Methods for Testing Pharmacodynamic Variables of Hemophilia and Inhibitor Therapy: Thrombin Generation Assay and Other Tests

of Hemophilia and Inhibitor Therapy:

Thrombin Generation Assay and Other Tests

K. Váradi, H. P. Schwarz and P.L. Turecek

Introduction

Prothrombin complex zymogens and enzymes are used as pharmaceutical agents for inhibitor-bypassing therapy. These agents include activated prothrombin com- plex concentrates, such as FEIBA (Baxter Healthcare, CA, USA), which are com- posed of vitamin-K-dependent coagulation factor zymogens and small amounts of their activated forms and preparations containing activated factor VII, like recom- binant activated FVII (rFVIIa; NovoSeven; Novo Nordisk, Glostrup, Denmark).

These products induce the activation of the clotting cascade by multiple reactions resulting in thrombin generation and thus achieving hemostasis independently from and bypassing factor VIII [1–3]. Because of the complex mechanism of action, no direct monitoring of the drug substance is available for either treatment regimes.

The measurement of the individual factor activities or their derivatives does not reflect the effectivity of these products. To measure the effect of FVIII-bypassing agents new types of assay systems, assessing the time-dependent changes in the hemostatic system are required.

Kinetics of Hemostasis. Mode of Action of FVIII-Bypassing Agents

Tissue injury triggers a series of reactions. Platelets adhere to damaged tissue at the site of injury providing an active surface for the hemostatic reactions. The complex formed by tissue factor (TF) and factor VII (FVII) activates factor X (FX) and factor IX (FIX). Enzyme-cofactor complexes assemble on the activated platelet surface, and small amounts of thrombin are generated resulting in fibrin formation. The various feedback effects of thrombin including the activation of factor V (FV), FVIII and fac- tor XI (FXI) produce a burst of thrombin generation, mostly after the clot is formed.

The fibrin clot is stabilized due to cross-linking of the fibrin chains by factor XIII (FXIII), activated by thrombin, and due to the retraction of activated platelets. The enhanced thrombin generation is controlled by the physiological inhibitor systems (TF pathway inhibitor, antithrombin, and the activated protein C-protein S system) slowing down thrombin activation by inactivating the active enzymes or degrading their cofa- ctors. Circulating inhibitors, such as a 2 -macroglobulin inactivate thrombin directly and thrombin concentration gradually decreases. Tissue injury also induces the fibri- nolytic system. Tissue plasminogen activator is liberated resulting in the generation of

I. Scharrer/W. Schramm (Ed.)

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Hemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

the active fibrinolytic enzyme, plasmin. To act effectively plasmin has to be bound to the fibrin clot and protected from inhibitors, some of which are activated by thrombin.

In the absence of FVIII the rate of reactions leading to thrombin generation and subsequent clot formation are very slow, which also delays all the thrombin-media- ted feedback reactions. The FVIII-bypassing agents increase the concentration(s) of some active enzymes, but the complex preparations like activated prothrombin com- plex concentrates (APCCs) also increase the concentrations of the potential zymo- gene substrates, and thus raise the rate of reactions independent of FVIII [4, 5].

General Assays to Assess the Effect of FVIII-Bypassing Therapy

To measure the effect of FVIII-bypassing agents an assay system is required that not only measures the activity of the single factors, but assesses the kinetics of those interactions described above. General screening assays just measuring the time of clotting cannot access the fine balance of these reactions. Therefore several attempts were made to modify the assays to increase their sensitivity and specificity for this purpose.

Thrombelastography

The thrombelastography (TEG) was first described more than 50 years ago [6]. This method measures the viscoelastic properties of blood induced to clot under low shear conditions, corresponding to venous flow. It evaluates the kinetics of formati- on, stabilization and subsequent lysis of the clot. TEG analyses whole blood and thus measures the effect of both cellular and plasmatic components on clot forma- tion. Inhibitor-bypassing agents both in vitro and in vivo reduce the clotting time of a FVIII-inhibitor plasma and thus also respond in thrombelastography. The main components of the TEG are a cylindrical cup and a pin hanging on a torsion wire.

Depending on the equipment system used, either the pin or the cup rotates in an oscillatory way at a low angle. When blood is added torque is transmitted as the clot forms linking the cup and pin together. The torque increases as the clot strengthens and decreases as the clot lyses. The changes in the clot elasticity measured by the TEG can be drawn as the function of time, giving specific, characteristic curves (Fig. 1). These curves assess the clotting time by distinguishing the time to initial clot formation and the time to reach an arbitrary clot strength. Thus some kinetic information on the initial clot formation and various physicochemical properties of the clot are provided. The rate of the clot strengthening, the maximum clot strength and the time at maximum clot strength are indicative of the combined effect of the coagulation factors and platelet functions. The clot strength measured at a defined time (e.g. 60 minutes) after the maximum is reached and the calculated clot lysis index, i.e. the percentual ratio of the two measured amplitudes, inform about the fibrinolytic activity of the blood sample.

The critical step for monitoring the FVIII-bypassing therapy is the rate of

thrombin generation, which also plays an important role even in the rate of clot

formation. To obtain further information on this step a new evaluation method, assessing the rate of clot formation in more detail was introduced recently by Sorensen et al. [7]. This method anticipates that the time-dependent changes in the clot strengthening indirectly reflect the course of thrombin generation. Even though the assay measures the thrombin activation, it only does so up to clot for-

-8000 -4000 0 4000 8000

elasticity (mm *100)

time a)

initial clotting time

clot formation time time at maximal clot strength rate of

clot

strengthening maximum

clot strength clot strength at time = t clot lysis index

time b)

-8000 -4000 0 4000 8000

elasticity (mm*100)

time at maximal clot strength clot formation time

initial clotting time

rate of clot strengthening

maximum clot strength

Fig. 1a, b. Characteristics of thrombelastogram. a) Schematic thrombelastogram of a normal

blood sample. b) Schematic thrombelastogram of a severe hemophilic blood sample. The

explanation of characteristic parameters is described in detail in the text. The tubes symboli-

ze the clot formation and lysis in whole blood during the time course of TEG.

mation and therefore cannot assess the whole kinetics of thrombin generation, which continues after the clot has been formed and leads to the main bulk of thrombin.

Thrombin Generation Assays

The first assay that measured the rate of thrombin generation was called the

»thrombin generation test« and was introduced by Macfarlane and Biggs in 1953 [8]. In this assay defibrinated plasma samples were activated, and the thrombin for- med determined by subsampling to a fibrinogen source, the natural substrate of thrombin. Hemker et al. (1986) were the first to describe the detailed kinetic analy- sis of prothrombin activation in plasma [9]. In 1989, a platelet-based thrombin generation assay for measuring the bioavailability of FEIBA was presented [10]. In the following year Gill et al. [11] were able to show clinical correlations between thrombin generation and clinical outcome after patients with hemophilia and inhi- bitors were treated with APCCs. In 1993, Sultan and Loyer [12] used such a throm- bin generation test for in vitro evaluation of the FVIII-bypassing activity of APCCs and FVIIa. Hemker et al. subsequently introduced a new form of thrombin genera- tion tests, using small peptide substrates for thrombin instead of fibrinogen. Recent thrombin generation assays (TGAs) measure the time-dependent changes in thrombin concentrations in platelet-rich or in platelet-poor plasma samples [13, 14]. In these assays thrombin generation is triggered by a low concentration of tis- sue factor, and the thrombin activity is measured continuously using a specific flu- orescence peptide substrate, which is cleaved by thrombin and liberates a fluoro- phore. The rate of development in the fluorescence intensity can be converted to thrombin-equivalent concentrations (nM) using a reference curve prepared by measuring the rate of substrate conversion by a thrombin calibrator.

The changes in the effective thrombin concentration can be drawn as the func- tion of time giving specific, characteristic curves. Figure 2 shows the typical throm- bin generation curves measured in normal human plasma and in a FVIII-deficient plasma with inhibitors.

The assay detects the time from trigger to starting and completing the initial clot formation, defined as the onset of thrombin generation and the kinetic lag phase, i.e. the time intercept of the maximum slope. The rate of thrombin generati- on, the highest thrombin concentration (peak thrombin) and the time required to reach it (peak time) is assessed. The assay informs about the rate of inactivation, and the amount of the residual thrombin concentration, reflecting the overall inhi- bitor capacity of the plasma. The thrombin potential, which is the area under the curve, defined first by Hemker at al. [15], is a measure of the total thrombin that could possibly have been formed after coagulation was triggered.

The absolute time parameters depend on the type and concentration of reagents

used to trigger the clot formation.

b)

peak time

0 time 50 100 150 200 250 300

thrombin (nM)

peak thrombin

thrombin potentia l

a)

TG onset

lag phase peak time

TG rate

inactivation

rate

residual thrombin

time 0

50 100 150 200 250 300

thrombin (nM)

TG onset lag phase

TG rate peak thrombin

Fig. 2a, b. Characteristics of the thrombin generation curves triggered by a low concentration

of tissue factor and phospholipid complex. a) Thrombin generation curve of a normal plasma

sample. b) Thrombin generation curve of a severe hemophilic plasma sample. The explanati-

on of characteristic parameters is described in detail in the text. The tubes symbolize the clot

formation in the plasma samples during the time course of thrombin generation.

Application of the Global Assays in the Monitoring of FVIII-Bypassing Therapy

In recent years there have been several new attempts to measure overall hemosta- sis, instead of measuring isolated factor activities, to assess the entire complex pro- cesses in which hemostatic components interact in various bleeding and thrombo- tic disorders and the results of their treatment.

Some »old assays« began their renaissance with modern computerized analy- tic techniques. TEG evaluates the kinetics of formation, stabilization and sub- sequent lysis of the clot. Yoshioka at al. [16] showed that after a single dose of rFVIIa the parameters reflecting the clotting times (time to initial clot formation and the time to reach an arbitrary clot strength) and also the maximum amplitude was corrected but the activated partial thromboplastin time (APTT) values did not normalize. The authors suggested that TEG was more suitable for monitoring treatment than APTT.

Clot waveform analysis is also a new technique for investigating the structural changes in clot formation during APTT or prothrombin time measurement [17].

The clot waveform analysis seems to be a very sensitive method for detecting minor changes in the low factor levels [18]. Clear changes were observed in the parameters of the APTT waveforms after a single infusion of rFVIIa, FEIBA or a FVIII concen- trate in a hemophilia patient with low titer inhibitor (2.6 Bethesda U/mL) [16]. The FVIII concentrate had a greater improvement effect than rFVIIa or FEIBA. In con- trast, when the results were compared using TEG analysis, the two bypassing agents had the more pronounced correction capacity. The differences were interpreted as arising from the different assay conditions used of whole blood or plasma.

New evaluation parameters, the maximum velocity and time of maximum velo- city of the initial clot formation of the TEG were published recently [19]. The cour- ses of whole blood clot formation seemed to be similar to the thrombin generation curves measured in plasma. Using this evaluation method the in vitro addition of recombinant FVIIa and APCC to the whole blood of hemophilia patients with inhi- bitor was shown to correct the diminished clot formation measured before [20].

Despite the apparent similarity of the transformed TEG curves to the thrombin generation ones, the assay measures the thrombin activation indirectly and only up to clot formation.

Thrombin is a multipotent enzyme that has many other roles apart from fibrin formation, all of which influence hemostasis. Physiologically the majority of throm- bin generation occurs after the clot has been formed. Therefore the information obtained from the TEG or from the waveform analysis cannot assess the whole kinetics of thrombin generation.

In contrast, the thrombin generation assay measures the actual thrombin con- centrations before and after the clot formation and is very sensitive to variations in individual factors or groups of coagulation factors.

Al Dieri at al. [21] showed a good correlation between thrombin potential and

bleeding tendency in rare coagulation disorders, showing that patients with a

bleeding tendency had a thrombin potential below 20% of normal. In vitro spi-

king of high-titer inhibitor plasma with increasing concentrations of FEIBA resul-

ted in the dose-dependent restoration of the thrombin-generating capacity of the

FVIII-inhibitor plasma. Normal thrombin generation was obtained with concen- trations corresponding to the expected concentrations that might have been achieved with the usual therapeutic doses of 50-100 U/kg FEIBA, calculated on the assumption of a 50 to 100% recovery [14]. The defective thrombin generation is also improved in patients with FVIII inhibitors within 30 minutes after a single injection of the therapeutical doses of FEIBA. The thrombin generation gradual- ly decreased back to baseline values with a biological half-life of approximately 5 hours for FEIBA [22].

The changes in the kinetics of thrombin generation measured before, during and after a substitution or FVIII-bypassing therapy reflect the pharmacological effect of the drugs. Thus, this assay enables monitoring of the in vivo effect of the therapy, giving the possibility of further optimizing and individualizing the treat- ment.

References

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