-survivors (4675±435 ng/ml, mean±SEM) compared to survivors of severe sepsis (3201±249 ng/ml; p<0.01). Septic patients with TIMP-1 values >3200 ng/ml were 4.5 times more likely to die than patients with lower values (RR = 4.5; 95% CI 1.14–17.6, p = 0.014). Our results indicate that MMP-9, TIMP-2 and TIMP-1 are elevated in severe sepsis. Furthermore, TIMP-1 may serve as a useful laboratory marker to predict the clinical outcome of patients presenting with severe sepsis.

CUTANEOUS REACTIONS TO ANTICOAGULANTS. RECOGNITION AND MANAGEMENT

Anticoagulant-induced skin reactions appear as allergic or necrotic responses to vitamin K antagonists or heparins. Cutaneous allergy has been reported with danaparoid sodium and flush reactions have been seen with hirudins. The pathogenesis of the reactions differs between drugs. Generally, they occur between days 3 to 10 after the start of treatment, but may also occur later. In patients experiencing necrosis with a vitamin K antagonist, concomitant protein C deficiency, protein S deficiency or lupus anticoagulant has been described, whereas the precise mechanism of the other reactions is unknown. In patients with allergic reactions to heparins, cutaneous tests may help to identify alternative anticoagulants. Such a test cannot be performed in patients with skin necrosis.In patients with heparin-induced skin reactions danaparoid sodium may be used after negative intracutaneous testing in some patients and a hirudin may be used without testing in all patients. Heparin-induced skin necrosis has been reported to be mediated by immunologic mechanisms and to be associated with a high frequency of heparin-induced thrombocytopenia type II. Surgical excision of the necrosis may be required. If further anticoagulation is indicated in any patient, extreme caution has to be taken when restarting oral anticoagulants. Because a large number of anticoagulants available today, safe treatment of all patients experiencing anticoagulant-induced skin reactions is feasible.

A new therapeutic option by subcutaneous recombinant hirudin in patients with heparin-induced thrombocytopenia type II: A pilot study

We prospectively studied 15 patients suffering from acute heparin-induced thrombocytopenia (HIT) type II with and without thromboembolic events and 4 patients with anamnestically known HIT type II recurrently requiring thromboprophylaxis in order to develop new therapeutic strategies by subcutaneous recombinant hirudin administration. Patients with acute venous or arterial thromboembolism were treated with aPTT-controlled intravenous (mean: 19.3 days) followed by subcutaneous r-hirudin (mean: 22.5 days). Patients without thromboembolism were treated with subcutaneous r-hirudin (mean: 25.9 days). Four patients were readmitted to subcutaneous r-hirudin (mean: 32 days). When r-hirudin was administered subcutaneously following intravenous treatment, mean baseline (prior to the injection) and mean peak (1.5-2.5 hours after the injection) aPTT ratios were 1.1 (+/-0.2) to 1.7 (+/-0.48) and 2. 48 (+/-0.43) to 2.52 (+/-0.4) times normal value, respectively. Mean baseline and mean peak ECT ratios were 1.2 (+/-0.12) to 1.9 (+/-0. 22) and 2.2 (+/-0.25) to 2.6 (+/-0.11) times the upper normal value, respectively. When r-hirudin was initially administered subcutaneously, mean baseline and mean peak aPTT ratios were 1.41 (+/-0.25) to 1.61 (+/-00.28) and 1.88 (+/-0.26) to 2.06 (+/-0.09) times the normal value, respectively. Mean baseline and mean peak ECT ratios were 1.25 (+/-0.2) to 1.5 (+/-0.38) and 2.01 (+/-0.21) to 2.23 (+/-0.25) times the upper limit of normal, respectively. Patients who received recurrent subcutaneous r-hirudin had mean baseline and peak aPTT values of 1.5 (+/-0.35) to 1.75 (+/-0.156) and 2.0 (+/-0.33) to 2.1 (+/-0.18) times the normal value, respectively. Mean baseline and peak ECT ratios were 1.3 (+/-0.26) to 1.65 (+/-0.09) and 1.94 (+/-0.256) to 2.7 (+/-0.23) times the upper limit of normal, respectively. The overall cumulative incidence of r-hirudin antibodies was 12/19 (63%) with a significant accumulation of r-hirudin in antibody-positive patients compared to antibody-negative patients (p<0.05). No patient suffered a new thromboembolic or major bleeding event. Subcutaneous administration of recombinant hirudin provides a long-term thromboprophylaxis regimen in HIT type II patients after passivation of acute thromboembolism.

Improved laboratory confirmation of heparin-induced thrombocytopenia type II. Time course of antibodies and combination of antigen and biologic assays.

We studied whether laboratory confirmation of heparin-induced thrombocytopenia (HIT) can be improved after antigen clearance by determining free antibody and combining the results of an antigenic and a biologic assay. Blood samples taken over 40 days in 14 patients with HIT with thromboembolism underwent fluorescence-linked immunofiltration and the carbon 14-serotonin release assays. Of the 14 patients, 11 showed positive results in both assays at day 1 after stopping heparin. The 3 patients with negative results seroconverted in one or both assays during the subsequent 7 days. Combining the positive results of the assays increased the sensitivity from 85% at day 1 to 100% at day 7. Assay results became negative in all patients within 40 days. The platelet count normalized between days 2 and 9 after withdrawal of heparin. It is assumed that the free antibody can be detected after withdrawal of heparin and after clearance of the platelet factor 4-heparin complex in patients with HIT.

Re-exposure to recombinant (r)-hirudin in antihirudin antibody-positive patients with a history of heparin-induced thrombocytopenia

Patients with a history of heparin‐induced thrombocytopenia (HIT) and antibodies to hirudin were re‐exposed to recombinant (r)‐hirudin for prophylaxis of thromboembolism. Four patients were re‐exposed to 2 × 25 mg of subcutaneous r‐hirudin for 8–27 d. Two patients were re‐exposed once, one patient twice and one four times. Re‐exposure was well tolerated in all patients and no thromboembolism occurred. Antihirudin IgG (4/4 patients), IgA and IgM (1/4 patients) antibody levels increased. Baseline ecarin clotting times showed high variability. Patients with antibodies to hirudin may be re‐exposed but anticoagulant monitoring is mandatory.

Quantitative Determination of PEG-Hirudin in Human Plasma Using a Competitive Enzyme-Linked Immunosorbent Assay

Polyethylene glycol (PEG) coupled r-hirudin mutein is determined by biological methods-the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-hirudin in human plasma. A PEG-hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-hirudin were between 12 and 22%. The correlation of the concentration of PEG-hirudin determined with the ELISA and the ecarin clotting time was r = 0.902. No interactions between unfractionated heparin, low molecular-weight heparin, or phenprocoumon and PEG-hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.

Niedermolekulare Heparine Prophylaxe und Therapie thromboembolischer Erkrankungen

LerninhalteDer Wert der Thromboembolieprophylaxe in der perioperativen Medizin und in der inneren Medizin ist eindeutig belegt. Eine Unterlassung der Prophylaxe bei Patienten mit erhöhtem Thromboembolierisiko stellt einen Behandlungsfehler dar. Die Therapie der akuten tiefen Beinvenenthrombose muß stationär erfolgen. Die subkutane Verabreichung von konventionellem Heparin in hoher Dosierung und aPTT-adjustiert ist der kontinuierlichen intravenösen Dauerinfusion gleichwertig. Subkutanes hochdosiertes niedermolekulares Heparin ist möglicherweise zur Therapie der frischen tiefen Venenthrombose dem konventionellen Heparin überlegen. Die Heparin-induzierte Thrombozytopenie Typ II ist eine seltene aber schwerwiegende Nebenwirkung. Maßnahmen zur Früherkennung dieser Nebenwirkungen gibt es nicht. Die Therapie besteht in einem Wechsel der Antikoagulation auf nicht heparinhaltige Antikoagulantien.

Purification of the monoclonal heparin antibody H-1.18

An antibody of the (immunoglobulin) IgG1 subclass against heparin was purified. Here we report on the purification of the heparin antibody. Ammonium sulfate precipitation was performed and showed a high purity of the precipitate. In the heparin radioimmunoassay it showed a high heparin binding. Capillary electrophoresis showed that albumin and other proteins were separated from the heparin antibody. The purification method allowed a large scale production of the heparin antibody.

Analysis of Heparin Binding to Human Leukocytes Using a Fluorescein-5-Isothiocyanate Labeled Heparin Fragment

The binding of LMWH-tyr-FITC to granulocytes, monocytes, and lymphocytes was analyzed by flow cytometry using a low-molecular-weight heparin (LMWH) labeled with fluorescein-5-isothiocyanate (FITC). FITC was covalently bound to tyramine, which was synthesized to LMWH by endpoint-attachment (Malsch et al.: Anal Biochem 217:255-264, 1994). The binding was rapid, specific, dose-dependent, saturable, and reversible. To investigate the molecular weight dependence of heparins, heparin-derived di- to dodecasaccharides were used. With decreasing molecular weight, the amount of oligosaccharides increased; these were bound to granulocytes, monocytes, and lymphocytes (r = -0.77). The degree of sulfation of non-heparin glycosaminoglycans influenced the binding to leukocytes. Decreasing the degree of sulfation decreased the binding. The pentasaccharide did not bind as strongly as the other heparin-derived oligosaccharides, indicating an AT III-independent mechanism. Two classes of heparin binding sites were identified on granulocytes and one class of binding sites on monocytes and lymphocytes. The lowest amount of LMWH-tyr-FITC detected was 1 ng on granulocytes, 0.18 ng on monocytes and 0.01 ng on lymphocytes. The data suggest that heparin and other sulfated polysaccharides may play a role in the physiology of thrombosis, arteriosclerosis, and inflammation by binding to granulocytes, monocytes, and lymphocytes.

Der Einfluss von Zytokinen auf die Endothelzellfunktion: das Endothel als Motor der Sepsis?

Unter physiologischen Bedingungen üben Endothelzellen eine Reihe von Funktionen aus, die zum Erhalt der Homöostase von Bedeutung sind: Hierzu gehören: 5 Hemmung der Blutgerinnung, 5 Koordination der Migration von Zellen aus dem Blut in das Gewebe, 5 Expression endothelialer Adhäsionsmoleküle, 5 Synthese chemotaktisch wirksamer Zytokine und Chemokine, 5 Regulation der Mikrozirkulation und der Vasopermeabilität.