VWF Propeptide Levels Research Articles

Biochemical and Functional Characterization of a Serum-Free rVWF Drug Candidate.

Recombinant VWF was co-expressed with recombinant FVIII in CHO cells. In order to obtain fully processed, mature rVWF, rVWF was exposed to recombinant CHO-cell-derived furin for VWF pro-peptide removal. Fermentation for both rVWF and furin and downstream processing were performed under serum-free conditions.Mature rVWF was highly purified by a series of conventional chromatography steps to a purity of > 95%. rVWF was formulated in a protein-free buffer.Several large scale batches of rVWF were produced and characterized in comparison to Humate-P, Wilfactin and a highly purified plasma-derived (pd)VWF, an experimental pd VWF product obtained from cryoprecipitate. Residual pro-VWF levels were approximately 2% of total VWF antigen compared with 0.12, 0.05 and 0.13 for the rVWF product, experimental pd VWF, Humate-P and Wilfactin, respectively. VWF pro-peptide levels were approximately 0.5 pmol per unit VWF antigen, compared with 0.6 and 0.9 for Humate-P and Wilfactin, respectively; in the experimental pd VWF pro-peptide was below the limit of detection. Residual FVIII content was approximately 0.02 units (chromogenic assay) per unit VWF antigen, compared with 0.4 units in Humate-P, and 0.04 units in Wilfactin, and ≤ 0.01 in the experimental pd VWF product.Ristocetin cofactor activity was around 0.7 VWF:RCo units per unit antigen for all rVWF and pd VWF products tested. Collagen-binding activity was also similar between rVWF and pd VWF controls (around 0.8 – 0.9 units per unit). FVIII-binding capacity of rVWF was compared by setting the mean pd VWF as 100% and was found to also be ± 100%. The VWF affinity for FVIII was similar in rVWF and pd VWF (Biacore). rVWF showed high molecular weight multimers in low resolution agarose gels, with a pre-dominance in the high molecular weight range. In contrast to pd VWF, rVWF showed homogenous multimer bands without triplet structure formation due to the non-exposure to ADAMTS13 during the manufacturing process.The pharmacokinetic properties of rVWF were compared with pd VWF in VWF knock-out mice. The half-life or rVWF in VWD mice was 4.3 and 3.5 hours for 2 batches investigated, which seems longer than the half-lives obtained for pd VWF, 2.9 hours for Humate-P and 2.2 hours for a highly purified pd VWF control. Both VWF preparations stabilized endogenous murine FVIII which rose over time to the levels observed in normal C57BI/6J mice.Overall, these results showed that the properties of mature, furin-processed rVWF in vitro and in vivo are equivalent to those of plasma-derived VWF.

Von Willebrand factor propeptide in malaria: evidence of acute endothelial cell activation

The pathogenicity of Plasmodium falciparum is thought to relate to the unique ability of infected erythrocytes to adhere to and subsequently activate the vascular endothelium. To study the state of endothelial activation during falciparum malaria, we measured plasma levels of both von Willebrand factor (VWF) and its propeptide, indices of chronic and acute endothelial cell perturbation, respectively. Results were correlated with clinical and biochemical markers of disease severity, including plasma lactate. Our data show that acute endothelial cell activation is a hallmark of malaria in children, indicated by a significant rise in VWF and VWF propeptide. The highest VWF and propeptide levels were seen in cerebral and non-cerebral severe malaria, and associations found between VWF propeptide level and lactate (P < 0.001). Mean VWF propeptide levels (nmol/l) were in cerebral malaria 33.4, non-cerebral severe malaria 26.3, mild malaria 22.1, non-malaria febrile illness 10.2, and controls 10.1. Differences between patient and control groups were highly significant (P < 0.005). Follow-up of 26 cerebral malaria cases showed that levels of VWF propeptide, but not VWF fell by 24 h, following the clinical course of disease and recovery. These novel findings potentially implicate acute, regulated exocytosis of endothelial cell Weibel-Palade bodies in the pathogenesis of Plasmodium falciparum malaria.

Acute Activation of Endothelium Induces Circulation of Active Von Willebrand Factor in HELLP Sydrome.

Von Willebrand Factor (VWF) is unable to interact spontaneously with platelets under physiological conditions. To induce this interaction, a conversion of the VWF A1-domain into a Glycoprotein Ibα (GpIbα)-binding conformation is required. We recently described a nanobody, designated AU/VWFa-11 that preferentially recognizes the GpIbα-binding conformation (Hulstein et al. 2005 Blood, in press). Using an AU/VWFa-11 based immunosorbant assay, we found that the active form of VWF circulates in VWD type 2B and Thrombotic Thrombocytopenic Purpura. Although these diseases have different phenotypical appearances, both are characterized by thrombocytopenia. Another disease associated with a low platelet count is HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome, a severe form of preeclampsia that compromises pregnancy. In this study, we have compared the levels of activated VWF in the plasma of healthy pregnant women (n=9), patients suffering from preeclampsia (n=6) and patients with HELLP syndrome (n=14) at similar gestational age. In HELLP syndrome, the levels of activated VWF were increased 1.8-fold compared to healthy volunteers (p=0.0001). Moreover, these levels were also increased 1.5-fold compared to the patients suffering from preeclampsia. In order to get insight into the origin of the increased activated VWF levels, a number of other parameters were determined. VWF antigen levels were found to be increased in normal pregnancy (165% ± 32.5), as expected. In preeclampsia and HELLP syndrome these levels were even further increased (178% ± 89.8 and 338% ± 89.3, respectively). In healthy pregnant women and in preeclampsia, VWF propeptide levels were concomitantly increased. In contrast, in HELLP syndrome propeptide levels were increased up to 3-fold compared to propeptide levels in healthy pregnant women (p<0.0001) and 1.8-fold compared to patients with preeclampsia (p<0.04). When we calculated the propeptide /VWF antigen ratio, this ratio was found to be within the normal range in healthy pregnant women (0.11 ± 0.04) and preeclampsia (0.12 ± 0.03). However, this ratio was significantly increased in HELLP syndrome (0.17 ± 0.05, p<0.05). Because these increased ratios may represent secretion of VWF from the Weibel Palade bodies, these data suggests that acute activation of the endothelium occurs in HELLP syndrome and is the source of circulating active VWF. Therefore, we determined the amounts of active VWF secreted by human umbilical vein endothelial cells (HUVEC) in vitro. Medium containing constitutionally secreted VWF and medium from PMA stimulated endothelial cells was collected and the amount of active VWF was measured in the AU/VWFa-11-based immunosorbant assay. A 1.6-fold increase in activated VWF levels was found in medium containing constitutionally secreted VWF compared to normal pool plasma (p<0.05). The amount of activated VWF was increased even up to 2.2-fold in medium of the stimulated endothelial cells (p<0.001) indicating that activation of endothelium in HELLP could indeed result in increased levels of activated VWF. In conclusion, acute activation of the endothelium in HELLP syndrome results in increased amounts of activated VWF. This might well explain the thrombocytopenia associated with HELLP syndrome.

Endothelial cell activation in thrombotic thrombocytopenic purpura (TTP): a prospective analysis

It is generally assumed that endothelial cell injury is the primary event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In this study, we have determined the extent of vascular perturbation during acute episodes of the disease. We performed a prospective, serial study of nine patients with relapsing TTP during hospitalization and treatment, and assessed the degree of endothelial cell involvement at admission, exacerbation and remission by measurement of von Willebrand factor (VWF) and VWF-propeptide levels. Measurement of both VWF and its propeptide enabled discrimination between acute and chronic perturbation of the endothelium. Elevated levels of both VWF and propeptide were found at admission. These levels decreased immediately upon plasma exchange therapy. However, plasma VWF and propeptide concentrations did not change, even at the time of acute exacerbation. These observations suggest that endothelial cell activation is not the primary event leading to TTP. Vascular perturbation seems to be a consequence, rather than a cause, of the disease.