THROMBOSIS
RESEARCH
Volume
1, Pages
315-318,
Pergamon
Press,
1977. Printed
in Gt. Britain.
COMMUNICATION
BRIEF
CHARACTERISTICS AND THROMBOGENICITY OF PROTHROMBIN COMPLEX CONCENTRATES
Nobuo Sakuragawa, Kaoru Takahashi, Mari Hoshiyama, Kenji Niiya, Masakazu Itoh, Matsuzo Matsuoka and Yoshihisa Dhnishi Department of Medicine and Department of Pathology Niigata University School of Medicine, Niigata, Japan
(Received 28.10.1976; in revised form 30.11.1976. Accepted by Editor W.H. Seegers)
For treatment of hemophilia A, fresh blood can be transfused to stop bleeding.
This can be associated with hypervolemia, polycythemia, hepatitis
and formation of antibody to platelets or white blood cells.
Coagulation
factor concentrates have been developed and offer many advantages.
For hemo-
philia B, prothrombin complex concentrates have been made available.
contain
prothrombin,
Factors
VII, IX and X as well as Protein C.
These
In the case
of hemophilia A with inhibitors, prothrombin complex preparations have been found effective (l-3).
In one study small amounts of Factor Xa and thrombin
were found in a prothrombin-complex concentrate (4). was not found (5).
In another analysis this
Recently, disseminated intravascular coagulation syndrome
(DIC) followed the infusion of prothrombin-complex preparation for stoppage of bleeding in liver cirrhosis (6).
On the other hand, it was completely
effective for arresting bleeding in cases of hemophilia A with inhibitor to Factor
VIII (7).
The characteristics of prothrombin complex preparations
varies appreciably, and we are reporting some observations of the one supplied in Japan by Nihon-Seiyaku Co. and Called PPSB. induction of
We investigated experimental
DIC by infusion of this prothrombin-complex preparation (PPSB)
and checked the characteristics of it. When the contents of one bottle of PPSB was dissolved in 200 ml of distilled water, 100% of prothrombin, 2% of Factor V, 180% of Factor VII, 1% of Factor
VIII,
105% of Factor IX, 125% of Factor X, 4.5% of Factor XI and 12.5%
PROTHROMBINCOMPLEX CONCENTRATE
316
Vol.lO,No.2
of Factor XII were found. In each case plasma is regarded as 100%. For estimating Factor Xa and thrombin, PPSB was dissolved in 10 ml of distilledwater. PPSB solution had 0.12 units/ml of thrombin and 0.03 units/ml of Factor Xa. Next, the correctingactivity of PPSB on hemophilicplasma was tested. The contents of one bottle was dissolved in 5 ml of distilled water and diluted serially with saline solution. Normal titrated plasma, hemophiliaA plasma and hemophiliaA with inhibitorplasma were used for the experiment. Each plasma in the amount of 0.1 ml was added to 0.1 ml of PPSB diluted and incubated at 37°C for 1 min.
Imnediatelyafter the incubation,the activated
partial thromboplastintime was performed (8). The normal plasma showed a response in 30.4 set, hemophiliaA plasma in 51.2 set and hemophiliaA with inhibitorplasma in 75.0 set as control studies. Addition of the straight PPSB solution caused no shortening in clotting time compared with the control studies, but the 64 times diluted specimen showed a reaction in 64.4 set in hemophiliaA plasma with inhibitorand in 49.9 set in hemophiliaA plasma, but did not reach the clotting time of normal plasma. The main reason for the longer clotting time in the high concentrationsis the effect of heparin which is added in the PPSB preparation. Heparin would make an immediateinhibitor by forming a complex with antithrombinIIIwhich exists in normal and hemophilic plasma. TABLE 1 Correctionof Plasma with PPSB PPSB Dilution Folds
Normal Plasma (set) 298.0
:
B i! 64
128 Control (Saline)
138.6 86.8 54.8 40.0 36.4 35.6 30.4 30.4
HemophiliaA Plasma (set)
HemophiliaA with Inhibitor (set)
10 min 226.8 118.8 105.4 78.8 67.2 49.9 54.0
10 min 225.6 140.2 120.6 94.4 90.4 64.4 74.6
51.2
75.0
In an unusual observation it was found that one prothrombincomplex out of three different kinds would convert slowly to thrombin in 2.5 M glycine solution (5). A PPSB preparationwas dissolved in 5 ml of 2.5 M glycine
PROTHROMBIN COMPLEX CONCENTRATE
Vol.lO,No.2
317
solution (pH 7.2) and kept at 4'C to assay the thrombin yield.
As shown in
Fig. 1, the full activation of prothrombin in PPSB solution was observed.
Units
FIG. 1 Generation of thrombin from PPSB in 2.5 M glycine solution (pH 7.2) at 4OC. Thrombin yield was 100%.
0
1
3
2
lncubotionTime(MonthsI (4” c, pH 12)
PPSB preparation was dissolved in 20 ml of distilled water, and 10 ml of it was infused into a rabbit by Rodriguez-Erdmann's technique (9). VII,
Factors II,
IX and X were increased initially, but they decreased promptly in a few
hours.
Factor VIII
infusion.
was decreased remarkably, but PTT was shortened after PPSB
Platelet numbers and fibrinogen were decreased, but the serial
thrombin time (8) and thrombin time were not changed significantly.
After
the experiment was over, the rabbit was sacrificed by air infusion, and many microthrombi were found in the lungs, kidneys, etc.
When black ink was in-
fused into the rabbit to block the reticuloendothelial system, the infusion of PPSB solution with the same condition mentioned above showed manifestations of DIC. When PPSB is infused into a rabbit, the coagulatiqn system may be activated and prothrombin is converted to thrombin by the action of small amounts of Factor Xa, and this in turn develops into DIC.
One possibility is that the
effective stoppage of hemorrhages in the case with hemophilia A with inhibitor is due to the Factor Xa or thrombin which are contained in the PPSB preparation.
The fact that DIC is not observed in the case with hemophilia A with
inhibitor is the resistance supplied by the inhibitor and secondly the normal function of the,reticuloendothelial system as a clearance mechanism.
We do
not know whether excessive PPSB infusion would cause DIC in cases of hemophilia A with inhibitors.
PROTHROMBIN COMPLEXCONCENTRATE
318
Vol. lO,No.2
REFERENCES 1. FEKETE, L.F., HOLST, S.T., PETOOM, F., and DeVEBER, L.L. Auto-factor IX concentration. A new therapeuticapproach to treatment of hemophilicA patients with inhibitors. 14th Int. Cong. Hematol., Sao Paulo (Abst. 295), 1972.
2.
KURCZYNSKI,EM. and PENNER, J.A. Activated prothrombinconcentratefor patients with factor VIII inhibitors. New Eng. J. Med. 291, 164, 1976.
3.
ABILGAARD, C.F., BRITTON, M., and ROBERTS, R. Prothrombincomplex concentrate (Konyne) for patients with factor VIII inhibitors. 17th Annual Meeting Amer. Sot. Hematol. (Paper B7), 1974.
4.
SAKURAGAWA,N., TAKAHASHI, K., HOSHIYAMA,M., ASHIZAWA, T., TAKAHASHI, H., and thromboMATSUOKA, M., and OHNISHI,Y. Studies on characteristics genicity of factor IX concentrates. The Saishin-Iqaku(In Press).
5.
SEEGERS, W.H., SAKURAGAWA,N., and MCCOY, L.E. Laboratoryanalysis of clotting-factorcomponentsconcentratedfor therapeuticuse. Thrombosis Res. 1, 33, 1972.
6.
SAKURAGAWA,N., TAKAHASHI, K., MATSUOKA, M., YOSHIDA, K., and OHNISHI, Y. Characterizationsof factor IX concentratesand a case of liver cirrhosis with disseminatedintravascularcoagulationsyndrome induced by infusion of factor IX concentrates. Reports of 15th Annual Meeting of Plasmin Research, Niigata, Japan, Daiich-SeiyakuCo., Tokyo, p. 272, 1975.
7. MATSUOKA, M. and ITOH, M. Treatmentof hemophiliaA patients with inhibitor with prothrombin-complexconcentrates. Abstracts of XIth Cong. Jap. Blood TransfusionMeeting, p. 78, 1976.
a. MATSUOKA, M. 1971. 9.
Laboratorymethods for blood coagulation. Kanahara,Tokyo,
RODRIGUEZ-ERDMANN,F. Uber eine einfache methode zur wiederholen blutgerinnungbeim kaninchen. Pflueger Arch. 269, 306, 1959.
RESEARCH
Volume
1, Pages
315-318,
Pergamon
Press,
1977. Printed
in Gt. Britain.
COMMUNICATION
BRIEF
CHARACTERISTICS AND THROMBOGENICITY OF PROTHROMBIN COMPLEX CONCENTRATES
Nobuo Sakuragawa, Kaoru Takahashi, Mari Hoshiyama, Kenji Niiya, Masakazu Itoh, Matsuzo Matsuoka and Yoshihisa Dhnishi Department of Medicine and Department of Pathology Niigata University School of Medicine, Niigata, Japan
(Received 28.10.1976; in revised form 30.11.1976. Accepted by Editor W.H. Seegers)
For treatment of hemophilia A, fresh blood can be transfused to stop bleeding.
This can be associated with hypervolemia, polycythemia, hepatitis
and formation of antibody to platelets or white blood cells.
Coagulation
factor concentrates have been developed and offer many advantages.
For hemo-
philia B, prothrombin complex concentrates have been made available.
contain
prothrombin,
Factors
VII, IX and X as well as Protein C.
These
In the case
of hemophilia A with inhibitors, prothrombin complex preparations have been found effective (l-3).
In one study small amounts of Factor Xa and thrombin
were found in a prothrombin-complex concentrate (4). was not found (5).
In another analysis this
Recently, disseminated intravascular coagulation syndrome
(DIC) followed the infusion of prothrombin-complex preparation for stoppage of bleeding in liver cirrhosis (6).
On the other hand, it was completely
effective for arresting bleeding in cases of hemophilia A with inhibitor to Factor
VIII (7).
The characteristics of prothrombin complex preparations
varies appreciably, and we are reporting some observations of the one supplied in Japan by Nihon-Seiyaku Co. and Called PPSB. induction of
We investigated experimental
DIC by infusion of this prothrombin-complex preparation (PPSB)
and checked the characteristics of it. When the contents of one bottle of PPSB was dissolved in 200 ml of distilled water, 100% of prothrombin, 2% of Factor V, 180% of Factor VII, 1% of Factor
VIII,
105% of Factor IX, 125% of Factor X, 4.5% of Factor XI and 12.5%
PROTHROMBINCOMPLEX CONCENTRATE
316
Vol.lO,No.2
of Factor XII were found. In each case plasma is regarded as 100%. For estimating Factor Xa and thrombin, PPSB was dissolved in 10 ml of distilledwater. PPSB solution had 0.12 units/ml of thrombin and 0.03 units/ml of Factor Xa. Next, the correctingactivity of PPSB on hemophilicplasma was tested. The contents of one bottle was dissolved in 5 ml of distilled water and diluted serially with saline solution. Normal titrated plasma, hemophiliaA plasma and hemophiliaA with inhibitorplasma were used for the experiment. Each plasma in the amount of 0.1 ml was added to 0.1 ml of PPSB diluted and incubated at 37°C for 1 min.
Imnediatelyafter the incubation,the activated
partial thromboplastintime was performed (8). The normal plasma showed a response in 30.4 set, hemophiliaA plasma in 51.2 set and hemophiliaA with inhibitorplasma in 75.0 set as control studies. Addition of the straight PPSB solution caused no shortening in clotting time compared with the control studies, but the 64 times diluted specimen showed a reaction in 64.4 set in hemophiliaA plasma with inhibitorand in 49.9 set in hemophiliaA plasma, but did not reach the clotting time of normal plasma. The main reason for the longer clotting time in the high concentrationsis the effect of heparin which is added in the PPSB preparation. Heparin would make an immediateinhibitor by forming a complex with antithrombinIIIwhich exists in normal and hemophilic plasma. TABLE 1 Correctionof Plasma with PPSB PPSB Dilution Folds
Normal Plasma (set) 298.0
:
B i! 64
128 Control (Saline)
138.6 86.8 54.8 40.0 36.4 35.6 30.4 30.4
HemophiliaA Plasma (set)
HemophiliaA with Inhibitor (set)
10 min 226.8 118.8 105.4 78.8 67.2 49.9 54.0
10 min 225.6 140.2 120.6 94.4 90.4 64.4 74.6
51.2
75.0
In an unusual observation it was found that one prothrombincomplex out of three different kinds would convert slowly to thrombin in 2.5 M glycine solution (5). A PPSB preparationwas dissolved in 5 ml of 2.5 M glycine
PROTHROMBIN COMPLEX CONCENTRATE
Vol.lO,No.2
317
solution (pH 7.2) and kept at 4'C to assay the thrombin yield.
As shown in
Fig. 1, the full activation of prothrombin in PPSB solution was observed.
Units
FIG. 1 Generation of thrombin from PPSB in 2.5 M glycine solution (pH 7.2) at 4OC. Thrombin yield was 100%.
0
1
3
2
lncubotionTime(MonthsI (4” c, pH 12)
PPSB preparation was dissolved in 20 ml of distilled water, and 10 ml of it was infused into a rabbit by Rodriguez-Erdmann's technique (9). VII,
Factors II,
IX and X were increased initially, but they decreased promptly in a few
hours.
Factor VIII
infusion.
was decreased remarkably, but PTT was shortened after PPSB
Platelet numbers and fibrinogen were decreased, but the serial
thrombin time (8) and thrombin time were not changed significantly.
After
the experiment was over, the rabbit was sacrificed by air infusion, and many microthrombi were found in the lungs, kidneys, etc.
When black ink was in-
fused into the rabbit to block the reticuloendothelial system, the infusion of PPSB solution with the same condition mentioned above showed manifestations of DIC. When PPSB is infused into a rabbit, the coagulatiqn system may be activated and prothrombin is converted to thrombin by the action of small amounts of Factor Xa, and this in turn develops into DIC.
One possibility is that the
effective stoppage of hemorrhages in the case with hemophilia A with inhibitor is due to the Factor Xa or thrombin which are contained in the PPSB preparation.
The fact that DIC is not observed in the case with hemophilia A with
inhibitor is the resistance supplied by the inhibitor and secondly the normal function of the,reticuloendothelial system as a clearance mechanism.
We do
not know whether excessive PPSB infusion would cause DIC in cases of hemophilia A with inhibitors.
PROTHROMBIN COMPLEXCONCENTRATE
318
Vol. lO,No.2
REFERENCES 1. FEKETE, L.F., HOLST, S.T., PETOOM, F., and DeVEBER, L.L. Auto-factor IX concentration. A new therapeuticapproach to treatment of hemophilicA patients with inhibitors. 14th Int. Cong. Hematol., Sao Paulo (Abst. 295), 1972.
2.
KURCZYNSKI,EM. and PENNER, J.A. Activated prothrombinconcentratefor patients with factor VIII inhibitors. New Eng. J. Med. 291, 164, 1976.
3.
ABILGAARD, C.F., BRITTON, M., and ROBERTS, R. Prothrombincomplex concentrate (Konyne) for patients with factor VIII inhibitors. 17th Annual Meeting Amer. Sot. Hematol. (Paper B7), 1974.
4.
SAKURAGAWA,N., TAKAHASHI, K., HOSHIYAMA,M., ASHIZAWA, T., TAKAHASHI, H., and thromboMATSUOKA, M., and OHNISHI,Y. Studies on characteristics genicity of factor IX concentrates. The Saishin-Iqaku(In Press).
5.
SEEGERS, W.H., SAKURAGAWA,N., and MCCOY, L.E. Laboratoryanalysis of clotting-factorcomponentsconcentratedfor therapeuticuse. Thrombosis Res. 1, 33, 1972.
6.
SAKURAGAWA,N., TAKAHASHI, K., MATSUOKA, M., YOSHIDA, K., and OHNISHI, Y. Characterizationsof factor IX concentratesand a case of liver cirrhosis with disseminatedintravascularcoagulationsyndrome induced by infusion of factor IX concentrates. Reports of 15th Annual Meeting of Plasmin Research, Niigata, Japan, Daiich-SeiyakuCo., Tokyo, p. 272, 1975.
7. MATSUOKA, M. and ITOH, M. Treatmentof hemophiliaA patients with inhibitor with prothrombin-complexconcentrates. Abstracts of XIth Cong. Jap. Blood TransfusionMeeting, p. 78, 1976.
a. MATSUOKA, M. 1971. 9.
Laboratorymethods for blood coagulation. Kanahara,Tokyo,
RODRIGUEZ-ERDMANN,F. Uber eine einfache methode zur wiederholen blutgerinnungbeim kaninchen. Pflueger Arch. 269, 306, 1959.
