THROMBOSIS RESEARCH 67; 41-48,1992 0049~3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
EPRECT
OR SODXDM P-S&R
POLYSULPHATR OR THR THROMROgRRICITY OR PR~I#cooQpLRYCOR~TRs
H.-P. Kl&kingl, g. Dornheim3 and a. Schulre-Riewaldl Inetitute of Pharmacology and Toxicology1 , Xedical Academy Erfurt, 0-5010 Erfurt, Rordldueer Str. 74 and Dietltict Institute of Blood Transfueion2 , O-6013 Suhl, AlbertSchweitzer Str. 14, BRg (Received 4.1 .1992; accepted in revised form 6.5.1992 by Editor J. Dyr) ABSTRACT
The addition of pentoeau polyaulphate eodium OmPPS) to thrmbogenic prothrmbin complex concentrates (PCC!) dose-dependently thZO&US reduce8 or aboliS&S formation in rat6 in the ataeie model act. to Weelrler. However, no reduction of thrombogenicity was found in PCC preparations manufactured in the presence of RaPPS.
INTRODUCTIOR Preriouely it was shown that addition of naturally occurring in oamplex conhirudin or reco&inant hirudin to procentratee (PCC!)aboliehed their thrombogenicity in the otaeicr model act. to Weasler in rats and rabbit6 (1,2,3).In this etudy the effect on thrombogenicity of eoditm~ pemtosan polysulphate (WaPPS) involved in PCC sanuf acturing was c-red to that of aubeequaat addition of the substance to already finished PCC.
Key words:
Prothrombin complex concentratee, tvicity, influence of sodium pentoean polysulphate
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MATERIALS Manufacturing prothrombia complex concentrates in the presence of sodium pentosan polysulphate (WaPPS; batch 230685): Adsorption Infusion solution E 154 (18 ml) containing 0.3 mg WaPPS/ml was given to the respective cryoprecipitate supernataut (mean volume 445 f 21 ml). The prothrombin complex was prepared by adsorption to DEAESephadex(a) A-50 (0.5 g DXAE-Sephadex/cryoprecipitate superfor 45 min at room temperature. natant) while being shaken Then, the DBAE-Sephadex gel was separated by centrifugation (1800 rpm, O-2 OC for 20 m&n) and the plasma superuatant was removed except for a residue of 30-35 ml. 48 cryoprecipitate supernatants were used per batch. Washing out of DEAE-Sephadex(X) A-50 The DEAB-Sephadex residue plasma suspensions, 12 at a time, were collected in a 500 ml blood preserving jar aud centrifuged again (1800 rpm, O-2 OC, for 15 min. The gel obtained was suspended in washing liquid (sodium chloride, 0.2 mol/l; sodium citrate , 0.01 mol/l; pH 6.85) and WaPPS, 10.6 mg/l. The DEAESephadex suspensions, 24 at a time, were given into a special filtration unit (4). Washing out of the DHAB-Sephadex was continued with another 4 1 of washing liquid. Elution of prothrombin complex Unnecessary washing liquid was removed by superpressure filtration (0.15 ama; 10 min). Elueut (20 ml; 3.95 mol/l sodium chloride, 0.1 auol/l sodium citrate, pH 6.85); eluent II (40 ml; 0.5 mol/l sodium chloride, 0.01 mol/l sodium citrate, pH 6.85) and WaPPS (1.85 ~1 were subsequently given into the filtration unit and were shaken for 20 min until the state of equilibrium was reached. The eluate was separated by superpressure as well as centrifugal filtration. Thus prothrombin cwlex concentrate (169 g) was obtained to which WaPPS (2.5 mg) was added. Of this concentrate samples of 7.4 ml each were prepared for every transfusion unit (TU) (reconstitution volume 20 ml). They were immediately frozen aud lyophilixed; a temperature of -10 OC was not exceeded during the ice phase. To determine the factors as well as the non-activated partial throlmboplastin time and the thrombin generation time the lyophilixed preparation (transfusion unit) was dissolved in 20 ml distilled water. This batch contained 22 units of Pactor IX/ml;
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25 units of Factor II/ml, and 22 units of Factor X/ml. The in vitro thmmbogenicity was characterized by a non-activated partial thrcmboplaetin time ratio of 0.64 and a thrombin generation time of 9.4 min. Prothrombin complex concentrates (PCC) (without heparin addition; batch 3606841: PCC was isolated by adsorption to DEAE-Sephadex(n)A-50 frcm human titrated plasma after separation of factor VIII by cryoprecipitation (4). The lyophilized preparation, dissolved in 20 ml dietilled water, contained 17 unite of FaCtOr IX/ml, II/ml and 21 units of Factor X/ml. The in 22.8 units of Factor vitro thrombogenicity was characterized by a non-activated partial thromboplastin time ratio of 0.44 and a tbrombin generation time of 4.4 min. sodium pentosan polysulpbata mU?PS)r Fibraxym (bene Arzneimittel GmbW, Munich, FRG) Experimental animals: Female Wistar rats (Sch&/Wi) with a bodyweight of 290 f 50 g, anaesthetixed with 1.2 g of ethyl urethaue intraperitoneally. Further experimeutal condftionsr For animal experiments the lyophilirred batches (trausfusion unit, UT) were dissolved in distilled water: in 20 ml for administration of a Factor IX dose of 25 or 50 U/kg; in 10 ml for the injection of 100 or 20 U/kg and in 5 ml for a dose of 400 U/kg.
In vitro procoagulant: Determination of thrombin generation time (TGt50) was performed act. to Sas et al. (5) and the determination of non-activated part&al thromboplastin time (WAPTT) (LB described by Ringdon et al. (6). In viva thrombogenicity: Thrombogenicity studies were carried out in female Wistar rats by mans of the stasis amdel act. to Wessler (7). A segment of the jugular vein of 10 mm in length was e%poBed and dimected free of surrouuding tissue. PCCs (1.0 ml/kg) was given via the cannulated femoral vein. The jugular vein 8-t
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was ligated at both ends, 30 8 after PCC administration. 10 sin later, the segment was removed and the thrombi formed were calculated macroscopically after rinsing out the contents of the segment into a sodium citrate-filled Petri dish. Each test result was recorded as positive or negative depending on the presence or absence of a thrombus in the isolated venous segment. The thrombus size was graded by a scoret 0 o fluid; 1~ a few macroscopic strauds of fibrin1 2s several small thrcmbi; 351 two or sore large thrcmbir 4= a single thrombus forming a cast of the isolated segment.
Intravenous administration of various guantities of PCC (Bactor IX units/kg bodyweight; batch no. 230685 cormpared to batch uo. 360984) into rats followed by stasis in a segment of the jugular vein led to the formation of clotting thrombi (Tabs. 1 and 2). From the axgerimentally obtained data a median ef fective dose (ED501 of 53 (30-93) Factor IX U/kg was calculated for PCC preparations manufactured in the presence of WaPPS (batch 230685); the XD50 for the PCC (without heparin addition; batch 360984) was 47 (26-83) Pactor IX U/kg). TABLE1 Thrombogenicity of prothrombin complex concentrate (PCC) adsorbed in the presence of sodium pentosan polysulphate (batch 230685) in rats in a stasis model act. to Wessler (7) Dose PCC
Thrtius incidence*
(U/kg) 25 50 100
200
Thrombus
size
Score
01234 l/5 3/6 4/6 6/6
41000 30300 20031 00222
*Wumber of animals with thrombi in relation animal8
0.2 1.0 2.2 3.0
to total number of
The ED50 value indicates that no thrcmbi were formed in 50 % of the animals at this concentration or activity, reeg.
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TABLE 2 Thrombogeuicity of prothrombin complex concentrate (PCC) (batch 360984) without addition of heparin in rats in a stasis model act. to Wessler (7) Dose PCC (U/kg)
Thrcmbus incidence*
Thrombus
size
Score
01234
25 50 100 200 400
53000 30221 10250 00044 00017
3/8 5/8 7/8 8/8 8/8
0.37 1.8 2.4 3.5 3.87
*see Table 1 HaPPS was added to PCC transfusion unit, batch no. 360984, before administration. The i.v. injection of PCC solution containing NaPPS led either to reduced size of thrcmbi or completely prevented thrombue formation, depending on NaPPS conceutration. ~hrcmbus formation was completely prevented on addition of 1.5 mg NaPPS to the transfusion unit (Table 3). This corresponds to a virtual dose of 0.7 mg XaPPS/kg into rats. TABLE 3 Influence of sodium gentosan golysulphate (aTaSPS addition to the PCC transfusion unit (TU)(batch 360984) on the thrombus formation by 200 Factor IX PCC/kg of this preparation in rats in a stasis model act. to Wessler (7) Addition NaPPS bg/TIJ)
of
0 0.25 0.5 1 1.5
*see Table 1
Thrombus incidence*
Throarbus size
Score
01234 8/9 8/8 8/8 l/6 O/6
00044 00233 01421 51000 60000
3.5 3.12 2.38 0.12 0
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An almost similar reduction of thrombogenicity was achieved by the addition of 1.5 mg BTaPPS to the PCC transfusion unit (batch 230685) (Tab. 4).
TABLE 4 Effect of WaPPS addition to the PCC transfusion unit (TD) on the thrombogenicity of a PCC adsorbed in the presence of WaPPS (batch 230685) in rats in a stasis model act. to Wessler (7) Addition WaPPS (ms/Tm
of
_
1.5
PCC
dose (U/kg,* 200 200
Thrormbus incidence*
Thrombus
size
Score
01234 6/6 l/6
00222 51000
3.0 0.17
*see Table 1
DISCUSSIOW
Sodium pentosan polysulphate (WaPPS) is a heparin-like compound which has been therapeutically used in thrombosis prophylaxis for more than 40 years because of its anticoagulant and profibrinolytic activity (reviewed by (8,911. The anticoagulant activity of BTaPPS ie partly due to the inthe hibition of factor Xa formation (8). That justified assqtion that the presence of WaPPS during the preparation of PCCs would already diminish the thrombogenicity of such PCCs. As demonstrated by the results , the in vivo thrombogenicity of PCCs could not be prevented by adding WaPPS to the different ghaSeS of PCC manufacturing (Table l).There was no significant difference in thrombogenicity between PCC adsorbed in the presence of NaPPS (batch 230685) and PCC adsorbed without any addition (batch 360984) (Tables 1 and 2). However, related to the thrombin generation time and the nonactivated partial thromboplastin time ratio, the in vitro thrombogenicity of PCC manufactured in the presence of WaPPS (batch 230685) was definitely reduced comagared to PCC adsorbed without any addition (batch 360984).0nly after the addition of 1.5 mg sodium pentosan polysulphate to the PCC transfusion unit (batch 230685) the PCC thrombogeuicity in rats was reduced to
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an average score of 0.17 (Tab. 4). The mme reduction of thrombogenicity was achieved by subsequent addition of NaPPS to already finished PCC (batch 360984). The addition of NaPPS to the PCC transfusion unit This became dose-dependeutly diminished thrombus f omation. particularly evident with the use of 200 Factor IX U/kg bodyweight (Tab. 4). The studies revealed that the thrombogenicity of PCCs may be prevented by the addition of small amounts of NaPPS after the manufacturing procedure of PCC had been finished. Such small quantities of NaPPS did not significantly prolong the activated partial thromboplaetin time measured ax viva. At the concentrations used NaPPS did neither prolong the bleeding time nor induce haemorrhagic coqlications (8). Compared to heparin, NaPPS did not require the presence of antithrombin III (10). Therefore, NaPPS is recommended as anticoagulant additive to PCCS.
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2 KLijCKING, H.-P., SCHGLZE-PIRWALD, H., MARKWARDT, F. Inhibitory effect of hirudin on thrombosis induced by prothrcmbin complex concentrates. Folia Haematol. 115, 106 -109, 1988. 3 lCtiClCING,H.-P. SCEULKE-BIWOALD, H., xAR%wARDT, F. Influence of hirudin on the thrombogeuicity of prothrombiu coxplex concentrates. Haemorrtasis 18, S 2, 134, 1988 (abstract). 4 DGRUKKIM, G., WKGNKR, H. Heretelluug und Charakterisierung eines neuen Prothrombiukomplexkonrentrates. Folia Haematol. 112, 614-628, 1985. 5 SAS,
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