Summary
Heparin possesses antimetastatic effects that were related to various molecular mechanisms
beyond anticoagulant activities. The ability of heparin to interfere with the function
of adhesion receptors in the metastatic course appears as a promising therapeutic
approach. This refers to numerous findings that heparin attenuates metastasis in a
selectin-dependent manner. We recently demonstrated that heparin interferes with the
integrin VLA-4 on murine melanoma cells binding to VCAM-1. To confirm this activity
and to obtain further insight into molecular recognition of heparin by VLA-4, we investigated
the inhibition of VLA-4 mediated binding of human melanoma MV3 cells to immobilized
VCAM-1 by different heparins. The size of heparin has an important impact on inhibition.
Unfractionated heparin (UFH) and tinzaparin, a low-molecular-weight heparin (LMWH)
representing a mean of about 18– 20 monomers, displayed high inhibitory activity.
Fractionating tinzaparin to 14– 18 monomers reduced inhibition slightly, while the
pentasaccharide fondaparinux was without effects. To confirm molecular recognition
of tinzaparin by VLA-4,a surface acoustic wave-biosensor was applied. A VLA-4 containing
membrane preparation of MV3 cells was immobilised at the sensors to allow for detection
of kinetic binding constants of tinzaparin compared to VCAM-1. Tinzaparin binds to
VLA-4 with affinity in the low micromolar range (4.61× 10−6 M), which clearly indicates specific molecular recognition. Furthermore, tinzaparin
displays a nearly identical koff compared toVCAM-1 (5.13× 10−3 s-1 versus 3.44× 10–3 s-1) which is evident for interference with the ligand binding. The data provide evidence
for a direct confirmation of heparin binding to VLA-4 and thus, contribute to understand
the antimetastatic activity of heparin.
Keywords
Heparin - melanoma cell adhesion - VLA-4 - antimetastatic activity
