Control of von Willebrand factor multimer size by a fibronectin related substance.

KEMPFER ANA C; FARÍAS CRISTINA; CARBALLO GONZALO A; AMARAL MARÍA M; LAZZARI MARÍA A

Birmingham

Congreso; XIX CONGRESS. THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS; 2003

THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS

We have previously found that fractions (F) II and III obtained by heparinsepharose separation of partially purified fibronectin (FN) with cathepsin D and F3 obtained like FIII (highest heparin affinity) but from untreated FN exerted activity (arFN) on unfolded purified von Willebrand factor (VWF) that controls VWF multimer size. Our aim was to evaluate the arFN of commercial 30-kDa (with heparin affinity), 45-kDa (gelatine affinity) and 70-kDa (gelatine and heparin affinity) FN products. In addition, we evaluated the activity in the following F: F3a obtained like F3 but from plasma substances of Mr in order of 90 kDa with gelatine affinity without FN compounds that had been removed by CNBr-Sepharose 4B with immobilized policlonal antibody against FN (a-FN) and different aliquots obtained from F3a separation on Sephadex G-50 column. The arFN was detected in 30-kDa, 45-kDa, 70-kDa products, F3a and F3b aliquot in the fractionation range of Sephadex column from F3a. Zymography studies (gelatin 1 mg mL-1 ) of products and F did not show matrix metalloproteinases (that copurified with FN in chromatography on Sepharoses conjugated with gelatine or heparin). Characterization studies of F3b in comparison with preceding aliquot from Sephadex G-50 column, revealed high concentration of a band of approximately 60 kDa and several bands of c. 30 kDa. Our data suggest that a fragment of Mr in order of 60 kDa that copurified with FN, with affinity to heparin and gelatine, has the arFN that controls VWF multimer size. Therefore, the arFN fragment can hardly be associated with von Willebrand factor cleaving protease due to its different Mr and to its binding to heparin-Sepharose column. The arFN fragment is unlikely to be associated with thrombospondin-1 because of its different Mr F3b would not be related to FN because its FN compounds were removed, however, the 30-kDa FN fragment from commercial 30 kDa FN product was not removed by a-FN. Further work will elucidate the role of Fb3 (free plasma fragment) in clinical pathology.