Vol. 152, No. 3, 1988

May 16, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1410-1415

NATIVE FLUORESCENCE OF PLATELETS FROM PATIENTS WITH

OCCLUSIVE ARTERIAL DISEASE

Wolfgang Lohmann and Chris Lohmann

Institut fur Biophysik der Justus-Liebig-Universitit

Giessen, W. Germany

Received April 12, 1988

Healthy platelets exhibit a fluorescence band with a peak at 475 nm if excited at 360 nm. This peak increases first with the progression of occlusive arterial disease (OAD) followed by a decrease at an advanced stage. Concomitantly, a new fluorescence band at 445 nm will appear, which increases steadily with the progression of OAD. These findings can be explained by the oxidation of NADH (fluorescence at 475 nm) to NAD (445 nm) and support, thus, the assumption that oxidative processes are involved in the formation of OAD. ©1988AcademicPress, Inc.

Numerous people, especially with increasing age, suffer

from occlusive arterial disease. Generally, it is assumed that

it might be caused directly or indirectly by stimulated

platelets. Despite intensive studies, little is known,

however, about either the chemical modifications of these

platelets or the molecular mechanism(s) resulting in the

formation of occlusive arterial disease.

Newer results favor the involvement of oxidative

reactions (i and references therein). It could be shown that

lipid peroxidation could initiate or promote the process of

atherosclerotic lesion formation by directly damaging

endothelial cells, and by enhancing the susceptibility of

platelets to aggregate (1,2). Lipid peroxides also strongly

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Vol. 152, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibit prostacyclin synthetase, which synthesizes

prostacyclin in the endothelial cells of the arterial wall

(3,4). Since prostacyclin is a very important inhibitor of

platelet aggregation, its diminished concentration could

mediate plaque formation.

Stimulation of platelets can also be achieved by

oxidation of cytosolic NADH via the malate-aspartate shuttle

(5). This oxidative process will supply the energy required

for platelet aggregation. It could be also shown that an

inhibition of this shuttle will result in an inhibition of

platelet aggregation. It is interesting to note that high

concentrations of malate and aspartate as well as high

activities of aspartate aminotransferase and malate

dehydrogenase have been determined in homogenates of human

platelets (6,7).

While the existence of a malate-aspartate shuttle in

platelets has been confirmed (5), no direct evidence is

available for the presence of NADH and its oxidation to NAD in

stimulated platelets. Since the fluorescence technique is very

sensitive and specific for detecting NADH and NAD (8), this

method was used for determining the presence of these

compounds in platelets of patients with occlusive arterial

disease.

MATERIALS AND METHODS

Washed platelet suspensions were obtained from citrate- anticoagulated blood (1:10) according to a method described by Bowry (9). Albumin was excluded, however, from Hank's balanced salt solution (HBSS), since it will interfere with the fluorescence spectra. An Ultra-Flo 100 Whole Blood Platelet Counter (Clay Adams, New Jersey, USA) was used for counting platelets in suspensions. The fluorescence spectra of these platelet suspensions were recorded, under 90 ° , with a Zeiss spectrofluorometer using a 450-W xenon high-pressure lamp XBO (Osram) for monochromatic excitation at 360 nm. Longer wavelengths (up to 500 nm) didn't show any fluorescence response at all.

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RESULTS AND DISCUSSION

The fluorescence spectrum obtained with healthy platelets

is shown in Fig. i, middle spectrum. This fluorescence band

with its peak at about 475 nm is caused by NADH (8). It should

be pointed out that this spectrum seems to be independent of

age (20-50 years) and sex. Its optimum position (~M) is always

the same, while its intensity depends on the platelet count,

at least for up to 66 x 104 platelets/~l. For healthy

controls, the standard deviation of the mean value for the

fluorescence intensity/platelet ratio is ----- i0 %.

At a relativ early stage of an occlusive arterial disease

(CAD), the fluorescence band at 475 nm increases with a

concomitant appearance of another band at shorter wavelengths

(s. Fig.l, CAD stage IIb, spectrum exhibits a shoulder at-~450

nm). With progression of the disease, this shoulder will be

Fig. i:

ul

C

PLATELETS HBSS, pH 7.4 kE= 360nm

OAD, stage II b

HEALTHY

OAD, stage Il l

sSo 68o XF (rim)

Fluorescence spectra of platelets obtained from healthy volunteers and from patients with occlusive arterial disease (CAD) at different stages. The platelets were kept in Hank's balanced salt solution (HBSS) at pH 7.4 and were excited monochromatically at k~ = 360 nm. The fluorescence intensities have to be multiplied by 6 (stage II b) and by 5 (stage III), resp. if compared with the spectrum of the healthy platelets.

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expressed finally as a fluorescence band located at about 445

nm (s. Fig. i, OAD stage III spectrum). This peak at 445 nm is

caused by NAD indicating that oxidative reactions are involved

in an occlusive arterial disease which either causes or is a

consequence of an oxidation of NADH to NAD. The shoulder at

around 420 nm cannot be assigned yet.

It should be pointed out that in the case of OAD the tel.

fluorescence intensity per platelet is about 5-10 times larger

than for healthy platelets. The fluorescence intensities of

the spectra shown in Fig. 1 have to be multiplied by 6 (stage

IIb) and by 5 (stage III), resp., if compared with the

control spectrum of the healthy platelets. At 475 nm, they

increase first with the progression of OAD followed by a

decrease, while at 445 nm the intensity increases continously.

To prove that the fluorescence band at 475 nm for healthy

platelets is caused by NADH, pyruvate and lactate

dehydrogenase (LDH; L-lactate: NAD-oxidoreductase EC 1.1.1.27)

were added to healthy platelets. According to the equation:

NADH + H ÷ + pyruvate L~ L-lactate + NAD ~

NADH is oxidized to NAD. As can be seen in Fig. 2, the peak at

475 nm for the healthy platelets (control) disappears and the

peak at 445 nm appears, caused by NAD. Again, the shoulder at

420 nm for the yet unidentified compound is also present. If

the same concentrations of pyruvate and LDH are used for OAD

platelets (stage IIb), the NADH peak decreases considerably

and the formation of the NAD peak is, at least, indicated

(s.Fig.2, solid lines). If larger concentrations of pyruvate

and LDH are used (~ 10 x), the disappearance of the 475 nm

peak is accompanied by a concomitant appearance of the 445 nm

peak.

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Fig. 2 :

PLATELETS / ~ HBSS, pH 14

~/ \ k E = 360 nm

/// ', \ • heotfhy / / f ' , \ OAD

- / / 2 , \ ' /U ",'%

~ > } CONTROL

\ ~ 1 +PYRUVATE [09 mM] , J , / and LOH [015uM]

400 450 500 600 XF (nm)

The e f fec t of pyruvate and lac ta te dehydrogenase (LDH) on the fluorescence spectra of platelets obtained from healthy volunteers and from patients with occlusive arterial disease (CAD, stage IIb). The platelets were kept in Hank's balanced salt solution (HBSS) at pH 7.4 and were excited monochromatically at )%~= 360 nm. For comparison, the fluorescence intensity of the CAD stage IIb platelets has to be multiplied by 7.

From the results obtained one might conclude that at

initiation and progression of the occlusive arterial disease

more antioxidants in form of e.g. NADH are required and

supplied from pools resulting initially in an increase in the

475 nm peak. The rapid turnover of NADH to NAD will result in

an exhaustion of the NADH pool present normally in biological

systems and in the appearance of the 445 nm band. At a very

advanced stage of the disease, the spectrum will consist only

of the 445 nm band. The fluorescence behavior of the NADH/NAD

redox system is, thus, a good indicator for normal and/or

abnormal metabolic reactions, at least, in some types of

diseases.

ACKNOWLEDGEMENT

This work was supported in part by grants from the Bundesministerium fur Forschung und Technologic, from the

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Deutsche Forschungsgemeinschaft, the Hoechst Co., and from the Fonds der Chemischen Industrie. We would like to thank Prof. G. M~ller-Berghaus for giving one of us (Ch.L.) the opportunity for preparing the platelets and Dr.S.K. Bowry for advice.

REFERENCES

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(1959) Thromb. Diath. Haemorrh. 3, 520-547. 7. Goebli, H., Bickel, H., Bode, Ch., Egbring, R. and Martini,

G. A. (1968). Klin. Wschr. 46, 526-533. 8. Lohmann, W., Lohmann, Ch. and Ibrahim, M. (1988) Natur-

wissenschaften, in print. 9. Bowry, S. K. and M~ller-Berghaus, G. (1986). Thromb.

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