Jack Levin

An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation

Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate.

Immunologic Studies in von Willebrand's Disease: EVIDENCE THAT THE ANTIHEMOPHILIC FACTOR (AHF) PRODUCED AFTER TRANSFUSIONS LACKS AN ANTIGEN ASSOCIATED WITH NORMAL AHF AND THE INACTIVE MATERIAL PRODUCED BY PATIENTS WITH CLASSIC HEMOPHILIA

Antihemophilic globulin (AHF, factor VIII) levels were measured by a standard coagulation assay and by an immunological technique before and serially after infusion of fresh frozen plasma or cryoprecipitate into patients with von Willebrands disease. Initial levels of AHF, measured both as procoagulant and as antigen, were low. Immediately after transfusions, the rise in levels of AHF-like antigen was compatible with the quantity of antigen present in the infused plasma or cryoprecipitate. Thereafter, levels of antigen declined rapidly and reached preinfusion values in approximately 24 hr. In contrast, procoagulant activity remained elevated, and sometimes continued to rise, for longer periods of time. One possible explanation of this finding is that the AHF molecule produced by patients with von Willebrands disease, in response to transfusion of as yet unidentified factors, lacks the antigenic site associated with the normal AHF molecule or the inactive molecule produced by patients with hemophilia A.

Platelet alpha-granule fibrinogen, albumin, and immunoglobulin G are not synthesized by rat and mouse megakaryocytes.

It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet alpha-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in alpha-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into alpha-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of alpha-granule fibrinogen is plasma.

Lack of Endotoxin in Borrelia hispanica and Treponema pallidum

Abstract Borrelia hispanica from infected guinea pigs and Treponema pallidum from testicular syphilomas of rabbits were assayed for the presence of endotoxin with the Limulus lysate test. A suspension of Borrelia, containing 1.3 × 108 spirochetes/ml, was nonreactive both when it was tested as intact organisms, and when tested after disruption of the spirochetes by sonication. Eight different suspensions of treponemes, ranging from 0.6 × 109 to 3 × 109 treponemes/ml, were negative at a 1:10 dilution and were no more active than control suspensions of normal rabbit testes. Therefore, it was concluded that T. pallidum, as well as the Borrelia, possessed no endotoxin.

Detection of thrombopoietic activity in plasma by stimulation of suppressed thrombopoiesis

Rabbits in which thrombocytosis had been produced by five daily transfusions of platelet concentrates had suppressed endogenous thrombopoiesis, as reflected by decreased incorporation of selenomethionine-(75)Se ((75)SeM) into the circulating platelet mass. Rabbits in which endogenous thrombopoiesis had been suppressed by transfusion-induced thrombocytosis were used to detect thrombopoietic activity in rabbit plasma. Thrombopoietic activity was demonstrated in the plasma of both normal and thrombocytopenic donor rabbits. A dose response relationship was observed between the incorporation of (75)SeM into platelets and the dose of plasma administered. Infusion of 20-150 ml of plasma from thrombocytopenic donors increased the incorporation of (75)SeM into platelets from 52 to 107% above control values. A dose response effect also was seen after infusion of normal plasma, but normal plasma produced less effect than comparable doses of plasma from thrombocytopenic donors. Rabbits with transfusion-induced thrombocytosis appear to be more sensitive assay animals for the detection of thrombopoietic activity than animals with normal platelet counts. Changes in the rate of appearance and levels of (75)SeM may primarily indicate changes in platelet protein or platelet size and are apparently more sensitive indicators of the state of thrombopoiesis than are alterations in the numbers of circulating platelets. The results strongly support the concept of a humoral agent, i.e. thrombopoietin, that acts on megakaryocytes to regulate platelet production.

Fractionation of Limulus amebocyte lysate. Characterization of activation of the proclotting enzyme by an endotoxin-mediated activator.

Limulus amebocyte lysate was fractionated by heparin-Sepharose chromatography into four components (fractions A, B, C and D). Major coagulation factors, i.e., proclotting enzyme, coagulogen, and proclotting enzyme activating factor precursor (proactivator) in the lysate were eluted, respectively, in fraction A, fraction B and fraction C. Clotting enzyme activity was detected only following recombination of fraction A and fraction C in the presence of endotoxin. The conversion of proactivator to its active form (activator) was an endotoxin-dependent reaction and was inhibited by polymyxin B. Either proactivator is an endotoxin-sensitive factor or another endotoxin-sensitive factor, which activates proactivator, is present in fraction C. Optimal pH for proclotting enzyme activation by activator was broad and ranged from pH 6.0 to 8.0, while that for the endotoxin-mediated activation of proactivator was pH 7.0. No initial latent period was observed during activation of the proactivator or proenzyme. The activator was inhibited by benzamidine, leupeptin, soybean trypsin inhibitor and diisopropyl fluorophosphate, suggesting that the activator is a trypsin-type serine protease. Trypsin, but not thrombin, urokinase, plasmin, papain or alpha-chymotrypsin activated the proclotting enzyme. Therefore, limited proteolysis, i.e., of an arginyl- or lysyl-X bond(s), of the proenzyme molecule is probably involved in its activation.

A method for the removal of endotoxin from purified colony-stimulating factor.

Summary Limulus lysate assays showed that several samples of purified L-cell CSF were markedly contaminated with endotoxin. Several pools of material contained 1 to 10 μg/ml of this bacterial lipopoly-saccharide. Ultracentrifugation in sucrose density gradients led to a 95% separation of CSF and endotoxin activities. In four studies, endotoxin content of the CSF preparations was reduced to levels varying from 0.05 to 0.0001 μg/ml. In vivo studies with known quantities of E. coli endotoxin were conducted in mice. Acute and repeated injections of 0.1 μg of endotoxin led to increased serum CSF activity and increased number of splenic CFU-C and CFU-E. In contrast, similar injections of 0.01 μg of endotoxin produced virtually no effect on these parameters of hemopoiesis. Thus, the removal of endotoxin from CSF as outlined herein will permit in vivo studies of the effects of CSF on hemopoiesis, without concern for the complicating effects of endotoxin. The authors gratefully appreciate the excellent technical assistance of Florence Boegel, Lea Einsporn, and Francine C. Levin.

Failure to Demonstrate Circulating Endotoxin in Malaria

Summary The possibility that endotoxin or an endotoxin-like substance plays a role in malaria has been suggested by the clinical similarity between human malaria and the febrile reaction to endotoxins, as well as the occurrence of endotoxin tolerance in humans infected with malaria. However, endotoxin or endotoxin-like activity was not demonstrable, using the Limulus test, in the plasma of humans or monkeys infected with plasmodia. The data indicate that the febrile paroxysm of malarial infection is not associated with detectable levels of endotoxin in the blood. This investigation could not have been possible without the cooperation of the inmate volunteers who willingly underwent experimental malarial infections. Madame Francine Corthesy provided excellent technical assistance. We are also grateful to Dr. W. E. Collins for his expert advice and assistance with the infections in monkeys.

Colony-Stimulating and Inhibiting Factors in Sera from Normal and Endotoxin-Treated CF1 Mice

Summary Sera from normal and endotoxintreated CF1 mice were fractionated by Sephadex chromatography. CSF activity was demonstrated in the prealbumin region in serum obtained from mice that had previously received endotoxin. Identical fractions from mouse embryo conditioned medium also displayed CSF activity. Normal serum had very low or no detectable CSF levels. An inhibitor was demonstrable in the high molecular weight fractions from both normal and endotoxin serums. Its effect is decreased by utilizing a double-layer technique. These results suggest that there is an increase in CSF levels after endotoxin administration rather than a decrease of serum inhibitory factors. Furthermore, the increase in CSF detected in sera after administration of endotoxin does not depend on the presence of endotoxin in such sera.

Tritiation of endotoxin

Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin.