Influence of dry heat treatment and anticoagulant on coupling of fibrinogen and fibronectin to von Willebrand factor in normal plasma

Abstract

In plasma from healthy subjects a coupling was identified between von Willebrand factor (vWf), fibrinogen (fg), and fibronectin (fn) that was dependent of anticoagulants heparin, Edta, and citrate.

Binding was quantitated by Elisa methodologies, based on polyclonal antibodies directed against the proteins studied, in order to express the percentage of moles of fg or fn bound to moles vWf, C[fg/vWf] or C[fn/vWf] (mol/mol)%. The intra-assay coefficients of variation (CV%) for fg and fn bound to vWf were 10.6% and 7.4% (n=10) respectively, and the inter-assay coefficients of variation were 24.4% and 22.2% (n=10). The largest degree of coupling was found in heat-treated lyophilized heparin plasma, where C[fg/vWf] andC[fn/vWf] were 12.9±1.4(mol/mol) % and 2.4±0.1 (mol/mol) % (mean±SD). Binding was further qualitatively demonstrated through experiments using gel filtration chromatography and agarose gel electrophoresis followed by immunoblotting. In all instances coupling of vWf with fg was higher than with fn. Lyophilisates of normal plasma that were subjected to dry heating (60 °C in 72 h) showed considerably increased coupling.

Previous investigators, studying reconstituted factor VIII concentrates by means of gel filtration, pointed out that an association between vWf, fg and fn was present in such therapeutic material. This investigation signifies that a coupling between these proteins may be present even in the source plasma, and that ‘dry heating’ increases binding. Implications of these results are discussed.

Key Words:

• aggregate
• binding
• fibrinogen
• fibronectin
• von Willebrand factor