THROMBOSIS RESEARCH 60; 63-70,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.

COMPARISON OF DIFFERENT PROTHROMBIN COMPLEX CONCENTRATES IN VITRO AND IN VIVO STUDIES

M. Kchler, M. Heiden, G. Harbauer*,

C. Miyashlta! S. Morsdorf,

B. Braun, P. ErneR*, E.Wenzel, S. Rose*, G. Pindur. Department of Clinical Haemostaseology *Department

and Transfusion Medicine

of Experimental Surgery

University of Saarland D- 6650 Homburg/Saar F.R.G.

(Received

19.7.1990;

accepted

in revised form 58.1990

by Editor H. Vinazzer)

ABSTRACT

The potency and tests for thrombogenicity

were studied prospectively

of each brand, A - G) prothrombin complex concentrates

in 7 different (two lots

(PCC). Human albumine (H) and a

factor IX concentrate (I), served as controls. The potency of coagulation factors and inhibitors varied considerably.

Two brands (E, F) contained no protein S, additionally

one brand con-

tained no protein C. Two preparations exhibited high amidolytic activities, especially towards the thrombin-sensitive

chromogenic

substrate S-2238, in vitro. These activities could be

quenched in part by the addition of hirudin or antithrombin

Ill. The heparin and antithrombin

Ill content of the PCCs was significantly different, and, after addition of antithrombin increase of thrombin-antithrombin

Ill an

Ill complexes in 2 preparations (B, D) was observed in vitro.

Additionally, three brands (8, D, F) caused more severe cardio-pulmonary

reactions in rabbits,

associated with an increase of fibrin split products for brands B and D. We conclude that the use of these preparations hypercoagulable

Key words: prothrombin

in patients, in whom an acquired

protein C or S defect, or a

state, can be suspected, cannot be recommended.

complex concentrate,

protein C, protein S, thrombogenicity,

animal model

64

PROTHROMBIN COMPLEX CONCENTRATES

Vol. 60, No. 1

INTRODUCTION Prothrombin complex concentrates (PCC) are frequently used for the treatment of hereditary coagulation factor deficiencies (factors II, VII, X, and IX) and even patients with protein C deficiency have received PCCs [I]. The high risk of transmitting viral infections has apparently been overcome by different virus inactivation procedures [for a review see 2, 31. The observation that virucidal treatment may also alter the protein structure of clotting factors is a cause for concern since a different biological behaviour, i.e. decreased half-life or increased thrombogenicity, may be the consequence [4]. The available clinical data on virusinactivated PCC’s, however, suggest a normal half-life in man [5, 6, 71. However, a problem hitherto unsolved is the thrombogenic potential of PCC’s. To date, more than 60 cases of disseminated intravascular coagulation (DIC), thrombosis or myocardial infarction in haemophiliacs attributable to the infusion of PCC have been reported to the ICTH subcommittee on FVIII/FIX [Lusher, personal communicarion 79891.The risk of developing thrombosis is even higher when treating patients with acquired defects of haemostasis such as those resulting from blood loss, oral anticoagulant therapy or severe liver disease. It is almost impossible, to determine the thrombogenicity of PCCs in these patients, thus numerous attempts have been made to measure thethrombogenicity invitro and in animal experiments. In order to control and compare the quality of virus-inactivated PCC we studied the in vitro potency and in vivo tolerance of these preparations in rabbits. METHODS 1. Design: Two lots of seven brands of PCCs were prospectively investigated in vitro (Al -Gl , A2-G2) and one lot of each brand was studied in an animal model (A2-G2). The brands are coded with letters A-G, the first series with 1, the second with 2, i.e. Al and A2. 2. Preparations: The PCC-preparations (A-G) were from the following manufacturers (or distributors): Alpha, Langen; Behringwerke, Marburg; Biotest, Dreieich; Immune, Heidelberg; Medac, Hamburg; Serapharm, Mijnster and Troponwerke, Win. Brands 6 and D were from one manufacturer. Additionally, albumin solution (H) and a factor IX concentrate (I), served as controls. The lyophilized prepations were dissolved in distilled water to obtain a factor IX activity of 25 U/ml (as declared by the manufacturers). This solution was injected i.v. in six rabbis. For in vitro studies further dilutions by Owren’s veronal buffer, pH 7.35, were carried out in order to meet the measuring range. 3. Assays: Coagulationfactorswere measured using one-stage assays and deficient plasmafrom Immune, Vienna, and a Schnitger Gross coagulometer as described previously [8]. The solution containing 25 U/ml FIX was further diluted using Owrens veronal buffer (usually 5 dilutions ranging from I:40 to 1:lOO). The calibration was performed using ‘Reference Plasma lOO%‘, lmmuno Vienna, which is calibrated against the WHO standards for factors II, VII, IX, and X (1st international standard). Protein C was measured by an enzyme immunoassay and a coagulometric assay [9] using reagents from Boehringer, Mannheim. Protein S was determined by electro-immunodiff usion and reagents from Immune, Heidelberg. The potency is given in percent of declared (factor IX) potency, i.e. when in 500 U PCC, as declared by the manufacturer, 400 U protein C antigen were found, the potency is stated to be 80% protein C. Thrombin generation time (TGt50) and non-activated partial thromboplastin time (NAPTT) were performed as decribed by Sas et a/. [9] and Prowse et a/. [lo], respectively. The chromogenic substrate assays were performed as follows: 0.1 ml of PCC (25 U/ml) and 0.8 ml assay buffer were incubated for 5 min at 37” C. Subsequently, 0.1 ml solution (3 mmol/l) of different chromogenic substrates was added. The chromogenic substrates utilized were S-2238, S-2222, S-2444, S-2368 and S-2251 from KabiVitrum, Munich, and the assay buffers were those recommended by KabiViirum. Antithrombin Ill and heparin activity were determined using chromogenic substrate test kits from Boehringer, Mannheim. Thrombin-antithrombin lllcomplexes (TAT) were measured using an enzyme immunoassay from Behringwerke, Marburg [I 11. Plasma D-dimer levels were determined by an enzyme immunoassay from Boehringer, Mannheim [13]. Prior to infusion of PCC, the mean D-dimer levels were 5,2 ng/ml in 54 rabbits (standard deviation 6,8 ng/ml). 3. lnhlbltlon studies: The activity against the chromogenic substrate S-2238 was determined to be equivalent less than 100 mu/ml thrombin. The amount of antithrombin Ill (AT Ill) or hirudin required to neutralize this amount of thrombin was added to PCC dilutions. Recombinant hirudin was kindly provided

Vol. 60, No. 1

PROTHROMBIN

COMPLEX CONCENTRATES

65

by Professor Markwardt, Erfurt. The preparation was dissolved in distilled water and tri-fluoric-acid and subsequently diluted in saline to obtain a final concentration of 1000 antithrombin units per ml. The reaction mixture consisted of 0.75 ml assay buffer, 0.05 ml hirudin solution, and 0.1 ml PCC (25 U/ml). After incubation at 37°C for 5 min, O.lml of chromogenic substrate solution (S-2236, 3mmol/l) was added and the color reaction recorded. AT Ill concentrate (KabiViirum, Munich) was diluted to a concentration of 1 U/ml and heparin (Roche, Grenzach) to 50 U/ml. Equal volumes of AT Ill/heparin solution (or buffer) and PCC were mixed and incubated for 30 min at 37” C. The activity against the chromogenic substrate and the concentration of TAT were determined as described above. 4. Animal studles: Each brand (A 2 - I 2) was administered to 6 rabbits. A dose of 50 U/kg body weight was injected into the marginal ear vein within 10 seconds [I 31. The following parameters were recorded for 10 min: blood pressure (BP), pulse rate (PR), ventilation frequency (VF) and electrocardiography. Additionally, a blood specimen (0.4 ml 0.109 mol/l sodium citrate and 1.6 ml blood) was drawn prior to and 10 min after the injection of PCC. In previous experiments, the limits of these parameters were established after injection of one lot of PCC into IO rabbits. The ‘normal range’ was defined as the mean values +/- 2 standard deviation (SD). Each measurement at 2, 5, and 10 min after injection of PCC outside these limits was counted as + 1. The final score is calculated by adding all counts in one rabbi (0= no change at all), and the score of one lot is the mean score obtained from 6 rabbits. When thrombin containing PCC’s or Wessler-serum is injected, a score >6 can be observed; the cardiopulmonary changes can be described by an initial rapid decrease of BP, associated by an increase of PR and VF, followed by an increase of BP. RESULTS 1. In vitro studies: The potencies of coagulation factors and inhibitors determined in our study are shown in table 1. Both lots of brands B and D had a low factor IX potency. Brands E and F had a low FVII potency, while brand G had a high content of FVII, when compared with the other concentrates. TABLE 1: Potency of coagulation factors, protein C activity, and protein S antigen concentration in % of declared potency of prothrombin complex concentrates (A-G). Additionally, 2 lots of a factor IX concentrate are shown. The results are expressed as percentage of declared (factor IX) potency. Brand

Factor

Protein

IX

II

VII

X

C

S

Al A2

106 157

123 109

25 59

266 336

115 360

106 46

Bl 82

31 22

227

62 99

232 174

116 200

147 21

Cl c2

134 96

139 161

50 27

142 155

144 360

140 126

Dl D2

46 37

226 267

72 70

216 196

66 192

110 16

El E2

62 69

102 125

16 11

131 105

102 126

10 16

Fl F2 Gl G2

107 176 119 150

116 206 126 221

4 4 137 116

124 62 151 119

5 2 102 256

5 4 213 167

I1 I2

124 166

Comparison of different prothrombin complex concentrates--in vitro and in vivo studies.

The potency and tests for thrombogenicity were studied prospectively in 7 different (two lots of each brand, A-G) prothrombin complex concentrates (PC...
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