Effect of FVIII on Clotting Time and Thrombin Potential in Tissue Factor-Activated Hemophilia A Plasma

Tissue Factor-Activated Hemophilia A Plasma

P. Fritsch, G.Cvirn, K. Baier, B. Leschnik and W. Muntean

Introduction

FVIII-deficiency (hemophilia A) is known to be associated with severe bleeding.

The quantity and intensity of bleeding in hemophiliacs can differ individually in patients despite similar factor VIII-concentrations. Also neonates have a lower bleeding tendency than older hemophiliacs. This could be the effect of factors like prothrombin, factor VII, but also of inhibitors like tissue factor pathway inhibitor (TFPI), antithrombin (AT) and protein C (PC) [1]. To measure the effect of these factors on hemostasis sensitive assays are necessary. We can not use standard assay systems via the intrinsic pathway (aPTT), because the plasma is too highly diluted and the activation is done with too high amounts of activator. However, it has been shown in recent years that plasma activation via the extrinsic pathway by addition of low amounts of lipidated tissue factor (TF) as activator is probably more compa- tible with the physiological milieu [2]. Butenas et al. [3] have shown, that low con- centrations of lipidated TF as trigger are suitable for a sensitive detection of the effects of different levels of pro- and anticoagulants on thrombin-generation. Cvirn et al. [4, 5] showed in their studies the influences of the above mentioned factors after activation with low amounts of TF in cord and adult plasma. Therefore, our study was performed to investigate whether low concentrations of TF as activator can also be used to determine the influence of factor VIII on clotting time and thrombin generation.

Materials and Methods

FVIII-deficient plasma (containing 0 % FVIII-clotting FVIII deficient plasma Dade Behring) was spiked with increasing amounts of purified FVIII-concentrate (HaemateP Aventis Behring) and the effect on clotting time (CT) and thrombin potential (TP) was evaluated. It was evaluated in the plasma of hemophilic patients.

For the activation we used different amounts of recombinant lipidated TF (Innovin Dade Behring).The concentration of TF was measured with TF-Elisa (American diagnostica).

380 micro liters of plasma with different FVIII-concentrations were incubated with 100 micro liters of buffer containing different amounts of TF for 2 min at 37°C.

The activation of the plasma samples was done by addition of 20 microliters CaCl ₂ .

I. Scharrer/W. Schramm (Ed.)

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Hemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

The CT were determined by means of the optomechanical coagulation analyzer Behring Fibrintimer II from Dade Behring Marburg, Germany, which applies the turbodensitometric measuring principle. For the determination of the TP we used additionally H-Gly-Pro-Arg-Pro-OH (Pefabloc FG). Also the subsampling method was used derived from a recently described technique [6]. 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 µmol L S-2238. The extinction of the plasma was measured in the Anthos micro plate-rea- der 2001, Salzburg, Austria [7].

Results

Effect of TF and FVIII on Clotting Time (CT)

Activation of plasma with high concentration of TF showed no significant increase of the CT with decreasing amounts of F VIII. With small amounts of TF (2.5 pM L ^–1 ) the CT in FVIII-deficient plasma was dose-dependently shortened when increasing amounts of FVIII-concentrate were added (Fig. 1).

Effect of FVIII on Thrombin Potential (TP)

There was no significant effect on the TP using high concentrations of TF in plas- ma with different F VIII-content. But using low concentration of TF (2,5 pM L ^-1 ), the TP was increased when the FVIII-content was successively raised (Fig. 2).

Fig. 1. Effect of activation with different TF-concentration in plasma with different FVIII-

content on clotting time. 13 pM L

TF (왔), 6 pM L

Clotting Time in Plasma from Hemophilia A Patients

Plasma of hemophiliacs with different FVIII-content, showed an increase of the CT with decreasing FVIII-content (Fig. 3).

Fig. 2. Effect of different FVIII-concentration on the thrombin generation after an activation with 2.5 pM L

TF. Plasma with 60 % F VIII (쮿), plasma with 15 % FVIII (쐌), plasma with 0 % FVIII (왖).

Fig. 3. Effects on CT in plasma of hemophiliacs with different FVIII-content after an activation

with 1.25 pM L

TF.

Thrombin Generation in Plasma from Hemophilia A Patients

In contrast to the CT, the height of the TP did not correlate that strictly as CT with the FVIII-content. While in general the TP of mild hemophiliacs were higher and the TP of the severe hemophiliacs were lower, in two patients with the same FVIII- content (both 20 %- 왖 and 왔 in Figure 4) had different high TP-levels (Fig. 4).

Discussion

Recent investigations showed that hemophiliacs with factor V Leiden disease [9, 10, 12] or protein C deficiency [13] had lower bleeding tendency than hemophiliacs with no prothrombotic risk factors. Some data even report of sporadic cases of symptomatic thromboembolism in patients with severe hemophilia and prothrom- botic risk factors after factor replacement therapy [12, 16–18]. This is not confirmed by others [11,19–21]. They postulate that there must be other variables, that influ- ence the bleeding tendency and the lower factor concentrate utilization in severe hemophiliacs. Van’t Veer [1] could show the influence of factors like TFPI and AT on the TG in plasma of healthy adults.

It is known that excessive bleeding during delivery and in the neonatal period is a relatively rare event in hemophiliacs. In a French study of 754 neonates with hemophilia, only 8 % showed clinically overt bleeding , in 71,5 % of patients the diagnosis was made after the neonatal period. Intracranial bleeding is found in only 3.9 % and 3.6 % of neonates with hemophilia [14, 15].

In previous works we have focused on the problem why healthy neonates do not

bleed easily in spite of low clotting factors: It was shown that after activation with

low concentrations of TF physiological low levels of natural inhibitors, like TFPI, AT

Fig. 4. Effects on TP in plasma of hemophiliacs with different FVIII-content after an activation

with 1.25 pM L

TF. Healthy proband (쮿), patients with 25 % FVIII (쐌), 20 % F VIII (왖), 20 %

FVIII (왔), 17 % FVIII (쏆), 8 % FVIII (+), 2 % FVIII (x), 1 % FVIII (*), 0.6 % FVIII (–).

and PC, compensate for low concentrations of clotting factors, like the prothrombin complex, and allow sufficient thrombin generation [4, 5].

Conventional clotting assays, that are used for factor VIII-measurement, are not sensitive enough for the detection of these factors. Davie et al. [2] showed in their work, that an activation via the extrinsic pathway, using small amounts of TF, is more physiological than the conventional clotting assays. The group of Mann [3]

showed that this method is sensitive enough to detect the effects of different levels of pro- and anticoagulants on the thrombin generation.

In our study there was no significant difference in plasmas with different FVIII-content using high concentrations of TF for the activation. Using low con- centrations of TF, the clotting time decreased dose-dependently and the TP increa- sed dose-dependently when the FVIII-content was successively raised by addition of increasing amounts of purified FVIII-concentrate to FVIII-deficient plasma.

First measurement of the TP in plasma of hemophiliacs showed the expected data. There was a correlation measuring the CT in plasma of hemophilia A patients to FVIII-content. But in accordance to Ingerslev [8] and »the FVIII and FIX Sub- committee of the International Society on Thrombosis and Haemostasis«, the TP showed a heterogeneity in patients with the same phenotypes (FVIII:C 0.2 IU mL- 1). An explanation might be that other compounds of the hemostatic system, e.g.

FII, FVII, tissue factor pathway inhibitor (TFPI), or antithrombin (AT) effectively influence thrombin generation in TF-activated plasma. Another explanation could be the different age of our patients (11 months to 18 years). As mentioned before, there is a discrepancy between the good hemostasis and the prolonged values in neonates [4, 5].

Our investigation suggest that low concentrations of TF can be used for a sensi- tive detection of prothrombotic and inhibitory variables in plasma of hemophiliacs.

Therefore, we intent to investigate the effects of these variables on the CT and TP in plasma of hemophilia A patients in the future, to possibly explain the difference in bleeding manifestation between patients, different age groups and especially be- tween neonates and older children.

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VIIa. Hemophilia