Comparison of the Anticoagulant Action of Recombinant Human Activated Protein C in Cord with that in Adult Plasma

Human Activated Protein C in Cord with that in Adult Plasma

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

Background

Recently, recombinant human activated protein C (rhAPC) has been shown to be a good candidate for the treatment of microvascular thrombosis, disseminated intra- vascular coagulation, and septicemia in both adults and neonates [1]. In the present study we focus our attention on the influence of tissue factor pathway inhibitor (TFPI) and antithrombin (AT) on the anticoagulant action of rhAPC. TFPI and AT have been shown to amplify the anticoagulant action of APC in a synergistic man- ner in plasma activated by addition of lipidated tissue factor (TF) [2, 3]. TFPI and AT levels in neonatal plasma are reduced to approximately 50% of adult value [4].

Thus, different anticoagulation action has to be expected between rhAPC admini- stered to neonates compared with rhAPC administered to adults.

Therefore, we comparatively evaluate in the present study the anticoagulant action of rhAPC in terms of prolongation of clotting time, suppression of thrombin potential (TP), and suppression of prothrombin fragment 1+2 (F1+2) generation in cord plasma containing TFPI and AT at neonatal levels, in cord plasma adjusted to contain TFPI and AT at adult levels, and in adult plasma. Clot formation is induced not only by addition of high (30 µM final concentration) amounts of lipidated tis- sue factor (TF), as usual in standard assay systems (activated partial thromboplas- tin time, prothrombin time) [5], but also by addition of low (20 pM final concen- tration) amounts of TF.

Methods

Collection and Preparation of Plasma

Cord blood was obtained immediately following the delivery of 21 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), 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 hemato- crit 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. Scharrer/W. Schramm (Ed.)

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Hemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

Preparation of cord Plasma Containing TFPI at Adult Level

The TFPI level of the pooled cord plasma was raised up to adult value by addition of 10.5 µL of TFPI stock solution to 989.5 µL of cord plasma.

Determination of the Plasma TFPI Level

TFPI activity in cord and adult plasma was determined by means of the chromo- genic assay ActichromeTM TFPI activity assay.

Preparation of cord Plasma Containing AT at Adult Level

AT level of pooled cord plasma was raised up to the respective adult value by addi- tion of 12.5 µl of the AT stock solution to 987.5 µl of cord plasma.

Determination of the AT Content

Determination of the AT content of cord and adult plasma was performed by means of a standard chromogenic method on a BM/Hitachi 917 from Boehringer Mann- heim (Vienna, Austria).

Activation of Plasma

Three hundred eighty µl of plasma were incubated with 20 µl of PBS containing dif- ferent amounts of rhAPC (0.1–16 µg mL-1 final concentration) for 1 min at 37°C.

Subsequently, 20 µl of PBS containing H-Gly-Pro-Arg-Pro-OH (Pefabloc FG, 1.0 mg ml-1 final concentration) to inhibit fibrin polymerization [6] were added.

Subsequently, 40 µl of PBS were added containing TF (20 pM final concentration) 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

Clotting times were determined by means of the optomechanical coagulation ana- lyzer Behring Fibrintimer II from Behring Diagnostics GmbH (Marburg, Germany).

Determination of Thrombin Generation

We used a subsampling method derived from a recently described technique [7].

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 PBS containing 255 µM pNAPEP 0238. Amidolysis of pNAPEP 0238 was stopped after 6 min by addition of 250 µl 50% acetic acid.

Results

Effect of Increasing Amounts of rhAPC on Thrombin Potential

Suppression of thrombin generation dose-dependently increased in the presence of increasing amounts of rhAPC in both cord and adult plasma (Fig. 1). Whereby, the anticoagulant action of rhAPC was significantly more pronounced in cord plasma under high coagulant challenge compared with that in adult plasma (P of differen- ces <0.01, panel A) and, in contrast, significantly more pronounced in adult plasma under low coagulant challenge compared with that in cord plasma (P of differences

<0.01, panel B).

Fig. 1. Effect of addition of

rhAPC on suppression of

thrombin potential in cord

(

) and adult (

) plasma in the

presence of 30 µM (A) or 20 pM

(B) tissue factor to induce clot

formation.

Anticoagulant action of rhAPC in cord plasma containing neonatal levels of TFPI and AT compared with that in cord plasma containing both inhibitors at adult levels

Raising TFPI and AT in cord plasma up to adult values resulted in significantly pro- longed clotting time (348 ± 16 vs. 282 ± 11 s, P <0.01), suppressed TP (234 ± 18 vs.

309 ± 23 nM min, P <0.01), and suppressed F1+2 generation (0.402 ± 0.03 vs. 0.512

± 0.04 µmol L-1, P <0.01) in the presence of 20 pM TF to initiate clot formation (Fig. 2). Significantly enhanced anticoagulant action of 0.2 µg mL ^–1 rhAPC was observed in cord plasma containing adult levels of TFPI and AT compared with cord plasma containing both inhibitors at neonatal levels: addition of 0.2 µg ml ^–1 rhAPC significantly prolonged clotting time by 35.1 % in cord plasma containing neonatal levels of TFPI and AT, and by 49.7% in cord plasma containing both inhibitors at adult levels (P of differences < 0.01). TP and F1+2 generation were suppressed by 26.2 and 15.2%, respectively, in cord plasma containing neonatal levels of TFPI and AT, and by 47.8 and 28.1%, respectively, in cord plasma containing both inhibitors at adult levels (P of differences < 0.01).

Fig. 2: Effect of TFPI and AT on the anticoagulant action of rhAPC in cord plasma. Free throm-

bin generation over time is displayed in reactions initiated by 20 pM TF in cord plasma con-

taining physiological levels of TFPI and AT in the absence of rhAPC ( 쐽 ), in the presence of

0.2 µg ml

rhAPC ( 쎲 ), and in cord plasma containing TFPI and AT at adult levels in the absen-

ce of rhAPC ( 왖 ), and in the presence of 0.2 µg ml

rhAPC (+). Results are expressed as means

(n = 3). SDs were not shown for clarity of graph reading but represented less than 10 % of the

mean.

Conclusions

We found that under both high and low coagulant challenge clotting time was dose- dependently prolonged, thrombin potential and prothrombin fragment 1+2 gene- ration were dose-dependently suppressed in the presence of increasing amounts of rhAPC in both cord and adult plasma. Whereby, cord plasma was significantly more susceptible to addition of rhAPC in the presence of high amounts of TF as trigger for clot formation and adult plasma was significantly more susceptible to addition of rhAPC in the presence of low amounts of TF. Whereas the elevated anticoagulant action of rhAPC in cord plasma under high coagulant challenge might be caused by the low neonatal levels of prothrombin, we demonstrate that the increased anti- coagulant efficacy of rhAPC in adult plasma under low coagulant challenge is attri- butable to the physiologically high levels of TFPI and AT present in adults.

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