Rudolf Egbring

Effect of elastase-like and chymotrypsin-like neutral proteases from human granulocytes on isolated clotting factors.

Abstract Elastase-like and chymotrypsin-like proteolytic enzymes isolated from human granulocytes were investigated for their influence on several purified clotting factors. The elastase-like proteases induce a rapid destruction of fibrinogen, factor VIII, XII and XIII activity whereas a moderate effect on factor V activity is observed. The chymotrypsin-like enzymes show a rapid inactivation of factor VIII, moderate effect on factor VII and XII and only weak activity against fibrinogen and factor XIII. Both groups of enzymes are ineffective against prothrombin whereas incubation with factor V leads to a transitory activation. The possible role of these enzymes in physiological and pathological hemostasis is discussed.

Participation and interactions of neutrophil elastase in haemostatic disorders of patients with severe infections

The prognosis of septicaemia depends on the occurrence of complications such as shock and coagulation defects. The damage to haemostasis is usually explained by the action of the main coagulation and fibrinolysis enzymes, thrombin and plasmin. This paper presents data concerning the role of a third protease, granulocytic elastase. 82 patients who had been admitted to our hospital with suspected septicaemia were examined. Septicaemia was proven in 22 patients by the growth of microorganisms in blood cultures, and was clinically diagnosed in 9 patients. The plasma levels of neutrophil elastase‐like protease complexed to a1antitrypsin (a1AT‐ELP) were measured by zone immunoelectrophoresis assay (ZIA). The a,AT‐ELP values were significantly increased in the 31 septic as compared to the 51 non‐septic patients. In patients with complicated septicaemia, negative correlations of a1AT‐ELP with factor XIII and the coagulation inhibitor antithrombin III were demonstrable. Among the patients with septic complications, the 3 who survived exhibited a dramatic decrease of a1AT‐ELP, whereas in the other 16 patients who died the levels remained elevated. It might be of therapeutic significance that in 9 patients receiving fresh plasma and AT Ill‐concentrate substitution for DIC the a1AT‐ELP levels dropped, whereas they remained high in the other septicaemia patients. There were no correlations between a1AT‐ELP and the a2antiplasmin‐plasmin complexes (a2AP‐Pl), but strong correlations with signs of coagulation. The data suggest an interaction of coagulation and elastase release, probably involving the Hageman factor.

Ulcerative colitis and Crohn's disease: factor XIII, inflammation and haemostasis.

An important role has been ascribed to plasma factor XIII (FXIII) in inflammation and wound healing. FXIII is necessary for fibrin stabilization and interacts with connective tissue and adhesive proteins and cells. In a prospective study, FXIII activity and parameters of coagulation, fibrinolysis and inflammation, were determined in patients with ulcerative colitis (UC; 13 active, 22 moderate) and Crohns disease (CD; 36 active, 45 moderate). FXIII levels were lower in active than in moderate UC and CD, and were < 70% of normal values in 7/13 patients with active UC, and in 7/36 patients with active CD, although the median values did not fall below the normal range. FXIII was somewhat higher in active UC patients responding to therapy. The FXIII levels were widely scattered, and low values appear to be due to greatly enhanced turnover. A correlation between FXIII and the systemic levels of markers of activation of haemostasis and inflammation was lacking, but there was a correlation with the extent of bowel involvement. FXIII levels were lower in the patients with involvement beyond the sigmoid colon in CU (p = 0.0045), and both small and large bowel segments in CD (p = 0.0223) patients. This points to local consumption and/or loss of FXIII within the inflamed tissue, and provides an argument for FXIII substitution in the treatment of acute episodes of inflammatory bowel diseases.

Die Bedeutung des Thrombin-Antithrombin III-Komplexes in der Diagnostik der Lungenembolie und der tiefen Venenthrombose — Vergleich mit Fibrinopeptid A, Plättchenfaktor 4 und β-Thromboglobulin

In 22 patients with suspected pulmonary embolism and 19 patients with suspected deep vein thrombosis, thrombin-antithrombin III complex (TAT) as an indicator of thrombin activation was measured using a newly developed ELISA. For comparison fibrinopeptide A (FPA), as a marker of an activated coagulation, as well as platelet factor 4 (PF4), and beta-thromboglobulin (beta-TG), as markers of platelet activation, were determined. In all patients in whom pulmonary embolism was confirmed by perfusion lung scan and in 15 of 16 patients in whom deep vein thrombosis was confirmed by phlebography, TAT exceeded the upper limit of normal (3.0 ng/ml). FPA was increased in 71% of the pulmonary embolism patients, PF4 in 53%, and beta-TG in 59%. The data for the patients with deep vein thrombosis were comparable. PF4 and beta-TG were increased in more than 25% of the normal controls, FPA in 17%, and TAT in 9%. TAT is very sensitive in detecting an activation of the coagulation system in patients with suspected thromboembolic events. The test, however, is not specific for thromboembolism; it only indicates an activation of the coagulation system. Acute pulmonary embolism or deep vein thrombosis would appear to be unlikely if TAT is normal. The measurement of TAT is easier and less susceptible to disturbances than that of FPA, PF4, and beta-TG.SummaryIn 22 patients with suspected pulmonary embolism and 19 patients with suspected deep vein thrombosis, thrombin-antithrombin III complex (TAT) as an indicator of thrombin activation was measured using a newly developed ELISA. For comparison fibrinopeptide A (FPA), as a marker of an activated coagulation, as well as platelet factor 4 (PF4), and β-thromboglobulin (β-TG), as markers of platelet activation, were determined. In all patients in whom pulmonary embolism was confirmed by perfusion lung scan and in 15 of 16 patients in whom deep vein thrombosis was confirmed by phlebography, TAT exceeded the upper limit of normal (3.0 ng/ml). FPA was increased in 71% of the pulmonary embolism patients, PF 4 in 53%, and β-TG in 59%. The data for the patients with deep vein thrombosis were comparable. PF 4 and β-TG were increased in more than 25% of the normal controls, FPA in 17%, and TAT in 9%.TAT is very sensitive in detecting an activation of the coagulation system in patients with suspected thromboembolic events. The test, however, is not specific for thrombembolism; it only indicates an activation of the coagulation system. Acute pulmonary embolism or deep vein thrombosis would appear to be unlikely if TAT is normal. The measurement of TAT is easier and less susceptible to disturbances than that of FPA, PF4, and β-TG.

Fibrinogen marburg a new genetic variant of fibrinogen

Summary A new case of congenital dysfibrinogenemia has been discovered in a 20 year old woman, who suffered from a severe postpartal hemorrhage after the delivery of her first child, followed by episodes of thrombosis. Coagulation studies revealed a prolongation of thrombin time, reptilase time was immeasurable. Thromboplastin time and partial thromboplastin time were slightly prolonged.Low fibrinogen levels were obtained by techniques, which depend on the coagulation velocity following addition of thrombin, while immunological procedures gave slightly diminished values of fibrinogen. Patients fibrinogen had a moderate inhibitory effect on the fibrin formation in normal plasma. However, inhibitors of the fibrinogen-fibrin conversion could not be detected. Coagulation factors were normal, fibrinolysis as well. The cause of the coagulation disorder was found to be a defect of the fibrinogen molecule, leading to an abnormal fibrin polymerization of patients fibrin monomers. The release of the fibrinopeptides in the paperelectrophoresis was normal. The defect of the fibrinogen molecule did not protect from thrombotic complications.The same defect could be found in the lower scale in patients father, 4 of her 7 brothers and sisters, and her son.ZusammenfassungIn Marburg wurde eine neue kongenitale Dysfibrogenaemie bei einer 20jährigen Patientin mit einer fakultativen haemorrhagischen Diathese entdeckt. Sie ist gemäß den beschriebenen bisherigen Untersuchungen charakterisiert durch eine deutlich verlängerte Thrombinzeit und eine unmeßbar verlängerte Reptilasezeit. Auch die Thromboplastinzeit und die partielle Thromboplastinzeit waren verlängert. Das Fibrinogen, mit Gerinnungsmethoden gemessen, war stark vermindert, während die immunologische und spektralphotometrische Bestimmung nur einen diskret erniedrigten Fibrinogenwert ergab. Plasma und isoliertes Fibrinogen der Patientin hatten auf die Gerinnung von Normalplasma und Fibrinogen einen geringen Hemmeffekt. Inhibitoren der Fibrinogen-Fibrinumwandlung ließen sich jedoch nicht nachweisen. Die isoliert bestimmten Gerinnungsfaktoren waren normal, ebenso die Fibrinolyse. Als Ursache der Gerinnungsstörung wurde ein isolierter Defekt des Fibrinogenmoleküls gefunden, der mit einer abnormen Fibrinpolymerisation der Patientenfibrinmonomere einhergeht. Die Freisetzung der Fibrinopeptide zeigte in der Papierelektrophorese keine Abweichung von der Norm. Die Anomalie schützte nicht vor thrombotischen Komplikationen. In geringem Ausmaß ließ sich diese Störung bei dem Vater der Patientin, bei dem Sohn und bei vier von sieben Geschwistern nachweisen.

Die vereinfachte radiologische Faktor-XIII-Bestimmung und ihre klinische Anwendung bei kongenitalem Faktor-XIII-Mangel (I)

ZusammenfassungDie Aktivitätsbestimmung des Faktor XIII durch Messung der Einbaurate von C-14-markiertem Putrescin in Casein ist eine Methode, die nach Änderung des Ansatzes und der Waschtechnik der für die Szintillationszählung vorbereiteten Proben auch im großen Rahmen durchfürhrbar ist. Die Waschung der Proben erfolgt nicht mehr in Milliporefiltern, sondern im sog. Batch-Verfahren. Die Faktor-XIII-Bestimmungen können bis zu 1% der Norm durchgeführt werden und bringen reproduzierbare Ergebnisse. Nach Substitution des Faktor XIII bei einem Patienten mit kongenitalem Faktor-XIII-Mangel kann die Plasmaaktivität mit 2 Ampullen des Faktor-XIII-Konzentrats auf 20% der Norm gebracht werden. Thrombozyten des Patienten nehmen nach Substitution keine meßbare Aktivität auf.SummaryThe method of factor XIII assay by incorporation of C-14 labelled putrescin into casein can be used as a large scale routine method if the concentration of the different components and the procedure for washing the samples have been changed.Instead of using millipore filter, washing was performed by the batch technique.This method is reproducible and allows the measurement of factor XIII concentration down to 1%.By employing a factor XIII concentrate for substitution in a case of congenital factor XIII deficiency the plasma level could be raised to 20% of the concentration present in normals. After substitution no measurable activity in the patients thrombocytes could be observed.

Degradation of human plasma fibrin stabilizing factor XIII subunits by human granulocytic proteinases.

Decreased activity of fibrin stabilizing factor XIII may occur in diseases with enhanced destruction of granulocytes. Haemorrhage and impaired wound healing may result. It has been shown by means of SDS-polyacrylamide gel electrophoresis that the neutral proteinases from human polymorphonuclear granulocytes, the Elastase Like Proteinase (ELP), and the Chymotrypsin Like Proteinase (CLP), are able to digest purified human plasma factor XIII. Both subunits, a and b, are affected at concentrations which might locally or systemically occur under pathophysiological conditions. Higher concentrations are required for the degradation of subunit b. Depending on the proteinases, the concentration used and the time of incubation, numerous split products were formed. To obtain comparable effects, the concentration of CLP had to be about twice that of ELP. Aprotinin had only a slight inhibitory effect on the two leukocyte proteinases. The results presented indicate that factor XIII is degraded and inactivated by granulocytic proteinases, both subunits being altered by these proteinases. Therefore the determination of subunit b may be helpful in differentiating between the proteolytic effect of thrombin which degrades only subunit a, and the granulocyte proteinases.

Veränderte Aktivitäten der Enzyme des Energiestoffwechsels in Thrombocyten von Patienten mit Lebercirrhose und Splenomegalie

Zusammenfassung1. Die Aktivitäten von 28 Enzymen aus verschiedenen Abschnitten des Energiestoffwechsels wurden in isolierten Thrombocyten von 17 gesunden Menschen und 15 Patienten mit gesicherter Lebercirrhose gemessen.2. Die für Gewebe bekannte Proportionskonstanz der Enzymaktivitäten des zentralen Segmentes der Glykolyse (TIM, GAPDH, PGK, GPM und EN) findet sich auch in den Thrombocyten als Ordnungsprinzip. Die mitochondrial lokalisierten Enzyme NAD-spezifische Isocitratdehydrogenase und Glycerophosphatoxydase wurden erstmalig in Thrombocyten mit relativ hoher Aktivität nachgewiesen.3. Die Plättchen von Patienten mit Lebercirrhose und Milzvergrößerung infolge portaler Hypertension zeigten signifikant erhöhte Enzymaktivitäten in allen untersuchten Stoffwechselwegen, insbesondere der mitochondrial lokalisierten Enzyme. Funktionell bedeutsam erscheinen der ausgeprägte Anstieg der Mg++-aktivierbaren ATPase, die gleichbleibende Aktivität der Fructose-6-Phosphatkinase und eine Verminderung der Lactatdehydrogenase. Bei der Patientengruppe mit Cirrhose ohne Milzvergrößerung lagen fast alle gemessenen Aktivitäten der Thrombocyten im Normbereich.4. Es wird ein Patient mit Lebercirrhose ohne Milzvergrößerung beschrieben, dessen Thrombocyten um den Faktor 8 höhere Aktivitäten fast aller gemessenen Enzyme aufwiesen. Auch in diesen Plättchen zeigte die Lactatdehydrogenase eine Aktivitätsverminderung gegenüber der Norm.5. Ein Enzymdefekt der Plättchen wie bei Thrombasthenie wurde bei Lebercirrhose nicht gefunden. Nach unseren Kenntnissen über die Enzymausstattung von Blutzellen handelt es sich bei den Thrombocyten mit höherer Enzymaktivität um eine jugendliche Zellpopulation. Die Pathogenese wird besonders hinsichtlich der Rolle der vergrößerten Milz diskutiert.Summary1. The activities of 28 enzymes from different pathways of energy producing metabolism were measured in the isolated platelets of 17 normal persons and 15 patients with proven cirrhosis of the liver.2. In mammalian tissues a constant proportion between the enzymes of the central segment of the glycolytic pathway (TIM, GAPDH, PGK, GPM, EN) has been described. This constant proportion has been demonstrated also in the platelets. The mitochondrially localized enzymes NAD-specific Isocitratdehydrogenase and Glycerophosphatoxidase have been measured for the first time in platelets with a high activity.3. The platelets of patients with liver cirrhosis and splenomegaly following portal hypertension showed significant higher enzyme activities in all investigated pathways, mainly in the citric acid cycle. Functionally important could be the marked increase of the Mg++ activated ATPase, the unaltered activity of the Phosphofructokinase and a lowered activity of the Lactatdehydrogenase in these platelets. The patients with cirrhosis but without a large spleen had normal enzyme activities of the platelets.4. One patient is described with cirrhosis without splenomegaly who had an elevation of nearly all measured enzymes by a factor 8 in his platelets. The LDH showed a decreased activity.5. No enzyme defect in the platelets of cirrhotic patients as in thrombasthenia was found. Basing on our knowledge of the enzyme equipment of blood cells the conclusion is drawn that the platelets with higher enzyme content represent a young cell population. The pathogenesis is discussed with special reference to the role of the enlarged spleen.

[Factor XIII deficiency in adults with acute leukemia: results of a substitution therapy with factor XIII (author's transl)].

A decrease in fibrin stabilizing factor (Factor XIII) is the most frequent coagulation disorder seen in adults with acute leukemia. Patients with prominent reduction of factor XIII (FSF) (less than 50%) were substituted with a factor XIII concentrate from human placenta, and factor XIII plasma concentration and bleeding tendency were followed up during the course of the disease. After substitution plasma, factor XIII activity went up to normal levels in most of the patients. As compared to the course of 12 patients with distinct factor XIII reduction without factor XIII therapy, there were less bleeding complications in 13 courses of patients with prominent reduction of factor XIII substituted with factor XIII concentrate and in 11 with normal or only slightly reduced factor XIII levels.SummaryA decrease in fibrin stabilizing factor (factor XIII) is the most frequent coagulation disorder seen in adults with acute leukemia. Patients with prominent reduction of factor XIII (FSF) (<50%) were substituted with a factor XIII concentrate from human placenta, and factor XIII plasma concentration and bleeding tendency were followed up during the course of the disease.After substitution plasma, factor XIII activity went up to normal levels in most of the patients. As compared to the course of 12 patients with distinct factor XIII reduction without factor XIII therapy, there were less bleeding complications in 13 courses of patients with prominent reduction of factor XIII substituted with factor XIII concentrate and in 11 with normal or only slightly reduced factor XIII levels.ZusammenfassungDie häufigste Form der Koagulopathie bei akuter Erwachsenen-Leukämie ist eine Verminderung von Fibrin stabilisierendem Faktor (Faktor XIII). Patienten mit deutlichem Faktor XIII-Mangel (FSF) (<50%) wurden mit einem Faktor XIII-Konzentrat aus menschlicher Plazenta substituiert und die Plasmakonzentration von Faktor XIII im Verlauf bestimmt. Durch die Substitution war fast immer eine schnelle Normalisierung des Faktor XIII-Spiegels zu erzielen. 13 Verläufe von Patienten mit deutlichem Faktor XIII-Mangel mit Substitution und 11 mit normalem oder nur mäßig vermindertem Faktor XIII zeigten weniger Blutungskomplikationen als eine Vergleichsgruppe von 12 Verläufen von Patienten mit deutlichem Faktor XIII-Mangel ohne Substitution.

Ein neuer Fall von schwerem Faktor XIII-Mangel

SummaryAn infant with congenital homozygous factor XIII deficiency demonstrated a severe retroperitoneal and intracerebral bleeding with development of a posthemorrhagic hydrocephalus in the first months of life. Factor XIII activity was not measurable by means of enzymatic method and the antiserum inhibition test. Quantitative immunoelectrophoresis according to Laurell presented absence of the subunit A, whereas the concentration of subunit S was reduced to 47 % the normal value. After replacement therapy factor XIII activity was estimated at 23 % and corresponded to the concentration of the subunit A, concentration of subunit S increased by 20 %. The turnover rate of fibrin stabilizing factor could be observed over a period of 39 days. The halflife was estimated at 4,7 days. The child developed normally after continous substitution with 250 units of factor XIII concentrate every 6 weeks.ZusammenfassungBei einem SÄugling mit angeborenem homozygoten Faktor XIII-Mangel traten in den ersten Lebensmonaten eine schwere retroperitoneale und eine intracerebrale Blutung mit Ausbildung eines posthÄmorrhagischen Hydrocephalus auf. Mit der Transglutaminationsreaktion und dem Antiserum-Hemmtest war keine Faktor XIII-AktivitÄt me\bar. In der quantitativen Immunelektrophorese nach Laurell war die eigentlich wirksame Untereinheit A nicht nachweisbar, wÄhrend die Konzentration der Untereinheit S auf 47 % der Norm vermindert war. Nach Substitution mit 250 E. Faktor XIII-Konzentrat wurde enzymatisch eine Faktor XIII-AktivitÄt von 23 % gemessen. Die immunologisch bestimmte Konzentration der Untereinheit A entsprach dem Anstieg der Faktor XIII-AktivitÄt, die Konzentration der Untereinheit S nahm um 20 % zu. Im Alter von 8 Monaten wurde durch wiederholte Bestimmungen der Faktor XIII-AktivitÄt über einen Zeitraum von 39 Tagen nach Substitution der Turnover des Fibrinstabilisierenden Faktors durch eingehende Gerinnungsanalysen bestimmt. Die Halbwertszeit berechnete sich auf 4,7 Tage. Unter einer Dauersubstitutionstherapie mit 250 E. Faktor XIII im Abstand von 6 Wochen blieb das Kind blutungsfrei und entwickelte sich normal.An infant with congenital homozygous factor XIII deficiency demonstrated a severe retroperitoneal and intracerebral bleeding with development of a posthemorrhagic hydrocephalus in the first months of life. Factor XIII activity was not measurable by means of enzymatic method and the antiserum inhibition test. Quantitative immunoelectrophoresis according to Laurell presented absence of the subunit A, whereas the concentration of subunit S was reduced to 47% the normal value. After replacement therapy factor XIII activity was estimated at 23% and corresponded to the concentration of the subunit A, concentration of subunit S increased by 20%. The turnover rate of fibrin stabilizing factor could be observed over a period of 39 days. The half life was estimated at 4,7 days. The child developed normally after continous substitution with 250 units of factor XIII concentrate every 6 weeks.