Publication Date:
2006-11-16
Description:
Circulating soluble fibrin (sFn) is a marker for ongoing disseminated intravascular coagulation and may have prognostic significance, especially in metastatic cancers. Anti-coagulant therapies have been effective in reducing metastasis in several cancers, but with increased risk of bleeding. The authors have previously demonstrated that soluble fibrin (sFn), which is elevated in many cancer patients, enhances metastasis in an experimental model, and increases platelet/tumor cell adherence by cross-linking platelet aIIbb3 to tumor cell CD54 (a receptor for two of the leukocyte b2 integrins aLb2 and aMb2). sFn also binds to monocyte aMb2 (Mac1), and the peptide sequences of the fibrin(ogen) binding sites for aMb2 and CD54 have recently been identified. It was, therefore, hypothesized that sFn binding to these receptors would result in inhibition of monocyte/tumor cell adherence, and consequently cytotoxicity, which may be reversed by inclusion of blocking peptides. To test this, monocyte adherence to A375 melanoma cells was quantified at physiologically relevant shear rates (35–560 s−1) using a laminar flow perfusion chamber mounted on a Leica DMIRB inverted microscope equipped with a computer controlled digital imaging camera. Effector and target cells were untreated, or incubated with sFn (fibrinogen (Fg), 0.5 mg/ml; fibrin polymerization inhibitor Gly-Pro-Arg-Pro amide (GPRPa), 4 mM; and human thrombin (0.125 U/ml)) in the presence or absence of specific single or combined blocking peptides directed against the sFn binding sites on CD54 (P1) and aMb2 (P2), or the sFn g-chain binding sites for CD54 (P3) and aMb2 (P4). The effect of peptides on thrombin (0.125 U/ml) induced clotting of purified Fg (0.5 mg/ml) was assessed visually. The effect of sFn on monocyte cytotoxicity (effector: target 20:1) against green fluorescent protein (GFP) transfected A375 melanoma cells was measured by GFP release from lysed cells using a Perkin-Elmer Victor-3 96-well fluorescence plate reader. Pre-treatment of tumor cells with sFn significantly (P0.05) on adherence. However, pre-incubation of both cells with sFn resulted in a significant (P
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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