Recombinant von Willebrand factor: preclinical development.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Preclinical evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease (vWD) have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of vWD in humans. We used this canine model of vWD to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with an rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 hours in one dog and 22.1 hours in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 hours, and LMW-rvWF, 9 hours. The in vivo recovery of vWF:Ag after administration of rvWF was 59, 64 and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78, 110 and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

[Preclinical investigation of alpha 1-acid glycoprotein (orosomucoid)]. / Präklinische Untersuchung von alpha 1-saurem Glykoprotein (Orosomucoid).

The molecular properties of alpha 1-acid glycoprotein are briefly discussed. This molecule has been shown in in vitro experiments to have both a stabilizing effect on vascular permeability and antiinflammatory properties. We were able to demonstrate these two effects in vivo in guinea pigs (skin, Evan's Blue extravasation) and in rats (paw, carrageenan induced inflammation). Further experiments were performed in rats relating to possible therapeutic indications for alpha 1-acid glycoprotein: (1) inhibitory effect on brain edema formation after experimental stroke, (2) therapeutic effect in the puromycin aminonucleoside-induced minimal change nephrosis, (3) improvement of vital parameters in hemorrhagic-hypovolemic shock, (4) increase in survival rate in septic peritonitis, and (5) promising effects in burn-induced remote lung injury. The high content of sialic acid and the high negative charge of alpha 1-acid glycoprotein are believed to be major contributors to its stabilizing effect on vascular permeability. The protein is bound to the glycocalyx of the endothelial cells (and presumably to structures of the glomerular basement membrane), thereby hindering the passage of other polyanionic molecules through the vascular wall. The antiinflammatory/immunomodulatory effect of alpha 1-acid glycoprotein appears mainly due to suppression of polymorphonuclear neutrophils. This action is dependent on the glycan part of the molecule, which is highly variable (microheterogeneity). It is obvious that there are differences between the different glycan forms as far as the antiinflammatory property of the protein is concerned. Together with data in the literature, the results presented here suggest a variety of potential indications for therapeutic use of alpha 1-acid glycoprotein in humans.

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.