A NEW SIMPLE AND RELIABLE METHOD FOR INVESTIGATING THE AFFINITY BETWEEN COLLAGEN AND VON WILLEBRAND FACTOR IN PLASMA SAMPLES

Abstract number: P-T-210

Karlman¹ M., Wiman¹ B.

¹Department of Clinical Chemistry, Karolinska University Hospital, Institution of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

How-to-cite Karlman M, Wiman B. A NEW SIMPLE AND RELIABLE METHOD FOR INVESTIGATING THE AFFINITY BETWEEN COLLAGEN AND VON WILLEBRAND FACTOR IN PLASMA SAMPLES. J Thromb Haemost 2007; 5 Supplement 2: P-T-210

Abstract

Introduction: The interaction between collagen and von Willebrand factor (vWf) is clinically important and might be used in clinical routine for the diagnosis of von Willebrand disease. Commercially available methods have not met the quality criteria for diagnostic use. Therefore we have developed a new ELISA system for use in plasma samples.

Methods: For this purpose microtiter plates have been coated with collagen type III from human placenta (C4407, Sigma). After washing, diluted plasma samples were added and incubated for 1 h. Then HRP-conjugated antibodies against vWf were added and used for quantification of bound vWf.

Results: If the samples were diluted to give similar final concentrations of vWf, the bound vWf was proportional to the affinity between collagen and vWf. The interaction diminished dramatically if pH was decreased below 6.5. This suggests the involvement of one or more His-residues in the interaction. To prove this, histidine/imidazole was added to the reaction mixtures. The binding of vWf decreased in a concentration dependent manner. At about 150 mmol/L of these ligands, 50% binding was obtained. Dipeptides with His as NH2-terminal or COOH-terminal amino acid acted in similar ways. Just an increase in ionic strength did not affect the binding at this concentration level. The collagen binding in individuals with a normal vWf concentration and collagen binding was determined as 100% 5.0% (SD).

Conclusions: A new simple and very reproducible method for analyzing the interaction between collagen and vWf in plasma samples has been developed. The binding of vWf to collagen involves one or more His residues.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

Session Details

Date:	01/08/2007
Time:	00:00-00:00
Session name:	XXIst ISTH Congress
Subject:
Location:	Oxford, UK
Presentation type:
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