Granulocyte-platelet interactions and platelet fibrinogen receptor exposure.

We have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase (0.5-1 microgram/ml). Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. Experiments performed by immunoblotting with use of polyclonal and monoclonal antibodies directed to GPIIIa showed that pretreatment of human platelets with granulocyte elastase resulted in the appearance of an additional proteolytic derivative of GPIIIa migrating with an apparent molecular mass of 120 kDa in a nonreduced system. GPIIIa appears to be the preferred substrate of elastase, since GPIIb was not degraded by human granulocyte elastase. We conclude that 1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and 2) human granulocyte elastase exposes active fibrinogen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen.

Modulation of endothelial cell functions by different molecular species of interleukin 1.

Different molecular species of interleukin 1 (IL 1) were examined for the spectrum of responses elicited in human endothelial cells (HEC), including synthesis of prostacyclin (PGI2), tissue-type procoagulant activity (PCA), platelet activating factor (PAF), and plasminogen activator inhibitor (PA-I). The IL 1 preparations utilized for the present study included a natural, partially purified IL 1, a preparation purified to homogeneity with extensive homology with the derived aminoacid IL 1 beta (pI7) sequence denominated "22K factor," murine recombinant IL 1 alpha, human recombinant IL 1 alpha (pI5) and beta (pI7). Natural, partially purified IL 1, a mixture of alpha and beta species, induced the entire spectrum of responses in HEC. Production of PA-I was elicited by all forms of IL 1 tested. PGI2 and PCA were elicited by "22K factor" and by human recombinant IL 1 beta and alpha but not by murine recombinant IL 1 alpha. PAF synthesis was stimulated by murine and human recombinant IL 1 alpha but not by human recombinant IL 1 beta and 22K factor. Thus the available different molecular forms of IL 1 elicit largely but not completely overlapping patterns of responses in HEC. The IL 1 pathway of regulation of HEC functions might provide a basis for novel strategies in therapeutically oriented research on vessel wall disorders.

Low T3 syndrome in patients following major surgery.

Time sequence and specificity of thyroid hormones and biochemical parameters were investigated in patients following major surgery. Serum concentration of triiodothyronine decreases significantly following operation, with a biphasic regression. There is a reciprocal change in serum reverse triiodothyronine levels, but serum thyroxine levels show no significant change after operation. The significant decrease in serum concentration of alpha-2-macroglobulin and antithrombin III during and after surgery is the result of consumption of these inhibitors because the reciprocal change in serum concentration of elastase-like protease has been recognized. According to the change of curvilinear regression of serum triiodothyronine levels, 14 patients were grouped into 3. The patients for whom the curvilinear regression resembled a polynomial of degree 3 and 2 had a good prognosis, but the remaining 4 with no significant curvilinear regression had major complications and 2 died. It is meaningful that the postoperative change of triiodothyronine levels relates to the clinical outcome, to some degree.

Alpha 2-plasmin inhibitor inactivation by human granulocyte elastase.

Plasminogen-binding human alpha 2-plasmin inhibitor is converted by human granulocyte elastase into its non-plasminogen-binding and finally into the inactive form of the inhibitor. This degradation of the plasmin inhibitor, described earlier as "spontaneously" occurring conversion, is shown in dodecyl sulfate polyacrylamide gel electrophoresis, in two-dimensional immunoelectrophoresis and by measuring the kinetics of plasmin inhibition. Experiments in the presence of normal human plasma required unphysiologically high concentrations of elastase to inactivate alpha 2-plasmin inhibitor, suggesting a role of elastase in this type of indirect fibrinolysis in a microenvironment only and not in systemic events.

Immunochemical discrimination of leukocyte elastase from plasmic degradation products of fibrinogen.

A rabbit antiserum was elicited to the D-like fragment (De) generated by cleavage of human fibrinogen with isolated leukocyte elastase. After absorption with intact fibrinogen and plasmic degradation products, the antiserum retained its capacity to recognize the De fragment in radioimmunoassays. Plasmic digests of fibrinogen, produced at various enzyme-to-substrate ratios, and purified plasmic degradation products of fibrinogen reacted poorly with the absorbed antiserum. In contrast, elastase digests of fibrinogen and purified elastase degradation products reacted well, although earlier derivatives expressed the recognized epitopes less well than the De fragment. These observations indicated the recognition of an elastase-elicited neoantigen that was not expressed by plasmic degradation products of fibrinogen. When elastase degradation products were added to normal plasma, the elastase-elicited neoantigen could be quantitatively detected in radioimmunoassay. Normal plasma was negative (less than or equal to 5 nM) for the elastase-elicited neoantigen. Of 30 pathological plasmas containing high concentrations of leukocyte elastase, four contained low levels of the elastase-elicited neoantigen (greater than 15 nM). These results suggest that under most circumstances the fibrinolytic activity of leukocyte elastase is well regulated but that under certain pathophysiological conditions, the leukocyte protease may participate in the fibrinolytic process.

Cytochemical determination of granulocyte elastase and chymotrypsin in human myeloid cells and its application in acquired deficiency states and diagnosis of myeloid leukemia.

Two cytochemical methods for detection of granulocytic elastase and chymotrypsin employing alanine and phenylalanine naphthyl esters were developed. Specificity of reaction with the ester substrates was proven by chloromethyl ketone inhibitors. The results of both staining methods were almost identical with the staining for naphthol AS-D chloroacetate (Cl Ac-O Nap AS-D) esterase, since Cl Ac-O Nap AS-D also reacts with granulocyte elastase and chymotrypsin. Mature neutrophils and myeloid precursors except myeloblasts are stained with all three substrates in peripheral blood and bone marrow. Mast cells, however, only react with Cl Ac-O Nap AS-D and the chymotrypsin substrate and not with the elastase substrate. In acute myeloid leukemia the three esterases appear in parallel at a somewhat later stage of maturation than myeloperoxidase. In blood smears from 380 hospital patients no hereditary elastase or chymotrypsin deficiency could be demonstrated. Staining for elastase and chymotrypsin was also normal in hereditary myeloperoxidase deficiency and chronic granulomatous disease. On the other hand 6% of the hospital patients and about two-thirds of patients with acute myeloid leukemia showed a partial elastase deficiency in more than 25% of the peripheral neutrophils.

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.

Cleavage of IgG by elastase-like protease (ELP) of human polymorphonuclear leukocytes (PMN): isolation and characterization of Fab and Fc fragments and low-molecular-weight peptides. Stimulation of granulocyte function by ELP-derived Fab and Fc fragments.

The elastase-like protease (ELP) from human polymorphonuclear granulocytes (PNM) is able to split human IgG into Fab and Fc-like fragments and smaller peptides. These fragments are similar but not identical to those produced by papain. They differ in their electrophoretical mobility as well as in their molecular weights. Both ELP-Fab and papain-Fab show antigen-binding capacity. In contrast to papain-derived split products of IgG, the ELP-generated Fab and Fc fragments could enhance granulocyte function. Both ELP-Fab and ELP-Fc increase the spontaneous reduction of nitroblue-tetrazolium (NBT) in granulocytes dose-dependent in a homologous as well as in an autologous system. Furthermore, the ELP-derived Fab and Fc induce an active release of ELP by PMN. It could also be demonstrated that Fab as well as Fc increased the peroxidase activity in granulocytes.

Fibrinogen degradation by two neutral granulocyte proteinases. Influence of calcium on the generation of fibrinogen degradation products with anticlotting properties.

Degradation of human fibrinogen by elastase-like proteinase, chymotrypsin-like proteinase and plasmin, was done in the presence and absence of calcium ions, respectively. The resulting fibrinogen degradation products were tested for their coagulant and anti-coagulant properties. The results show that 1. fibrinogenolysis is delayed in the presence of calcium ions. Higher enzyme concentrations are required to get unclottable split products when calcium ions are present. 2. The fibrinogen fragments obtained in the presence of calcium are different in their molecular weights and anticoagulant activities compared to those obtained in the absence of calcium ions. This effect of calcium is most striking during fibrinogen cleavage by chymotrypsin-like proteinase. Elastase and plasmin-induced fibrinogenolysis was substantially influenced by calcium only at a late degradation stage.

[Ascertainment of physical fitness for work in persons applying for annuities in the Federal Republic of Germany (author's transl)]. / Feststellung der Leistungsfähigkeit im Erwerbsleben beim Rentenantragsverfahren in der Bundesrepublik Deutschland.

Annuities granted because of unfitness for work in general or unfitness to perform particular professional work are being paid in Germany by a state insurance agency. Unfitness for work has to be ascertained in each individual case as has been ruled by jurisdiction of the Highest German Court. It is the task of the medical expert in the course of this procedure to register the clinical status of the applicants and to reach a medical judgment in regard to fitness for work. This judgment does not rely upon the diagnosis or prognosis of a particular disease; a decreased fitness for work because of a particular disease can only be assumed if the disease has led to long-term irreversible deficient organ functions. Annuities for a limited time period may be granted if there is a chance for improvement of health. Problems of finding expert judgement are reported.

[Prenatal diagnosis of alpha-1-antitrypsin phenotype. Case record and prognosis in severe alpha-antitrypsin deficiency Pi ZZ (author's transl)]. / Pränataler Ausschluss eines Alpha-1-antitrypsinmangels. Kasuistik und Prognose bei Phänotyp Pi Z.

A mother had a child with cirrhosis of the liver and alpha-1-antitrypsin deficiency. In a subsequent pregnancy the fetal phenotype Pi MZ was detected by isoelectrofocusing in the amniotic fluid. Quantitative assay of alpha-1-antitrypsin gave results in the normal range. Umbilical vein blood analysis confirmed the antenatal findings. In this case it has been possible to rule out the disease before birth. In this context the clinical importance of alpha-1-antitrypsin deficiency is stressed, its frequency in the European and North-American population and the prognosis with phenotype Pi Z.

A radioassay for proteolytic cleavage of isolated cartilage proteoglycan. 2. Inhibition of human leukocyte elastase and cathepsin G by anti-inflammatory drugs.

20 non-steroidal anti-inflammatory drugs and other agents were evaluated for their effectiveness in directly inhibiting the proteolytic activity of human leukocyte elastase and cathepsin G. The proteolysis of hide powder azure by leukocyte granule extracts was used for initial testing, and selected drugs were then studied further using a radioassay of the proteolysis of isolated proteoglycan by purified leukocyte elastase and cathepsin G. The results indicated that at drug concentrations likely to be attained to vivo, phenylbutazone may significantly inhibit elastase, while gold thiomalate and mucopolysaccharide polysulfonic acid ester (MPSE; Arteparon) could limit the action of cathepsin G. Oleic acid may provide a useful starting point for development of agents specifically designed to inhibit cartilage erosion.

ACTH-like activity in immune complexes of patients with oat-cell carcinoma of the lung.

Immune complexes could be isolated from sera of 7 patients with oat-cell carcinoma of the lung, but not from 5 normal controls, using zonal ultracentrifugation. After ultracentrifugation, fractions containing macromolecular IgG were absorbed on a protein A-sepharose column and the immune complexes were eluted and dissociated by glycin-HCl buffer at pH 3.5. The eluates were tested for the presence of tumour-associated proteins as carcinoembryonic antigen (CEA), non-specific crossreacting antigen (NCA), alpha2 pregnancy associated antigen (alpha2PAG) and isoferritin. Whereas none of these tumour-associated antigens could be demonstrated, an ACTH-like activity was detected in the immune-complex fractions of 4 patients with oat-cell carcinoma, by radioimmuno- and bioassay. Polyacrylamide electrophoresis of an immune-complex fraction from a patient with Cushing syndrome showed ACTH-like activities, with mol. wt of 110,000, 75,000, 30,000 and less than 20,000 (all glycoproteins) indicating the presence of different subfractions of big ACTH.