In vitro effect of phospholipase C from bacillus cereus on tissue thromboplastin from different species

ThrombosisRESE!&XH 28; 93-101, 1982 0049-3838/82/190093-09$03.00/O Printed in the USA. Copyright(c) 1982 PergamonPress Ltd. All rights reserved.

IN VITRO EFFECT OF PHOSPHOLIPASE C FROM BACILLUS CEREUS ON TISSUE THROMBOPLASTIN FROM DIFFERENT SPECIES.

8. Hetland, T.L. Janson and B. Johnsen Research Institute Oslo 1, Norway.

for Internal Medicine, University

of Oslo,

(Received13.4.1982;in revised form 12.7.1982. Acceptedby Editor U. Abildgaard)

ABSTRACT Purified phospholipase C from Bacillus cereus caused a significant loss in the procoagulant activity of thrombo. plastin preparations from man, rabbit, sheep, cow, rat and mouse. However, marked differences were observed with respect to the degree of inactivation. Rat, mouse, bovine and one type of rabbit preparations (prepared from acetone powdered brain) were markedly more sensitive to attack by phospholipase C than were preparations of human, sheep and standard rabbit preparations. The relative amounts of the individual phospholipids in thromboplastin preparations showed only minor variations among the species. The effect of phospholipase C on each of these phospholipids in the various thromboplastin preparations showed some significant differences.

INTRODUCTION Tissue thromboplastin consists of an integral plasma membrane protein (apoprotein III) surrounded by phospholipids (1,2,3) and may under certain conditions be exposed or released

Key words: Phospholipase C, Bacillus cereus, tissue thromboplastin, tissue factor, species specificity.

93

94

PHOSPHOLIPXX C AND THROHBOPL.~IIN

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to the circulation causing intravascular coagulation via the extrinsic system (4,5,6). The apoprotein of human thromboplastin is a glycoprotein of M about 52000 containing about 6% carbohydrate. The phospholfpid moiety of crude or semipurified thromboplastin consists predominantly of phosphatidylcholine and phosphatidylethanolamine with minor amounts of phosphatidylserine, sphingomyelin and phosphatidylinositol (2).

Exposure of thromboplastin to phospholipase C (Bacillus cereus) results in rapid loss of the procoagulant activity caused by hydrolysis of the essential phospholipids (7). Gollub et a1.(8) demonstrated a protective effect of phospholipase C from Bacillus cereus against thromboplastin infusions in rabbits. Protection of rats against otherwise lethal doses of thromboplastin (8,9) and pregnant mice against abortion and intrauterine foetal death induced by endotoxin (11) have stimulated a further study of the enzyme as a possible prophylactic agent against certain folrms of thrombosis.

In order to select appropriate animal models for such studies, the effect of phospholipase C on thromboplastin preparations from six different species has been investigated.

MATERIALS AND METHODS Tissue thromboplastin Crude preparations of thromboplastin were prepared from brains of the various species according to Hjort (12) and diluted to optimal activity. Human and boving thromboplastin preparations were lyophilized and stored at -20 C until use. she other thromboplastins were stored as suspensions at -20 C for less than 4 weeks before use. Rabbit thromboplastin was also prepared from rabbit brain acetone powder (Pel-Freeze Biologicals, Rogers, Ar., USA) by homogenization and saline extraction at 45 c. The extract was centrifuged at 2000 g for 20 min at room temperature. Phospholipase C Phospholipase C (PLC) was purified from Bacillus cereus ATC 10987 (MS-l) as previously described (13,14). Purified enzyme was stored in barbital-buffered saline (12) containing 1 mmol/l ZnSO The enzyme was homogeneous by polyacrylamide gel electrop d' oresis both in the presence (15) and absence (16) of sodium dodecyl sulphate. Assay for thromboplastin activity Thromboplastin was assayed by measuring the clotting time in a one-stage test system consisting of 0.1 ml homologous titrated plasma, 0.1 ml of the solution to be tested for thromboplastin, 0.1 ml barbital-buffered saline (in some experiments containing PLC) and 0.1 ml 0.030 mol/l CaCl . The test solution was mlxed with buffe& (or buffer containing PLC) and incubated for 3 minutes at 37 C; then plasmaoand CaC12 (both prewarmed separately for 3 minutes at 37 C) were added in rapid succession.

Reference curves were obtained bl; testing serial dilutions of each crude thromboplastin solution, th$ activity of the undiluted samples was arbitrarily taken as 100%. Protein was determined by a modified Lowry-method (17). Extraction of phospholipids Phospholipids were extracted from thromboplastin preparations according to (18). Thromboplastin suspension (one volume) was added to 5.5 volumes of isopropanol and left for one hour at room temperature with occasional mixing, Chloroform (3.5 volumes) was added with mixing and the resulting suspension was left for another hour. The suspension was filtered through glass fibre filters prewashed with isopropanol/chlorofogm. The filtrate was evaporated to dryness under nitrogen at 45 C. The residue was dissolved in 4 ml chloroform:methanol (2:l) and washed twice with 1 ml 0.05 mol/l KCl. The aqueous phase was removed and the organic phase dried by adding a few mg of anhydrous sodium sulphate. The supernatant was decanted off and evaporated to dryness under nitrogen at 45OC. The residue was finally dissolved in 0.05 ml chloroform:methanol 9:l. Aliquots were analyzed for total lipid phosphorus by digestion and for individual phospholipids by thin lager chromatography on Silica FI plates (Merck) activated at 120 C for one hour. The chromatographic system consisted of chloroform:methanol:acetic acid:water (50:25:7:3) (19). The individual spots were visualized by iodine vapour, scraped off and analyzed for phosphorus afteh digestion with 0.4 ml perchloric acid for 20 minutes at 200 C according to a modification (20) of the method of Bartlett (21).

RESULTS The activity of crude thromboplastin preparations from man, cow, sheep, rabbit, rat and mouse remaining after a standardized treatment with PLC varied markedly (figure 1). Incubation with PLC at two different concentrations suggested that thromboplastin preparations from the species investigated fall into two groups with respect to resistance to PLC-inactivation. The lower concentration of PLC (0.25 w/ml) inactivated less than 50% of the thromboplastin from human and sheep whereas 80-90% of the activity was destroyed in the preparations from rat, mouse and cow under identical treatment. Similar results were found at the higher PLC concentration (1.0 m/ml) (figure 1). The inactivation of rabbit thromboplastin depended on how it had been prepared, that prepared according to Hjort (12) being as sensitive as the preparations from rat, mouse and cow, whereas that extracted from acetone powdered brain was much more resistant. Dilution curves were prepared for each thromboplastin preparation, these were essentially linear at least to 1:lOO dilution. The difference in steepness of the slopes of these dilution curves was insignificant and could not explain the varying susceptibility to phospholipase C. The difference in susceptibility to attack by PLC might be caused by differences in the total amount or nature of the phos-

?BOSP:IOLIPASEC AXJ C-ii!OXEiOPLASTTX

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PHOSPHOLIPASE C AND XXOK3OPLASTi~

FIG.

1

Effect of phospholipase C on crude preparations of brain thromboplastin from various sources. Remaining thromboplastin activity An per cent of initial activity after treatment for 3 min at 37 C with phospholipase C at final concentrations of 0.25 tg/;;mL;otted bars) or 1.0 pg/ml (hatched bars). : B: Sheep, C: Rabbit (acetone powder), D: Rabbit, E: Bovine, 6: Rat, G: Mouse

pholipids surrounding apoprotein III. The total amount of phospholipid expressed as mm01 lipid-P/l showed a 30-fold variation (Table 1). The rabbit (acetone brain) preparation contained the highest total amount of phospholipid per ml and the bovine preThe lipid/protein ratios were remarkably paration the smallest. similar (0.13-0.20) for four of the preparations, rabbit (acetone brain) and human preparations were much higher and lower, respectively (Table I). The four major phospholipids in the thromboplastin preparations were determined (Table I). Although small differences were observed, the overall picture was the same in all preparations. Phosphatidylcholine and phosphatidylSmaller amounts of ethanolamine were the major phospholipids. phosphatidylserine + phosphatidylinositol (not separated by the method used) and sphingomyelin were found in all preparations

98

PHOSPHOLIPGiE C MB

TABLE

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THROMBOPL.ASTIH

~,.i

II

Hydrolysis by Phospholipase C of Phospholipids in crude Thromboplastin Preparations in per cent of control Values. RAT

MOUSE

SHEEP

cow

MAN

Total phospholipids

68.0

70.3

50.8

42.3

69.1

68.2

Sphingomyelin

2.7

14.1

4.6

10.2

5.5

0

Phosphatidyl choline

80.5

78.3

56.0

52.6

79.1

75.7

65.9

Phosphatidyl serine + inositol

25.1

45.3

30,9

19.9

41.0

32.7

26.0

Phosphatidylethanolamine

76.5

81.6

47.5

52.6

77.2

59.2

78.0

RABBIT

RABBIT* 63.3 8.9

* prepared from brain acetone powder. Thromboplastin suspensions were incubated with ghosphofipase C (final concentration 0.5 pg/ml) for 3 min at 37 C. Each value is the mean of triplicate determinations in two different experiments.

Sixty to seventy per cent of total phospholipid was hydrolysed by PLC in all preparations except ovine and bovine where 40-50% was broken down (Table II). About 80% of phosphatidylcholine and phosphatidylethanolamine was destroyed in the specimens from rat and mouse, and only 50% for sheep and bovine thromboplastin.

DISCUSSION The rapid loss of procoagulant activity of human thromboplastin caused by phospholipase C ("thromboplastinase") from Bacillus cereus was observed by Gollub et al. (7) using a crude Since then, several reports have preparation of the enzyme. appeared on the use of phospholipase C to destroy the activity of thromboplastin in vitro as well as in vivo (6,8,9,10). We report here that thromboplastin preparations from different species seem to fall into two categories with respect to phospholipase C susceptibility, human and ovine thromboplastin The explanation for the resistance to being quite resistant. PLC of rabbit thromboplastin prepared from acetone powder is probably trivial, these preparations contained 4-5 times as much Similarly, phospholipid per protein as the other preparations. the reason for the susceptibility of bovine thromboplastin may

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PHOSPHOLIPASEc AND

l3EmMEiOP~TiN

99

be the extremely low total amount of phospholipid in these preparations. The susceptible rat and mouse thromboplastins have average phospholipid/protein ratios and their phospholipids are no more susceptible to hydrolysis than those of human thromboplastin which, however, is quite resistant in spite of a very low phospholipid/protein ratio. Sheep thromboplastin has a slightly lower total phospholipid/protein ratio but a rather lower susceptibility of phosphatidylethanolamine and phosphatidylcholine to PLC than for the corresponding phospholipids in murine and rat thromboplastins. This may explain its higher residual procoagulant activity. The reduced susceptibility remains unexplained. In conclusion, crude thromboplastin prepared from the same source by different methods as well as by the same method from different sources show significant variations in phospholipid:protein ratios, phospholipid composition and susceptibility to inactivation by phospholipase C. The residual activity appears related neither to hydrolysis of total phospholipid nor to any class of phospholipid since: - sheep and cow thromboplastins are both less hydrolyzed than the others and present different resistances to PLC. - human and sheep thromboplastins, both the most resistant to PLC, are differently hydrolyzed one from the other. The lack of correlation between the composition of the preparations and susceptibility to PLC may be due to an influence of breakdown products (diglycerides) on the procoagulant activity. Alternatively, there may be inherent differences with regard to quantity or type of phospholipid required by apoproteins from different species. The available data for human and bovine apoprotein (2,3,22) may support this explanation.

ACKNOWLEDGEMENT This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council on CardioWe thank one of the referees for pertinent vascular Diseases. comments.

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