Glycosaminoglycans as Tools to Decipher the Platelet Tumor Cell Interaction: A Focus on P-Selectin

Abstract

Tumor cell-platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.

Keywords: 2-O-desulfated heparin; P-selectin; RO-heparin; decasaccharide heparin fragment; hexasaccharide heparin fragment; low molecular weight heparin; platelet aggregation; platelet secretion; platelets; tumor metastasis; unfractionated heparin.

Figure 1

Tumor cell-induced platelet secretion and aggregation. (A,B) Representative traces showing platelet-tumor cell aggregation in response to increasing concentrations of (A) MDA-MB-231 cells for 20 min (n = 5) or (B) MCF-7 cells for 30 min; (C) quantification of ATP release from resting platelets, 1 × 10⁴ MDA-MB-231 cells/mL, platelets co-incubated with 1 × 10⁴ MDA-MB-231 cells/mL for 30 min, platelets treated with 1% Triton X-100, or MDA-MB-231 cells (1 × 10⁴ cells/mL) treated with 1% Triton X-100, respectively; (D) quantification of ATP release from resting platelets, MCF-7 cells, platelets co-incubated with MCF-7 cells for 30 min, platelets treated with 1% Triton X-100, or MCF-7 cells (1 × 10⁴ cells/mL) treated with 1% Triton X-100, respectively; (E) representative traces showing platelet aggregation in response to MDA-MB-231 cells (1 × 10⁴/mL) or MCF-7 cells (1 × 10⁴/mL) (n = 5). Platelets were preincubated with 1 IU/mL UFH (left part of the figure) (n = 5). Quantification of ATP release from MDA-MB-231 (1 × 10⁴/mL) cell or MCF-7 (1 × 10⁴/mL) cells stimulated platelets preincubated with 1 IU/mL UFH (n = 5) (right part of the figure); (F) representative traces showing platelet aggregation in response to MDA-MB-231 cells (1 × 10⁴/mL) or MCF-7 cells (1 × 10⁴/mL) (n = 5). Platelets were preincubated with 775 ng/mL fondaparinux (left part of the figure) (n = 5). Quantification of ATP release from MDA-MB-231 (1 × 10⁴/mL) cell or MCF-7 cells (1 × 10⁴/mL) stimulated platelets preincubated with 775 ng/mL fondaparinux (n = 5) (right part of the figure). *** p < 0.001 indicated statistical significance.

Figure 2

Impact of heparin derivatives on platelet aggregation and secretion. (A,B) Representative traces showing platelet-tumor cell aggregation in response to MCF-7 cells. Platelets were preincubated for 30 min with (A) 100 µg/mL RO-heparin or (B) 100 µg/mL 2-O-desulfated heparin; (C) quantification of ATP release from MCF-7 cell stimulated platelets preincubated with 100 µg/mL RO-heparin or 100 µg/mL 2-O-desulfated heparin; (D,E) representative traces showing platelet-tumor cell aggregation in response to MCF-7 cells, platelets were preincubated with (D) 100 µg/mL hexasaccharide or (E) 100 µg/mL decasaccharide (n = 5); (F) quantification of ATP release from MCF-7 cell stimulated platelets preincubated with 100 µg/mL hexasaccharide or 100 µg/mL decasaccharide; (G) representative traces showing platelet-tumor cell aggregation in response to MCF-7 cells. Tumor cells were preincubated with 1 µg/1000 cells recombinant human P-selectin (n = 5); (H) representative traces showing platelet-tumor cell aggregation in response to MCF-7 cells. Platelets were preincubated with 100 µg/mL P-selectin inhibitor (n = 5); (I) quantification of ATP release from MCF-7 cell (preincubated with 1 µg recombinant human P-selectin/1000 cells in some experiments) stimulated platelets preincubated with 100 µg/mL P-selectin inhibitor. *** p < 0.001 indicated statistical significance.

Figure 3

Impact of heparin derivatives on platelet aggregation and secretion after platelet shape change. (A) Platelets were stimulated with TRAP-6, MDA-MB-231, or MCF-7 cells, respectively, and P-selectin expression was determined by flow cytometry; (B) representative traces showing platelet-tumor cell aggregation in response to PAR-1 receptor agonist TRAP-6 (41 µM or 10 µM, respectively). Platelets were preincubated for 30 min with 100 µg/mL P-selectin inhibitor (n = 5); (C) representative traces showing platelet-tumor cell aggregation in response to MDA-MB-231 or MCF-7 cells, respectively. P-selectin inhibitor (100 µg/mL) was added 5 min (10 min in case of MCF-7 cells) after tumor cell addition when platelet shape change was in progress (n = 5); (D–F) representative traces showing platelet-tumor cell aggregation in response to MDA-MB-231, or MCF-7 cells, respectively, when RO-heparin (100 µg/mL) (D), 2-O-desulfated heparin (100 µg/mL) (E), or fondaparinux (775 ng/mL) (F) were added 5 min/10 min after tumor cell addition when platelet shape change was in progress (n = 5).

Figure 4

P-selectin inhibition impacts platelets’ secretion. Heparanase secretion from platelets activated with TRAP-6, MDA-MB-231 cells, or MCF-7 cells, respectively. Platelets were preincubated with 1 IU/mL UFH or 100 µg/mL P-selectin inhibitor, for 30 min in some cases. Proteome profiler mediated quantification of the platelet secretion due to MDA-MB-231 or MCF-7 cell (1 × 10⁴ cells/mL) induced secretion. Platelets were preincubated with P-selectin inhibitor for 30 min, as indicated in the figure, and PDGF-AA, PDGF-AB/BB, Serpin E1, CXCL5, CCL3/CCL4, BDNF, MIF, and angiopoietin were quantified in the releasates (n = 1).

Figure 5

P-selectin mediated signaling in platelets. (A) Immunoblot analysis using an anti-phosphoserine/phosphothreonine/phosphotyrosine antibody of lysates from resting platelets, platelets after activation with 41 µM TRAP-6, after co-incubation with MDA-MB-231 or MCF-7 cells (1 × 10⁴ cells/mL), respectively. Platelets were preincubated with 100 µg/mL P-selectin inhibitor for 30 min in some samples. Standard from the same gel at an earlier time was inserted in the figure to avoid overexposure. The orange box indicates changes in platelet phosphoproteome; (B) proteome profiler Phospho-Kinase array kit of lysates from platelets co-incubated with P-selectin inhibitor or without coincubation and stimulated with MCF-7 cells. Twelve kinases (as indicated in the figure) involved in platelet signaling were quantified simultaneously. The orange boxes indicate changes in phosphorylation of different kinases due to P-selectin inhibition; (C) immunoblot analysis of lysates using an anti-pERK1/2 antibody from resting platelets, platelets after activation with 41 µM TRAP-6, after coincubation with MDA-MB-231, or MCF-7 cells (1 × 10⁴ cells/mL), respectively. Platelets were preincubated with 100 µg/mL P-selectin inhibitor in some samples.