A comparative evaluation of a new fully automated assay for von Willebrand factor collagen binding activity to an established method.

INTRODUCTION: Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ristocetin cofactor and collagen binding (VWF:CB) assays. AIM: We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme-linked immunosorbent assay (ELISA) method. METHODS: The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2-step immunoassay that uses magnetic particles coated with a type III collagen triple-helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3). RESULTS: Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method). CONCLUSION: The new assay is rapid and simple to use, with its ready-to-use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.

Mitogen-induced p53 downregulation precedes vascular smooth muscle cell migration from healthy tunica media and proliferation.

The tumor suppressor protein p53 plays an important role in the cell-cycle G(1) and G(2) checkpoints. In response to DNA damage, p53 can induce the transcription of p21, which inhibits the activation of various G(1) cyclin/cyclin-dependent kinase complexes. It is not known whether p53 plays a role in the initial migration of vascular smooth muscle cells from the arterial tunica media (mVSMCs). In this study, we have investigated whether mVSMC migration from healthy tunica media of young pigs and proliferation are regulated by p53. After 6 hours of incubation in mitogen-rich medium, explanted porcine tunica media tissue showed complete downregulation of p53 protein and p53 mRNA. The blockage of gene activity was not due to DNA methylation at the 5' control region of the gene. The mVSMC outgrowth did not show p53 expression. Mitogen-depletion of cultured p53(-)/mVSMCs did not restore p53 expression. Incubation of explanted porcine tunica media tissue in mitogen-deprived medium increased p53 protein content and blocked mVSMC outgrowth from the explant. As in p53-deficient rodent cells, mVSMCs incubated with colcemid overrode the spindle-dependent checkpoint, giving polyploidy and chromosomal pairing. UV-induced DNA damage in mVSMCs incubated with mitogen-free medium induced p53 expression and apoptotic cell death showing DNA nucleosomal laddering. However, UV-irradiated mVSMCs incubated in mitogen-rich medium did not express p53 and did not show cell death. In conclusion, our results demonstrate that early mVSMC migration from the tunica media requires mitogen-induced suppression of p53 that is highly expressed in contractile mVSMCs residing in the healthy vessel wall.