Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features

Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis

Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin alpha IIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb(-/-)) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb(-/-) mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb(-/-) mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb(-/-) mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases

Platelet-tumor-aggregate formation <i>in vivo</i>.

<p><b>a)</b> GFP-transfected B16-D5 melanoma cells were injected intravenously into wildtype mice. After 1 hour, lung tissue was obtained for immunofluorescence analysis. Photomicrographs show mouse lung tissue stained with antibodies directed against platelet GPIIb (CD41, red) and B16-D5 (GFP, green); nuclei were stained with DAPI (blue). Bars, 40μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. <b>b-d)</b> Arrest of DCF-tagged B16-D5 melanoma cells was visualized in the pulmonary vasculature by intravital confocal videofluorescence microscopy (IVM) in GPIIb^+/+ and GPIIb^-/- littermate mice immediately and after 1 hour. <b>b)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- mice compared to its WT littermate (n = 5–6 experiments per group; *<i>P</i><0.01 acute; **<i>P</i><0.001 after 1 hour). <b>c)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- mice compared to its WT littermate (n = 5–6; *<i>P</i><0.05 after 1 hour). <b>d)</b> Photomicrographs show representative IVM images obtained in GPIIb^+/+ and GPIIb^-/-. In GPIIb^+/+ mice, arrest of large multicellular aggregates is frequently observed (left). Arrest of DCF-tagged B16-D5 is visualized in precapillary vessels (right). Bars, 20μm. <b>d e-f)</b> GPIIb^+/+ (WT) or GPIIb^-/- platelets were injected into GPIIb^-/- mice just prior to administration of DCF-labeled B16-D5. IVM was performed immediately and after 1 hour. <b>e)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 4; *<i>P</i><0.05). <b>f)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 3; P = n.s.).</p

Effect of platelet GPIIb on melanoma metastasis formation.

<p><b>a)-c)</b> B16-D5 melanoma cells were injected intravenously into GPIIb^+/+ or GPIIb^-/- littermate mice. After 10 days, metastasis formation was analyzed. <b>a)</b> Photomicrographs show representative lung images. Bars, 0.5 cm. <b>b)</b> Quantification of metastatic nodules in the lung (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. <b>c)</b> Quantification of Pmel mRNA expression. Individual Pmel data normalized to beta-actin is indicated as 2^(-ΔCt) (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. <b>d-e)</b> Pulmonary melanoma proliferation was determined by analyzing Ki67 expression in HMB-45-positive melanoma cells 10 days after intravenous seeding (n = 5; *P<0.05). Photomicrographs show fluorescence microscopy images of mouse lung tissue stained with antibodies directed against melanoma HMB-45 (green) and Ki67 (red); nuclei were stained with DAPI (blue). Images show 3μm optical sections. Left image, overview of lung tissue section; right image, magnification. Bars, 100μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x and 40x objective. <b>f)</b> Effect of platelets on M21 mitogenesis <i>in vitro</i>. Serum-starved M21 cells were grown in the absence or presence of increasing concentrations of washed human platelet and BrdU uptake was quantified (*P<0.05, **P<0.01). <b>g-h)</b> Effect of platelet GPIIb on subcutaneous melanoma growth. <b>g)</b> B16-D5 cells were injected subcutaneously in the right flank of wildtype and GPIIb^-/- littermate mice and tumor growth surveyed using a digital scaliper. <b>h)</b> 21 days after tumor cell seeding tissues were explanted. Tumor weight was measured indicating no difference in subcutaneous tumor growth (P = n.s.).</p

Platelet-tumor-endothelium interaction under flow.

<p><b>a)</b> Photomicrographs show platelet and M21 aggregate formation under flow (1000/s). Co-perfusion of platelets and M21 cells induced formation of aggregates which did not firmly adhere and are therefore only partly focused (middle picture; arrows indicate platelet-M21 aggregates). Aggregation is virtually abolished in the absence of platelets (left picture) or the presence of c7E3 Fab (right picture). Bars 0.2mm. Images were taken using a Zeiss Axiovert 100 microscope, 20x objective. <b>b)</b> HUVEC (resting or TNF-α/IFN-γ-stimulated) were perfused with M21 or αv-deficient M21L melanoma cells, that lack the expression of both αvβ3 and αvβ5 integrins, in the absence and presence of platelet-rich plasma (10min, shear rate 1000/s). Results are shown as number of firmly adherent M21 and M21L (n = 5; *<i>P</i><0.05 for M21 vs. M21+PRP on activated HUVEC). <b>c)</b> Frequency of M21 and M21L aggregates in the absence and presence of platelets and c7E3 antibody. Results are given as number of melanoma cell-aggregates in % of all perfused tumor cells (n = 5; *<i>P</i><0.001 for M21 in the absence vs. presence of platelets). <b>d)</b> Size distribution of M21 aggregates in the absence and presence of platelets and c7E3. <b>e-g)</b> M21 melanoma cells were incubated for 15 minutes in the absence and presence of platelet-rich plasma. <b>e)</b> Scanning electron microscopy indicates single melanoma cells in the absence (left picture) and melanoma cell aggregates in the presence of platelets (right picture). Bars, 5μm. <b>f)</b> Transmission electron microscopy indicates direct platelet-melanoma interaction. Bar, 1μm. <b>g)</b> Photomicrographs show representative fluorescence microscopy images of M21-platelet aggregates stained with phalloidin (red) and anti-GPIIb (CD41) mAb (green). Bars, 20μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. <b>h-i)</b> Analysis of mouse GPIIb^-/- or WT platelet interaction with B16 melanoma under flow conditions (1000/s). <b>h)</b> Percentage of platelet-positive B16 cells (n = 4; *<i>P</i><0.05). <b>i)</b> Representative FACS contour plots indicate CD42-staining on B16 cells in the absence (control) and presence of GPIIb^+/+ or GPIIb^-/- platelets.</p