The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways

Abstract

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

Keywords: VEGF; heparin; metastatic niche; platelet; thrombin; tumor.

Figure 1

Flow cytometric analysis of TF expression by different tumor cells. (A) Human MDA-MB-231 breast cancer cells possess a strong TF expression. (B) Human MV3 melanoma cells display a certain capacity to express TF but they rank behind the indicated breast cancer cells. (C) The PC-3 prostate cancer cells exhibit nearly no fluorescence signals indicative of TF expression. Figures illustrate representative data of at least three identical experiments (n = 3).

Figure 2

Thrombin generation by tumor cells and the interference by anticoagulants. (A) The addition of TF to the fluorigenic thrombin generation assay induces a signal that can be diminished by UFH, tinzaparin and fondaparinux, but not by the non-anticoagulant RO-heparin. Thrombin generation by (B) MDA-MB-231 cells, (C) MV3 melanoma cells, and (D) PC-3 prostate cancer cells and the inhibitory effects of the heparin derivatives, or fondaparinux, respectively. While UFH and tinzaparin prevent thrombin generation nearly completely in the individual approaches, and the inability of RO-heparin confirms the non-anticoagulant properties of this derivative, the restricted activity of fondaparinux remains elusive and probably refers to other activation pathways. The data are representative illustrations of at least three identical experiments.

Figure 3

Aggregation of platelets as an activation parameter, measured by light transmission aggregometry in platelet buffer. TRAP-6 was applied as a functional control in the individual experiments (orange). (A) MDA-MB-231 cells trigger a massive platelet aggregation, which can be prevented by adapted therapeutic concentrations of UFH or tinzaparin, or RO-heparin (100 µg/mL). FXa inhibitor fondapariux is ineffective. Similar findings were obtained in the case of MV3 cells (B). (C) PC-3 cells have no impact on platelet aggregation. Data are representative of three identical experiments (n = 3).

Figure 4

ATP release from platelets’ dense granules as indicator of activation, detected by luminescence measurements. The effect of TRAP-6 as a functional control was considered as 100% standard. MDA-MB-231 and MV3 cells induce a strong ATP release that can significantly be prevented by therapeutically relevant concentrations of UFH and tinzaparin; RO-heparin, while fondaparinux is ineffective. PC-3 cells display again no capacity to activate platelets (n = 3). Asterisks indicate statistical significance: *** p < 0.001.

Figure 5

Detection of VEGF release from α-granules of platelets induced by tumor cells. MDA-MB-231 (left) and MV3 cells (middle) trigger a massive release of VEGF from α-granules, which can be significantly prevented by RO-heparin or therapeutic relevant concentrations of UFH or tinzaparin, while fondaparinux is unable to interfere with this activation route. Asterisks indicate statistical significance: * p < 0.05; ** p < 0.01.

Figure 6

Tube formation assay to illustrate the impact of platelets, activated by tumor cells on the angiogenic activity of endothelial cells. (A) EA.hy926 endothelial cells were seeded on a geltrex matrix, coincubated with platelet releasates and the sprouts were quantified after 24 h microscopically. Releasates of platelets that have been activated by MDA-MB-231, MV3, or PC-3 cells are presented and the impact of the indicated anticoagulants at therapeutic concentrations thereon. UFH and tinzaparin possess significant effects to reverse the endothelial tube formation by MDA-MB-231 and MV3 cells treated platelets, while RO-heparin is less effective, fondaparinux is ineffective. (B) Representative images of the angiogenetic approach showing EA.hy926 cell cultivations triggered by VEGF or platelets releasates activated by MDA-MB-231 cells without inhibition “TC+Plt” or MDA-MB-231 cell activated platelet releasates after administration of the indicated anticoagulants. The tube formation density is evidently affected by UFH and tinzaparin. Asterisks indicate statistical significance: * p < 0.05; ** p < 0.01.