Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growthfactor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observ ation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recentl y been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cel l-platelet adherence and tumor cell TF activity on platelet VEGF release; a nd (2) the effects of GpIlb/IIIa blockade on tumor cell-induced platelet VE GF release, tumor cell-induced thrombocytopenia and experimental metastasis . Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) c ells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of V EGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich pla sma with or without anti-GpIIb/IIIa (Abciximab(TM)) was added to A375 monol ayers and supernatant VEGF measured by ELISA, Tumor cell-induced platelet a ctivation and release were determined by CD62P expression and serotonin rel ease respectively. Ire vitro, tumor cell-platelet adherence was evaluated b y flow cytometry, In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcin oma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rat s. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 c ells had adhered to platelets, Abciximab(TM) significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry, Incubati on of A375 cells with platelets induced VEGF release in a time-dependent ma nner. This release was significantly inhibited by Abciximab(TM) (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced b y A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001), Omitting fibrinogen or Fn, fr om the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/III a blockade with murine 7E3 F(ab')(2) reduced LL2 tumor cell-induced thrombo cytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude th at TF-bearing tumor cells can activate platelets largely via thrombin gener ation, and that such activation is associated with release of VEGF. This ma y enhance metastasis, possibly by increasing extravasation at points of adh esion to vascular endothelium.