TrallsfusiOI/ Medicil/i?, 2005, 15, 319-322

ORIGINAL ARTICLE

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doi: 1O.1111íj.l365-3148.2005.00594.x. _'_n. -. - é"";:;

The effect of continuous-flow automated plateletpheresis onfibrinolytic activity of danar plasmaP. Radziwon*t, B. Boczkowska-Radziwon*,A. Lipska*, M. Smoktunowicz*and J. Kloczkot*Regiollal Ce11lrefor Transfusiol/ Medicine, BialJ'stok, and tDeptart11lent of Hael7latology, Medical Uni\'ersity of BialJ'stok, Bialystok, Poland

Receil'ed 26 Janllary 2005; acceptedfor publicatiol/ 7 March 2005

SUMMARY.Blood circulating in extracorporealcircuit of lhe apheresis seis has a contact with anartificial surface. The data on lhe influence ofplateletpheresis on fibrinolytic activity are very lim-ited and difficult to interpret. The aim of our studywas to estimate lhe effect of plateJetpheresis on lheactivation of fibrinolysis. Plateletpheresis was per-formed in 17 hea1thy blood donors using continu-ous-flow cell separator COM.TEC (Fresenius, BadHomburg, Germany). Before and after plateletpher-esis, blood samples were taken and markers of fibri-nolysis (PAP, t-PA, PAI-I) as well as factor XII

activity have been l11easured. We observed statist-ically significant decrease in t-PA and factor XIIactivities anel' plateletpheresis. There were no signifi-Gani changes in concentrations of t-PA, PAI-l andPAr as well as PAI-l activity anel' plateletpheresis.Plateletpheresis perforl11edby COM.TEC cell separa-tor has very little, if any, effect on lhe activation offibrinolysis. The mechanism of lhe inhibition of t-PAactivity needs further investigations.

Key words: COM.TEC, factor XII, fibrinolysis, PAI-I,PAP, plateletpheresis, t-PA.

During extracorporeal circulation occurring in hae-modialysis, cardiopuhnonary bypass or apheresisbloud colllacl,; wilh lhe artiCicÜl1, on!y in parI bio-

compatible anel reJativcly large surface of disposablcseis. Plasma proteins, e.g. coagulation factors, adsorbon lhe foreign surface (Vroman et aI., 1971; Forbes,1981). Protein deposition is followed by lhe adhesionof blood cells: platelets, leucocytes and erythrocytes(Vroman, 1987; Koepke et aI., 1981).The interactionwith lhe surface affects enzyme activity (induces fac-tor XII activation) and cell functions (MulvihiII et ai.,1990). There are reports suggesting that fibrinogen,vou Willebrand factor and fibronectin stimulateplatelet interaction with artificial surfaces, whereasalbumin, ceruloplasmin and transferrin inhibit it(Kim et ai., 1977; Elam & Nygren, 1992).Kobayashi et aI. (l9?3) observed increased coagul-aliou activity anel' plateletpheresis performed with

Correspondence: Piotr Radziwon, MD, PhD, Regional Centre forTransfusion Medicine, Sklodowskiej 23, PL - 15-950Bialystok,rolandoTe!.: +48 85 7447002;

fax: +48 85 7447133;

e-mail: piotr.radziwon@wp.p1

~ 2005 BlackweIl Publishing Ltd

continuous-flow blood separator CS-3000 (BaxterHealthcare, Deerfield, IL, USA). In contrary,Slohlawctz c/ oi. (1999) cliel nol detecl significantchangcs in blooel coaglllalion inelllcecl by plalcJel-pheresis perforl11ed on lhe two other blood separators- AMICUS (Fenwal Division, Baxter Healthcare)and MCS 3p (Haemonetics, Braintree, MA, USA).The data on lhe influence of plate1etpheresis on fibri-nolytic activity are very limited and are difficult tointerpret (Kobayashi et ai., 1993; Stohlawetz et ai.,1999). There are no reports about lhe effects of pla-teletpheresis on t-PA concentration and activity andon PAI-l activity.

The aim of this study was to estimate lhe effect ofplateletpheresis performed on lhe new automatedblood separator - COM.TEC (Fresenius, BadHomburg, Germany) - on lhe main activator andlhe main inhibitor of fibrinolysis - t-PA and PAI-las well as on factor XII activity.

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MA TERIALS AND METHODS

Seventeen hea1thy donors, aged 21-40 years, who hadnot taken any drugs for at least 2 weeks prior to bloodsampling, underwent standard thrombapheresis proce-dure. The automated continuous-flow cell separator

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The concentration and activity of toPA were meas-ured using commerciaIly available kits (COALIZA High-molecular-weight kininogen, prekaIlikrein, fac-toPA and COATEST toPA, respectively) from ter XII and factor XI recognized as lhe 'contactChromogenix-Instrumentation Laboratory SpA, system' require contact with artificial, negatively

.. -,_It~IY'd charged surfaces for zymogene activation in vitro.~~~~~tJ : -:w :

';:~-;'F"'rabl~'2. T~e data of measuredparameters of fibrinolysis(mean1['80,':'; = 17)

Parameter

320 P. Radzi1l'01let a!.

Table 1. The main parametersof performed thrombapher-esis (11= 17)

Parameter Mean :I: SD

Blood flow (mL min-l)Blood volume processed (mL)Donation time (min)ACD consull1ption (mL)PLT yieId (x 1011)

46.60 :I: 122601.5 :I: 314.4

58.9 :I: 14.3338:1: 423.2 :I: 0.3

COM.TEC (Fresenius) with single needle apheresis sei(S5L) were used. Table I shows lhe main parameters ofperformed procedures. Whole blood was anticoagu-lated with ACD-A at average blood : ACD-A fatia of9 : I. The investigations were perfonl1ed with approvalof lhe Committee on lhe Ethics of Research in Hul11anExperimentation at Medical Academy in Bialystok,Poland and under lhe guidelines of lhe HelsinkiDecIaration for human research.

Blood collectioll and p/'epa/'atioll o/ platelet poorplasma (PPP)

Venous blood was drawn without stasis from donorsbefore and 5 min after thrombapheresis, into syringescontaining either sodium citrate (0.32% final concen-tration) for lhe measurement of topA, PAIol andPAP concentrations or 0.5 M citrate buffer, pH 4.3(StabilyteTM Biopool, Bray, Ireland) for lhe measure-mcnt ol' toPA and PAI. 1 activities, Platclet poorplasma was prepared by centrifugatian af wholeblood at 2000 x g for 10 min

toPA

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The concentration and activity of PAI-I were meas-ured using COALIZA PAIol and COATEST PAIfrom Chromogenix-Instrumentation LaboratorySpA, Italy, respectively.

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PAP

Concentration of PAP was measured using ImuclonePAP Elisa kit (American Diagnostica, Greenwich,CT, USA),.

Factor XII actil'ity ,

Coagulation factor XII deficient plasma (human)(Dade Behring, Marburg, Gerl11any) and activatedpartial thromboplastin assay has been applied forlhe meaSUrel11entof fartar XII activity. The resultswere expressed in percentage of lhe narro. '

Statistics

The data are presented as mean value :f: standarddeviation (SD). For statistical evaluation of lheresults Wilcoxon's test was used.

Changes in l11easured parameters were correctedfor haemodilution.

RESUL TS

We observed significant decrease of toPA ano factorXII activity after thrombapheresis, whereas its con-centration remained unchanged. There were no sig-nificant changes in lhe concentratians of PAP anoPAIol as well as PAIol activity. The data of lhemeasured parameters are presented in Table 2.

DISCUSSION

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PAP concentration (ng mL-I)toPA concentration (ng rol-I)toPA activity (IU mL-I)PAI-1 concentration (ng mL-I)PAI-1 activity (AV mL-I)Factor XII activity (%)

@ 2005 Blackwell Publishing Lld, TrallsfusíOIlMedícille, 15,319-322

Before After Significance

21.15 :I: 11.9 21.00:1: 11.7 NS3.70:1:11.2 3.18:1:0.8 NS5.36:1: 3.3 1.17:1: 0.6 P < 0.05

46.18:1: 30.50 42.82:1: 29.21 NS6.88 :I: 4.58 6.21 :I: 4.31 NS

99.20:1: 16.45 78.69 :I: 14.92 P < 0.05

These surfaces are thought to be a substitute for

i, possible physiological receptors on lhe biologicalrf activating surface. The 'contact proteins' have beenfi ascribed to have a role in lhe initiation of açuleW inflammatory responses following tissue injury as

~: we~1 ~s in t?e activation of plasma fibrinolysisf'~ (Nlewlarowsh & Prou-Wartelle, 1959; Colman

et aI., 1975; Colman & Schmaier, 1997). Factor

~, XlIa and kallikrein cleave plasminogen directly, butI; less efficiently than t-PA or u-PA (Colman, 1969;.:' Goldsmith et aI., 1978; Mandle & Kaplan, 1979).

I,., Bradykinin selectively induces t-PA release from

'.~ endothelial ceIls (Smith et aI., 1985). However, not" only lhe electronegativity of lhe surface but algo

other physicochemical parameters could influencelhe activation of lhe contact phase system of plasma(Renaux et aI., 1999). The 'damage' to lhe blood isalgo directly related to lhe duration of extracorporealcirculation.

Extracorporeal circulatory systems applied in ceIlseparators differ depending on lhe principIe of lheapheresis method (e.g. constant or intermittent flow)and on lhe material of lhe disposable set. The pIate-letpheresis performed on blood separator CS 3000induced . significant activation of coagulation,decrease of Clrantiplasmin concentratiol1 and hadno effect on plasmin-plasmin inhibitor complex(Kobayashi et aI., 1993). Unfortunately, in thatstudy, t-PA and PAI-I had not been measured.Another published report revealed no effeet 01'extra-corporeal circulation of blood separators: AMICUSand MCS 3p on lhe activation of coagulation, on lheconcentration of plasmin-plasmin inhibitor complexas weIl as on lhe PAI-I concentration (Stohlawetzet aI., 1999). However, these two studies are notcomparable. They used different cell separators andthere were differences in lhe whole blood : antico-agulant (ACO-A) fatias, blood volume processedanel lhe time of extracorporeal circulation. In lhestudy of Stohlawetz et aI. (1999), markers of coagul-ation were measured in blood taken from lhe inflow-ing and outflowing blood of cell separator afterprocessing a certain flow volume. Kobayashi et ai.(1993) in their stuciy meas\lred lhe effect of thromba-pheresis in blood drawn from coIlection line of danarimmediately after lhe start and just before lhe end ofapheresis. In our study, we investigated lhe effect ofplateletpheresis performed with continuous-flowblood separator COM.TEC on t-PA and PAI-l con-centration and activity and plasmin-antiplasmincompIex in platelet donors. We detected decreasedfactor XII activity, but did not detect significantchanges in PAr, PAI-I and t-PA concentrations aswell as PAI-l activity after plateletpheresis. The

(j) 2005 Blackwell Publishing Ltd, Transfllsion Medicine, 15, 319-322

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Plateletpheresis and jibrinolysis 321

mechanism of lhe observed decreased activity oft-PA is difficult to explain. In our opinion, it maybe due to lhe inhibitory effect of citrate infusion ont-PA activity/release. Nevertheless, thrombapheresisperformed with COM.TEC presents a save procedurefor lhe danar in terms of activation of fibrinolysis.

ACKNOWLEOGMENT

This study was supported by Medical University ofBialystok - Grant no. 3-52839L.

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