Research Paper Mediators of Inflammation, 8, 205–209 (1999)

In a previous work we have shown that heparin, in the presence of ozone (O3), promotes a dose-dependent platelet aggregation, while after Ca2+ chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF), transforming growth factor beta1 (TGF-beta1) and interleukin-8 (IL-8) are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limb ischemia treated with O3 autohaemoteraphy (O3-AHT).


Introduction
Ozone (O 3 ) c an promote plate le t aggre gation particularly 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 lation 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 (O 3 -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 rleukins (IL). 6 PDGF and TGFb 1 promote w ound healing and if, in the cours e of O 3 AHT, the re infus ed plate lets inc re ase their re lease , it c an be e nvisaged how this compleme 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 O 3 w as ge ne rate d from me dic al grade O 2 using ele ctric al corona arc dis charge in the last gene ration O 3 ge ne rator (Model Ozonosan PM100K, Hansler GmbH, Iffe zheim, Ge rmany) w hich allow s the gas flow rate and O 3 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 O 3 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 purposes (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 ave rage plate let c ount of 3 3 10 8 /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 rnatant p late le t-fre e plasma used for biochemic al dete rmination s.

O 2 and O 3 delivery to biological samples
A pre de te rmine d volume of the O 2 /O 3 gas mix ture at thre e O 3 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 disposable 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 O 3 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 O 2 . 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 rmining 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 ecifie 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 according 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 dissolve 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 immunoreactive form) w e re carrie d out using Quantikine immunoas say kits p roduc ed by R&D Syste m (Minne apolis, USA

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 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 renc 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 O 3 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 renc e at a medium (40 m g/ml) and at a high (80 m g/ml) O 3 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 O 2 -O 3 w as very simple, prac tic al and fre e of risk of contaminatio n. Sinc e that time , c ountle ss O 3 -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, O 3 -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 mechanisms of ac tion and ex plain w hy O 3 -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 O 3 , can promote plate let aggre gatio n w hile , in contras t, Ca 2+ che latio n is prac tic ally ine ffe ctive . We the n w ent to suspe ct that promotion of 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 O 3 . 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 O 3 c onc entrations of eithe r 40 or 80 m g/ml, re spe ctive ly. The forme r O 3 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 O 3 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 O 3 , w hile is promoted by a te mporar y rise of H 2 O 2 14-16 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 prostaglandin 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 O 3 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 O 3 -AHT conc entratio n of 40 m g/ml.