Fibrinopeptide a release by factor-IX concentrates and feiba fraction (factor eight inhibitor bypassing activity)

THROMBOSISRESEARCH20; 623-631,1980 0049-3848/80/230623-09$02.00/O Printed in the USA. All rights reserved. Copyright(c) 1981 PergamonPress Ltd

FIBRINOPEPTIDE A RELEASE BY FACTOR-IX CONCENTRATES AND FEIBA FRACTION (FACTOR EIGHT INHIBITOR BYPASSING ACTIVITY)

Victor Hofmann*, and P. Werner Straub Department of Medicine, Inselspital, University of Berne, Switzerland

(Received25.6.1980;in revisedform 20.11.1980. Acceptedby Editor C.R.M. Prentice) ABSTRACT

Factor IX-concentrates (FC) can cause in vivo activation of the coagulation system. To detect either thrombin presence or generation in vitro, the release of fibrinopeptide A (FPA) from fibrinogen added to FC or activated FEIBA fractions has been measured by a specific radioimmunoassay. In none of 8 batches thrombin was detected. However, the addition of CaC12 led to thrombin generation, which was high in 3 and moderate in 5 concentrates. Prior addition of heparin could fully inhibit the CaCl -mediated FPA release from fibrinogen. In contrast, FESBA fractions were able to split FPA from fibrinogen in the absence of CaC12, indicating the presence of preformed thrombin in these fractions. The addition of CaC12 led to further thrombin generation, which could be prevented by high amounts of heparin or hirudin. Thus, in FC thrombin is either absent or blocked by the heparin added by manufacturers. However, rapid thrombin generation is observed upon incubation with CaC12. The coagulant activity of FEIBA is due to preformed thrombin rather than to a "factor VIII inhibitor bypassing activity". *) Present address: Department of Medicine, University Hospital of Zurich, 8091 Zurich, Switzerland. Requests for reprints: P.W. Straub, M.D., Department of Medicine, Inselspital, 3010 Berne, Switzerland. KEYWORDS:

Factor IX-concentrates, FEIBA fraction, thrombogenicity, Fibrinopeptide A. 623

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INTRODUCTION Factor IX-concentrates (FC) were developed for the treatment of patients with Christmas disease (1). Since these concentrates also contain factors II, VII and X, their clinical use was extended to other conditions associated with an acquired deficiency of the vitamin K dependent factors such as oral anticoagulant overdosage and severe liver disease (2). However, several reports of arterial thrombosis, venous thromboembolism and disseminated intravascular coagulation (3, 4, 5, 6, 7, 8, 9, 10) suggested that these concentrates activate coagulation in vivo. Recently the use of so-called activated FC has been advocated for the treatment of patients with acquired factor VIII inhibitors (11, 12). The postulated mechanisms of action were that factor IX substitutes for factor VIII as a cofactor for platelet aggregation and further that factor Xa directly activates prothrombin, thus bypassing factor VIII in vivo (13). In vitro investigations of the clot-promoting activity of these concentrates produced evidence for the presence of activated factors IXa and Xa (14, 15). The addition of calcium has been supposed to amplify the formation of thrombin (15). The present study was undertaken following the observation that fibrinopeptide A (FPA) levels in vivo markedly increased from 0.4 - 2.4 to 5.6 - 80 ng/ml after the infusion of FC in 5 of our patients with hemophilia B, suggesting either the presence of thrombin in these preparations or thrombin formation in vivo after FC-infusion (16). Using fibrinogen as a substrate and the release of FPA as an index for a thrombin mediated proteolytic activity, we have compared commercially available FC concentrates and the activated FC FEIBA fraction in vitro.

MATERIALS AND METHODS 1

Factor IX-concentrates

and FEIBA fraction

5 FC were generously supplied by the Swiss Red Cross. They contained 8 U of heparin per 20 U of factor IX added by the manufacturer during the preparation steps. 3 factor IX-concentrates devoid of heparin and 2 FEIBA fractions were a gift of Immuno AG, Vienna, Austria. All preparations were lyophilized. They were dissolved and diluted to 20 U/ml, according to the instructions of the supplier, and used immediately upon dilution. 2. Fibrinogen 1 gram of lyophilized fibrinogen grade L (Kabi AB, Stockholm) with a clottability greater than 90% was dissolved in 100 ml distilled water and dialysed against 2 1 of buffer for 24 hrs at 4oC. The fibrinogen solution was further diluted with buffer to give a final concentration of 2 mg/ml, subsampled in 5 - 10 ml portions and stored at -25OC until

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use. Heparin (Liquemin, Hoffmann-La Roche, Basle), 5000 USP/ ml, and Hirudin (Pentapharm, Basle) 2000 ATU (antithrombin units) were used as specified under methods and results. The buffer used was 0.05 M Tris, 0.1 M NaCl, pH 7.4.

All materials were stored and all experiments carried out in plastic tubes. 3. Fibrinopeptide

A release

a)

Influence of preincubation time with calcium: 0.2 ml of FC were preincubated for 5, 15, 30, 60 and 180 min with either 0.2 ml of 0.025 M CaCl or 0.2 ml of buffer subsequently added to 0.2 ml of $he fibrinogen solution. After exactly 60 set 100 U of heparin were added.

b)

2 hrs preincubation: 8 FC and FEIBA fractions were preincubated for 120 min with or without calcium and subsequently added to fibrinogen directly or after addition of 100 U of heparin or larger amounts for the fraction FEIBA (see text). Controls were carried out simultaneously.

cl

Influence of time on FEIBA: 1.5 ml of FEIBA fraction were added to 3.0 ml of buffer and subsequently to either 1.5 ml of plasma or fibrinogen. 600 ul of this mixture were sampled after 5, 30 and 60 min axd added to 25 U of hirudin.

4. Measurement

of FPA

All samples were adjusted to an end volume of 4 ml by addition of a 0.15 M Tris NaCl buffer, pH 8.5. 1 ml of this solution was then dialysed against 3 ml of the same buffer containing 1 mg/ml of ovalbumin (Sigma Chemical Company, St. Louis, Missouri). The dialysis step was slightly modified in comparison to previous publications by the use of a new dialysis membrane with a molecular weight cutoff of 10'000 (HP 101000, Diachema, Rtischlikon, Switzerland) giving a recovery of FPA of 75 - 80% after 21/2hrs rotational dialysis at room temperature. The radioimmunoassay was performed as previously described (18). All results were expressed as % FPA release= FPA release total FPA release upon thrombin addition' 100% indicating complete thrombin proteolysis of the AM-chain of the fibrinogen substrate.

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RESULTS

In figure 1 FPA release from fibrinogen is shown to be dependent on the preincubation time of the FC with calcium.

100

80 % FPA release 60

20

0 I

3

1

30

60 FIG.

120

160 min

1

FibrinopeptideAreleasedependsontheprekcubation tima of Fc with Cac12 0.2 ml of FC were preincubatedfor 5, 15, 30, 60 and 180 min with either 0.2 ml of 0.025 M CaCl or 0.2 ml of buffer and subsequentlyadded to 0.2 ml of fibrinogen(~mg/ml.)??. After exactly60 sec100 Uofheparin wereadded. Controls:JX+CaCl A, FibrincgenKaCl 0, Fc+Fg A. FPArelease is shown tobe dependen$on the preincubatioztineof the Fcwith calcium.

To test the hypothesis of a time-dependent thrombin generation initiated by calcium, 8 different FC were subsequently analysed for their capacity to release FPA after a 2 hrs preincubation with calcium. Out of 8 FC, 3 generated between 42 and 80% of FPA, whereas in 5 the generation was below 10% (Fig. 2).

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?? ? ? ??

II

0

X FPA release 0 ??

.

. t

A

0”. .

a

nn b

C

FrG.2 Ccqarisonof theFPAgenerationof8different Fc 8 Fcwere~incubatedfor12Ominwithorwithoutcdlci~ and subsequently added to fibrinogendirectlyor after additionof 100 U of heprin. a) 0.2 ml of EC + 0.2 ml of buffer.After 120 min + 0.2 ml of fibrinogen for 60 set, then 100 U of heparin. b) 0.2 ml of FC + 0.2 ml of CaC12. Then as describedunder a). c) 0.2 ml of FC + 0.2 ml of cacl . After 120 nlin+ 100 u of heparin. IiTlnadiately thereafter+ 0.2 ml o$ fibrinogenfor 60 set, then 100 U of heparin. In the absence of calcium insignificant amounts of FPA were released. The calcium-dependent FPA release could be prevented by addition of heparin to the preincubation mixture before transfer to the fibrinogen solution. 2 FEIBA fractions released 80 and 88% resp. of FPA in the absence of calcium and this effect could also be prevented by heparin. However, when calcium was added to FEIBA, 100 U of heparin produced no inhibition. Inhibition was then obtained only with either an ll-fold amount of he-

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parin or with 100 U of hirudin, suggesting that calcium considerably amplified the FPA release. The time dependent FPA release from fibrinogen, either Pu'cified or in plasma is shown in figure 3. 100

80 56FPA release

0

FIG. 3 FPArelease frampurified fibrinogenoor fibrq incitratedplasmaouponadditionof FEIBAfraction 1.5 ml of FEDA fractionwere added to 3.0 ml of buffer and subsequentlyto either 1.5 ml of plasma or fibrirqen. 600 ulofthismixture WSe sanpled after5,3Oand60minandaddedto25Uof fLd.irL Fvenintheabsence of calciuma rapid releaseof FPA was observ@ in both sys+==, amnmtingti100%withfibrir0genandtoakout50%inplasmaat 60 min. Even in the absence of calcium a rapid FPA release was observed in both systems, amounting to 100% with fibrinogen and to about 50% in plasma at 60 min.

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DISCUSSION

FC Concentrates were initially introduced in clinical practice for the treatment of Christmas disease (1) and later for acquired deficiencies of vitamin K dependent factors (2). Evidence for in vivo activation of the coagulation system, however, restricted their clinical usefulness. Although thrombin presence or formation had been hypothesized (16, 171, it has not yet been demonstrated. In the present study FPA release from the AoGchain of the fibrinogen molecule has been used to detect either thrombin presence or generation in vitro. In all 8 FC no thrombin activity was demonstrable, probably due to addition of heparin during preparation steps of FC. Heparin complexes with antithrombin III (AT III) and thus inactivates formed thrombin and possibly also factors IXa and Xa (19). The addition of calcium, however, was followed by high thrombin generation in 3 FC and low generation in 5. This release was time-dependent and probably due to slow activation of prothrombin by factors IXa and Xa (15). Thrombin progressively formed then probably participates in further rapid conversion of prothrombin (20, 211, explaining the sharp increase after longer preincubation times. Heparin rapidly inhibits thrombin in the presence of AT III (22). Since calcium-initiated thrombin formation could be fully inhibited by heparin, AT III must be present in sufficient amounts in these concentrates. The possibility that high amounts of heparin could directly interfere with the thrombin-fibrinogen reaction has, however, not been excluded. FEIBA on the other hand produced an immediate and rapid FPA release even in the absence of calcium, suggesting that preformed thrombin is present in this product. The further increase of thrombin activity after reconstitution of the FEIBA is probably due to the conversion of prothrombin through factors IXa and Xa, via activation of factors V and VIII. In citrated normal plasma FPA release was only half that observed in purified fibrinogen, probably as a consequence of naturally occurring inhibitors against thrombin and other clotting factors. Following the addition of a physiological amount calcium to FEIBA, much larger amounts of heparin were required for inhibition of FPA release. Although an extrapolation from "in vitro" to "in viva" thrombogenicity is not possible (231, it must be remembered that the FEIBA fraction is exposed to physiological calcium concentrations after injection into the circulating blood. The "autoactivation" of the FEIBA fraction may be of interest for its clinical use. Since progressive thrombin formation is observed rapidly after reconstitution of the lyophilized powder, thromboembolic complications may be the consequence of delay in the application rather than of the initial thrombin content of

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the preparation. (Preliminary results have been presented at the Annual Meeting of the Swiss Society of Hematology, Lausanne, May 25 - 26, 1978, and were published in abstract form in Schweizerische Medizinische Wochenschrift 108: 1600, 1978.)

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