in Cord and Adult Plasma: an in-vitro Examination
M. Köstenberger, S. Gallistl, G. Cvirn, B. Leschnik and W. Muntean
Abstract
The aim of our study was to compare the effects of the new thrombin inhibitor melagatran on markers of thrombin generation in cord and adult plasma activated with either high or low amounts of tissue factor (TF). The new direct thrombin inhi- bitor melagatran acts, in contrast to the most frequently administered anticoagulant heparin, independent of the antithrombin content in plasma. In the absence of melagatran adult plasma clotted significantly prior to cord plasma when activated with high amounts of TF, while in contrast clotting of adult plasma is significantly delayed when compared to clotting times of cord plasma under low activation.
Under both high and low activation of plasma clotting times dose-dependently increased when melagatran concentrations were successively elevated. Increasing amounts of melagatran resulted in dose-dependently decreased F 1.2 and TAT gene- ration in both cord and adult plasma under both high and low amounts of TF.
Our in vitro results cannot be used for determination of clinical efficacy but assessing the degree of thrombin generation inhibition may be useful for selecting effective doses of melagatran for in vivo studies in newborn infants with thrombo- embolic complications.
Introduction
The hemostatic system of neonates has unique features when compared with adults [1, 2]. In the newborn, plasma concentration of the vitamin k-dependent coagulati- on factors, i.e. prothrombin, and of the anticoagulants antithrombin (AT), protein C (PC), protein S (PS), and tissue factor pathway inhibitor (TFPI) are reduced to approximately 50 % of adult amounts [3–6]. Physiological low levels of anticoagu- lant proteins in neonatal plasma might compensate for low levels of coagulation fac- tors and allow sufficient thrombin generation.
Infants less than one year old constituted the age group at highest risk for thromboembolic complications among children, mainly due to serious associated conditions such as central venous lines [7]. Until there is convincing data to the contrary they do require anticoagulant therapy. Usually newborns with thrombo- embolic complications are treated with heparin. It is uncertain whether the relative deficiency of AT in neonatal plasma limits the antithrombotic effects of heparin in
I. Scharrer/W. Schramm (Ed.)
34thHemophilia Symposium Hamburg 2003
” Springer Medizin Verlag Heidelberg 2005
this age group. On one hand inhibition of thrombin in heparinized plasma was sug- gested to be less effective than in adult plasma due to neonatal AT deficiency [8], on the other hand Chan et al. [9] demonstrated that reduced concentrations of UH and LMWH were required to inhibit thrombin generation in cord compared to adult plasma.
Direct thrombin inhibitors (DTI), such as melagatran, were developed to im- prove the efficacy/bleeding ratio to that of heparin and to overcome the inability of the heparin/antithrombin complex to inactivate bound thrombin [10]. DTIs pro- duce a more predictable anticoagulant response than heparin because, unlike hepa- rin, they do not bind to plasma proteins or require antithrombin as a cofactor.
Therefore DTIs may be of particular interest in newborns with thromboembolic complications. We, therefore, investigated the potential significance of the physio- logical AT deficiency in neonatal plasma for the antithrombotic effects of melaga- tran when compared to adult plasma.
The lower levels of coagulation factors in newborns are reflected in standard assays as a prolonged prothrombin time (PT) and prolonged activated partial thromboplastin time (APTT). However, prolongation of PT and APTT in neonatal plasma give only a simplified picture of the relative importance of various compo- nents of the coagulation process. Davie et al. [11] suggested that coagulation activa- tion via the extrinsic pathway with low amounts of TF is probably more physio- logical than the plasma activation commonly used in standard assays. Low TF concentrations have been shown to be suitable for sensitive detection of the effects of different levels of pro- and anticoagulants on thrombin generation [12].
We, therefore, investigated the effect of melagatran on prothrombin activation, thrombin-antithrombin generation, and clotting time activated with either high or low amounts of tissue factor in cord versus adult plasma.
Materials and Methods
Melagatran, the active form of the oral direct thrombin inhibitor ximelagatran
(Exanta), was kindly supplied by AstraZeneca R&D (Mölndal, Sweden). The Imu-
bind Tissue Factor ELISA Kit for quantitative determination of TF concentration
was obtained from American Diagnostica Inc. (Greenwich, CT, USA). H-Gly-Pro-
Arg-Pro-OH (GPRP; Pefabloc) was purchased from Pentapharm LTD (Basel,
Switzerland). Testkit prothrombin fragment 1.2 (F 1.2) micro for determination of
F 1.2, and testkit thrombin-antithrombin (TAT) micro for determination of TAT
were purchased from Behring Diagnostics (Marburg, Germany). Stopping solution
for F 1.2 and TAT determination consisted of Trasylol/EDTA/Na-citrate 8:1:1 and
110 µmol/l PPACK (D-Phe-Pro-Arg chloromethyl ketone) from Sigma (Vienna,
Austria). Trasylol from Bayer (Vienna, Austria), contained the protease inhibitor
aprotinin. Buffer A contained 0.05 mol/L Tris-HCl (pH 7.4), 0.1 mol/l NaCl and
0.5 mol/l bovine serum albumin.
Collection and Preparation of Plasma
Cord blood was obtained immediately following the delivery of 24 full term infants (38-42 weeks gestational age). Newborns with Apgar scores of 9 or less five minutes after delivery were excluded from the study. Blood (2.7 ml) was collected into pre- citrated S-Monovette pre-marked tubes from Sarstedt, Nümbrecht, Germany, containing 300 µl 0.106 M citrate, centrifuged at room temperature for 15 min at 2800 x g, pooled and stored at -70°C in propylene tubes until assayed. The hema- tocrit of cord blood was slightly, but not significantly, elevated over adult values. FV and FVIII activities were elevated over the respective adult values, other pro- and anti- coagulant factors were in the normal range for neonates. I.e., cord plasma contained 162 µg/ml AT. In the same way blood from 18 healthy adults was collected from the antecubital vein, prepared, and checked. I.e., adult plasma contained 264 µg/mL AT.
Activation of Plasma
Four-hundred µl plasma were incubated with 30 µl of buffer A containing melaga- tran at desired concentration for 2 minutes at 37°C in flat-bottomed plastic tubes while stirring. After subsequent incubation with 20 µl buffer A containing H-Gly- Pro-Arg-Pro-OH (Pefabloc FG, 1.0 mg/mL final concentration) to inhibit fibrin polymerization, plasma samples were activated by addition of 50 µl of an activa- tion-mixture composed of 0.5 M CaCl
2and diluted Thromborel S as a source of TF.
Final concentration of TF were 1.84 ng/ml TF for »high coagulant challenge« and 4.6 pg/ml TF for »low coagulant challenge«, respectively.
Determination of AT Plasma Concentrations
Determination of the AT content of cord plasma was performed by means of a stan- dard chromogenic method on a BM/Hitachi 917 from Boehringer Mannheim (Vienna, Austria).
Determination of TF Concentrations
TF concentrations were quantitatively determined via an enzyme-linked immuno- assay. TF levels were determined by measuring solution absorbency’s at 450 nm and comparing the values with those of a standard curve.
Determination of Clotting Time
Clotting times were determined by means of the optomechanical coagulation ana-
lyzer Behring Fibrintimer II from Dade Behring (Marburg, Germany), which
applies the turbodensitometric measuring principle. Plasma samples were prepared
as described above except that Pefabloc was replaced by buffer A.
Determination of F 1.2
Plasmas were prepared and activated as described above. Prothrombin activation was determined by subsampling 10 µl aliquots, taken from plasmas 10 minutes after explosive thrombin generation has occurred, into 490 µl stopping solution. After subsequent 1:10-dilution in stopping solution, the amount of F 1.2 generated was quantified by using an immuno-enzymatic kit.
Determination of TAT
Plasmas were prepared and activated as described above. TAT activation was deter- mined by subsampling 10 µl aliquots, taken from plasmas 10 minutes after explosive thrombin generation has occurred, into 490 µl stopping solution. After subsequent 1:20-dilution in stopping solution, the amount of TAT generated was quantified by using an immuno-enzymatic kit.
Statistical Analysis
The effects of different concentrations of melagatran on TAT and F 1.2 generation were analyzed using paired t-test. For comparison of TAT and F 1.2 values after activation of plasma at high and low coagulant challenge Mann-Whitney U test was performed. The significant level of P-values was set at P<0.05.
Results
All results obtained in cord and adult plasma are expressed as means (± SD) of five experiments.
Effect of Melagatran on Clotting Time in Cord and Adult Plasma
Under high coagulant challenge (cc) adult plasma clotted significantly prior to cord plasma in the absence of the direct thrombin inhibitor melagatran (Fig. 1a). In con- trast, under low coagulant challenge clotting of adult plasma is significantly delay- ed when compared to clotting times of cord plasma in the absence of melagatran (Fig. 1b).
Under both high and low cc clotting times dose-dependently increased when
melagatran concentrations were successively elevated. The clotting-time-prolon-
ging effect of melagatran in cord plasma was comparable to that in adult plasma.
Effects of Melagatran on Prothrombin Fragment 1.2 Values in Cord and Adult Plasma
Addition of increasing amounts of melagatran resulted in dose-dependently decreased F 1.2 generation in both cord and adult plasma under high (Fig. 2a) and low (Fig. 2b) coagulant challenge.
Melagatran revealed comparable capability to suppress F 1.2 formation in both cord and adult plasma under high (Fig. 3a) and low (Fig. 3b) coagulant challenge, calculated in percent inhibition referring to F 1.2 values in the absence of the drug.
Effects of Melagatran on TAT Generation in Cord and Adult Plasma
Addition of increasing amounts of melagatran resulted in dose-dependently decreased TAT generation in both cord and adult plasma under high (Fig. 4a) and low (Fig. 4b) coagulant challenge.
0 0,01 0,05 0,1 0,25 0,5 1
0 200 400 600 800 1000 1200
Time [s]
Melagatran [ µmol/l]
0 0,01 0,05 0,1 0,25 0,5 1
0 50 100 150 200 250
a
b
Fig. 1a, b. Effect of various melagatran concentrations on prolongation of clot- ting time in cord and adult plasma under high coagulant challenge a) and low cc b). Black columns:
Melagatran, adult
plasma. White
columns: Melagatran,
cord plasma. Results
shown are expressed
as means (n = 10).
Melagatran revealed comparable capability to suppress TAT formation in both cord and adult plasma under high (Fig. 5a) and low (Fig. 5b) coagulant challenge, calcu- lated in percent inhibition referring to F 1.2 values in the absence of the drug.
Discussion
The coagulation system in full term neonates is immature at birth and evolves post- natally toward the mature adult system. The plasma concentration of both pro- coagulants and inhibitors significantly differ in healthy neonates compared with adults [1]. The capacity to generate thrombin is decreased and delayed in plasmas from healthy newborns compared with adults [2]. Despite physiological low levels of procoagulant proteins hemostasis in the neonate is as least as good as in adults.
Physiological low levels of inhibitors might compensate for low levels of procoagu- lants, and, thus, allow sufficient thrombin generation. Results from our previous stu-
0 0,01 0,05 0,1 0,25 0,5 1 2 0,7
0,8 0,9 1,0 1,1 1,2
a
b
