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Effects of N-acetyl glucosamine on platelet aggregation
THROMBOSIS RESEARCH 23; 289-300, 1981 0049-3848/81/150289-12$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
EFFECTS OF N-ACETYL CLLJCCGAMINEON PLATELET AGGREGATION*
J. Bertram, B.H. Ragats, W. Baldwin, and P.G. Iatrides From the Departments of Biochemistry and Physiology Northwest Center for Medical Education Indiana University School of Medicine Gary, Indiana 46408, U.S.A.
(Received 5.7.1980; in revised form 14.4.1981. Accepted by Editor E.W. Salzman. Received in final form by Executive Editorial Office 30.7.1981) ABSTRACT N-acetyl glucosamine (NAG) alone did not aggregate platelets but in low concentrations shortened the delay phase of the Staphylococcus aureus induced platelet aggregation in a concentration dependent manner, which suggests that NAG may be one of the components of the 2. aureus cell wall responsible for platelet aggregation. NAG, on the other hand, inhibited collagen induced platelet aggregation and the secondary platelet aggregation induced by epinephrine and ADP. This inhibition appears to be due to impurities that occur at higher levels in some NAG lots than in others. NAG has also been shown to decrease the length of time before the release of ATP from platelets when S. aureus is the aggregating agent.
INTRODUCTION
Disseminated intravascular coagulation (DIC) is a clinical disorder, characterized by generalized microthrombosis and hemorrhagic diathesis resulting in circulatory collapse (31). Both gram negative and gram ositive septicemias have been reported to be associated with DIC (7, 12, 25P . In animal models a number of investigators (13, 21, 27) have shown that endotoxin is able to aggregate platelets -in vivo and thus trigger the intrinsic clotting mechanism by release of platelet factor 3 and by the activation
*Key Words for subject indexing- platelet aggregation, Staphylococcus aureus, N-acetyl glucosamine. 289
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of the contact system of blood coagulation (15, 30). Whole gram negative bacteria as well as a variety of endotoxins have been shown to induce platelet aggregation in vitro (17,20, 25). Furthermore, whole gram positive bacteria may be one of the causes of DIC. It has been shown that endotoxin from gram negative bacteria (26)and the cell surface of gram positive bacteria (28) have some similarities. The cell surface of gram Positive bacteria consists of a cell wall made up of peptidoglycan. Chemically, peptidoglycan consists of a polymer made up of alternating N-acetyl glucosamine and N-acetyl muramic acid crosslinked through the number 3 carbon of N-acetyl muramic acid by a peptide bridge (28). Endotoxin is a complex polysaccharide containing some of the components found in peptidoglycan. The endotoxin polysaccharide contains, among other sugars, glucosamine (11) which may be acetylated to form N-acetyl glucosamine (NAG) or the nitrogen may be substituted with a fatty acid to form the lipid A component of endotoxin (11). The present study was undertaken to determine the effects of NAG on platelet aggregation and its importance in bacteriainduced platelet aggregation.
MATERIAIS AND METHODS
Preparation of Bacteria. A derivative of 5. aureus ATCC 2323 was prepared by a modification of the method of Clawson and White (3). S. aureus was grown on brain heart infusion broth (DIFCO) to either mid or iate logarithmic phase and harvested in a Sorvall RC2-B centrifuge adapted for continuous flow at 13,000 rpm (4OC). These cells were washed once with distilled H 0 (3,000 x g for 10 minutes at 4'C) and resuspended in distilled H 0. T&ee ml aliquots were heat killed at 70°C for 30 minutes (3). The gells were washed two more times in distilled H20, lyophilized and stored at -2O'C. Cells were gram positive cocci and colonies were uniform at each stage of the preparation. The lyophilized preparation was resuspended 24-72 hours before use in 0.85 NaCl. Platelet Aggregation. Human blood was collected from the antecubital veins of healthy subjects into 0.1 volume of 3.2$ trisodium citrate and was centrifuged to prepare platelet rich plasma and platelet poor plasma as described previously (16). Platelet aggregation was initiated by addition of 0.05 mls of an inducer 15 seconds after the addition of the same volume of saline or a modifier of platelet qgregation to 0.4 mls of PRP stirred at 900 rpm in siliconized cuvettes at 37 C. The platelet concentration of PRP was standardized at 300,0OO/ml by dilution with PPP. Aggregation was monitored by continuous recording of light transmission (LT) as a function of time. In these experiments a dual channel Payton Aggregometer was used. Release of ATP. Platelet aggregation was performed as described above. At selected points the PRP was transferred from the aggregometer to a fluorimeter. MgS04 (0.02 mls of 154 mM) and luciferin luciferase (0.05 mls of 145 mg/ml from DuPont) was added. The luminescence was measured and two aliquots of ATP were added as standards. This was a modification of the method of Feinman -et al. (9). Aggregating agents investigated y&re 2. aureus, adenosine at a final con entration of 10 M, epiane (Parke and collagen suspension (bovine tendon Davis) at a concentration of 10-% from Sigma, lOug/ml). Collagen suipension was prepared by vortexing and filtering it through cheesecloth. N-acetyl glucosamine (Sigma), in 0.8% NaCl was used at a final concentration of l-100 mM. NAG in this concentration range had no effect on plasma PH.
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Purity of NAG was evaluated by descending paper chromatography, ninhydrin reactivity, and by its ultraviolet spectrum. Chromatograms were developed on Whatman #l filter paper using isobutanol:pyridine:H20 (10:10:5),a modification of the method of Moreno -et al. (22). The method of Trevelyan et al. (29) for detecting reducing sugars was used. Ninhydrin spray (0.9 in ethanol), which reacts with many primary amines was used to detect glucosamine on the paper chromatogram. The content of ninhydrin reactive amine was measured by adding 1 ml of the sugar solution to 1 ml of 0.9 ninhydrin in ethanol and heating at 100°C for 15 minutes. Glucosamine was used as a standard and results are reported as mmoles of glucosamine/mole NAG in solution. Absorption was measured at 570 nm. Absorption spectra were done with a Perkin-Elmer Lambda 3 spectrophotometer
RESULTS Purity of NAG. 250 ug of NAG from lots 84c-0101, 107C-0121, and 31C-3000 were chromatographed as described in methods. When developed with a reagent specific for reducing sugars only one spot appeared. No spots were detected with ninhydrin spray indicating that no reactive amines were present. Glucose and glucosamine were used as standards. Glucosamine reacted with ninhydrin and migrated at .555 relative to NAG. Although ninhydrin reactive amines were not detectable on the paper chromatogram they could be detected in solution. The ninhydrin test described in methods was performed on the different NAG lot numbers. Glucosamine was used as a standard and the absorption at 570 nm was linear in the range of 0.251.00 mM glucosamine. Lot 84c-0101 contains the equivalent of 9.3 mmoles of glucosamine/mole NAG while the other NAG lot numbers contain only minimal amounts of ninhydrin reactive amines. (Table 1).
Table 1 Ninhydrin Reactivity of Different NAG Lots NAG Lot # 84-C-0101
mmoles glucosamine/mole 9.3
107C-0121
1.0
31c-3000
1.0
A scan of each of the NAG lot numbers was performed as described in methods The spectrum of lot 84.~~01 was considerably different from the other lots or glucosamine (Fig. 1). NAG lot 84c-0101 had an absorption peak at 272 nm where neither glucosamine nor the other NAG lots absorb. S. aureus induced platelet aggregation. In Figure 2, saline was added to PRP 15 seconds prior to the addition ofg. aureus suspension. The ratio of bacteria to platelets was as follows: in Curve A 8:I (100 ug/ml), in Curve B 6.5:1 (80 ug/ml), in Curve C 4:l (50 ug/ml), and in Curve D 3:l (40 &ml). The results are very similar to those presented by Clawson and White (3), that is, following the addition of 2. aureus there was a "contact" phase which was followed by a narrowing of the ban, termed the shape change phase, and
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finally by a rapid aggregation of the platelets. Figure 2 shows that the "contact" phase was proportional to the ratio of bacteria to platelets. Above a ratio of 8 bacteria/platelet the turbidity of the bacterial suspension began to interfere with the assay. Strong aggregations were obtained with a ratio of 2.5 bacteria/platelet and sometimes as low as 1 bacteria/platelet. Effects of NAG alone on Platelet bgregation. In Figure 3, NAG was added to PRP at a final concentration of 10 mM (Curve B), or 100 mM (Curve C) 15 seconds prior to the addition of saline. In Curve A, saline was added 15 seconds prior to the addition of S. aureus at a bacteria/platelet ratTo 0f 2.5. In Curve D, NAG at a final concentration of 10 mM was added 15 seconds urior to the addition of S. aureus ai a ratio of 2.5 bacteria7 platelet. As it can be seen. although s. aureus can induce platelet aggre-, NAG alone did not cause platelet aggregation at the concentrations tested (Curves B and C). However, NAG in combination with 2. aureus (Curve D) shortened the contact phase of S. aureus induced platelet aggregation. NAG at concentrations of 100 mM causes a drop in the baseline of the recording. Subsequent addition of ADP at sufficient concentration to induce platelet aggregation failed to induce an increase in light transmission which is cheracteristic of platelet aggregation. Effects of NAG on S. aureus induced platelet aggregation. In Figure 4, NAG or saline was added to PRP 15 seconds prior to the addition of S. aureus at a bacteria/ to platelet ratTo of4. The results cleerly show that here at NAG concentrations of 10 mM (Curve A) and 1 mM (Curve B) the delay phase of 2. aureus induced platelet e.ggregatiE shorter than the control (Curve G). The addition of 0.1 mM NAG, however, did not show any effects on S. aureus induced platelet aggregakmve C). The effect of NAG on S. aureus induced platelet aggregation Was a concentration dependent phenomenon, that is, increasing concentrations of NAG re-
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QI A@$.
1
-0
a00
360
Figure 1 Ultraviolet spectrum of N-acetyl glucosamine. A, 250 mM NAG lot++ 84~-0101; B, 25O mM NAG lot#107C0121; C, 25O mM glucosamine; D, H20.
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TIME
293
IN MINUTES Figure 2
s.,aureus induced plateletagregation. Saline was added to PRP 15 seconds prior to the addition of 2. aureus at a bacteriato plateletratio of 8:l 4:l (50 &ml) C, or 3:l (40 ug/ml) D. (100 &ml) A, 6.4:1(80 ug/ii LT= Light Transmission.
LT 41
4
1
1
TIME
3
4
s
6
7
IN MINUTES Figure 3
The effects of N-acetylglucosaminealone on plateletaggregation. Saline (A) or N-acetyl glucosamineat a final concentrationof 10 ml4(D) was added to PRP 15 seconds prior to the additionofg. aurea at a final concentration of 2.5 bacteria/platelet.NAG at concentrationsof 10 mM (B) and 100 mJ4(C) was added 15 seconds prior to the additionof saline.
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LT 4
1
2
3
TIME
4
5
6
7
8
IN MINUTES Figure 4
The effects of NAG on 2. aureus induced platelet aggregation. Saline, (G), or NAG at concentrations of 100 mM (D), 0.1 mM (C), 1 mM (B), or 10 mM (A) was added to PRP 15 seconds prior to the addition ofS_. aureus at a ratio of 4 bacteria/platelet (50 ug/ml).
LT 4
/
1234$6 TIME IN
MINUTES
Figure 5 The effects of NAG on collagen induced platelet aggregation. Saline (A), or 10 mM NAG from lot #84c-0101 (C) or lot #107C-0121 (B) was added to PRP 15 seconds prior to the addition of collagen (10 ug/ml).
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L'1
4 3 2 1
Figure 6 The effects of NAG on epinephrineinducedplateletaggregation. Saline (A), 10 mM NAG lot # 107C-0121 (B), or 10 mM NAc610t#84~-0101 (C) was added to PRP 15 seconds prior to the addition of 10 M epinephrine. sulted in successivelyshorter delay phases. At NAG concentrationsof 100 mM (Curve D), S. aureus induced plateletaggregationwas c mpletelyinhibited. This inhibiTionwas not overcomeby the additionof lo-% ADP. Furthermore, the light transmissionof Curve D decreasedbelow the initialbaseline. NAG lot 84c-0101or lot 107C-0121gave the same results. Table 2 shows the differencein the delay phase between2. aureus induced platelet aggregationboth in the presenceand absence of 10 mM NAG. The results of multiple measurementson two differentPRP preparationsare reported. Although the length of the delay phase in the controldiffers from experimentto experiment,a reductionin the length of this phase is consistentlyobserved upon the addition of NAG. A reductionof 3% in trial #I and 3% in trial# 2 was obtained. Effects of NAG on collagen,epinephrineand ADP inducedplateletaggregation. Saline, or 10 mM NAG from either lot #84&0101 or lot#107C-0121 was added to PRP 15 seconds prior to an aggregating-sent. The aggrggatingagent was either collagen (10 &ml), epinephrine(10 M), or ADP (10 )I). Figure 5 clearly shows that the rate and degree of plateletaggregationinducedby collagen are reduced in the presence of NAG lot#8&0101 but not lot #107c-0121.Figures 6 and 7 show that NAG lot #84~-0101 inhibitedsecondary platelet aggregationinduced by epinephrineor by ADP but lO7C-OlZldid not. The effects of NAG on ATP release from platelets. Saline or 10 mM NAG (lO7C-OlZl)was added to PRP 15 seconds prior to the additionof S. aureus at a bacteria to platelet ratio of 4:l (9 &ml) and the aggrega%.onswere interruptedand the luminescencedue to ATP releasedfrom plateletswas measured as described in methods. The largestdifferencebetweenthe control and the experimentalwas obtainedwhen aggregationwas almost completein the NAG-PRP and just beginning in the control. Table 3 shows that the release at that point is much greater in the samples to which NAG has been added.
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Table 2 The Effect of NAG on the Delay Phase of 2. aureus Induced Platelet Aggregation Delay (seconds) Trial #l* Control
332*10
10 mM NAG
234*12
Trial #2* Control
242+13
10 mM NAG *A bacteria to platelet ratio of 4:1
4
ly+ (9
ug/ml final concentration) was used,
LT 4 3 2 1
I
I
1
2
TIME
,
I
3
4
I
5
6
IN MINUTES Figure 7
The effects of NAG on ADP induced platelet aggregation. Saline (A), or IO mM NAG lot# lO7C-0121 (B), or 10 mM N$j lot# 84c-0101 (C) was added to PRP 15 seconds prior to the addition of 10 M ADP. Table 3 The Effects of NAG on ATP Release from Platelets
Trial #l
ATP (nmoles/l) Control Experimental 22*15 n=4 79*10 n=4
Trial #2
21*10 n=5
47*10 n=5
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DISCUSSION Clanson and White first described S. aureus induction of platelet aggregation. They divided the process &to four phases. The first phase after the addition of the bacteria was the contact or delay phase in which there was no change in a broad baseline produced by stirred discoid platelets. This was followed by a rapid narrowing of the baseline, called the platelet shape change phase. Next, there was an early aggregation and finally a plateau referred to as irreversible aggregation. The length of the contact phase increased with a decrease in the number of bacteria added. Our system behaved in a similar manner although slightly higher numbers of bacteria were required to produce rapid aggregation with an acceptably short contact phase. Clawson -et al. (6) were able to show that N-ethylmaleamide, a sulfhydryl inhibitor, blocked the platelet release reaction induced by S. aureus in a manner similar to that observed with adenosine. 2. aureus iyduced platelet aggregation is probably mediated by the release reaction since the enzyme system phosphoenol pyruvate-pyruvate kinase, which converts ADP to ATP, inhibited both the release and the platelet aggregation induced by 2. aureus (6). The nature of the chemical component of the cell surface of S. aureus that interacts with platelets is not certain. Hawiger et al. (10) have suggested -7 that the 2. aureus induced platelet aggregation is mediated by protein A and the F porti=IgG. These investigators showed that soluble protein A inhibzted release and aggregation and that the addition of IgG was necessary for the binding of protein A to was ed platelets. They also showed that protein A could induce the release of % serotonin. In contrast to their findings Clawson and White (4) showed that washed platelets could aggregate in response to 2. aureus. It has been suggested that IgG might have been absorbed to the surface of these washed platelets (personal communication). Another possible candidate for the cell surface feature of bacteria which is responsible for platelet aggregation is suggested by the work of Rotta et al. (24). These investigators showed that synthetic peptidoglycan subunits induced lysis of rabbit blood platelets. These subunits consisted of the sugar components plus peptide side chains. It is not clear from their data whether this lysis is analogous to the release reaction and this investigator did not do aggregation studies. Amino sugars have been shown to influence platelet aggregation in a negative fashion. Treatment of collagen with glucosamine has been reported to inhibit platelet aggregation (19). N-acetyl neuraminic acid has been shown to inhibit platelet aggregation induced by a variety of agents (2, 18). In previous work we have reported that NAG and glucosamine inhibited collagen induced platelet aggre ation and secondary platelet aggregation induced by epinephrine and ADP (17 . In the present study we have investigated the purity of NAG by several methods. Descending paper chromatography of the different NAG lots revealed one spot with reagents capable of detecting reducing sugars. No ninhydrin reactive material was detectable. In NAG solutions, however, nine times as much ninhydrin reactive material was detected in lot # 84c-0101 as in the other lot #s. The ultraviolet spectrum of this NAG lot also had a peak at 2.72nm in contrast to glucosamine and the other NAG lots. This is the same lot number that inhibited platelet aggregation due to collagen, epinephrine and ADP. There appears to be one or more impurities in NAG lot # 84C-0101 which are not precisely characterized. The impurity that absorbs at 272 nm is not glucosamine. The impurity that reacts with ninhydrin is probably an amine. The inhibition of collagen, epinephrine, and ADP induced platelet aggregation is probably due to a contaminant in lot # 84c-0101 since the other lot #s do not cause this inhibition. 2. aureus induced platelet aggregation was not measurably affected by the contmm. The reason for this
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is unclear. It may be that the contaminant has no effect on the system or that the effect is masked by other factors. We have shown that NAG reproducibly shortens the delay phase of 2. aureus induced platelet aggregation. We have also shown that it shortens the length of time necessary for the release reaction to occur. This is of significance since 2. aureus induced platelet aggregation is probably release mediated (6). We were unable to induce platelet aggregation using NAG alone, even at high concentrations (100 mM). The amount of hexosamine in the peptidoglycan of sufficient S. aureus to induce platelet aggregation is much higher than this. Addition of-high concentrations of NAG as a monomer probably interferes with platelet aggregation through an osmotic effect (8). Use of a NAG polymer to circumvent the osmotic problems has been attempted in our laboratory but technical difficulties with the behavior of poly NAG in solution have prevented us from using it in aqueous systems. The suggestion that NAG may be one of the components of the bacterial cell wall responsible for platelet aggregation is not conclusively proven by the data presented here. However the fact that NAG shortens both the delay of aggregation and the delay of the release reaction in S. aureus induced platelet aggregation but has no affect on collagen, epynephrine, and ADP induced platelet aggregation (in its purer form) suggests that this phenomenon is not based on a general effect on platelet aggregation but is related only to bacterial platelet aggregation. Whether NAG enhances platelet aggregation es pert of the cell wall in bacteria is not certain. Further studies exe needed to elucidate the important effects of NAG on platelet aggregation.
REFERENCES
1.
BERTRAM, J., B.H. RAGATZ, W. BALDWIN, and P.G. IATRIDES, 1980. The effects of Glucosamine and N-acetyl on platelet aggregation. _ glucosamine Fed. Proc. 2:426.
2.
BORN, G.V.R., and I.B. KOVACS, 1978. Inhibition by N-acetyl neuraminic acid of platelet thrombogenesis induced by laser injury to rat and hamster venu64:301-304. les. Br. J. Pharmac. -
3.
CLAWSON, C.C., and J.G. WHITE, *Am. J. Path. 6J:367-380.
1971. Platelet interaction with bacteria I.
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CLAWSON, C.C., and J.G. WHITE, Am. J. Path. 65:381-395.
1971. Platelet interaction with bacteria II.
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CLAWSON, C.C., 1973. Platelet interaction with bacteria III, ultrastructure Am. J. Path. z:&+472.
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CLAWSON, C.C., M.D. GUNDU, H.R. RAO, and J.G. WHITE, 81:411-419. action with bacteria IV. Am. J. Path. .-
7.
CORRIGAN, J., L. WALKER, N. MAY, 1968. Changes in the blood coagulation system associated with septicemia. New Eng. J. Med, 2'JJ:851-856.
8.
COWAN, D.H., 1980. Effect of alcoholism on hemostasis. Seminars in Hematology. 17(2):137-147.
1975. Platelet inter-
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FEINMAN, R.D., J. LUBOWSKY, I.CHARO, and M.P. ZABINSKI, 1977. The lumiaggregometer; a new instrument for simultaneous measurement of secretion and aggregation by platelets. J. Lab. Clin. Med. p-:125-129.
10. HAWIGER, J., S. STECKLEY, D. HAMMOND, C. CHENG, S. TIMMONS, A. GLICK, and R. DEPREZ, 1979. Staphylococci-induced human platelet injury mediated by protein A and Immunoglubulin G Fc receptor. J. Clin. Invest. 3:931-937.
11. HEATH, E.C., 1971. Complex polysaccharides. Ann. Rev. Biochem. -@:2936.
12. HOERDER, M.H., and R. MANRIQUE, 1965. Akterserung der blutgerinnung durch ein bacterielles endotoxin beim menschen unter kumarin therapie. Xlin. IJschr. Q:397.
13. HOROWITZ, H.I., R.M. DEPREZ, and E.W. HOOK, 1962. Effects of bacterial endotoxin on rabbit platelets II. Enhancement of platelet factor 3 activity -in vitro and -in vivo. J. Exp. Med. 116:610-633. 14. HOVIG, T., 1963. Release of platelet aggregating substance (ADP) from rabbit blood platelets induced by saline extract of tendons. Thrombos. Diath. Haemorrh. (Stuttg.1 9:26b-278.
15. IATRIDES, P.G., J.H. FERGUSON and S.G. IATRIDES, 1964. Surface factor mechanisms in relation to blood platelets: evidence that activated Hageman factor is present on the surface of platelets. Thrombos. Diath. Hameorrh. (Stuttg.) 11:355-371.
16. IATRIDES, S.G., P.G. IATRIDES, S.G. MARKIDOU, and B.H. RAGATZ. 1975. 2,3-Diphosphoglycerate; A physiological inhibitor of platelet aggregation. -Sci. 187~259-261.
17. JOHNSON, H., and PER MAGNUS NIKLASSAN, 1976. Effects of menigococcal and Escherichia coli polysacchexide on human and dog platelet aggregation J. ClKPath. 2:438-440.
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18. KOVACS, I.B., and P. GOROG, 1979. Inhibition by N-acetyl neuraminic (sialic) acid of platelet aggregation induced by different stimuli. Thrombos. Haemostas. (Stuttg.) 2:1187-1192.
19. LEGRAND, Y., J.P. CAEN, and L. ROBERT, 1968. Effect of glucosamine on platelet collagen reaction.
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20. MACINTYRE, D.E., A.P. ALLEN, K.J.I. THORNE, A.M. GLAUERT, and J.L. GORDON, 1977. Endotoxin induced platelet aggregation and secretion. J. Cell Sci. %:211-223.
21. MCKAY, D.G., E.N. MARGAREIT, and I. CSAVOSSY, 1966. An electron microscopic study of the effects of the bacterial endotoxin on the blood vascular system. &ab. Invest. ~181~1829. 22.
MORENO, R .E., FUMINORI KANETSUMA, and L.M. CARBONELLI, 1969. Isolation of chitin and glucan from the cell wall of the yeast form of Paracoccidioides brasiliensis. Arch. Biochem. Biophys. 13:212-m
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23. NAGAYAMA, M., M.B. ZUCKER, and F.K. BELLER, 1971. Effects of a variety of endotoxins on human and rabbit platelet function. Haemorrh. (Stutt.) 26:467-473. 24.
Thrombos Diath.
ROTTA, J., M. RYC, K. MASEK, and M. ZAORAL, 1979. Biological activity of synthetic subunits of Streptococcus peptidoglycan. I pyrogenic and thrombocytolytic activity. Exptl. Cell Biol. a 258-268.
25. RYTEL, M.W., T.H. DEE, J. FERSTENFELD, and G. HENSLEY, 1974. Possible pathogenic role of capsular antigens in fulminant pneumococcal disease with disseminated intravascular coagulation (DIC). Am. J. Med. 57:889-
896. 26. SHARON, N., 1975. Complex carbohydrates, pp. 318-336. Addison Westley Publishing Co., Reading, MA.
27. STETSON, C.A., 1951. Studies on the mechanism of the Schwartzman phenomenon. Certain factors involved in the production of the local hemorrhagic necrosis. J. Exp. Med. =:489-504.
28. TIPPER, D.J., and J.L. STROMINGER, 1965. Mechanism of action of penicillin.
PNAS 54:1133-1141. m-
29. TREVELYAN,W.E., D.P. PROCTER, and J.S. HARRISON, 1950. Detection of sugars on paper chromatograms. Nature 166:~445. --
30. WALSH, P.N., 1972. Platelets, blood coagulation and hemostasis, PP.
23-43 in Platelets and Thrombosis, Sol Sherry and Alexander Scriabine,
1974, ed. UniversityPerk Press, Baltimore, MD. 21202.
31. YOSHIKAWA, T., K.R. TANAKE, and L.B. GUZE, 1971. Infection and disseminated intravascular coagulation. -Med. 50:237-2%.
EFFECTS OF N-ACETYL CLLJCCGAMINEON PLATELET AGGREGATION*
J. Bertram, B.H. Ragats, W. Baldwin, and P.G. Iatrides From the Departments of Biochemistry and Physiology Northwest Center for Medical Education Indiana University School of Medicine Gary, Indiana 46408, U.S.A.
(Received 5.7.1980; in revised form 14.4.1981. Accepted by Editor E.W. Salzman. Received in final form by Executive Editorial Office 30.7.1981) ABSTRACT N-acetyl glucosamine (NAG) alone did not aggregate platelets but in low concentrations shortened the delay phase of the Staphylococcus aureus induced platelet aggregation in a concentration dependent manner, which suggests that NAG may be one of the components of the 2. aureus cell wall responsible for platelet aggregation. NAG, on the other hand, inhibited collagen induced platelet aggregation and the secondary platelet aggregation induced by epinephrine and ADP. This inhibition appears to be due to impurities that occur at higher levels in some NAG lots than in others. NAG has also been shown to decrease the length of time before the release of ATP from platelets when S. aureus is the aggregating agent.
INTRODUCTION
Disseminated intravascular coagulation (DIC) is a clinical disorder, characterized by generalized microthrombosis and hemorrhagic diathesis resulting in circulatory collapse (31). Both gram negative and gram ositive septicemias have been reported to be associated with DIC (7, 12, 25P . In animal models a number of investigators (13, 21, 27) have shown that endotoxin is able to aggregate platelets -in vivo and thus trigger the intrinsic clotting mechanism by release of platelet factor 3 and by the activation
*Key Words for subject indexing- platelet aggregation, Staphylococcus aureus, N-acetyl glucosamine. 289
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of the contact system of blood coagulation (15, 30). Whole gram negative bacteria as well as a variety of endotoxins have been shown to induce platelet aggregation in vitro (17,20, 25). Furthermore, whole gram positive bacteria may be one of the causes of DIC. It has been shown that endotoxin from gram negative bacteria (26)and the cell surface of gram positive bacteria (28) have some similarities. The cell surface of gram Positive bacteria consists of a cell wall made up of peptidoglycan. Chemically, peptidoglycan consists of a polymer made up of alternating N-acetyl glucosamine and N-acetyl muramic acid crosslinked through the number 3 carbon of N-acetyl muramic acid by a peptide bridge (28). Endotoxin is a complex polysaccharide containing some of the components found in peptidoglycan. The endotoxin polysaccharide contains, among other sugars, glucosamine (11) which may be acetylated to form N-acetyl glucosamine (NAG) or the nitrogen may be substituted with a fatty acid to form the lipid A component of endotoxin (11). The present study was undertaken to determine the effects of NAG on platelet aggregation and its importance in bacteriainduced platelet aggregation.
MATERIAIS AND METHODS
Preparation of Bacteria. A derivative of 5. aureus ATCC 2323 was prepared by a modification of the method of Clawson and White (3). S. aureus was grown on brain heart infusion broth (DIFCO) to either mid or iate logarithmic phase and harvested in a Sorvall RC2-B centrifuge adapted for continuous flow at 13,000 rpm (4OC). These cells were washed once with distilled H 0 (3,000 x g for 10 minutes at 4'C) and resuspended in distilled H 0. T&ee ml aliquots were heat killed at 70°C for 30 minutes (3). The gells were washed two more times in distilled H20, lyophilized and stored at -2O'C. Cells were gram positive cocci and colonies were uniform at each stage of the preparation. The lyophilized preparation was resuspended 24-72 hours before use in 0.85 NaCl. Platelet Aggregation. Human blood was collected from the antecubital veins of healthy subjects into 0.1 volume of 3.2$ trisodium citrate and was centrifuged to prepare platelet rich plasma and platelet poor plasma as described previously (16). Platelet aggregation was initiated by addition of 0.05 mls of an inducer 15 seconds after the addition of the same volume of saline or a modifier of platelet qgregation to 0.4 mls of PRP stirred at 900 rpm in siliconized cuvettes at 37 C. The platelet concentration of PRP was standardized at 300,0OO/ml by dilution with PPP. Aggregation was monitored by continuous recording of light transmission (LT) as a function of time. In these experiments a dual channel Payton Aggregometer was used. Release of ATP. Platelet aggregation was performed as described above. At selected points the PRP was transferred from the aggregometer to a fluorimeter. MgS04 (0.02 mls of 154 mM) and luciferin luciferase (0.05 mls of 145 mg/ml from DuPont) was added. The luminescence was measured and two aliquots of ATP were added as standards. This was a modification of the method of Feinman -et al. (9). Aggregating agents investigated y&re 2. aureus, adenosine at a final con entration of 10 M, epiane (Parke and collagen suspension (bovine tendon Davis) at a concentration of 10-% from Sigma, lOug/ml). Collagen suipension was prepared by vortexing and filtering it through cheesecloth. N-acetyl glucosamine (Sigma), in 0.8% NaCl was used at a final concentration of l-100 mM. NAG in this concentration range had no effect on plasma PH.
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Purity of NAG was evaluated by descending paper chromatography, ninhydrin reactivity, and by its ultraviolet spectrum. Chromatograms were developed on Whatman #l filter paper using isobutanol:pyridine:H20 (10:10:5),a modification of the method of Moreno -et al. (22). The method of Trevelyan et al. (29) for detecting reducing sugars was used. Ninhydrin spray (0.9 in ethanol), which reacts with many primary amines was used to detect glucosamine on the paper chromatogram. The content of ninhydrin reactive amine was measured by adding 1 ml of the sugar solution to 1 ml of 0.9 ninhydrin in ethanol and heating at 100°C for 15 minutes. Glucosamine was used as a standard and results are reported as mmoles of glucosamine/mole NAG in solution. Absorption was measured at 570 nm. Absorption spectra were done with a Perkin-Elmer Lambda 3 spectrophotometer
RESULTS Purity of NAG. 250 ug of NAG from lots 84c-0101, 107C-0121, and 31C-3000 were chromatographed as described in methods. When developed with a reagent specific for reducing sugars only one spot appeared. No spots were detected with ninhydrin spray indicating that no reactive amines were present. Glucose and glucosamine were used as standards. Glucosamine reacted with ninhydrin and migrated at .555 relative to NAG. Although ninhydrin reactive amines were not detectable on the paper chromatogram they could be detected in solution. The ninhydrin test described in methods was performed on the different NAG lot numbers. Glucosamine was used as a standard and the absorption at 570 nm was linear in the range of 0.251.00 mM glucosamine. Lot 84c-0101 contains the equivalent of 9.3 mmoles of glucosamine/mole NAG while the other NAG lot numbers contain only minimal amounts of ninhydrin reactive amines. (Table 1).
Table 1 Ninhydrin Reactivity of Different NAG Lots NAG Lot # 84-C-0101
mmoles glucosamine/mole 9.3
107C-0121
1.0
31c-3000
1.0
A scan of each of the NAG lot numbers was performed as described in methods The spectrum of lot 84.~~01 was considerably different from the other lots or glucosamine (Fig. 1). NAG lot 84c-0101 had an absorption peak at 272 nm where neither glucosamine nor the other NAG lots absorb. S. aureus induced platelet aggregation. In Figure 2, saline was added to PRP 15 seconds prior to the addition ofg. aureus suspension. The ratio of bacteria to platelets was as follows: in Curve A 8:I (100 ug/ml), in Curve B 6.5:1 (80 ug/ml), in Curve C 4:l (50 ug/ml), and in Curve D 3:l (40 &ml). The results are very similar to those presented by Clawson and White (3), that is, following the addition of 2. aureus there was a "contact" phase which was followed by a narrowing of the ban, termed the shape change phase, and
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finally by a rapid aggregation of the platelets. Figure 2 shows that the "contact" phase was proportional to the ratio of bacteria to platelets. Above a ratio of 8 bacteria/platelet the turbidity of the bacterial suspension began to interfere with the assay. Strong aggregations were obtained with a ratio of 2.5 bacteria/platelet and sometimes as low as 1 bacteria/platelet. Effects of NAG alone on Platelet bgregation. In Figure 3, NAG was added to PRP at a final concentration of 10 mM (Curve B), or 100 mM (Curve C) 15 seconds prior to the addition of saline. In Curve A, saline was added 15 seconds prior to the addition of S. aureus at a bacteria/platelet ratTo 0f 2.5. In Curve D, NAG at a final concentration of 10 mM was added 15 seconds urior to the addition of S. aureus ai a ratio of 2.5 bacteria7 platelet. As it can be seen. although s. aureus can induce platelet aggre-, NAG alone did not cause platelet aggregation at the concentrations tested (Curves B and C). However, NAG in combination with 2. aureus (Curve D) shortened the contact phase of S. aureus induced platelet aggregation. NAG at concentrations of 100 mM causes a drop in the baseline of the recording. Subsequent addition of ADP at sufficient concentration to induce platelet aggregation failed to induce an increase in light transmission which is cheracteristic of platelet aggregation. Effects of NAG on S. aureus induced platelet aggregation. In Figure 4, NAG or saline was added to PRP 15 seconds prior to the addition of S. aureus at a bacteria/ to platelet ratTo of4. The results cleerly show that here at NAG concentrations of 10 mM (Curve A) and 1 mM (Curve B) the delay phase of 2. aureus induced platelet e.ggregatiE shorter than the control (Curve G). The addition of 0.1 mM NAG, however, did not show any effects on S. aureus induced platelet aggregakmve C). The effect of NAG on S. aureus induced platelet aggregation Was a concentration dependent phenomenon, that is, increasing concentrations of NAG re-
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QI A@$.
1
-0
a00
360
Figure 1 Ultraviolet spectrum of N-acetyl glucosamine. A, 250 mM NAG lot++ 84~-0101; B, 25O mM NAG lot#107C0121; C, 25O mM glucosamine; D, H20.
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TIME
293
IN MINUTES Figure 2
s.,aureus induced plateletagregation. Saline was added to PRP 15 seconds prior to the addition of 2. aureus at a bacteriato plateletratio of 8:l 4:l (50 &ml) C, or 3:l (40 ug/ml) D. (100 &ml) A, 6.4:1(80 ug/ii LT= Light Transmission.
LT 41
4
1
1
TIME
3
4
s
6
7
IN MINUTES Figure 3
The effects of N-acetylglucosaminealone on plateletaggregation. Saline (A) or N-acetyl glucosamineat a final concentrationof 10 ml4(D) was added to PRP 15 seconds prior to the additionofg. aurea at a final concentration of 2.5 bacteria/platelet.NAG at concentrationsof 10 mM (B) and 100 mJ4(C) was added 15 seconds prior to the additionof saline.
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LT 4
1
2
3
TIME
4
5
6
7
8
IN MINUTES Figure 4
The effects of NAG on 2. aureus induced platelet aggregation. Saline, (G), or NAG at concentrations of 100 mM (D), 0.1 mM (C), 1 mM (B), or 10 mM (A) was added to PRP 15 seconds prior to the addition ofS_. aureus at a ratio of 4 bacteria/platelet (50 ug/ml).
LT 4
/
1234$6 TIME IN
MINUTES
Figure 5 The effects of NAG on collagen induced platelet aggregation. Saline (A), or 10 mM NAG from lot #84c-0101 (C) or lot #107C-0121 (B) was added to PRP 15 seconds prior to the addition of collagen (10 ug/ml).
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L'1
4 3 2 1
Figure 6 The effects of NAG on epinephrineinducedplateletaggregation. Saline (A), 10 mM NAG lot # 107C-0121 (B), or 10 mM NAc610t#84~-0101 (C) was added to PRP 15 seconds prior to the addition of 10 M epinephrine. sulted in successivelyshorter delay phases. At NAG concentrationsof 100 mM (Curve D), S. aureus induced plateletaggregationwas c mpletelyinhibited. This inhibiTionwas not overcomeby the additionof lo-% ADP. Furthermore, the light transmissionof Curve D decreasedbelow the initialbaseline. NAG lot 84c-0101or lot 107C-0121gave the same results. Table 2 shows the differencein the delay phase between2. aureus induced platelet aggregationboth in the presenceand absence of 10 mM NAG. The results of multiple measurementson two differentPRP preparationsare reported. Although the length of the delay phase in the controldiffers from experimentto experiment,a reductionin the length of this phase is consistentlyobserved upon the addition of NAG. A reductionof 3% in trial #I and 3% in trial# 2 was obtained. Effects of NAG on collagen,epinephrineand ADP inducedplateletaggregation. Saline, or 10 mM NAG from either lot #84&0101 or lot#107C-0121 was added to PRP 15 seconds prior to an aggregating-sent. The aggrggatingagent was either collagen (10 &ml), epinephrine(10 M), or ADP (10 )I). Figure 5 clearly shows that the rate and degree of plateletaggregationinducedby collagen are reduced in the presence of NAG lot#8&0101 but not lot #107c-0121.Figures 6 and 7 show that NAG lot #84~-0101 inhibitedsecondary platelet aggregationinduced by epinephrineor by ADP but lO7C-OlZldid not. The effects of NAG on ATP release from platelets. Saline or 10 mM NAG (lO7C-OlZl)was added to PRP 15 seconds prior to the additionof S. aureus at a bacteria to platelet ratio of 4:l (9 &ml) and the aggrega%.onswere interruptedand the luminescencedue to ATP releasedfrom plateletswas measured as described in methods. The largestdifferencebetweenthe control and the experimentalwas obtainedwhen aggregationwas almost completein the NAG-PRP and just beginning in the control. Table 3 shows that the release at that point is much greater in the samples to which NAG has been added.
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Table 2 The Effect of NAG on the Delay Phase of 2. aureus Induced Platelet Aggregation Delay (seconds) Trial #l* Control
332*10
10 mM NAG
234*12
Trial #2* Control
242+13
10 mM NAG *A bacteria to platelet ratio of 4:1
4
ly+ (9
ug/ml final concentration) was used,
LT 4 3 2 1
I
I
1
2
TIME
,
I
3
4
I
5
6
IN MINUTES Figure 7
The effects of NAG on ADP induced platelet aggregation. Saline (A), or IO mM NAG lot# lO7C-0121 (B), or 10 mM N$j lot# 84c-0101 (C) was added to PRP 15 seconds prior to the addition of 10 M ADP. Table 3 The Effects of NAG on ATP Release from Platelets
Trial #l
ATP (nmoles/l) Control Experimental 22*15 n=4 79*10 n=4
Trial #2
21*10 n=5
47*10 n=5
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DISCUSSION Clanson and White first described S. aureus induction of platelet aggregation. They divided the process &to four phases. The first phase after the addition of the bacteria was the contact or delay phase in which there was no change in a broad baseline produced by stirred discoid platelets. This was followed by a rapid narrowing of the baseline, called the platelet shape change phase. Next, there was an early aggregation and finally a plateau referred to as irreversible aggregation. The length of the contact phase increased with a decrease in the number of bacteria added. Our system behaved in a similar manner although slightly higher numbers of bacteria were required to produce rapid aggregation with an acceptably short contact phase. Clawson -et al. (6) were able to show that N-ethylmaleamide, a sulfhydryl inhibitor, blocked the platelet release reaction induced by S. aureus in a manner similar to that observed with adenosine. 2. aureus iyduced platelet aggregation is probably mediated by the release reaction since the enzyme system phosphoenol pyruvate-pyruvate kinase, which converts ADP to ATP, inhibited both the release and the platelet aggregation induced by 2. aureus (6). The nature of the chemical component of the cell surface of S. aureus that interacts with platelets is not certain. Hawiger et al. (10) have suggested -7 that the 2. aureus induced platelet aggregation is mediated by protein A and the F porti=IgG. These investigators showed that soluble protein A inhibzted release and aggregation and that the addition of IgG was necessary for the binding of protein A to was ed platelets. They also showed that protein A could induce the release of % serotonin. In contrast to their findings Clawson and White (4) showed that washed platelets could aggregate in response to 2. aureus. It has been suggested that IgG might have been absorbed to the surface of these washed platelets (personal communication). Another possible candidate for the cell surface feature of bacteria which is responsible for platelet aggregation is suggested by the work of Rotta et al. (24). These investigators showed that synthetic peptidoglycan subunits induced lysis of rabbit blood platelets. These subunits consisted of the sugar components plus peptide side chains. It is not clear from their data whether this lysis is analogous to the release reaction and this investigator did not do aggregation studies. Amino sugars have been shown to influence platelet aggregation in a negative fashion. Treatment of collagen with glucosamine has been reported to inhibit platelet aggregation (19). N-acetyl neuraminic acid has been shown to inhibit platelet aggregation induced by a variety of agents (2, 18). In previous work we have reported that NAG and glucosamine inhibited collagen induced platelet aggre ation and secondary platelet aggregation induced by epinephrine and ADP (17 . In the present study we have investigated the purity of NAG by several methods. Descending paper chromatography of the different NAG lots revealed one spot with reagents capable of detecting reducing sugars. No ninhydrin reactive material was detectable. In NAG solutions, however, nine times as much ninhydrin reactive material was detected in lot # 84c-0101 as in the other lot #s. The ultraviolet spectrum of this NAG lot also had a peak at 2.72nm in contrast to glucosamine and the other NAG lots. This is the same lot number that inhibited platelet aggregation due to collagen, epinephrine and ADP. There appears to be one or more impurities in NAG lot # 84C-0101 which are not precisely characterized. The impurity that absorbs at 272 nm is not glucosamine. The impurity that reacts with ninhydrin is probably an amine. The inhibition of collagen, epinephrine, and ADP induced platelet aggregation is probably due to a contaminant in lot # 84c-0101 since the other lot #s do not cause this inhibition. 2. aureus induced platelet aggregation was not measurably affected by the contmm. The reason for this
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is unclear. It may be that the contaminant has no effect on the system or that the effect is masked by other factors. We have shown that NAG reproducibly shortens the delay phase of 2. aureus induced platelet aggregation. We have also shown that it shortens the length of time necessary for the release reaction to occur. This is of significance since 2. aureus induced platelet aggregation is probably release mediated (6). We were unable to induce platelet aggregation using NAG alone, even at high concentrations (100 mM). The amount of hexosamine in the peptidoglycan of sufficient S. aureus to induce platelet aggregation is much higher than this. Addition of-high concentrations of NAG as a monomer probably interferes with platelet aggregation through an osmotic effect (8). Use of a NAG polymer to circumvent the osmotic problems has been attempted in our laboratory but technical difficulties with the behavior of poly NAG in solution have prevented us from using it in aqueous systems. The suggestion that NAG may be one of the components of the bacterial cell wall responsible for platelet aggregation is not conclusively proven by the data presented here. However the fact that NAG shortens both the delay of aggregation and the delay of the release reaction in S. aureus induced platelet aggregation but has no affect on collagen, epynephrine, and ADP induced platelet aggregation (in its purer form) suggests that this phenomenon is not based on a general effect on platelet aggregation but is related only to bacterial platelet aggregation. Whether NAG enhances platelet aggregation es pert of the cell wall in bacteria is not certain. Further studies exe needed to elucidate the important effects of NAG on platelet aggregation.
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