Effects of Bacterial Endotoxin on Rabbit Platelets

Prez, Roger M. Des

doi:10.1084/jem.120.2.305

Cited by 23 publications

(3 citation statements)

References 7 publications

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“…While a superficially similar form of platelet aggregation and release of platelet factors is produced by thrombin, previous studies have shown that the effects of endotoxin and of thrombin on platelets differ in several important respects (6). The blood of most mammals contains antibodies to endotoxin (7).…”

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Effects of Bacterial Endotoxin on Rabbit Platelets

Prez

Bryant

1966

The Journal of Experimental Medicine

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When endotoxin is injected by vein it is immediately fixed onto platelets (1). Both in vivo and in vitro studies have further shown that endotoxin causes rapid aggregation and disappearance of blood platelets, accelerates coagulation, and promotes release of several intracellular platelet factors with physiological activity (2-5). It has thus appeared likely that this platelet-endotoxin ~nteraction may play a role in the earliest phases of removal of endotoxin from the blood stream.While a superficially similar form of platelet aggregation and release of platelet factors is produced by thrombin, previous studies have shown that the effects of endotoxin and of thrombin on platelets differ in several important respects (6). The blood of most mammals contains antibodies to endotoxin (7). Because antigen-antibody interactions also produce platelet aggregation and release of intraceUular components (8-11), the present studies explored the thesis that platelet-endotoxin interaction might be a special instance of platelet damage by antigen-antibody complexes. These experiments compare the divalent ion requirements of these two types of platelet injury. They have led to the tentative hypothesis that antigen-antibody and endotoxin injury may be identical in nature, and secondary to platelet phagocytosis. Materials and MethodsRabbit blood obtained by cardiac puncture was delivered directly through plastic tubing into cold sterile siliconized centrifuge tubes containing anticoagulant solutions to provide a final mixture of one part anticoagulant to nine parts blood. Piatelet-rich plasma and plateletpoor plasma were prepared by centrifugation as previously described and contained from 4 to 8 X 10 s platelets per milliliter (2). Platelet-rich plasma prepared directly from heparinized blood contained a much lower concentration of platelets due to spontaneous platelet clumping in this anticoagulant under the conditions employed. Accordingly, heparinized platelet-rich plasma was prepared by collection of blood in NaEDTA, preparation of platelet-rich plasma,

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Effects of Bacterial Endotoxin on Rabbit Platelets

Prez

Bryant

1966

The Journal of Experimental Medicine

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“…In vitro studies with rabbit platelets have shown that relatively small concentrations of heparin are capable of preventing thrombin induced-platelet injury whereas very large amounts of the anticoagulant are necessary to prevent similar damage by bacterial endotoxin (1). In vivo investigations have demonstrated that the usual anticoagulating amounts of heparin do not prevent the thrombocytopenia in the generalized Shwartzman reaction (gSr) ( 2 ) in rabbits and a similar phenomenon has been observed in humans with septic shock treated with heparin (3,4).…”

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Effect of Heparin on Endotoxin-Induced Thrombocytopenia

Corrigan

1971

Experimental Biology and Medicine

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In vitro studies with rabbit platelets have shown that relatively small concentrations of heparin are capable of preventing thrombin induced-platelet injury whereas very large amounts of the anticoagulant are necessary to prevent similar damage by bacterial endotoxin (1). In vivo investigations have demonstrated that the usual anticoagulating amounts of heparin do not prevent the thrombocytopenia in the generalized Shwartzman reaction (gSr) ( 2 ) in rabbits and a similar phenomenon has been observed in humans with septic shock treated with heparin (3, 4). The gSr is elicited by giving rabbits two properly spaced intravenous injections of endotoxin and is characterized by the production of diffuse intravascular coagulation with the subsequent occurrence of bilateral renal cortical necrosis ( 5 ) . I t therefore provides an in vivo model for the study of both endotoxin-and thrombin-induced hematologic changes.The purpose of this investigation was to evaluate the in vivo effect of large amounts of heparin on endotoxin-induced thrombocytopenia, leukopenia and hypofibrinogenemia using the gSr as the experimental model. Material and Methods. E . coli 0127:B8 (Boivin type; Difco Laboratories, Detroit, Michigan) was prepared fresh daily in pyrogen-free normal saline solution.Aqueous heparin, 20,000 units/ml, was purchased from the Upjohn Company, Kalamazoo, Michigan.Rabbits, white hybrid of either sex weighing 1.0 kg were housed in an air-conditioned animal room and fed Purina rabbit pellet and , Ariz. 85721.water, ad lib.The generalized Shwartzman reaction was produced in the classical manner (5). Animals received two intravenous injections of endotoxin (0.1 mg/kg) 24 hr apart. Twentyfour hr after the second endotoxin injection, they were sacrificed and the frequency of bilateral renal cortical necrosis noted.Blood for white blood cell (WBC), platelet, and fibrinogen determination was obtained by two-syringe technique from the heart, and anticoagulated with 3.8% sodium citrate. Plasma was obtained by centrifuging the blood at 8000 rpm at 0" for 20 min. Blood was obtained immediately before (0 hr) and again 4 hr after the second (provocative) injection and the results were compared. WBC counts were done by standard technique, platelet counts by phase microscopy ( 6 ) , and fibrinogen concentation by the method of Ratnoff and Menzie (7).Results. Previous investigations had shown that rabbits are anticoagulated with concentrations of heparin of 5 mg (500 units)/kg or larger when administered intravenously (2). Des Prez reported that in vitro 5.0 mg of heparin/ml of platelet-rich plasma was necessary to inhibit the endotoxin effect on rabbit platelets ( 1). In these in vivo studies, concentrations of heparin ranging from 1000 units to 25,OOO/kg were used. The rabbits weighed 1.0 t 0.1 kg, with hematocrits ranging from 42-45%.All animals received the first injection of endotoxin. Twenty-four hr later they received either (a) endotoxin alone, (b) saline and no endotoxin or heparin, (c) heparin and no endotoxin, or (d) simulta...

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“…Previous studies have demonstrated that 5-hydroxytryptamine (5HT) released from blood platelets by bacterial endotoxin (lipopolysaccharide; LPS) requires the presence of calcium ion and plasma factor and that heparin inhibits its release (1)(2)(3)(4)(5). Recently, Nomura and Takagi (6) have separated the 5HT releasing factor from rabbit plasma by gel filtration on a Sephadex G-150 column.…”

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Studies on Plasma Factor Involved in 5-Hydroxytryptamine Release From Blood Platelets by Bacterial Endotoxin

Nomura

1972

Jpn.J.Pharmacol

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Previous studies have demonstrated that 5-hydroxytryptamine (5HT) released from blood platelets by bacterial endotoxin (lipopolysaccharide; LPS) requires the presence of calcium ion and plasma factor and that heparin inhibits its release (1-5).Recently, Nomura and Takagi (6) have separated the 5HT releasing factor from rabbit plasma by gel filtration on a Sephadex G-150 column. They have suggested that the plasma factor with 5HT releasing activity is a protein with a molecular wt. of approx. 110,000 and that 5HT release induced by LPS could be mediated through antagonism by LPS to the inhibitory effect of heparin on the 5HT releasing plasma factor, and also that activity of the 5HT releasing factor in plasma could be regulated by heparin and the anti-5HT releasing factor existing in the albumin fraction.
The present report describes studies on the further purification of the 5HT releasing factor in rabbit plasma and its properties.METHODS 1) Preparation of rabbit plasma: Eight rabbits of both sexes (wt. 2.0 to 3.5 kg) were used.Blood was obtained by cutting the carotid artery and mixing with 1/10 volume of 3.5 sodium citrate solution and centrifuged 1,400 g for 20 min at 4-C twice. The supernatant was pooled and used as rabbit plasma.2) Preparation of platelet suspension: Rabbit platelets isolated by similar methods previously described were suspended in 0.02 M Tris-HC1 buffer (pH 7.5) containing salt solution, the composition being as follows: NaCI, 8.33 g; KCI, 0.35 g; CaC12.2H2O, 0.37 g; MgCl2.6H2O, 0.224g; glucose, 2.O g in 1,000 ml of distilled water.3) Assay of 5HT releasing activity: Platelet suspension with or without heparin (2 units/ml) was incubated in the presence of each separated fraction of plasma factor at 37°C for 30 min under nitrogen atmosphere. Gentle shaking was employed. The mixture was then centri gufed at 1,200 g for 30 min at 4°C. Contents of 5HT in the supernatant and the pellet were determined spectrophotofluorimetrically. Methods described by Bertler (11) and Bogdanski

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Effects of Bacterial Endotoxin on Rabbit Platelets

Cited by 23 publications

References 7 publications

Effects of Bacterial Endotoxin on Rabbit Platelets

Effects of Bacterial Endotoxin on Rabbit Platelets

Effect of Heparin on Endotoxin-Induced Thrombocytopenia

Studies on Plasma Factor Involved in 5-Hydroxytryptamine Release From Blood Platelets by Bacterial Endotoxin

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