Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogene (procarboxypeptidase B) which can decrease fibrinolysis and thus act as a haemostatic factor. TAFI is now extensively studied in many complications as well as in physiological and complicated pregnancy. The question we posed in the present study was whether TAFI antigen is present in cord blood plasma. The study group consisted of 38 parturient women, 26 primiparous and 12 multiparous with normal course of pregnancy and delivery. The cord blood was sampled from the cord vein, and the mother's blood from the antecubital vein. 3.2% sodium citrate was used as an anticoagulant. TAFIa/ai antigen was measured by ELISA method. TAFIa/ai antigen was identified in all samples of cord blood plasma. Its level was 91.50 ng/ml (range: 71.76 - 160.77 ng/ml) vs. 55.46 ng/ml (range: 39.77 - 68.54 ng/ml ) in the mother's blood, which means that the level of TAFIa/ai antigen was significantly higher in fetal blood than in maternal blood (

Góralczyk, Krzysztof

Uszyński, Mieczysław

Uszyński, Waldemar

Zekanowska, Ewa

English

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IntroductionThrombin Activatable Fibrinolysis Inhibitor - TAFI,also called procarboxypeptidase B or carboxypepti-dase U and carboxypeptidase R, is formed in the liveras a unichain glycoprotein of molecular weight of 60000 Da and circulates in the blood in the form ofzymogene (pro-TAFI). It is activated by the throm-bin/thrombomodulin complex, functioning on thewalls of vessels (thrombomodulin exposed on theendothelium is a receptor of thrombin and forms com-plexes with it) [Fig. 1]. Activated pro-TAFI, that isTAFIa (m.w. 35 000 Da), is a proteolytic enzymewhich changes the fibrin structure in such a way thatit becomes less susceptible to fibrinolysis. This phe-nomenon consists in splitting off of C-final lysine andarginine which decreases adsorption of tPA (tissueplasminogen  activator) and plasminogen on fibrin,leading in consequence to a decrease of plasmin gen-eration and thus to prolongation of fibrinolysis [4,17].TAFI is treated as a link connecting coagulation withfibrinolysis [5]. Anti-inflammatory effect is anotherproperty of TAFI [6]. TAFI has beneficial influenceon hemostasis, which is supposed to be an effect ofantifibrinolytic activity; this has been observed duringtherapeutic application of recombined factor VIIa inhemophilia [2,11]. Increased level of TAFI antigenwas described for many diseases. The increase of thelevel of TAFI and of fibrinolysis inhibitor of type PAI-1 in the coronary disease create together a high risk ofcardiac infarction [9,12]. In hepatocirrhosis the levelsof TAFI, antithrombin and alpha-2-antiplasmin godown, which is explained by weaker synthesis[10,15]. In disseminated intravascular coagulation(DIC), TAFI level is lower, which can be a conse-quence of consumption [18]. A lower level of TAFIantigen was observed in women in severe preeclamp-sia  and in intrauterine growth retardation syndrome(IUGR) [1].  But nothing is known about TAFI in thefetal blood. The aim of the present study was to findout whether and at what level TAFI antigen appears incord blood. FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 45, No. 1, 2007pp. 33-36Thrombin activatable fibrinolysis inhibitor (TAFI) in cord bloodWaldemar Uszyñski1,  Mieczys³aw Uszyñski2, Ewa ¯ekanowska3, Krzysztof Góralczyk31Obstetric-Gynecological Ward of the Regional Hospital in W³oc³awek, 2Department of Propedeutics of Medicine, Nicolaus Copernicus University in Toruñ - Collegium Medicum in Bydgoszcz, and 3Department of Pathophysiology, Nicolaus Copernicus University in Toruñ - Collegium Medicum in Bydgoszcz, PolandAbstract: Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogene (procarboxypeptidase B) which candecrease fibrinolysis and thus act as a haemostatic factor. TAFI is now extensively studied in many complications as wellas in physiological and complicated pregnancy. The question we posed in the present study was whether TAFI antigen ispresent in cord blood plasma. The study group consisted of 38 parturient women, 26 primiparous and 12 multiparous withnormal course of pregnancy and delivery. The cord blood was sampled from the cord vein, and the mother's blood from theantecubital vein. 3.2% sodium citrate was used as an anticoagulant. TAFIa/ai antigen was measured by ELISA method.TAFIa/ai antigen was identified in all samples of cord blood plasma. Its level was 91.50 ng/ml (range: 71.76 - 160.77 ng/ml)vs. 55.46 ng/ml (range: 39.77 - 68.54 ng/ml ) in the mother's blood, which means that the level of TAFIa/ai antigen was sig-nificantly higher in fetal blood than in maternal blood (p<0.00001).  TAFIa/ai antigen is an integral component of cord bloodplasma. The concentration of TAFIa/ai antigen is about two times higher in fetal blood than in maternal blood.Key words: TAFI - cord blood - fibrinolysisCorrespondence: M. Uszyñski, Dept. of Propedeutics ofMedicine, Collegium Medicum, ul. Œwiêtojañska 20, 85-077 Bydgoszcz, Poland; e-mail: kizproped@cm.umk.pl Materials and methodPatients. The study groups consisted of 38 parturient women, atthe age of 21.3±3.2 years, including 26 primiparous and 12 multi-parous with a single fetus and a physiological course of pregnancy(cases with hypertension and restricted growth of the fetus wereexcluded). The control group consisted of 20 healthy women of21.1±0.9 of age, in the 18-25 day of menstrual cycle. All thepatients were informed about the aim of the study and gave theirpermission for blood sampling. The permission of the RegionalBioethics Committee was also obtained. Sampling of material. Cord blood was sampled immediately afterdelivery of the fetus, before omphalotomy, by puncturing the cordvein and aspirating blood into a syringe with 3.2% sodium citrate(anticoagulant/blood proportion: 1:9). The blood was next trans-ferred to a test-tube for centrifugation, put into water with ice andtaken to the laboratory where centrifugation was done (2000 × gfor 20 min, temp. +4°C). The blood plasma was put in test-tubes in200 µl portions, tightly closed and kept until next examination (4-6 weeks) at -70°C. The maternal blood and the blood of womenfrom the control group was sampled from the antecubital vein andthen processed in the same way as described above for cord blood.Measurement of the level of TAFI antigen. The TAFI antigenlevel was measured using the immunoenzymatic (ELISA) methodwith the laboratory kit IMUBIND TAFIa/ai Antigen ELISA(American Diagnostica Inc.). That test measures TAFIa antigen(active enzyme) and TAFIai antigen (inactivated enzyme).According to the manufacturer, the sensitivity of that method is10 ng/ml. In our laboratory the interassay and intraassay coeffi-cients of variability were lower than 10%.Statistical analysis. The program STATISTICA for Windows byStatSoft was used for statistical analysis.A characteristic feature of the AgTAFI variable in cord bloodand in the maternal blood was decomposition, different from nor-mal, and that is why that variability was described by a median(Me) and lower (Q1) and upper (Q3) quartiles. In the control groupthe distribution of the feature under analysis was close to normal,yet, for the uniformity of analysis it was also described as Me andQ1 and Q3. The statistical analysis of dependent variables wasdone with Friedman test. The value p<0.05 was adopted as a sta-tistically significant level.Results The level of TAFIa/ai antigen in cord blood was sta-tistically significantly higher than in maternal blood(p<0.0001). Accordingly, in cord blood  (n=38)  thelevel was 91.50 ng/ml (median), range:  71.76 -160.77 ng/ml (Q1-Q3)  vs. the level in the maternalblood  (n=38): 55.46 ng/ml (median),  range: 39.77 -68.54 ng/ml (Q1-Q3).The level of TAFIa/ai antigen in the blood of thecontrol group (non pregnant women n=20) was72.55 ng/ml, range: 67.50-76.69 ng/ml (Q1-Q3). Itwas statistically significantly higher than in parturi-ent women (p<0.0004). Fig. 2 presents those resultsin a graphic formDiscussionAt the beginning of the discussion it seems appropriateto comment on the terminology concerning TAFI. Notso long ago, American and European authors used var-ious terms. Now, the following unified terms are com-monly used: pro - TAFI, i.e.  procarboxypeptidase B,thus TAFI proenzyme; TAFIa is carboxypeptidase B inthe form of an active enzyme, while TAFIai  is theinactivated form of carboxypeptidase B. In the presentstudy we measured the total level of TAFI antigen,both as an active enzyme and as an inactivated enzyme(TAFIa + TAFIai).TAFI antigen was present in all samples of thecord blood. We take that result to be evidence thatTAFI is an integral component of the blood. This is anew observation. The total level of TAFI (TAFIa +TAFIai) in the fetal blood is almost two times higherthan in the maternal blood (91.50 ng/ml vs. 55.46ng/ml). But, as we demonstrated in our report at theXXIII Congress of the Polish Gynecological Society,the activity of TAFI in cord blood is about 50% lowerthan in the maternal blood [16]. Most probably, theinactivated TAFI (TAFIai) is a dominant componentof the total level. In contrast to research on fetal blood, the level ofthe antigen and/or TAFI activity in the pregnantwomen blood has been the subject of research ofmany authors [1-3,7,8,13,19]. It has been found that34 W. Uszyñski et al. Fig. 1. The mechanism of procarboxypepti-dase B activation by thrombin/antithrombincomplex. * on the left side - thrombin for-mation, and on the right side - TAFI forma-tion, * TF - tissue factor, ** TAFI - throm-bin activatable fibrinolysis inhibitor in the course of pregnancy the level of the antigenand TAFI activity increase gradually, though moder-ately. According to Chabloz et al. [7] the level of theantigen in the third trimester of pregnancy constitutes130% of the level in the first trimester; Mousa et al.[13] found the level of the antigen to be 6.6±2 µg/mlat the beginning of pregnancy, and 9.6±2 µg/ml in thethird trimester.An increase of activity in the course of pregnancywas also reported by Watanabe et al. [19], with plateauon the 20th week. After delivery, already in the firstand second day, the level of the antigen and TAFIactivity falls down to the values found in non-pregnantwomen [13,19].It is not known what factors influence the increasea relatively high level of TAFI in the fetus. It may bedue to an unknown role of that factor in the develop-ment of the fetus? Likewise, it is not known whenTAFI appears in the fetal blood and when in theextrauterine life the level drops down to that of adults.It is possible that the increase of the level depends onthe high level of some of the hormones in the pregnantwoman's organism? Perhaps Chettaile et al. [8] pro-vided an argument for such an assumption when theyreported an increase of the level of TAFI antigen inwomen using oral contraceptives.TAFI in maternal blood was not the target of ourstudy; maternal blood was only a reference point ofthe gold standard type thanks to which we couldrelate our results. It was important because the resultsof other authors differ among each other (quantita-tively, not qualitatively). Most probably, the cause ofthose differences is the application of different mono-or polyclonal antibodies, as well as insufficient stan-dardization of measurements of TAFI in biologicalfluids. There are reasons, then, to postulate furtherstudies.ConclusionsThrombin activatable fibrinolysis inhibitor (TAFI) isan integral component of cord blood and it can beidentified in all samples of the cord blood. The level of thrombin activatable fibrinolysisinhibitor (TAFI) is statistically significantly higher incord blood than in maternal blood (about two timeshigher). References[ 1] Antoviæ JP, Hamad RR, Antoviæ A, Blombäck M, Bremme K.Does thrombin activatable fibrinolysis inhibitor (TAFI) con-tribute to impairment of fibrinolysis in patients withpreeclampsia and/or intrauterine fetal growth retardation?Thromb Haemost, 2002; 88: 644-47[ 2] Antoviæ JP, Antoviæ A. Does recombinant factor VIIa, apartfrom overall hemostasis, regulate TAFI dependent fibrinoly-sis? In vitro analysis using overall hemostasis potential(OHP) assay. Thromb Haemost,  2003; 90: 620-27[ 3] Alacacioglu IA, Ozcan MA, Oglu AA, Polat M, Yuksel F,Demirkan F, Piskin O, Ozgenc Y., Ozsan GH, Undar B. Plas-ma levels of thrombin activitable fibrinolysis inhibitor in nor-mal and preeclamptic pregnant women. Thromb Res, 2004;114: 155-59[ 4] Bajzar L, Morser J, Neisheim ME. Purification and character-ization of TAFI, a thrombin activatable fibrinolysis inhibitor.J Biol Chem, 1995; 270: 14477-84[ 5] Booth NA. TAFI meets the sticky ends. Thromb Haemost,2001; 85: 1-2[ 6] Campbell W, Okada N, Okada H. Carboxypeptidase R is aninactivator of complement-derived inflammatory peptides35TAFI in cord bloodFig. 2. The level of TAFI antigen(thrombin activatable fibrinolysisinhibitor) in the cord blood plasma.TAFI a/ai - enzymatically activeand inactive forms: TAFI(a) andTAFI(ai).and an inhibitor of fibrinolysis. Immunol Rev, 2001; 180:162-67[ 7] Chabloz P, Reber G, Boehlen F. TAFI antigen and D-dimerlevels during normal pregnancy and at delivery. Br J Hae-most, 2001; 115: 150-52[ 8] Chetaille P, Kouassi AD, Morange PE, Juhan-Vague I. PlasmaTAFI antigen variations in healthy subjects. Thromb Hae-most, 2000; 83: 902-05[ 9] Lau HK, Segev A, Hegele RA, Sparkes JD, Teitel JM,ChisholmRJ, Strauss BH.  Thrombin-activitable fibrinolysisinhibitor (TAFI): a novel predictor of angiographic coronaryrestenosis. Thromb Haemost, 2003; 90: 1187-91[10] Lisman T, Leebeek F-W, Mosnier L-O, Bouma BN, MeijersJC, Janssen HL, Nieuwenhuis HK, de Groot PG. Thrombin-actavatable fibrinolysis inhibitor deficiency in cirrhosis is notassociated with increased plasma fibrinolysis. Gastroenterol-ogy, 2001; 121: 131-9[11] Lisman T, Mosnier LO, Lambert T, Mausner-Bunschoten EP,Meijers JC, Nieuwenhuis HK, de Groot PG. Inhibition of fib-rinolysis by recombinant factor VIIa in plasma from patientswith severe hemophilia A. Blood, 2002; 99: 175-9[12] Morange PE, Juhan-Vague I, Scarabin PY, Alessi MC, Luc G,Arveiler D, Ferrieres J, Amouyel P, Evans A, Ducimetiere P,PRIME Study group. Association between TAFI antigen andAla I 47Thr polymorphism of the gene and the angina pec-toris insidence. Thromb Haemost, 2003; 89: 554-60[13] Mousa HA, Downey C, Alfirevic Z, Toh Ch-H. Thrombinactivitable fibrinolysis inhibitor and its fibrinolytic effect innormal pregnancy. Thromb Haemost, 2004; 92: 1025-31[14] Stromqvist M,Schatteman K, Leurs J, Leurs J, Verkerk R,Anderssen JO, Johansson T, Scharpe S, Hendriks D. Immuno-logical assay for the determination of procarboxypeptidase Uantigen levels in human plasma. Thromb Haemost, 2001; 35:12-17[15] Van Thiel DH, George M, Fareed J. Low levels of thrombinactivatable fibrinolysis inhibitor (TAFI) in patients withchronic liver disease. Thromb Haemost, 2001;  85: 667-70[16] Uszyñski W, ¯ekanowska E, Szymañski W, Uszyñski M.Thrombin activatable fibrinolysis inhibitor (TAFI) in cordblood  and blood of mother during labour (in Polish). XXIIIKongres Polskiego Towarzystwa Ginekologicznego. GinekPol, 2003; 74: 1329-34[17] Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. Astudy of the mechanism of fibrinolysis by activated thrombin-activitable fibrinolysis inhibitor. J Biol Chem, 1998; 273:27176-81[18] Watanabe R, Wada H, Watanabe Y, Sakakura M, Nakasaki T,Mori Y, Nishikawa M, Gabazza EC, Nobori T, Shiku H.Activity and antigen levels of thrombin - activatable fibrinol-ysis inhibitor in plasma of patients with disseminatedintravascular coagulation. Thromb Res, 2001; 104: 1-6[19] Watanabe T, Minakami H, Sakata Y, Matsubara S, Sato I,Suzuki M. Changes in activity of plasma thrombin activitablefibrinolysis inhibitor in pregnancy. Gynecol Obstet Invest,2004; 58: 19-21Received: October 17, 2006Accepted after revision: November 16, 200636 W. Uszyñski et al. 

Thrombin activatable fibrinolysis inhibitor (TAFI) in cord blood.

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Thrombin activatable fibrinolysis inhibitor (TAFI) in cord blood.

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