Higher Concentrations of Heparin and Hirudin are Required to Inhibit Thrombin Generation in Tissue Factor-Activated Cord Compared to Adult Plasma

to Inhibit Thrombin Generation in Tissue Factor-Activated Cord Compared to Adult Plasma

K. Baier, G. Cvirn, P. Fritsch, M. Köstenberger, S. Gallistl, B. Leschnik and W. Muntean

Abstract

In standard clotting assays plasma samples are markedly diluted and high amounts of initiator are added to trigger clot formation. Under these conditions, adult plas- ma clots faster than neonatal plasma and only 30–50% of peak adult thrombin activity can be produced in neonatal plasma. It was reported that decreased con- centrations of heparinoids are required to inhibit thrombin generation in plasma from newborns to the same extend as in adults due to reduced neonatal thrombin potential (TP). Plasma activation by addition of low amounts of lipidated tissue fac- tor (TF) in combination with only slight dilution of plasma samples probably bet- ter reflects the conditions in vivo and allows sensitive detection of the effects of anticoagulants on thrombin generation. We have shown recently that under these conditions cord plasma clots faster and higher amounts of thrombin are generated compared to adult plasma. In accordance, we show in the present study that higher amounts of the anticoagulants heparin and hirudin are required in cord plasma for effective inhibition of thrombin generation compared to adult plasma: Prolongation of clotting time and suppression of TP were significantly more pronounced in adult plasma under low coagulant challenge compared with that in cord plasma. In con- trast, prolongation of clotting time and suppression of TP were significantly more pronounced in cord plasma under high coagulant challenge. Our results suggest that in neonates effects of anticoagulants very much depend on the type of acti- vation used to initiate clotting and doses of anticoagulants can not be derived from studies done in adults, and that higher doses of thrombin inhibitors may be required.

Introduction

The hemostatic system of neonates is different to that of children and adults. The risk for thromboembolic complications is considerably less for newborns and child- ren than for adults for any given insult, because neonates have lower levels of pro- coagulant factors, especially prothrombin and the other factors of the prothrombin complex. This is reflected in prolonged prothrombin time (PT) and activated part- ial thromboplastin time (APPT). Physiological low levels of anticoagulant proteins in neonatal plasma might compensate for low levels of coagulation factors and allow

I. Scharrer/W. Schramm (Ed.)

34^thHemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

sufficient thrombin generation. Neonatal plasma levels of antithrombin (AT) are reduced to approximately 60% of adult values [1]. It has been shown that these phy- siological low AT levels result in an impaired ability of neonatal plasma to inhibit thrombin compared to adult plasma [2].

It has been suggested that decreased concentrations of heparinoids are required to inhibit thrombin generation in plasma from newborns to the same extent as in adults due to reduced neonatal thrombin potential (TP) [3]. However, thrombin generation is only decreased and delayed in newborn plasma compared to adults when plasma samples are markedly diluted due to addition of high amounts of activator, as is usually done in the conventional clotting assays activated partial thromboplastin time (APTT) or prothrombin time (PT). Under these conditions only 30-50% of peak adult thrombin activity can be produced in neonatal plasma [4].

Plasma activation due to addition of low amounts of tissue factor (TF) probably better reflects the conditions in vivo [5] and allows sensitive detection of the effects of pro- and anticoagulants on thrombin generation [6]. In previous work we have shown that under such a low coagulant challenge, in contrast to the findings in the presence of high amounts of activator, thrombin generation starts faster than that in adult plasma and higher amounts of free thrombin can be generated in cord com- pared with adult plasma [7]. This elevated thrombin-forming capacity of cord com- pared with adult plasma is caused by the low anticoagulant action of the natural inhibitors tissue factor pathway inhibitor (TFPI), antithrombin (AT), and activated protein C (APC) in newborns [7, 8]. Thus, one might assume that higher concentra- tions of anticoagulants might be required in neonates to inhibit thrombin genera- tion to the same extent as in adults in the presence of low amounts of initiator of clot formation due to increased neonatal thrombin potential (TP).

Therefore, it was the aim of our study to evaluate the anticoagulant action of the anticoagulants heparin and hirudin under low coagulant challenge (better reflec- ting the conditions in vivo) in comparison to that under high coagulant challenge (reflecting the conditions prevailing in standard clotting assays). We investigated both the effect of an indirect and a direct thrombin inhibitor. We measured the effects of increasing amounts of heparin or hirudin on prolongation of clotting time and on suppression of TP in both cord and adult plasma in the presence of high (30µM final concentration) or low (30 pM final concentration) amounts of TF; in a effort to better understand the mechanisms of anticoagulation with indirect and direct thrombin inhibitors in neonates and provide a rationale for dose finding in neonates.

Material and Methods Reagents

Buffer A contained 0.05 M Tris-HCl at pH 7.4, 0.1 M NaCl and 0.5 M bovine serum

albumin. Buffer B was analogous to buffer A but contained in addition 20 mM

EDTA. Full length lipidated recombinant human tissue factor (a stock solution was

prepared by dissolving 50 ng of the lyophilized lipoprotein in 4.5 ml buffer A, aliquots of 500 µl were stored at –70°C), and Actichrome TF activity assay were obtained from American Diagnostica Inc. (Greenwich, CT, USA). Heparin (1000 IE/mL) was obtained from Baxter Hyland Immuno, Vienna, Austria. A stock solu- tion of 10 IU/ml was prepared by 1:100 dilution with distilled water. Hirudin was obtained from Sigma, Vienna, Austria. 500 IE were dissolved in 1 mL of distilled water and subsequent 1:10 dilution in buffer A. For strong activation of plasma samples we used Thromborel S as a source of TF, obtained from Behring Diagnostics GmbH (Marburg, Germany), containing human placental thrombo- plastin and calcium chloride. The fibrin polymerization-inhibitor H-Gly-Pro-Arg- Pro-OH (GPRP, Pefabloc FG) was purchased from Pentapharm LDT (Basel, Switzerland). The chromogenic substrate used for thrombin determination was H- D-Phe-Pip-Arg-pNA.2HCl (pNAPEP 0238) from CoaChrom Diagnostics (Vienna, Austria). Twenty-five mg of the lyophilized substrate were dissolved in 7 ml of dis- tilled water to a concentration of 5.7 mM.

Collection and Preparation of Plasma

Cord blood was obtained immediately following the delivery of 16 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 premarked tubes from Sarstedt (Nümbrecht, Germany), con- taining 300 µl 0.106 M citrate, centrifuged at room temperature for 10 min at 4100 x g, pooled and stored at –70°C in propylene tubes until assayed. The hematocrit of cord blood was similar to that of adult blood. FV and FVIII activities were elevated over the respective adult values, other pro- and anticoagulant factors were in the normal range for neonates, i.e. cord plasma contained 49 µg/ml prothrombin, 22 ng/ml TFPI and 172 µg/ml AT. In the same way plasma from 14 healthy adults was collected from the antecubital vein, prepared, and checked. Adult plasma contained 103 µg/ml prothrombin, 58 ng/ml TFPI, and 294 µg/ml AT.

Determination of TF Concentration

TF procoagulant activity was quantitated by means of the Actichrome TF activity assay.

Activation of Plasma

Three hundred eighty µl of plasma were incubated with 20 µl of buffer A containing different amounts of heparin (0–0.5 IU/ml) or hirudin (0–2.5 IU/ml) for 1 min at 37°C. Subsequently, 20 µl of buffer A containing H-Gly-Pro-Arg-Pro-OH (Pefabloc FG, 1.0 mg/ml final concentration) to inhibit fibrin polymerization [12] were added.

Subsequently, 40 µl of buffer A were added containing TF (30 pM final concentra-

tion) or Thromborel S (30 µM final concentration of TF) for 1 minute at 37°C.

Finally, 20 µl of 0.5 M CaCl

2

were added to trigger clot formation.

Determination of Clotting Time

Plasma was activated as described above except that Pefabloc FG was replaced by buffer A. Clotting times were determined by means of the optomechanical coagula- tion analyzer Behring Fibrintimer II from Behring Diagnostics GmbH (Marburg, Germany), which applies the turbodensitometric measuring principle.

Determination of Thrombin Generation

We used a subsampling method derived from a recently described technique [4–5, 14]. Plasmas were prepared and activated as described above. At timed intervals 10 µl aliquots were withdrawn from the activated plasma and subsampled into 490 µl buffer B containing 255 µM pNAPEP 0238. Amidolysis of pNAPEP 0238 was stopped after 6 min by addition of 250 µl 50% acetic acid. The amount of thrombin genera- ted was quantitated by measuring the absorbency by double wave length (405–690 nm) in the Anthos microplate-reader 2001, from Anthos Labtec Instruments GmbH, Salzburg, Austria. The total amidolytic activity measured is caused by the simulta- neous activity of free thrombin and the alpha 2-macroglobulin-thrombin complex.

Free thrombin generation curves were calculated by mathematical treatment of total amidolytic activity curves using a method developed by Hemker et al.

The area under the free thrombin generation curve has been called »thrombin potential (TP)«. The TP has been shown to be a suitable parameter to reflect the thrombogenic potency in a given plasma sample [9].

Statistical Analysis

Results obtained in cord and adult plasma were compared by means of Mann Whitney U test. The effects of different concentrations of heparin or hirudin on clotting time, and TP were analyzed using Wilcoxontest. The significance level of p- values was set at 5%. Calculations were performed using Winstat 3.1 (Kalmia Co.

Inc., Cambridge, MA, USA).

Results

Effect of Increasing Amounts of Heparin on Clotting Time

Under both low and high coagulant challenge (30 pM and 30 µM of TF were applied

to trigger clot formation, respectively) clotting time was dose-dependently pro-

longed in both cord and adult plasma in the presence of increasing amounts of

heparin (Fig. 1). Whereby, prolongation of clotting time was significantly more pro- nounced in adult plasma under low coagulant challenge compared with that in cord plasma (P of differences < 0.01, panel A). On the contrary, prolongation of clotting time was significantly more pronounced in cord plasma under high coagulant chal- lenge compared with that in adult plasma (P of differences < 0.01, panel B).

Effect of Increasing Amounts of Heparin on Thrombin Potential

Under both low and high coagulant challenge suppression of thrombin generation dose-dependently increased in the presence of increasing amounts of heparin in both cord and adult plasma (Fig. 2). Whereby, the anticoagulant action of heparin was significantly more pronounced in adult plasma under low coagulant challenge compared with that in cord plasma (P of differences < 0.01, panel A). On the con- trary, suppression of thrombin generation due to addition of heparin was signifi- cantly more pronounced in cord plasma under high coagulant challenge compared with that in adult plasma (P of differences < 0.01, panel B).

Fig. 1a, b. Effect of addi-

tion of heparin on pro-

longation of clotting

time in cord (쮿) and

adult plasma (쐌) in the presence of 20 pM (panel a) and 30 µM (panel b) of tissue factor to induce clot formation. No clot for- mation within 15 min was observed in adult plasma under low co- agulant challenge at heparin concentrations exceeding 0.025 IU/ml and in cord plasma at heparin concentrations exceeding 0.125 IU/ml (panel a). No clot for- mation within 15 min was observed in adult plasma under high co- agulant challenge at heparin concentrations exceeding 0.5 IU/ml and in cord plasma at hepa- rin concentrations exceeding 0.4 IU/ml (panel b).

Effect of Increasing Amounts of Hirudin on Clotting Time

After activation with high and small amounts of TF the clotting time was dose- dependently prolonged in both cord and adult plasma in the presence of increasing amounts of hirudin (Fig. 3). Whereby, prolongation of clotting time was signifi- cantly more pronounced in adult plasma after activation with small amounts of TF compared with that in cord plasma (P of differences < 0.01, Fig. 3 panel a). On the contrary, prolongation of clotting time was significantly more pronounced in cord plasma after activation with high amounts of TF compared with that in adult plas- ma (P of differences < 0.01, Fig.3 panel b)

Effect of Increasing Amounts of Hirudin on Thrombin Potential

After activation with high and small amounts of TF suppression of thrombin gene- ration dose-dependently increased in the presence of increasing amounts of hiru- Fig. 2a, b. Effect of

addition of heparin on

suppression of throm-

bin potential in cord

(쮿) and adult (쐌)

plasma in the presence

of 20 pM (panel a) and

30 µM (panel b) of

tissue factor to induce

clot formation. Virtually

no thrombin formation

within 15 min was

detected in adult plas-

ma under low coagulant

challenge at heparin

concentrations exceed-

ing 0.025 IU/ml and in

cord plasma at heparin

concentrations exceed-

ing 0.175 IU/ml (panel

a). Virtually no throm-

bin formation within

15 min was observed in

adult plasma under

high coagulant chal-

lenge at heparin con-

centrations exceeding

0.5 IU/ml and in cord

plasma at heparin con-

centrations exceeding

0.4 IU/ml (panel b).

din in both cord and adult plasma (Fig. 4). Whereby, the anticoagulant action of hirudin was significantly more pronounced in adult plasma after activation with small amounts of TF compared with that in cord plasma (P of differences < 0.01, Fig. 4 panel a). On the contrary, suppression of thrombin generation due to addition of hirudin was significantly more pronounced in cord plasma after activation with high amounts of TF compared with that in adult plasma (P of differences < 0.01, Fig. 4 panel b).

Discussion

The excellent hemostasis in neonates despite physiological low levels of procoagu- lant proteins is not completely understood so far [10]. Physiological low levels of inhibitors might compensate for low levels of procoagulants, and thus allow suffi- cient thrombin generation. Results from our previous studies confirm this assump- tion: the combined physiological low anticoagulant action of the three inhibitors of

0,0 0,5 1,0 1,5 2,0 2,5

0 50 100 150 200 250 300

b

Hirudin [IU/ml]

0,0 0,5 1,0 1,5 2,0 2,5

0 50 100 150 200 250 300 a

Prolongation of Clotting Time [%]

Fig. 3a, b. Effect of addition of hirudin on prolongation of clotting time in cord ( 쮿) and adult plasma ( 쎲 ) in the presence of 20 pM (panel a) and 30 µM (panel b) tissue factor to induce clot formati- on. No clot formation within 15 min was observed in adult plas- ma under low coagulant challenge at hirudin concentrations exceed- ing 2.0 IU/ml and in cord plasma at hirudin concentrations exceed- ing 2.5 IU/ml (panel a).

Both cord and adult

plasma clotted within

15 min under high co-

agulant challenge when

hirudin concentrations

up to 2.5 IU/ml were

added.

TFPI, AT and activated protein C (APC) in cord plasma allows enhanced thrombin formation associated with shorter clotting times compared with adult plasma when low amounts of TF are applied to initiate clot formation [11]. These findings are in good agreement with the clinically observed well- functioning hemostasis.

In conventional clotting assays plasma samples are markedly diluted and high amounts of initiator are applied to trigger clot formation. Under these conditions adult plasma clots faster than neonatal plasma and the capability of neonatal plas- ma to generate thrombin is delayed and reduced as compared with that in adult plasma, due to decreased prothrombin and increased a

₂

-macroglobulin [1-2, 12]. It has been shown that under these conditions lower amounts of anticoagulants are required in neonatal plasma to suppress thrombin generation to the same extent as in adult plasma [3].

The high coagulant challenge applied in standard clotting assays probably does not reflect the conditions prevailing in the clinical situation. Marked dilution of plasma samples completely alters the kinetics of the enzymatic reactions on which the blood coagulation process is based on [13]. Additionally, the high amounts of

0,0 0,5 1,0 1,5 2,0 2,5

0 5 10 15 20 b

Hirudin [IU/ml]

0,0 0,5 1,0 1,5 2,0 2,5

-10 0 10 20 30 40 50 60 70 80 90 100 a

Suppression of Thrombin Potential [%]

Fig. 4a, b. Effect of

addition of hirudin on

thrombin generation

in cord (쮿) and adult

plasma ( 쎲 ) in the

presence of 20 pM

(panel a) and 30 µM

(panel b) tissue factor

to induce clot forma-

tion. Virtually no

thrombin formation

within 15 min was

observed in adult

plasma under low

coagulant challenge at

hirudin concentrations

exceeding 2.0 IU/ml

and in cord plasma at

hirudin concentrations

exceeding 2.5 IU/ml

(panel a).

initiator (thromboplastin applied in conventional clotting assays contains TF in the micromolar range) might not be present in the clinical situation. Thus, activation of plasma samples by means of low amounts of initiator (i.e. TF in the picomolar range) is applied in the majority of laboratory investigations in last years [6, 7, 13].

It has been shown recently that under this low coagulant challenge, in contrast to the findings under high coagulant challenge, cord plasma clots faster than adult plasma and higher amounts of thrombin are generated in cord compared to adult plasma [4]. We show in the present study that under this low coagulant challenge significantly higher amounts of the anticoagulants heparin (indirect thrombin inhi- bitor) and hirudin (direct thrombin inhibitor) are required to inhibit thrombin generation in cord plasma to the same extent as in adult plasma. Completely cont- radicting the dosage requirements derived from experiments performed in the pre- sence of high amounts of initiator [3].

However, a reservation has to be stated. Our experiments were performed in cord and not in neonatal plasma due to ethical reasons. The coagulation system has been shown to be at an activated state immediately after birth. I.e., elevated FV, FVII, and FVIII activities have been detected in cord plasma [14], not present in neonatal plasma. Additionally, cord and venous blood differ in their hematocrit [15, 16], influencing the concentrations of citrate and both pro- and anticoagulant proteins in plasma.

In conclusion, whereas decreased amounts of anticoagulants are required in neo- natal plasma to suppress thrombin generation to the same extent as in adults under high coagulant challenge, results from our in vitro study suggest that increased amounts of anticoagulants are required to suppress thrombin generation to the same extent as in adults under low coagulant challenge. If we assume that low coagulant challenge reflects in vivo situation better, our study might suggest that higher doses of thrombin inhibitors, both direct and indirect, may be required in neonates.

Acknowledgements. This study was supported by a grants from the »Gesellschaft zur Förderung der Gesundheit des Kindes (INVITA)« and the »Franz-Lanyar- Stiftung«. We thank Bettina Leschnik for technical and Yvonne Gallistl for secre- tarial assistance.

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