Introduction. Keywords: antithrombin, hemophilia, newborn, thrombin generation, tissue factor, tissue factor pathway inhibitor.

Transcription

1 Journal of Thrombosis and Haemostasis, 4: ORIGINAL ARTICLE Thrombin generation in factor VIII-depleted neonatal plasma: nearly normal because of physiologically low antithrombin and tissue factor pathway inhibitor P. FRITSCH,* G. CVIRN,* C. CIMENTI,* K. BAIER,* S. GALLISTL,* M. KOESTENBERGER,* B. ROSCHITZ,* B. LESCHNIK* and W. MUNTEAN* *Department of Pediatrics, Ludwig Boltzmann Research Institute for Pediatric Hemostasis and Thrombosis, Graz; and Institute for Medical Chemistry and Pregl-Laboratory, Department of Chemistry, Medical University of Graz, Graz, Austria To cite this article: Fritsch P, Cvirn G, Cimenti C, Baier K, Gallistl S, Koestenberger M, Roschitz B, Leschnik B, Muntean W. Thrombin generation in factor VIII-depleted neonatal plasma: nearly normal because of physiologically low antithrombin and tissue factor pathway inhibitor. J Thromb Haemost 6; 4: Summary. Background: Bleeding in hemophilic neonates has a low incidence. A possible explanation for this could be the peculiarities of the neonatal hemostatic system, especially low levels of the inhibitors tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Objective: We investigated the influence of an elevation of these inhibitors to adult levels on the thrombin generation (TG) in normal neonatal plasma and factor (F) VIII-depleted neonatal plasma by means of incubation with anti-fviii-antibodies. Patients/methods: TG was measured after activation with low amounts of tissue factor (TF) by using Calibrated Automated Thrombography. Results: TG in FVIII-depleted neonatal plasma was nearly as high as in normal neonatal plasma. TG decreased after elevation of AT in both neonatal plasmas. After elevation of TFPI TG decreased much more in FVIII-depleted neonatal plasma than in normal neonatal plasma. After elevation of both inhibitors their synergistic effect led to a stronger decrease of TG in FVIIIdepleted neonatal plasma. TG measured in plasma of one hemophilic newborn showed the same pattern as in FVIIIdepleted neonatal plasma. Conclusion: Our observation provides a biochemical basis for the rare bleeding in hemophilic neonates and shows the important role of the natural inhibitors in the hemostatic system of hemophilic patients. Keywords: antithrombin, hemophilia, newborn, thrombin generation, tissue factor, tissue factor pathway inhibitor. Correspondence: Wolfgang E. Muntean, Department of Pediatrics, Ludwig Boltzmann Research Institute for Pediatric Hemostasis and Thrombosis, Medical University of Graz, Auenbruggerplatz 3, 836 Graz, Austria. Tel.: ; fax: ; wolfgang. muntean@meduni-graz.at Received 28 October 5, accepted 13 February 6 Introduction In hemophilia, excessive bleeding during delivery and in the neonatal period is a rare event. Vaginal delivery is recommended for routine delivery in hemophilic neonates by the American Hemophilia Foundation [1]. This postulation is corroborated by the fact that the majority of hemophilic neonates show no bleeding during vaginal delivery and that hemophilia, especially without family history, is often diagnosed after the neonatal period. Studies on intracranial hemorrhage report an incidence of 1 3.9% in neonates with hemophilia [1,2]. These findings are surprising as the trauma of a normal vaginal delivery may result in bleeding such as ecchymosis or hematomas even in healthy neonates and should be enough to trigger major bleeding in neonates suffering from hemophilia. After delivery in healthy neonates and in neonates with mild hemophilia the factor (F) VIII level is higher than in later life [3], but must be low in neonates with severe hemophilia. The reasons for the low incidence of bleeding in patients with hemophilia in the neonatal period probably are the peculiarities of the neonatal hemostatic system [4]. Despite low levels of vitamin K-dependent factors, neonates show no easy bruising, no increased bleeding during surgery and good wound healing. Physiological low levels of anticoagulant proteins, such as antithrombin (AT) and tissue factor pathway inhibitor (TFPI), might compensate for low concentrations of clotting factors [5,6]. In previous work we have focused on this discrepancy between clinically excellent hemostasis and prolonged conventional clotting tests, and have shown that these physiologically low levels of the natural inhibitors AT and TFPI in neonatal plasma compensate for low concentrations of clotting factors by allowing sufficient thrombin generation (TG) despite low prothrombin levels [6 8]. Different quantity and intensity of bleeding in adult hemophilic patients with similar FVIII levels led to the assumption that factors other than FVIII alone are responsible

2 172 P. Fritsch et al for the expression of severity of hemophilia. Hemophilic patients with the FV Leiden (FVL) mutation [9 11] or Protein C (PC) deficiency [12] had a lower bleeding tendency than hemophilic patients without prothrombotic risk factors, but as the incidence of the FVL mutation is only 2 7%, there must be other variables of influence such as AT, TFPI and PC. van Ôt Veer et al. [13] also postulated that these variables influence the bleeding tendency and are responsible for the lower factor concentrate utilization in some severe hemophilic patients. A recent study of Dargaud et al. [14] showed that 25% of their patients did not present a close correlation between FVIII levels and TG. They also showed that the correlation between TG and the clinical presentation was closer than between FVIII levels and the clinical presentation: one of their patients suffering from severe hemophilia and AT deficiency had a nearly normal TG. The influence of TFPI in hemophilic patients has not yet been reported, but Dahm et al. [15] reported that the risk for venous thrombosis was increased 2-fold with TFPI levels below the fifth percentile in patients without other bleeding disorders. There is also a report of deep venous thrombosis in patients with severe hemophilia, in whom no predisposing thrombophilic risk factor was identified [16]. Conventional in vitro clotting assays such as activated partial thromboplastin time (APTT) and prothrombin time give a very incomplete picture of the relative importance of various components of the clotting system. In recent years it has been shown that coagulation activation in plasma via the extrinsic pathway, by addition of low amounts of lipidated tissue factor (TF) as activator, is probably more compatible with the physiological circumstances [17] and suitable for a sensitive detection of the effects of different levels of pro- and anticoagulants, including different levels of FVIII from 5% to 15%, on TG [18]. The group of Hemker [14] showed that the TG decreases with declining FVIII concentrations and that TG correlates with the incidence of bleeding. Furthermore, TG allows a detailed analysis of hemostasis, because it allows the differentiation of four major parameters: the lag phase; the time to peak height (TTP); the peak height; and endogenous thrombin potential (ETP). The aim of this study was to measure TG after activation with small amounts of TF in FVIII-deficient neonatal plasma, to investigate whether physiologically low levels of TFPI and AT in neonatal plasma may explain the low bleeding incidence in neonates with hemophilia. In absence of enough hemophilic neonatal plasma, normal neonatal plasma was pooled and FVIII depleted. Then the effect of raising TFPI and AT to adult plasma levels was investigated. TG of one hemophilic newborn was also measured. Material and methods Reagents Full-length recombinant human TFPI activity standard was purchased from American Diagnostica (Greenwich, CT, USA). Five micrograms of the glycoprotein were dissolved in 5 ll buffer A. Buffer A contained.5 mol L )1 Tris HCl,.1 mol L )1 NaCl and.5 mol L )1 bovine serum albumin at ph 7.4; after subsequent 1:1 dilution in buffer A 5 ll aliquots were stored at )7 C). The Total TFPI ELISA Kit from Imubind TM was used, and the TFPI activity assay from Actichrome TM (American Diagnostica). ATenativ Ò,anAT concentrate for clinical application, was purchased from Pharmacia & Upjohn GmbH (Vienna, Austria). Then IU AT from the stock solution were added to 2 ml of distilled water and stored as aliquots of 5 ll at)7 C. Fluobuffer contained 2 mm HEPES and 6 mg ml )1 bovine serum albumin, at ph 7.35, both purchased from Sigma (St Louis, MO, USA). Working buffer consisted of 14 mm NaCl, purchased from Merck (Darmstadt, Germany), 2 mm HEPES and 5mgmL )1 human serum albumin, purchased from Sigma. The fluorogenic substrate Z-Gly-Gly- Arg-amino-methyl-coumarin was purchased from Bachem (Bubendorf, Switzerland), and was dissolved in pure dimethylsulfoxide, which was purchased from Sigma. Calcium chloride was purchased from Merck [19 22]. TF containing platelet-poor plasma (PPP) reagent and the thrombin calibrator were purchased from Synapse/Thrombinoscope BV (Maastricht, the Netherlands). Collection and preparation of plasma This study was approved by the Ethics Committee of the Medical University of Graz (protocol number: ex3/4). Cord bloods were obtained immediately following the delivery of 28 full-term infants (38 42 weeksõ gestational age). Newborns with Apgar scores of 9 or less 5 min after delivery were excluded from the study. Blood was collected into.1 M citrate using a two syringe technique, centrifuged at room temperature for 1 min at 4 g. The plasma was pooled and stored at )7 C in propylene tubes until assayed. Pro- and anticoagulant factors were in the normal range for neonates. All plasmas from the neonates were pooled. All measurements were carried out in the plasma taken of the pool. In the same way plasma from healthy adults was collected from the antecubital vein, pooled, prepared, and checked (FVIII-level, APTT, PT, and platelets). Plasma from one newborn with hemophilia was collected on day 3 together with the sample for the diagnosis. FVIII level was lower than 1%. Antibodies and preparation of FVIII-depleted plasma Neonatal and adult plasmas were incubated with purified IgG anti-fviii antibodies from a hemophilic patient with 18 BU ml )1. FVIII depletion was verified by one stage FVIII activity measurement. Preparation of plasma with different TFPI levels The TFPI level of the pooled cord plasma was increased by addition of 4 ll buffer A containing 1 ll of human TFPI standard to 96 ll plasma.

3 Thrombin generation in FVIII-depleted neonates 173 Determination of the TFPI plasma concentrations Tissue factor pathway inhibitor antigen levels were determined by means of the Imubind TM Total TFPI ELISA Kit, TFPI activity was determined by means of the chromogenic assay Actichrome TM TFPI activity assay. Preparation of plasma with different AT levels Antithrombin levels of pooled cord plasma were increased by addition of 4 ll buffer A containing 15 ll of AT concentrate to 96 ll plasma. Determination of the AT content Determination of the AT content of cord plasma was performed by means of a standard chromogenic method on a BM/Hitachi 917 from Roche (Vienna, Austria). Determination of TG Measurement of TG was performed using Calibrated Automated Thrombography. For each experiment a fresh mixture of 2625 ll fluobuffer and 3 ll 1 M CaCl 2 solution was prepared and incubated for 5 min at 37 C. After these 5 min, 75 ll of the fluo-dimethylsulfoxide solution were added, mixed and incubated again for 5 min. The resulting clear solution was referred to as FluCa. Platelet-poor plasma-reagent was solubilized with 2 ml deionized water. Twenty microliters of PPP reagent or 2 llofthethrombin calibrator, after reconstitution with 1 ml sterile water, were put into each sample well of a 96-well round-bottom microtiter plate made of polypropylene, purchased from Nunc (Roskilde, Denmark). Finally, 8 ll of PPP containing different concentrations of anticoagulant agents were put into each well. All reagents were warmed up to 37 C before starting the experiment. The 96-well plate was put in the fluorometer (Fluoroskan Ascent, Thermolabsystems OY, Helsinki, Finland) with an excitation filter at 39 nm and an emission filter at 46 nm. The automated dispensing of 2 ll FluCastarted the measurement process. The final concentration contained 5pM TF and 4 lm phospholipids. During 4 min each well was measured every 2 s. Upon completion of the measurement, we used the Analysis Software from Thrombinoscope BV (Maastricht, the Netherlands) to analyze our results [23,24]. Results TG in neonatal and adult FVIII-deficient plasma Activated partial thromboplastin time was normal in the pooled adult plasma with a FVIII activity of 86%. APTT was slightly prolonged in the plasma of the healthy neonate with a FVIII activity of 142%. In the FVIII-depleted adult plasma (FVIII level < 1%) and also in the FVIII-depleted neonatal plasma (FVIII level < 1%), the APTT was prolonged to more than 14 s and showed a significant prolongation compared with the normal neonatal and adult plasma, respectively, but was nearly the same in both FVIII-depleted plasmas (Table 1). Thrombin generation in the adult plasma after activation with low amounts of TF showed no difference of the lag phase and a shorter TTP than in FVIII-depleted adult plasma; the peak height and ETP in FVIII-depleted adult plasma was lower than in the adult plasma (Table 1, Fig. 1A). In contrast, in FVIII-depleted neonatal plasma, lag time and TTP was only slightly prolonged compared with that of normal neonatal plasma, the peak height was nearly equal and the ETP was higher than in normal neonatal plasma (Table 1, Fig. 1B). In addition, TG in neonatal and adult FVIII-depleted plasma showed a nearly equal peak height, but a lower ETP level in FVIII-depleted neonatal plasma, whereas the lag phase and the TTP were significantly shorter than in the FVIII-depleted adult plasma. TG in both neonatal plasmas showed a lower peak height than normal adult plasma. However, TTP and lag phase were significantly shorter in both neonatal plasmas (Table 1, Fig. 1A,B). Effect of the inhibitors TFPI and AT in normal and FVIII-depleted plasma Thrombin generation in normal and FVIII-depleted neonatal plasma was measured after activation with 5 pm TF. All plasmas were taken from the same pool of neonatal plasma. Antithrombin level was raised by addition of AT from the neonatal level of 58% to the adult level of 13%. In normal neonatal plasma AT spiking resulted in a decrease of the ETP by 64% and the peak height by 33%, with only a slight prolongation of the TTP and no prolongation of the lag phase. Table 1 Data of thrombin generation (TG) measurement inneonatal andadultplasma: TGwasmeasured afteractivation with5pm TF. Results are shown as mean (range) of five different experiments Neonatal plasma FVIII-depleted neonatal plasma Adult plasma FVIII-depleted adult plasma FVIII level (%) 142 < 1 86 < 1 APTT (s) Lag phase (min) 1.56 ( ) 1.8 ( ) 2.56 ( ) 2.67 ( ) ETP (nm min )1 ) 1197 ( ) 1511 ( ) 2195 ( ) 18 ( ) Peak height (nm) 243 ( ) 237 ( ) 46 ( ) 23 ( ) TTP (min) 3.33 (3 3.67) 3.67 ( ) 4.2 ( ) 7.33 ( ) FVIII, factor VIII; APTT, activated partial prothrombin time; ETP, endogenous thrombin potential; TTP, time to peak (height).

4 174 P. Fritsch et al A B Fig. 1. TG in normal and FVIII-depleted adult and neonatal plasma. (A) Thrombin generation (TG) in normal and factor (F) VIII-depleted adult plasma: TG measured after activation with 5 pm TF in normal (full diamond) and FVIII-depleted (open square) adult plasma. (B) TG in normal and FVIII-depleted neonatal plasma: TG measured after activation with 5 pm TF in normal (full diamond) and F VIII-depleted (open square) neonatal plasma. In FVIII-depleted neonatal plasma, raising the AT level to 15% did not result in a prolongation of the lag phase and the TTP. The peak height decreased by 4% and the ETP by 69%. There were very few differences in the changes of TG between normal and FVIII-depleted neonatal plasma (Table 2, Fig. 2A,B; full diamond and full triangle). TFPI antigen level was raised after substitution of TFPI from neonatal level of ng ml )1 to the adult level of ng ml )1. TFPI activity level was raised from neonatal level of.81 U ml )1 to the adult level of 1.24 U ml )1.In normal neonatal plasma, raising TFPI resulted in a prolongation of the lag phase and the TTP, whereas the increase of TFPI in normal neonatal plasma had virtually no effect on the peak height and the ETP. In contrast, to the normal neonatal plasma, raising of TFPI in FVIII-depleted neonatal plasma resulted in a strong decrease of the peak height by 77%, a greater than 3-fold prolongation of the TTP of more than 8 min and also a prolongation of the lag phase of more than 2 min, and a decrease of the ETP by 4%. The prolongation of the TTP in the FVIII-depleted neonatal plasma was 8 times longer than in normal neonatal plasma (Table 2, Fig. 2A,B; full diamond and open square). In the normal neonatal plasma, raising AT and TFPI resulted in an increase of the lag phase of.7 min and the TTP of.89 min, and a decrease of the peak height by 35% and the ETP by 67% as compared with unspiked neonatal plasma. In FVIII-depleted plasma raising AT and TFPI decreased the peak height by 9%, prolonged the TTP more than 6 min and Table 2 Data of TG measurement in normal and factor (F) VIII-depleted neonatal plasma with different inhibitor levels: TG was measured in neonatal and FVIII-depleted neonatal plasma after activation with 5 pm TF. Results are shown as mean (range) of five different experiments FVIII-depleted neonatal plasma Neonatal plasma FVIII-depleted +AT +TFPI +AT + TFPI neonatal plasma +AT +TFPI +AT + TFPI Neonatal plasma Lag phase, range (min) 1.33 ( ) 1.33 ( ) 2.11 ( ) 2 ( ) 1.67 ( ) 1.67 ( ) 3.56 ( ) 2.67 ( ) ETP, range (nm min )1 ) 1197 ( ) 439 ( ) 1237 ( ) 395 (361 45) 146 ( ) 436 ( ) 854 ( ) 343 ( ) Peak height, range (nm) 243 ( ) 164 (157 17) 249 ( ) 156 ( ) 237 ( ) 141 ( ) 55 (37 78) 23 (11 25) TTP, range (min) 2.78 ( ) 3 ( ) 4 ( ) 3.67 ( ) 3.89 ( ) 3.33 ( ) ( ) 1.22 ( ) AT, antithrombin; TFPI, tissue factor pathway inhibitor; TTP, time to peak (height).

5 Thrombin generation in FVIII-depleted neonates 175 B A Fig. 2. TG in normal and FVIII-depleted neonatal plasma with different inhibitor levels. (A) TG in normal neonatal plasma with different tissue factor (TF) pathway inhibitor (TFPI) and antithrombin (AT) levels: TG measured after activation with 5 pm TF in normal plasma (full diamond), after spiking of AT (full triangle), after spiking of TFPI (open square) and after spiking of both inhibitors (open diamond). (B) TG in factor (F) VIIIdepleted neonatal plasma with different TFPI and AT levels: TG measured after activation with 5 pm TF in normal FVIII-depleted plasma (full diamond), after spiking of AT (full triangle), after spiking of TFPI (open square) and after spiking of both inhibitors (open diamond). the lag phase more than 1 min compared with unspiked FVIIIdepleted neonatal plasma. The reduction of the peak height after raising both inhibitors in normal neonatal plasma was 35%, whereas the reduction in the FVIII-depleted neonatal plasma was 9%. The prolongation of the TTP after raising both inhibitors was.89 min in normal neonatal plasma, in FVIII-depleted neonatal plasma the prolongation was 6.33 min, nine times longer (Table 2, Fig. 2A,B; full diamond and open diamond). TG in plasma of hemophilic newborn One plasma sample of a hemophilic newborn was available. FVIII activity in this patient was < 1%. TG in this plasma showed only a small prolongation of the lag phase and the TTP, and a decrease of the peak height by 25% when compared with the TG of a plasma sample of a healthy newborn (Fig. 3). Discussion In this investigation we show that the peak height of the TG in FVIII-depleted neonatal plasma is nearly as high as in normal Fig. 3. Thrombin generation in healthy and hemophilic newborn: TG measured after activation with 5 pm TF in hemophilic (open square) and normal newborn plasma (full diamond). neonatal plasma, whereas the peak height of the TG in FVIIIdepleted adult plasma is only 5% of that of normal adult plasma. The TG observed in the one available plasma sample from a severe hemophilic newborn essentially showed the same pattern as obtained with neonatal plasma artificially depleted by means of an anti-fviii-antibody. There was almost no difference in the APTT of FVIII-depleted neonatal and adult plasmas, whereas the peak height of the TG in neonatal FVIIIdepleted plasma was nearly 2% higher, and the TTP was 5% shorter than in adult FVIII-depleted plasma, although the ETP was higher in the adult plasma (Table 1, Fig. 1A,B). These findings are in agreement with the clinical observation of a low bleeding tendency in neonatal hemophilic patients and suggest that measurement of the TG is a valuable tool in the clinical assessment of patients with hemophilia. The ETP alone seems to be not as important as the peak height, the TTP and the lag phase. Clotting occurs when TG starts to increase, at the end of the lag time. The efficiency of the clotting probably correlates with the length of the lag phase, the peak height and TTP. The ETP, the area under curve, is dependent on the peak height and the time point at which the curve reaches zero again. In hemophilia typically the increase and the peak height of the TG is lower but the time point when the curve reaches zero is postponed, resulting in a relatively high ETP (Fig. 1A, Table 1). We also show that the TG in FVIII-depleted neonatal plasma after raising AT and TFPI, or especially both inhibitors, to adult levels is more strongly decreased than in normal neonatal plasma. In normal neonatal plasma, raising of the inhibitors showed again the previously described effects on the TG [6,7]. In FVIII-depleted neonatal plasma, raising AT showed a reduction of the TG, but there was no significant difference between normal and FVIII-depleted neonatal plasma. In contrast to that, raising TFPI in FVIII-depleted neonatal plasma showed a 4.5-fold reduction of the peak height, whereas there was no reduction in normal neonatal plasma; and furthermore, it showed a strong prolongation of the lag phase and the TTP compared with the normal neonatal plasma (Table 2, Fig. 2A,B). Raising the amount of both inhibitors, their synergistic effects led to a decrease of the peak height to

6 176 P. Fritsch et al 65%, compared with normal neonatal plasma without inhibitors, and to a decrease of the peak height to 1% and a strong increase of TTP, compared with FVIII-depleted plasma without raised inhibitors. All peaks were lower in neonatal plasma than in the corresponding adult plasma, probably because of the lower prothrombin levels in neonatal plasma [6,7]. Our data show that the influence of TFPI on the TG, especially on the peak height and the TTP, is much stronger when FVIII levels are low. Effects on the TG were most pronounced when both inhibitors, AT and TFPI, were increased and resulted in a stronger decrease in FVIII-depleted than in normal neonatal plasma. This effect can be explained by synergistic inhibition of the two phases of the TG: the Ôinitiation phaseõ and the Ôpropagation phaseõ.vanôtveeret al. [25] showed that high amounts of TFPI extends the Ôinitiation phaseõ by delaying the FV activation, which normally builds the Ôprothrombinase (FXa/FVa)Õ complex and protects FX that way from the binding to AT. They emphasized the importance of TFPI in FVIII absence by showing that the inhibition of the TG is significantly higher in the absence of FVIII, showing a decrease of TG of more than 75% when TFPI levels were twofold higher. Thus TFPI and AT together are about 7-fold more potent than each of them alone and the two inhibitors, acting in concert, seem to provide a synergistic inhibitory effect. The formation of the Ôtenase (FIXa/FVIIIa)Õ complex, which assembles with the Ôprothrombinase (FXa/FVa)Õ complex on the platelet surface, in hemophilic patients is suppressed or completely lacking. This results in a failure of FXa generation, associated with a suppressed TG and a strong bleeding tendency. In neonates there are two possible mechanisms extending the ÔFXa liveõ: the first is that higher amounts of TF in neonatal plasma [26] generate higher FXa levels; and the second that the combined action of TFPI and AT is reduced to approximately 5%, because of physiologically low inhibitor levels. We show in our work the efficacy of the latter mechanism. Our results provide further evidence for the notion that different inhibitor levels might be an explanation for the different clinical phenotypes in hemophilic patients, who show no correlation between the severity of clinical bleeding and FVIII plasma level. They support case reports about lower bleeding tendency in patients with FVL mutation, PC deficiency or AT deficiency. In addition, although no clinically relevant TFPI deficiency has reported so far, the TFPI level might be of interest in hemophilic patients [15,25]. The determination of TFPI was very complex in the past when only ELISA assays were available, but a novel TFPI activity assay to measure plasma TFPI activity has been developed by the group of Dahl et al. [27]. In conclusion, our results showing that neonatal FVIIIdepleted plasma with physiologically low TFPI and AT levels has a TG nearly as good as normal neonatal plasma, and a better TG than adult FVIII-depleted plasma, providing a biochemical basis for the observation that hemophilia rarely becomes manifest in the neonatal period. Our results also provide further evidence for the important role of natural inhibitors for the expression of the severity of bleeding in hemophilic patients. Acknowledgements This study was supported by the Bayer Hemophilia Awards 4. We thank B. Leschnik, who established the laboratory work (test kit and CAT) and Y. Gallistl for her organisational work. References 1 Kulkarni R, Lusher JM, Henry RC, Kallen DJ. 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