The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives

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• 作者：Martin Schlesinger^a ; ^{mschlesi@gmx.de} ; Patrick Schmitz^a ; Reiner Zeisig^b ; Annamaria Naggi^c ; Giangiacomo Torri^c ; Benito Casu^c ; Gerd Bendas^a
• 关键词：ECM ; extracellular matrix ; EDC ; N-ethyl-N-(dimethylaminopropyl)-carbodiimide ; FCS ; fetal calf serum ; HRP ; horseradish peroxidase ; KD ; equilibrium dissociation constant ; koff ; dissociation rate constant (off-rate) ; kon ; association rate constant (on-rate)
• 刊名：Thrombosis Research
• 出版年：2012
• 期刊代码：247_00493848
• 类别：med
• 出版时间：May, 2012
• 卷：129
• 期：5
• 页码：603-610
• 文件大小：717 K

摘要

Introduction

The integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated.

Materials and methods

A series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin.

Results

Experimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin`s 伪-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50%6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range.

Conclusions

These findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50%6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo.