BritishJournal ofHaematology, 1976,33,213.

Activation of Intravascular Coagulation by Endotoxin: the Significance of Granulocytes and Platelets GERTMULLER-BERGHAUS, ELISABETHBOHNAND WILFIUED HOBEL

Department of Medicine and Department of Medical Statistics and Documentation, ]ustus Liebig- Universitat, Giessen, Germany (Received 6 October 1975; acceptedfor publication 7 November 1975) SUMMARY. The importance of granulocytes and/or platelets in endotoxin-induced generalized intravascular coagulation was studied in neutropenic as well as thrombocytopenic rabbits. Neutropenia and thrombocytopenia were induced by oral administration of busulphan. Generalized intravascular coagulation, as indicated by renal glomerular microclot formation, was initiated by two intravenous injections of endotoxin. Granulocyte counts before the second injection of endotoxin were most significantly related to activation of intravascular coagulation whereas platelet counts either before the first or second injection of endotoxin were not definitely related to the activation process. Renal glomerular microclots occurred in rabbits after two injections of endotoxin even when the platelet counts were between 500 and 5000/pl. These experiments indicated that granulocytes but not platelets are essential to the activation of endotoxin-induced intravascular coagulation. The intravenous injection of bacterial endotoxin causes activation of intravascular coagulation (for review see McKay, 1964; MiiIler-Berghaus & Lasch, 1975). The method by which intravascular coagulation is triggered leading to the generalized Shwartzman reaction (GSR) and human equivalent diseases has been much debated, but is unresolved. Recent investigations have focused on the role of platelets and/or leucocytes in endotoxin-induced generalized intravascular coagulation (Horn & Collins, 1968; Niemetz & Fani, 1973 ; Niemetz & Marcus, 1974; Lipinski et al, 1974; Miiller-Berghaus & Eckhardt, 1975; Eckhardt & MiillerBerghaus, 1975). The significance of platelets and leucocytes in activation of intravascular coagulation, however, has not yet been unequivocally explained. After the intravenous injection of endotoxin, platelets as well as leucocytes disappear from the peripheral circulating blood within minutes (Sanarelli, 1924; Shwartzman & Gerber, 1948; Stetson, 1951;McKay & Shapiro, 1958; Shimamoto et a!, 1958; Kleinmaier et a], 1959). The endotoxin becomes fixed on the surface of platelets as well as leucocytes (Herring ct a / , 1963 ; Braude et al, 1955). Damage to the platelets by endotoxin becomes manifest in vivo, because the platelets that remain in the circulation lose the major part of thcir constituents like platelets which have undergone a release reaction (Lasch et nl, 1960; Weber et al, 1963). Platelet factor 3 (PF3) activity can be detected in the plasma after the intravenous administration of endotoxin (Horowitz et al, 1962). Although platelets react markedly to endotoxin the Correspondence: Dr Gert Muller-Berghaus, Department of Medicine, Justus Liebig-Universitat, Klinikstr 36, 63 Giessen, Germany.

213

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G. Miiller-Berghaus, E. Bohn and W. Hobel

precise role of platelets in triggering intravascular coagulation has not been clarified because of contradictory results. On the one hand the infusion of purified PF3 or frozen and thawed platelets did not produce the generalized Shwartzman reaction (GSR) (Muller-Berghaus et al, 1967; Evensen & Jeremic, 1970), but on the other hand immunologically induced thrombocytopenia protected against the occurrence of the GSR after endotoxin injection (Margaretten& McKay, 1969). The particular importance of leucocytes in the development of the GSR following endotoxin was recognized by Thomas & Good (1952). They showed that endotoxin does not provoke the GSR if granulocytopenia has been induced in the animals by pretreatment with nitrogen mustard. Detailed investigations of Horn & Collins (1968), Niemetz (1969, 1972) as well as those ofLerner et a1 (1971)demonstrated the procoagulant activity of the leucocytes. In particular, these authors showed that peritoneal leucocytes of rabbits develop thromboplastin-like activity in vitro when the cells are incubated with endotoxin. Most interestingly this procoagulant activity of the leucocytes can increase many times, if platelet membranes are present in the reaction mixture (Niemetz & Marcus, 1974). If leucocytes with this procoagulant activity are infused intra-aortically into rabbits together with endotoxin, the GSR occurs (Niemetz & Fani, 1971, 1973). For unknown reasons the infusion of leucocytes ‘activated’ by endotoxin only leads to the GSR if the infusion is given into the aorta, and not if the intravenous route is used. The present study was designed to investigate whether granulocytes are solely involved in endotoxin-induced activation of intravascular coagulation, or whether platelets contribute to the activation process. MATERIALS AND METHODS Animals. Male and non-pregnant female rabbits of mixed New Zealand breeds weighing between 2.1 and 2.6 kg were used in this study. The animals were purchased from local breeders and fed rabbit pellet food and water ad libitum. 8 or 10d before injecting the first dose of endotoxin, blood was taken from a marginal ear vein and busulphan was fed to the animals by a stomach tube. Before injecting the first dose of endotoxin another blood sample was withdrawn from an ear vein. 22 h thereafter blood was again taken and the second dose of endotoxin injected intravenously. 6 h after the second injection of endotoxin surviving animals were killed by an overdose of sodium pentobarbital and necropsies were performed immediately. Induction of thrombocytopenia and leucopenia. Thrombocytopenia and leucopenia were induced by feeding busulphan as described previously (Eckhardt & Miiller-Berghaus, 1975). Busulphan was dissoved in polyethylene glycol 400 (Roth, Karlsruhe, Germany) to a concentration of 5 mg/ml. The solution was administered to the animals by means of a stomach tube using a disposable Tiemann catheter (Charriere’s gauge: 12). Two doses of 25 mg per kg of body weight of busulphan were given at an interval of 72 h. Production ofglomerular microclots by two injections of endotoxin. Glomerular microclots were produced by two injections of endotoxin (Salmonella enteritidis endotoxin, Lipopolysaccharide B, Difco Laboratories, Detroit, U.S.A.) 22 h apart. The rabbits received 2s pg of endotoxin as the first and I so ,ug of endotoxin as the second injection. Both the doses of endotoxin were contained in I ml sterile, pyrogen-free isotonic saline.

Endotoxin and Intravasmlar Coagulation 215 Haemutological studies. Platelets were counted directly by phase contrast microscopy using procaine hydrochloride (3.5 g/dl) as diluent. Leucocytes were directly counted by a conventional method. With few exceptions differential counts were obtained on IOO leucocytes in blood films stained with May-Grunwald-Giemsa stain. In a few cases leucocyte counts were extremely low so that only 50 leucocytes were classified. Microhaematocrits were determined using standard heparinized tubes (Clay Adams, New York, U.S.A.). Histologic studies. Organs were fixed in neutral 5% formalin. Sections of the kidneys were stained with haematoxylin and eosin, and fibrin was identified by the dimethylaminobenzaldehyde (DMAB)-nitrite reaction for the histochemical demonstration of tryptophane according to the method of Adams (1957). Statistical evaluation. The results were subjected to discriminatory analysis according to Anderson (1958). The variables used were platelets, leucocytes and granulocytes before the first as well as before the second injection of endotoxin. Discrimination analysis was performed using the iterative procedure. Discrimination was tried first with the variate which best separates the groups with positive and negative histological results. Thereafter the best separating variate together with the preceding one is taken into account, etc. For each step the Mahalanobis distance and its increase was computed. A further statistical analysis was performed to compute the minimum misclassification error rate which indicates the percentage of positive results listed as negative ones and vice versa. RESULTS Induction of Thrombocytopenia and Leucopenia The alkylating agent busulphan was administered to rabbits in order to cause thrombocytopenia and leucopenia to different extents. Busulphan lowered the mean platelet count from 358-t IOI (mean, SD) to 46k 40 x 1o9/I. (13% of the initial value) when platelet counts were compared before and 10 d after feeding the first dose of the drug (Table I). 8 d after busulphan administration the mean platelet counts had decreased from 357+ 96 (mean+ SD) to r46+ 68 x 10~11.(41% of the initial value). Busulphan administration equally lowered mean peripheral leucocyte counts from 6.880+2.530 (mean? SD) before to 3.040+ 1.380 x 10~11.10d after the first dose of the drug (44% of the initial value) whereas 8 d after feeding busulphan the mean leucocyte count was 3.770+ 1 . 8 3 0 ~10~11. (53% of the initial value). In close relation to the W B C the differential leucocyte counts also changed. 10d after busulphan administration the mean neutrophil cell count decreased from 2.487 before to 0 . 2 9 8 ~10~11.after feeding busulphan (12% of the initial value). 8 d after busulphan administration the mean neutrophil cell count was 0.589 x 10~11. (27% of the initial value). Busulphan did not have a very pronounced depressive effect on erythropoiesis but some animals developed spontaneous haemorrhages. Therefore, the mean PCV values were slightly decreased from 32.9 before to 30.8% 10 d after busulphan administration (Table I).

Efect ofBusuIphan on the Occurrence ofMicroc/ots a f t r T w o Injections ofEndotoxin One hundred and seventeen rabbits fed with busulphan were injected with endotoxin. Twenty-four of these animals died after the first injection of endotoxin. 22 h later surviving animals received a second dose of endotoxin. Eighteen of the 93 animals injected with two

G.Muller-Berghaus, E. Bohn and W. Hobef

216

TABLE I. Changes in platelet, WBC and PMN counts as well as PCV 8 and 10 days after oral administration of busulphan (2 x 25 mg/kg 3 days apart) ;number of animal experiments (n) as well as means and standard deviations of means (SD) are given

Bdore busulplrafl

administration Platelets ( x 10~11.) n MeankSD

41 357-!-96

Platelets ( x 10~11.)n MeankSD

76 358k101

WBC ( x 109/l.)

n

MeankSD WBC ( x 109/l.)

41 7.140+2.290

Mean -!- SD

76 6.880f2.530

PMN ( x 10~11.) n Meanf SD

28 2.216k0.959

PMN ( x 109/l.)

n Mean f SD

39 2.487 k 1.602

pvc (%)

n MeankSD

41 35.4f2.8

p c v (%)

n

fl

Meanf SD

Eight days afrr busuiphaii administration

Ten days after busulphan administration

41 146f68

< 0.001 76 46 f 40

< 0.001

41 3.77of 1.830

< 0.001 76 3.040k 1.380

28 0.589+0.462

< 0.001

< 0.001 39 0.298f.0.303

< 0.001

41 32.8k4.I

72 32.gk3.0

P

< 0.002 72 30.8f4.05

'

< 0.001

doses of endotoxin died within 4 h after the second injection of endotoxin, probably because of massive haemorrhage and/or haemorrhagic shock. At autopsy extravasated blood was found in the pleural cavities as well as in the intestine. Frequently the animals exhibited a haemopericardium. Animals dying within 4 h of the second dose of endotoxin were excluded from the statistical evaluation since up to this time the development of renal glomerular microclots is not yet completed. If animals demonstrate glomerular microclots this phenomenon certainly occurs at the latest between 4 and 6 h after the second dose of endotoxin. For that reason surviving animals were killed 6 h after the second dose of endotoxin. Except for one rabbit dying at 5 h the remaining 74 animals were killed 6 h after the second dose of endotoxin. The incidence of microclot formation in these busulphan-pretreated TABLE 11. Incidence of renal glomerular microclots after two injections of endotoxin into busulphan-pretreated and untreated rabbits; only animals living longer than 4 h after the second dose of endotoxin are listed

Incidence of glomerular microclots

Untreated rabbits

Birsulphan-treated rabbits

I2/12*

I7/75*

* Expressed as the number of positive results over the total number of rabbits studied.

Endotoxin and Intravascufar Coagulation

217

rabbits was much reduced in comparison to normal control rabbits in which 12 out of 12 animals exhibited glomerular microclots (Table 11). In order to elucidate the influence of PMN as well as platelets on the occurrence of glomerular microclot formation PMN counts were plotted against platelet counts on a diagram. It is seen from Fig I that the incidence of glomerular microclot formation is predominantly 2ependent on the granulocyte counts because fibrin-rich microclots were only detected if the granulocyte counts were above 0 . 0 2 7 ~10~11. Platelet counts before the second dose of endotoxin did not decisively influence the occurrence of glomerular microclots as these were observed in animals with extremely low platelet counts between 0.5 and 5 x 10~11.

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FIG I . The influence of different polymorphonuclear leucocyte and platelet counts before the second injection of endotoxin on the occurrence of renal glomerular inicroclots. 0 , Animals with glomerular microclots; 0,animals without glomerular microclots.

In order to define the effect of busulphan treatment on the occurrence of glomerular microclots after two injections of endotoxin a discriminatory analysis was performed with regard to the influence of platelet, leucocytc and granulocyte counts before the first as well as before the second dose of endotoxin. The statistical analysis of 64 animal experiments, presented in Fig 2 , demonstrates that the occurrence of renal glomerular microclots is mainly dependent on the granulocyte counts before the second injection of endotoxin. Leucocyte counts before the second dose and even granulocyte counts before the first dose of endotoxin contribute to the discrimination between positive and negative results. Platelet counts before the first as well as before the second dose of endotoxin only slightly influenced the incidence

218

G. Miiller-Berghaus, E. Bohn and W . Hobel

FIG 2. Discriminatory analysis of the data of 64 animal experiments. On the abscissa the variates are listed in order of decreasing influence on separating positive (animals with glomerular microclots) and negative results (animals without glomerular microclots). The Mahalanobis distance is plotted on the ordinate indicating the extent of the influence of the variates. PMN-Ea,polymorphonuclear leucocyte count before the second injection of endotoxin; WBC.EI, white blood cell count before the first injection of endotoxin; Plat.E,, platelet count before the second injection of endotoxin; etc. Platelet counts before both the first and the second injection of endotoxin do not markedly contribute to the discrimination between positive and negative results as is indicated by the flattening ofthe slopc ofthe curve.

of glomerular inicroclot formation because the minimum misclassification error rate was only reduced from 38% to 36% ifplatelet counts were included in the discriminatory analysis. The minimum misclassification error rate of platelet counts only amounted to 63%.

DISCUSSION It is recognized that it has been difficult to prove that platelets are decisively involved in triggering intravascular coagulation. In the present study, of the different approaches tried, the discriminatory analysis of 64 animal experiments resulted in an unequivocal conclusion. The statistical analysis revealed a very pronounced correlation between the occurrence of glomerular microclots and granulocyte counts before the second injection of endotoxin. Renal glomerular microclots did not develop if the granulocyte counts before the second dose of endotoxin were below 0.028 x 10~11.(below 2% relative PMN counts). These figures imply leucocyte counts before the second dose of less than 1.3 x 10~11.In the present study neutropenia was induced by busulphan. Similar results were obtained if neutropenia was caused by injection of nitrogen mustard. In a previous study it has been shown that glomerular

Endotoxin and Intravascular Coagulation

219

microclot formation failed to develop after two injections of endotoxin if leucocyte counts were below 0.8 x 10~11. and granulocyte counts were below 0.07x 10~11.(Muller-Berghaus & Eckhardt, 1975).Corresponding figures were published by Forman et al(1969)and Gaynor (1973).Most interestingly microclot formation occurred in some animals with platelet counts below 5 x 1o9/I.,one of these animals exhibiting only 0.5 x 10~11.platelets before the second dose of endotoxin. Platelet counts before both the first or second injection of endotoxin were not definitely related to microclot formation. Despite platelet counts above 10x 1o9/I.microclots did not form in some of the animals because granulocyte counts were low at the same time. These experiments indicate that platelets are not involved in triggering intravascular coagulation by endotoxin. Experiments reported by Margaretten & McKay (1969)seem to conflict with the present data because immunologically induced thrombocytopenia protected against the occurrence of the generalized Shwartzman reaction after endotoxin injection. This effect of the platelet antiserum, however, may not depend on the antiserum-induced thrombocytopenia but on the immune reaction with consumption of complement factors since hypocomplementaemia inhibits the occurrence of the generalized Shwartzman reaction (Fong & Good, 1971). ACKNO WZEDGMENTS

This work was supported by the Deutsche Forschungsgemeinschaft,Bonn-Bad Godesberg, Germany. The authors wish to thank Mrs A. Chandoni and Miss E. Lohmann for excellent technical assistance. REFERENCES ADAMS,C.W.M. (1957) A p-dimethylaminobenzaldehyde-nitrite method for the histochemical demonstration of tryptophane and related compounds. Journal of Clinical Pathology, 10, 56. ANDERSON, T.W. (1958) A n Introduction to Multivariate Statistical Analysis. Wiley, New York. BRAUDE,A.I., CAREY,F.J. & ZALESKY, M. (1955) Studies with radioactive endotoxin. 11. Correlation of physiologic effects with distribution of radioactivity in rabbits injected with lethal doses of E. coli endotoxin labelled with radioactive sodium chromate. Journal of Clinical Investigation, 34, 8 5 8 . ECKHARDT, T. & MULLEX-BERGHAUS, G. (1975) The role of blood platelets in the precipitation of soluble fibrin by endotoxin. Scandinavian Journal of Haematology, 14,181. EVENSEN. S.A. & JEREMIC, M. (1970) Platelets and the triggering mechanism of intravascular coagulation. Briti~hJournal of Haematology, 19, 33. FONG,J.S.C. & GOOD,R.A. (1971) Prevention of the localized and generalized Shwartzman reactions by an anticomplementary agent, cobra venom factor. Journal of Experimental Medicine, 134, 642. FORMAN,E.N., ABILDGAARD, C.F., BOLGER,J.F., JOHNSON, C.A. & SCHULMAN, I. (1969) Generalized

Shwartzman reaction: role of the granulocyte in intravascular coagulation and renal cortical necrosis. BritithJournal ofHuernatology,.16, 507. GAYNOR, E. (1973) The role of granulocytes in endotoxin-induced vascular injury. Blood, 41,797. HERRING, W.B., HERION,J.C., WALKER,R.I. & PALMER, J.G. (1963) Distribution and clearance of circulating endotoxin. Journal of Clinical Investigation, &79. HORN,R.G. & COLLINS, R.D. (1968) Studies on the pathogenesis of the generalized Shwartzman reaction. The role of granulocytes. Laboratory Investigation, 18, 101. HOROWITZ, H.I., DES PREZ,R.M. & HOOK,E.W. (1962) Effects of bacterial endotoxin on rabbit platelets. 11. Enhancement of platelet factor 3 activity in vitro and in vivo. Journal of Experimental Medicine, 116,619. KLEINMAIER, H., GOERGEN, K., LASCH,H.G., KRECKE, H.-J. & BOHLE,A. (1959) Untersuchungen zur Frage der Gerinnungsstorung beim SanarelliShwartzman-Phhomen (sog. generalisierten Shwartznian-Phkomen) des Kaninchens. Zeitschrij f i i r die Gesamte Experimentelle Medizin, 132, 275.

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LASCH, H.G., RODRfGUEZ-ERDMANN, F. & KRECKE, H.-J. (1960) Quantitative und qualitative Verkderungen der Thrombocyten beim Sanarelli-Shwartzman-Phbomen. Verhandlungen der Deutschen Gesellschaftfir Znnere Medizin, 66, 992. LBRNER,R.G., GOLDSTEIN, R. & CUMMINGS, G. (1971) Stimulation of human leukocyte thromboplastic activity by endotoxin. Proceedings of the Society for Experimental Biology and Medicine, 138,145. A. & GUREWICH, V. (1974) The LIPINSKI, B., NOWAK, organ distribution of 1251-fibrinin the generalized Shwartzman reaction and its relation to leucocytes. British Journal ofHaematology, 28, 221. MARGAREXTEN, W.& MCKAY,D.G. (1969) The role of the platelet in the generalized Shwartzman reaction. Journal of Experimental Medicine, 129, 585. MCKAY,D.G. (1964) Disseminated Intravascular Coagulation. A n Intermediary Mechanism of Disease. Hoeber, Harper & Row, New York. MCKAY,D.G. & SHAPIRO, S.S. (1958) Alterations in the blood coagulation system induced by bacterial endotoxin. I. In vivo (generalized Shwartzman reaction).Journal ofExperimental Medicine, 107,3 5 3 . MULLER-BBRGHAUS, G. & ECKHARDT, T. (1975) The role of granulocytes in the activation of intravascular coagulation and the precipitation of soluble fibrin by endotoxin. Blood, 45, 631. MULLER-BERGHAUS, G. & LASCH,H.G. (1975) Microcirculatory disturbances induced by generalized intravascular coagulation. Handbook of Experimental Pharmacology, XVI/3, 429. MULLER-BERGHAUS, G., GOLDFINGER, D.,MARGARETTEN, w. & MCKAY,D.G. (1967) Platelet factor 3 and the generalized Shwartzman reaction. Thrombosis et Diathesis Haemorrhagica, 18. 726. NIEMBTZ, J. (1969) Thromboplastic activity of leukocytes from rabbits receiving endotoxin. (Abstract). Federation Proceedings, 28, 442. NIEMETZ, J. (1972) Coagulant activity of leukocytes.

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NIEMETZ, J. & FANI,K. (1971) Role of leukocytes in blood coagulation and the generalized Shwartzman reaction. Nature: N e w Biology, 232, 247. NIBMETZ, J. & FAN, K. (1973) Thrombogenic activity of leukocytes. Blood, 4, 47. NIBMETZ, J. & MARCUS,A.J. (1974) The stimulatory effect of platelets and platelet membranes on the prodoagdant activity of leukocytes. Journal of Clinical Investigation, 54, 1437. SANARELLI, G. (1924) De la pathogtnie du choldra (Neuvitme mdmoire). Le choltra expdrimental. Annales de I'Institut Pasteur, 38, I I . SHIMAMOTO, T., YAMAZAKI, H., SAGAWA, N., IWAHARA, s., KONISHI,T. & MAEZAWA, H. (1958) Effect of bacterial endotoxins on platelets and release of serotonin (yhydroxytryptamine). 11. Appearance of platelet-agglutinating substance and serotonin (S-hydroxytryptamine) in the plasma of rabbit by administration of bacterial endotoxin. Proceedings oj'the Japan Academy, 34, 450. SHWARTZMAN, G. & G m m , I.E. (1948) Hemorrhagic manifestations of bacterial and virus infections: experimental studies and pathological interpretations. Annals ofthe N e w York Academy ofsciences, 49, 627. C.A., JR (1951) Studies on the mechanism of STETSON, the Shwartzman phenomenon. Certain factors involved in the production of the local hemorrhagic necrosis.Journal of Experimental Medicine, 93,489. THOMAS, L. & GOOD, R.A. (1952) Studies on the generalized Shwartzman reaction. I. General observations conc+ng the phenomenon. Journal of Experimental Medicine, %, 605. WEBFIR, E., MALBSSA, S. & LASCH,H.G. (1963) Veranderungen der freien Thrombozytennukleotide beim Sanarelli-Shwartzman-Phhomen. Thrombosis et Diathesir Haemonhagica, 9, 304.

Activation of intravascular coagulation by endotoxin: the significance of granulocytes and platelets.

The importance of granulocytes and/or platelets in endotoxin-induced generalized intravascular coagulation was studied as well as thrombocytopenic rab...
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