Three small animal models of bleeding are described and used to evaluate the effects of preparations intended for therapy of human bleeding disorders. We modified techniques for the assessment of bleeding to be able to reproducibly quantify blood loss and rate of blood flow in addition to the measurement of bleeding time.
Temporary hemophilia was induced in a rabbit model by injection of high titer human inhibitor plasma [>1000 Bethesda units (BU)/ml]. A decrease in rabbit FVIII from normal values to below the limit of detection was observed within 30 min, cuticle bleeding time changed from normal (approx. 10 min) to steady state bleeding (>30 min), and the rate of blood flow increased from 4 to >30 µl blood/min. Infusion of an activated prothrombin complex concentrate (FEIBA STIM4, Immuno) at doses between 75 and 150 U/kg normalized the rate of blood flow, while infusion of FVIII/vWF concentrate resulted in partial correction. Administration of FVIIa, both recombinant and plasma-derived, failed to correct bleeding, however. In an analogous murine model, FVIII/ vWF inhibitor plasma was obtained by immunizing goats with a purified human FVIII/vWF complex. This plasma cross-reacted with mouse vWF in vitro. Injection of the anti-FVIII/vWF inhibitor plasma into mice caused a decrease in vWF antigen, in some animals with a complete loss of vWF multimers comparable to severe von Willebrand disease. A specific anti-vWF inhibitor plasma obtained by immunization of goats with recombinant vWF was used in a further murine model, resulting in a gradual but substantial decrease in FVIII as well as in intensive bleeding. The infusion of a FVIII/vWF concentrate (IMMUNATE, IMMUNO) normalized the rate of blood flow in both murine models. The same assessment methods were used to characterize bleeding in a natural mouse model of von Willebrand disease (strain RIIIS/J). The use of quantitative techniques of assessment of blood loss and rate of blood flow appears to be a helpful tool for characterizing hemorrhagic situations and evaluating the capacity of therapeutic preparations to correct hemostatic defects.
References
1
Doutremepuich C,
Toulemond F,
Bousquet F,
Bonini F.
Comparison of the haemorrhagic effects of unfractionated heparin and a low molecular weight heparin fraction (CY 216) in rabbits. Thromb Res 1986; 43: 0691-0695
2
Johnstone MT,
Andrews T,
Ware JA,
Rudd MA,
George D,
Weinstein M,
Loscalzo J.
Bleeding time prolongation with streptokinase and its reduction with l-desamino-8-D-arginine vasopressin. Circulation 1990; 82: 2142-2151
3
Chabbat J,
Hampikian-Lenin S,
Toully V,
Gaillandre A,
Péjaudier L,
Stein-buch M.
A human factor Vlla concentrate and its effects in the hemophilic A dog. Thromb Res 1989; 54: 0603-0612
4
Mertens K,
Briët E,
Giles AR.
The role of factor VII in haemostasis:Infusion studies of factor Vila in a canine model of factor VIII deficiency. Thromb Haemost 1990; 64: 0138-0144
6
Ni H,
Giles AR.
Normalization of the haemostatic plugs of dogs with haemophilia A (factor VIII deficiency) following the infusion of a combination of factor Xa and phosphatidylcholine/phosphatidylserine vesicles. Thromb Haemost 1992; 67: 0264-0271
9
Pijnappels MIM,
Briët E,
van derZwet GT,
Huisden R,
van TilburgNH,
Eulderink F.
Evaluation of the cuticle bleeding time in canine haemophilia A. Thromb Haemost 1986; 55: 0070-0073
10
Brinkhous KM,
Hedner U,
Garris JB,
Diness V,
Read MS.
Effect of recombinant factor Vila on the hemostatic defect in dogs with hemophilia A, hemophilia B, and von Willebrand disease. Proc Natl Acad Sci USA 1989; 86: 1382-1386
11
Lei Bi,
Lawler AM,
Antonarakis SE,
High KA,
Gearhart JD,
Kazazian Jr HH.
Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nature Genetics 1995; 10: 0119-0121
15
Uehlinger J,
Rose E,
Aledort LM,
Lerner R.
Successful treatment of an acquired von Willebrand factor antibody by extracorporeal immunoadsorption. N Engl J Med 1989; 320: 0254-0255
17
Erhardtsen E,
Madsen MT,
Ezban M,
Hedner U,
Diness V,
Glazer S,
Nordfang O.
Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced hemophilia A. Thromb Haemost 1993; 69: 0556
18
Nichols WC,
Cooney KA,
Mohlke KL,
Ballew JD,
Yang A,
Bruck ME,
Reddington M,
Novak EK,
Swank RT,
Ginsburg D.
Von Willebrand’s disease in the RIIIS/J mouse is caused by a defect outside of the von Wille-brand factor gene. Blood 1994; 83: 3225-3231
19
Sweeney JD,
Novak EK,
Reddington M,
Takeuchi KH,
Swank RT.
The RIIIS/J inbred mouse strain as a model for von Willebrand disease. Blood 1990; 76: 2258-2265
20
Mohlke KL,
Nichols WC,
Cooney KA,
Novak EK,
Swank RT,
Ginsburg D.
Fine-scale genetic mapping of a novel gele modifying von Willebrand factor levels in the RIIIS/J mouse. Blood 1995; 86: 0280a (abs)
24
Bradford MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 0248-0254
26
Fischer BE,
Schlokat U,
Mitterer A,
Reiter M,
Mundt W,
Turecek PL,
Schwarz HP,
Domer F.
Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin. FEBS Lett 1995; 375: 0259-0262
27
Novak EK,
Sweet HO,
Prochazka M,
Parentis M,
Soble R,
Reddington M,
Cairo A,
Swank RT.
Cocoa:A new mouse model for platelet storage pool deficiency. Brit J Haematol 1988; 69: 0371-0378
30
Garabedian HD,
Gold HK,
Leinbach RC,
Svizzero TA,
Finkelstein DM,
Guerrero JL,
Collen D.
Bleeding time prolongation and bleeding during infusion of recombinant tissue-type plasminogen activator in dogs:Potentiation by aspirin and reversal with aprotinin. J Am Coll Cardiol 1991; 17: 1213-1222
31
Marder VJ,
Shortell CK,
Fitzpatrick PG,
Kim C,
Oxley D.
An animal model of fibrinolytic bleeding based on the rebleed phenomenon:Application to a study of vulberability of hemostatic plugs of different age. Thromb Res 1992; 67: 0031-0040
32
Mertz ET.
The anomaly of a normal Duke’s and very prolonged saline bleeding time in swine suffering from an inherited bleeding disease. Am J Physiol 1942; 136: 0360-0362
34
Mielke CH,
Kaneshiro MM,
Maher IA,
Weiner JM,
Rapaport SI.
The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood 1969; 34: 0204-0215
35
Hilgartner MW,
Knatterud GL.
FEIBA Study Group The use of factor eight inhibitor by-passing activity (FEIBA Immuno) product for treatment of bleeding episodes in hemophiliacs with inhibitors. Blood 1983; 61: 0036-0040
