Research Paper
Mediators of Inflammation, 8, 205–209 (1999)
IN a p re vious w o rk w e h ave s h o w n th at h ep arin , in
th e p r es e nce of ozo ne (O3 ), p r om ote s a do s e-dep en d-
en t p latele t aggre gation , w h ile afte r Ca 2+ ch e lation
w ith citr ate , p latele t aggr ega tion is alm o s t ne gligible.
Th es e r es ults led us to th in k th at aggr ega tio n m ay
en h an ce th e r ele as e of p latelet co m po ne nts . We h ave
h er e s h o w n th at in de ed s ign ific an tly h igh e r am ount
of p late le t-der ived gr ow th factor (PDGF), tr an s fo r m -
in g gr ow th facto r b 1 (TGF-b 1) and in terle ukin -8(IL-8)
ar e r eleas ed in a do s e-dep en de nt m ann er afte r ozo na-
tion of h ep ar in is e d p latele t-r ich p las m a s am p les .
Th es e fin din gs m ay e x p lain th e en h anced h ealin g of
torp id ulce rs in p atie n ts w ith ch ro nic lim b is ch em ia
tre ated w ith O3 autoh ae m oter ap h y (O3-AHT).
Key w or ds : ozone , platelets, aggre gation, grow th factors,
inteleukin-8
Introduction
Ozone (O3 ) c an promote plate le t aggre gation partic -
ularly w hen he parin is used as an antic oagulant 1: this
observatio n is not surprising in the light of pre vious
re sults 2 show ing the role of re active ox ygen spec ie s
(ROS) in plate le t activatio n. In contras t, Ca 2+ che la-
tion w ith c itrate marke dly inhibits aggre gation. 1 Thus,
the sele ction of the most appropriate antic oagulant
bec omes crucial w he n blood is inte nde d to be use d
for autotrans fusion (O3-AHT) afte r being brie fly
ex pose d to a gas mix ture c ompose d of about 97%
ox ygen and 3% ozone .3 It is know n that plate lets are
a ric h sourc e of several grow th fac tors such as
plate let-de rive d grow th fac tor (PDGF), 4 trans forming
grow th fac tor b 1 (TGF-b 1),5 e icosanoids and inte r-
leukins (IL).6
PDGF and TGFb 1 promote w ound healing and if, in
the cours e of O3 AHT, the re infus ed plate lets inc re ase
their re lease , it c an be e nvisaged how this com-
pleme ntar y the rap y, be side s improving ox ygenatio n
of hypox ic tis sues, enhanc es he aling of torp id ulce rs
in chronic limb ische mia. For this re as on w e have
now inve stigate d w he ther the use of heparin or
citrate adde d to blood, be fore ozonatio n, affe cts the
re lease of plate let fac tors diffe re ntly.
Materials and methods
Ozone generation and measurement
O3 w as ge ne rate d from me dic al grade O2 using
ele ctric al corona arc dis charge in the last gene ration
ISSN 0962-9351 print/ISSN 1466-1861 online/99/040205-05 © 1999 Taylor & Francis Ltd
Studies on the biological effects of
ozone: 10. Release of factors from
ozonated human platelets
G. Valacchi and Velio BocciCA
Institute of General Physiology, University of Siena,
53100 Siena Italy
CA
Corresponding Author
Te l: (+39) 577 234217
Fax : (+39) 39 577 234219
Email: fis ge n@unisi.it
O3 ge ne rator (Model Ozonosan PM100K, Hansler
GmbH, Iffe zheim, Ge rmany) w hich allow s the gas
flow rate and O3 conc entratio n to be contro lle d in
re al time by photome tric de te rminatio n at 253.7 nm
as re comme nde d by the Standardis atio n Committe e
of the Inte rnatio nal O3 Associatio n.
Reagents
Antic oagulants w e re e ithe r he parin (calc ium salt,
30 IU/ml blood) normally used for the rapeutic pur-
poses (Calc iparina, Italfarmac o) or ACD (Citric ac id,
Na citrate , Gluc ose) (Hae mone tic s, Braintre e, USA).
Ade nos ine diphosphate (ADP) w as a product from
Sigma Che mical Co. (St. Louis, Mo) and for the
studying aggre gation a 0.5 mM solution w as fre shly
pre pare d.
Preparation of platelet rich plasma (PRP)
samples
Both ACD and hep arinis e d plate le t rich plasma (PRP)
w ere p re pare d from the same blood samples (60 ml)
draw n, afte r info rme d conse nt, from five fasting (12
hours) non-smoke rs volunte e rs be tw een the ages of
23 and 27 years, w ho w e re c ons ide re d to be healthy
and had not inge ste d plate let-ac tive medic atio n for at
least tw o w e eks.
Nine parts blood w ere antic oagulate d w ith e ither
one part ACD or w ith one part of saline containing
he parin so that its final conce ntration w as 30 IU/ml.
Blood w as c entrifuge d at 200 3 g for 20 min and
plate lets w e re measure d w ith a Coulte r c ounte r. An
205
G. Va la cchi a nd V. Bo cci

ave rage plate let c ount of 3 3 108/ml plasma w as

used. A furthe r c entrifug ation of PRP at 6000 3 g for
15 se c gave a plate let-c ontaining pellet and a supe r-
natant p late le t-fre e plasma used for biochemic al
dete rmination s.

O2 and O3 delivery to biological samples

A pre de te rmine d volume of the O2/O3 gas mix ture at
thre e O3 c onc entratio ns (20, 40 and 80 m g/ml per ml
of PRP) w as c ollec te d w ith a silic one c oate d dispos-
able syringe and immediate ly intro duce d into a
se cond syringe c ontaining an equivale nt volume of
PRP via a ‘y’ conne c tor. Final gas p re ssure re maine d at
normal atmosphe ric pre ssure . In orde r to obtain
re produc ible re sults, it ne eds to be emphas ised that
O3 is a very re active gas so that ex tre mely rapid and
pre c ise handling is re quire d. The PRP samples w ere
gently but c ontinuo usly mix ed w ith the gas for up to
30 se c and afte rw ards the y w ere dispe nsed into te st
tubes for various analys is . Contro l samples w ere
eithe r untre ate d or mix e d w ith an e qual volume of
O2 . Afte r inc ubatio n e ach sample w as immediate ly
ce ntrifuge d at 10,000 3 g for 20 min at 2°C and the
supe rnatant plate let-fre e plasma w as used for dete r-
mining variation s of thiobarbituric ac id re ac tive
substanc es (TBARS),7 the total antio x idant status
(TAS) 8 and of prote in thiol group s (PTG) acc ording to
Hu.9 An aliquot of the plasma samples w as froze n at
–70°C until dete rminatio ns of several fac tors sp eci-
fie d be low w ere carrie d out.

Biochemical determinations
1. Thiobarbituric acid re ac tive substanc es (TBARS)
dete rmination : in orde r to e valuate the re levanc e
of lipid pe rox idatio n, TBARS w ere assessed accord-
ing to Pompella e t a l. 7
2. Total antio x idant status (TAS) in plasma samples
w as assessed ac cording to Rice-Evans and Mille r.8
3. Prote in thiol groups (PTG) w e re me asure d in
plasma acc ording to Hu 9 using proce dure 1 w ith
5,59 -Dithio -bis(2-Nitrobe nzoic acid) DTNB dis-
solve d in absolute me thano l.
Immunoassay
Immunoas says of e ithe r human PDGF-AB or TGFb 1
(afte r activatio n of the late nt TGFb 1 to the immunor-
eactive form) w e re carrie d out using Quantikine
immunoas say kits p roduc ed by R&D Syste m (Minne a-
polis, USA). On the bas is of pre liminary te sts hep-
arinis ed PRPs w ere dilute d 1:20 w hile c itrate PRPs
w ere dilute d 1:1 only. Possible re lease of thrombox -
ane A2, a vasoconstric tor and aggre gation-e nhanc er
fac tor, w as monitore d by me asuring the stable com-
pound thrombox ane B2 (TXB2) by using an immu-
noas say kit produc ed by R&D Syste ms. For this assay,
206 Mediators of Inflammation · Vol 8 · 1999
FIG. 1. Effect of 30 sec exposure of either O2 or O2 –O3 (20, 40
and 80 m g/ml per ml of plasma) on total antioxidant status
(TAS), protein thiol group (PTG) and thiobarbituric acid
reactive substances (TBARS) of the same human platelet rich
plasma samples collected either in heparin or in ACD. The
statistical significance has been indicated with a (*) for
intergroup and with a (+) for intragroup analysis.
plasma samples w e re dilute d 1:5. Immunoassay of IL-
8 w as carrie d out using Cytosc re en kit p roduc ed by
Biosource Inte rnational (Camarillo , CA, USA). Plasma
samples w e re dilute d 1:1 w ith the app ropriate
dilue nt. A 3-c yc le automatic w ashing w as routine ly
performe d. Samples have bee n te ste d at le as t in
duplic ate agains t the appropriate standards .
Statistical analysis
Results obtaine d from five donors have be en
ex pre sse d as the mean ± the standard deviatio n of the
me an (SD). A softw are package w as used for data
collection and statis tic al analys is (Statvie w SE, Abacus
Conc epts Inc ., Berke le y, California). The signific anc e
of the diffe re nc es be tw ee n the means at diffe re nt
 Effe cts o f o zo n e o n pla te le ts

FIG. 2. Release of factors from human platelets during 1, 2 and 4 h incubation. The same PRP samples collected either in
heparin or ACD were either not exposed (control), or exposed to O2 alone, or O2 –O3 at 20, 40 and 80 m g/ml concentration for
30 sec before incubation. The statistical significance has been indicated with a (*) for intergroup and with a (+) for intragroup
analysis.

Mediators of Inflammation · Vol 8 · 1999 207

G. Va la cchi a nd V. Bo cci
time s in e ach group w as analys ed by one -w ay analys is
of varianc e (ANOVA). The signific anc e of the diffe r-
enc es be tw e en means for the tw o groups at diffe re nt
time s w as analys ed by Stude nt’s t-te st. The le vel of
statis tic al signific anc e w as se t at p <0.05 for both inte r
and intrag roups analys is.
Results
Partic ularly PTG value s de cre ase d in approx imate
re lation w ith the O3 c onc entratio n w hile TBARS
value s inc re as ed se veral folds (Fig.1). More ove r w e
have observe d that ox idatio n of PTG is far higher in
he parinis ed that Ca 2+ chelate d samples sugge sting
that physiological Ca 2+ levels favour ROS ac tivity.
Inde e d intragro up analys is show ed a signific ant diffe r-
enc e at a medium (40 m g/ml) and at a high (80 m g/ml)
O3 c onc entration.
Fig.2 show s the striking ly and signific ant diffe re nt
re lease of PDGF AB, TGFb 1 and IL-8 from hep arinis e d
in comparis on to Ca 2+ che late d PRP samples. For the
firs t tw o cytokine s the diffe re nc e is cle ar at all time s,
w hile for IL-8 it be comes e vide nt only afte r 4 hours of
inc ubatio n. As far as the re le as e of TXB2 is c onc erne d,
he parin doe s not appe ar to have a de te rminant role
and both antic oagulants have yie lde d signific ant
diffe re nc es afte r ozonatio n of PRPs.
Discussion
A modifie d form of AHT, by irradiating blood w ith
ultravio let light, w as firstly propose d by Wehrli and
Ste inbart 10 but AHT bec ame popular afte r Wolff 11 had
show n that dire c t ex posure of blood to a know n dose
of O2 –O3 w as very simple, prac tic al and fre e of risk of
contaminatio n. Sinc e that time , c ountle ss O3-AHT
se ssions have bee n performed in Europe and in spite
of a lack of double -blind, rando mise d studie s, it see ms
that this approac h c an be use ful in vasc ular dise ase s,
partic ularly in chronic limb ische mia. Rokitans ky e t
a l 12 and Werkmeiste r 13 had show n that e ve n at late
stage s (III and IV grade ) of the dis eas e, O3-AHT,
combine d to topic applic atio n of ozone , can spare
amputation and favour healing of torpid ulce rs and
ne crotic are as. It is unfortunate that the re sults of
these studie s have be en re porte d in a rath er ane cdotal
form, so that during a re vision 14 –15 of this fie ld, it w as
pointe d out that not only it is urgent to perform
controlle d studie s but to cle arly unde rstand mecha-
nisms of ac tion and ex plain w hy O3-AHT e nhanc e s
he aling of ulcers.
A first important point that has ne ve r be en clarifie d
w as w hich type of anti-coagulant: he parin or the
usual sodium c itrate w ould be more suitable. Inde e d
in a pre vious w ork 1, w e have show n that heparin, in
the pre se nce of O3 , can promote plate let aggre gatio n
w hile , in contras t, Ca 2+ che latio n is prac tic ally ine ffe c-
tive . We the n w ent to suspe ct that promotion of
208 Mediators of Inflammation · Vol 8 · 1999
aggregatio n w ould favour the re le as e of an array of
intrac e llular c ompone nts from plate lets and w e
thought w orth w hile to carr y out a pre liminar y
inve stigatio n.
We have now show n that tw o important he aling
fac tors, name ly PDGF and TGFb 1, inc re as e marke dly
during inc ubatio n partic ularly in heparinis e d PRP
samples ex pose d to 40 and 80 m g/ml of O3 . If this
happe ns in vivo, afte r re infusion of ozonate d blood in
patie nts w ith chronic limb is chemia, it may inde e d
favour he aling of ne c rotic ulce rs. How ever, this
assumption must be te mpere d by the pre vious
finding 1 that plate let aggre gation c orre sponds to
eithe r 20±6% or as much as 68±14% for O3 c onc entra-
tions of eithe r 40 or 80 m g/ml, re spe ctive ly. The
forme r O3 conce ntration still does allow an important
re lease of grow th fac tors w ith no ris k of blood
coagulatio n and there fore may re pre se nt the optimal
O3 c onc entration.
The fairly late re lease of IL-8 has bee n inte rpre te d as
due to the time lag ne cessary for the synthe sis. It is
know n that induc tion of IL-8 by O3 , w hile is
promoted by a te mporar y rise of H2O214–16 in
cytoplasmic w ate r via the activatio n of nuc le ar fac tor
(NF)-kB, is inhibite d by ROS sc ave nge rs.17 As this
che mokine is capable of initiating the che motac tic
gradie nt that draw s leukoc yte s from c irc ulatio n into
tis sues, it may ex ert the additio nal role of favouring
phagoc ytosis of bac te ria and ne crotic tissue pre se nt
in torpid ulce rs.
Re lease of TXB2, as the stable compound de rive d
from thrombox ane A2, ap pears as a draw back but w e
canno t draw a conclusion unle ss w e carry out
dete rmination of other eic osanoids such as prosta-
glandin E2 and pros tac yclin that induc e vas odilatio n
and inhibit aggre gation. By using endothe lial cells,
w ork now in progre ss aims to clarify the role of O3
activatio n of c ycloox ygenase and nitric ox ide
synte thas e.
On the bas is of these re sults, w e w ould like to
e valuate comparative ly the effe ctive ne ss of AHT in
patie nts w ith chronic limb ische mia tre ate d w ith
eithe r citrate d or heparinis ed blood ex posed to the
mild O3-AHT conc entratio n of 40 m g/ml.
ACKNOWLEDGMENTS: This w ork has been supporte d by Murs t grant (40%,
national and 60% local funds). The c are ful pre paration of the manuscript by
Mrs. Patrizia Marrocche si is grate fully acknow le dge d.
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Received 4 July 1999;
accepted (revised) 2 August 1999
Mediators of Inflammation · Vol 8 · 1999 209