A fully recombinant partial prothrombin complex effectively bypasses fVIII in vitro and in vivo.
The development of inhibitory antibodies is a serious complication in hemophilic patients, severely compromising therapeutic success. Bleeding episodes in affected patients are controlled by treatment with a plasma-derived prothrombin complex concentrate (PCC), activated PCC (APCC) or recombinant activated factor VII. We hypothesized that a recombinant two-component agent consisting of recombinant prothrombin (rfII) and activated factor X (rfXa) would have substantial fVIII bypassing activity and could be a safe alternative therapeutic option. To test this hypothesis we assembled an agent in vitro solely consisting of rfII and rfXa at a molar ratio of 37,500:1. These factors are believed to be responsible for the activity of APCC preparations. Recombinant fX, used as the source for fXa generation, and rfII were purified from serum-free and protein-free conditioned media of stably transfected CHO and BHK tissue culture cells, respectively. Activation of rfX to rfXa was accomplished by the plant protease ficin, obviating the need for a protease derived from a human or animal source. We found that in vitro the complex reduced the abnormally prolonged activated partial thromboplastin time (APTT) of a high-titer fVIII inhibitor plasma similar to an APCC preparation. Furthermore, addition of increasing amounts of rfII/rfXa to inhibitor plasma resulted in a linear dose-dependent increase in the rate of thrombin generation. In a rabbit fVIII inhibitor model, treatment with rfII/rfXa statistically significantly reduced the intensity of the abnormal cuticle bleeding. In the Wessler test, rfII/rfXa showed no thrombogenicity. These data show that a well-defined, particularly safe and efficacious agent with fVIII bypassing activity can be generated from recombinant fII and fXa.
Himmelspach
Michèle
M
Baxter BioScience, Vienna, Austria.
Richter
Günter
G
Muhr
Evelyn
E
Varadi
Katalin
K
Turecek
Peter L
PL
Dorner
Friedrich
F
Schwarz
Hans Peter
HP
Schlokat
Uwe
U
D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D005361:Ficain; D006801:Humans; D066298:In Vitro Techniques; D010314:Partial Thromboplastin Time; D011516:Prothrombin / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D012449:Safety; D013917:Thrombin / Q000096:biosynthesis; D013927:Thrombosis / Q000209:etiology; D014162:Transfection
D011994:Recombinant Proteins; D011516:Prothrombin; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa; D005361:Ficain