A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.
OBJECTIVE
Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone.


METHODS
The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model.


RESULTS
The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA.


CONCLUSIONS
The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.
Tomokiyo
K
K
The Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Okubo, Kumamoto, Japan. tomokiyo@kaketsuken.or.jp
Nakatomi
Y
Y
Araki
T
T
Teshima
K
K
Nakano
H
H
Nakagaki
T
T
Miyamoto
S
S
Funatsu
A
A
Iwanaga
S
S
D000818:Animals; D001760:Bleeding Time; D001777:Blood Coagulation / Q000187:drug effects; D001779:Blood Coagulation Factors / Q000627:therapeutic use* / Q000633:toxicity; D005164:Factor IX / Q000276:immunology*; D005169:Factor VIII / Q000276:immunology*; D015942:Factor VIIa / Q000627:therapeutic use* / Q000633:toxicity; D005170:Factor X / Q000627:therapeutic use* / Q000633:toxicity; D006041:Goats; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy* / Q000276:immunology; D002836:Hemophilia B / Q000097:blood / Q000188:drug therapy* / Q000276:immunology; D006801:Humans; D007518:Isoantibodies / Q000276:immunology* / Q000633:toxicity; D008252:Macaca fascicularis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D010314:Partial Thromboplastin Time; D011817:Rabbits; D013916:Thrombelastography; D013917:Thrombin / Q000096:biosynthesis; D013927:Thrombosis / Q000139:chemically induced
D001779:Blood Coagulation Factors; D007518:Isoantibodies; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; C065655:anti-inhibitor coagulant complex; D015942:Factor VIIa; D013917:Thrombin