Plasma concentrations of (A) D-dimer, (B) F1+2, (C) TAT, and (D) PAP in healthy volunteers during and after autologous serum transfusion.

<p>Transfusion of 50 mL serum was started at t = 0 and completed at t = 0.5 h. Data points show the median of n = 15 probands, error bars show the interquartile range. Smooth curves show best least squares fit exponential decay functions.</p

The mechanisms how heparin affects the tumor cell induced VEGF and chemokine release from platelets to attenuate the early metastatic niche formation

<div><p>Metastasis is responsible for the majority of cancer associated fatalities. Tumor cells leaving the primary tumor and entering the blood flow immediately interact with platelets. Activated platelets contribute in different ways to cancer cell survival and proliferation, e.g. in formation of the early metastatic niche by release of different growth factors and chemokines. Here we show that a direct interaction between platelets and MV3 melanoma or MCF7 breast cancer cells induces platelet activation and a VEGF release in citrated plasma that cannot be further elevated by the coagulation cascade and generated thrombin. In contrast, the release of platelet-derived chemokines CXCL5 and CXCL7 depends on both, a thrombin-mediated platelet activation and a direct interaction between tumor cells and platelets. Preincubation of platelets with therapeutic concentrations of unfractionated heparin reduces the tumor cell initiated VEGF release from platelets. In contrast, tumor cell induced CXCL5 and CXCL7 release from platelets was not impacted by heparin pretreatment in citrated plasma. In defibrinated, recalcified plasma, on the contrary, heparin is able to reduce CXCL5 and CXCL7 release from platelets by thrombin inhibition. Our data indicate that different chemokines and growth factors in diverse platelet granules are released in tightly regulated processes by various trigger mechanisms. We show for the first time that heparin is able to reduce the mediator release induced by different tumor cells both in a contact and coagulation dependent manner.</p></div

Impact of heparin on platelet-derived VEGF release.

<p>(A) Impact of UFH or fondaparinux, respectively, on MV3 cell induced VEGF release from platelets. (B) Impact of UFH or fondaparinux, respectively, on MCF7 cell induced VEGF release from platelets. (C) Impact of UFH on tube formation of EA.hy926 cells induced by MV3 and MCF7 mediated platelet activation. (D) Representative pictures of tube formation of EA.hy926 cells induced by releasates from platelets incubated with MV3 or MCF7 cells (for 12 min) previously. In some cases platelets were preincubated with UFH. Bars correspond to 100 μm.</p

Tumor cell-induced platelet activation in defibrinated plasma.

<p>(A) Thrombin generation in recalcified PPP and recalcified, defibrinated PPP due to TF addition. (B) MV3 cell mediated effect on VEGF release in PRP and defibrinated PRP after preincubation with UFH and fondaparinux, respectively, and recalcification (FF, fibrin free plasma). (C) MV3 cell mediated effect on CXCL5 release in PRP and defibrinated PRP after preincubation with UFH and fondaparinux, respectively, and recalcification (FF, fibrin free plasma). (D) MV3 cell mediated effect on CXCL7 release in PRP and defibrinated PRP after preincubation with UFH and fondaparinux, respectively, and recalcification (FF, fibrin free plasma).</p

Tumor cell induced platelet-derived CXCL5 and CXCL7 release.

<p>(A) Platelets in citrated plasma were treated with ADP and TRAP-6 and CXCL5 release was quantified by ELISA. (B) Platelets were incubated with MV3 or MCF7 cells, respectively, and CXCL5 release was determined by ELISA. (C) Platelets, preincubated with UFH or fondaparinux, respectively, were incubated with MV3 cells. CXCL5 release was quantified by ELISA. (D) Platelets, preincubated with UFH or fondaparinux, respectively, were incubated with MCF7 cells. CXCL5 release was measured by ELISA. (E) Platelets were either treated with ADP or TRAP-6 and CXCL7 release was quantified by ELISA. (F) Platelets were incubated with MV3 or MCF7 cells, respectively, and CXCL7 release was determined by ELISA. (G) Platelets, preincubated with UFH or fondaparinux, respectively, were incubated with MV3 cells. CXCL7 release was determined by ELISA. (H) Platelets, preincubated with UFH or fondaparinux, respectively, were incubated with MCF7 cells. CXCL7 release was measured by ELISA.</p

Tumor cell characterization concerning thrombin generation.

<p>(A) Thrombin generation of MV3, MCF7 cells and freshly isolated monocytes, respectively, was determined in platelet-rich plasma (PRP) without recalcification and compared to the effect of tissue factor (TF) (5 pM). No thrombin formation was detectable. (B) Thrombin generation of MV3, MCF7 cells was determined in recalcified PRP and compared to 5 pM TF. (C) Thrombin generation of MV3, MCF7 cells and monocytes, respectively, was quantified in recalcified PPP and compared to 5 pM TF. (D) Thrombin generation of MV3, MCF7 cells and monocytes, respectively, was quantified in recalcified Factor VII deficient PPP and compared to the effect of TF (5 pM).</p

Tumor cell induced platelet activation.

<p>Platelets in citrated plasma were incubated with ADP, TRAP-6, or MV3 melanoma cells, respectively, and P-selectin or integrin α_IIbβ₃ (CD41/CD61) were labeled either with anti-P-selectin mAbs or FITC-fibrinogen as ligand for activated integrin α_IIbβ₃. P-selectin presentation and integrin α_IIbβ₃ activation were determined by flow cytometry and compared to unstimulated platelets.</p

Interaction of tumor cells with platelets.

<p>(A) Representative pictures of Calcein-AM labeled platelets interacting with confluent layers of MV3 cells (black background). Scale bar 25 μm. (B) Representative pictures of ADP or TRAP-6 activated and Calcein-AM labeled platelets adhering to confluent layers of MV3 cells (black background). Scale bar 25 μm. (C) Quantification of platelet (Calcein-AM labeled) tumor cell (MV3 and MCF7) interaction with a plate reader. In some cases platelets were activated either with ADP or TRAP-6. (D) Impact of UFH or fondaparinux, respectively, on platelet (Calcein-AM labeled) MV3 cell interaction. (E) Impact of UFH or fondaparinux, respectively, on platelet (Calcein-AM labeled) MCF7 cell interaction.</p

Thrombin generation parameters of FVIII variants.

<p>FVIII concentration for each variant was adjusted to 5 ng/ml in complete medium. Subsequently each variant was diluted 1:1 in FVIII deficient plasma and triggered with 4 μM of phospholipids. The mean is calculated from three independent experiments performed in duplicate. (WT: wild-type; ETP: Endogenous thrombin potential; ttpeak: time to peak; SD: Standard deviation of mean).</p

The cleavage site region interface.

<p>The panels A and B in this image illustrate a close up view of the Arg³⁹¹ (a1-A2 junction), Arg⁷⁵⁹ (a2-B junction) cleavage site regions and their putative interactions in the crystal structure of FVIII. The cleavage site regions Arg³⁹¹ (a1-A2 junction) and Arg⁷⁵⁹ (a2-B junction) have been remodeled. Both panels show the same view but are colored differently. In both panels the backbone is depicted in ribbon format. Panel A illustrates the region surrounding the Arg³⁹¹ (a1-A2 junction) as a red ribbon while the Arg⁷⁵⁹ (a2-B junction) region is depicted in blue ribbon format. In Panel B both regions are colored based on their secondary structure (i.e. blue: helix, cyan:coil,yellow:short helix, red:beta sheet). The cleavage site P1 and P1´ residues are depicted in stick format. In Panel A they are colored green while in Panel B they are colored based on atom (i.e. white:hydrogen, blue: nitrogen and cyan:carbon). In both panels the hydrogen bonds are depicted as yellow colored dots extending from their donor to the acceptor atom. The interface region between the two cleavage site regions is marked with a blue shaded area.</p