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Tissue factor (TF) expression by tumor cells correlates with metastasis clinically and supports metastasis in experimental settings. However, the precise pathways coupling TF to malignancy remain incompletely defined. Here, we show that clot formation by TF indirectly enhances tumor cell survival after arrest in the lung, during experimental lung metastasis, by recruiting macrophages characterized by CD11b, CD68, F4/80, and CX 3 CR1 (but not CD11c) expression. Genetic or pharmacologic inhibition of coagulation, by either induction of TF pathway inhibitor expression or by treatment with hirudin, respectively, abrogated macrophage recruitment and tumor cell survival. Furthermore, impairment of macrophage function, in either Mac1-deficient mice or in CD11b-diphtheria toxin receptor mice in which CD11b-positive cells were ablated, decreased tumor cell survival without altering clot formation, demonstrating that the recruitment of functional macrophages was essential for tumor cell survival. This effect was independent of NK cells. Moreover, a similar population of macrophages was also recruited to the lung during the formation of a premetastatic niche. Anticoagulation inhibited their accumulation and prevented the enhanced metastasis associated with the formation of the niche. Our study, for the first time, links TF induced coagulation to macrophage recruitment in the metastatic process. (Blood. 2012;119(13): 3164-3175) IntroductionClinical and experimental studies over the past 30 years have established that the coagulation system actively supports tumor progression and metastasis. Consistent with these observations, expression of procoagulants by tumor cells, among them tissue factor (TF), cancer procoagulant, 1 and selectin ligands, correlates with advanced disease and poor outcome for multiple cancer types. 2,3 TF (also known as coagulation factor III or CD142) is the protease receptor that initiates coagulation after injury through the extrinsic pathway. Under normal physiologic conditions, TF expression is limited to extravascular sites that only become exposed to blood after trauma. In this case, the exposed TF binds to and activates the blood-borne coagulation factor FVII, triggering clot formation through a cascade of proteolytic events that results in thrombin formation, activation of platelets, and fibrin deposition. 4 In addition to triggering coagulation, the binding of FVIIa to TF activates intracellular signaling pathways through the TF cytoplasmic domain, by activating G-protein-coupled protease activated receptors (PARs), especially PAR2. 4 These signaling pathways support tumor angiogenesis 5,6 and regulate tumor progression. 7 Intracellular signaling pathways can be distinguished experimentally from the extracellular coagulative roles of TF by specific antibodies 7 or deletion of the cytoplasmic domain that eliminates many forms of TF signaling but still triggers coagulation. 6,8,9 TF enhances tumor growth and angiogenesis, [4][5][6][7]10 and specifically plays an important role in some experiment...

Delayed-onset heparin-induced thrombocytopenia is increasingly being recognized. To avoid disastrous outcomes, physicians must consider heparin-induced thrombocytopenia whenever a recently hospitalized patient returns with thromboembolism; therapy with alternative anticoagulants, not heparin, should be initiated.

Tissue factor (TF)

Summary. Background: Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. Methods: TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitrogenerated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. Results: Tumor cellderived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. Conclusions: MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Summary. Background: Treatment with Bevacizumab has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Objective: We investigated whether thrombosis might be the result of platelet activation mediated via the FccRIIa (IgG) receptor -which is not present on mouse platelets -and aimed to identify the functional roles of heparin and platelet surface localization in Bev-induced FccRIIa activation. Methods and results: We found that Bev immune complexes (IC) activate platelets via FccRIIa, and therefore attempted to reproduce this finding in vivo using FccRIIa (hFcR) transgenic mice. Bev IC were shown to be thrombotic in hFcR mice in the presence of heparin. This activity required the heparin-binding domain of BevÕs target, vascular endothelial growth factor (VEGF). Heparin promoted Bev IC deposition on to platelets in a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia. When sub-active amounts of ADP or thrombin were used to prime platelets (simulating hypercoagulability in patients), Bev IC-induced dense granule release was significantly potentiated, and much lower (sub-therapeutic) heparin concentrations were sufficient for Bev IC-induced platelet aggregation. Conclusions: The prevailing rationale for thrombosis in Bev therapy is that VEGF blockade leads to vascular inflammation and clotting. However, we conclude that Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of the platelet FccRIIa receptor, and that this provides a better explanation for the thrombotic events observed in vivo.

Anti-CD40L immunotherapy in systemic lupus erythematosus patients was associated with thromboembolism of unknown cause. We previously showed that monoclonal anti-CD40L immune complexes (ICs) activated platelets in vitro via the IgG receptor (FcγRIIa). In this study, we examined the prothrombotic effects of anti-CD40L ICs in vivo. Because mouse platelets lack FcγRIIa, we used FCGR2A transgenic mice. FCGR2A mice were injected i.v. with preformed ICs consisting of either anti-human CD40L mAb (M90) plus human CD40L, or a chimerized anti-mouse CD40L mAb (hMR1) plus mouse CD40L. ICs containing an aglycosylated form of hMR1, which does not bind FcγRIIa, were also injected. M90 IC caused shock and thrombocytopenia in FCGR2A but not in wild-type mice. Animals injected with hMR1 IC also experienced these effects, whereas those injected with aglycosylated-hMR1 IC did not, demonstrating that anti-CD40L IC-induced platelet activation in vivo is FcγRIIa-dependent. Sequential injections of individual IC components caused similar effects, suggesting that ICs were able to assemble in circulation. Analysis of IC-injected mice revealed pulmonary thrombi consisting of platelet aggregates and fibrin. Mice pretreated with a thrombin inhibitor became moderately thrombocytopenic in response to anti-CD40L ICs and had pulmonary platelet-thrombi devoid of fibrin. In conclusion, we have shown for the first time that anti-CD40L IC-induced thrombosis can be replicated in mice transgenic for FcγRIIa. This molecular mechanism may be important for understanding thrombosis associated with CD40L immunotherapy. The FCGR2A mouse model may also be useful for assessing the hemostatic safety of other therapeutic Abs.

Thromboembolic disease is a frequent complication in cancer. Tissue factor (TF), shown to be involved in tumor growth and metastasis, is also considered to play a central role in the pathogenesis of cancer-associated thrombosis. Circulating TF-bearing microparticles (TF+ MPs) have been found in the plasma of patients with different malignancies and are thought to contribute to their hypercoagulable state. Although numerous studies have focused on TF+ MPs derived from blood cells, there is no information available on the pathological relevance of MPs originating from tumor cells. We conducted a study to detect, enumerate and characterize the procoagulant activity (PCA) of MPs released specifically from tumor cells. MPs from high (MDA-231) and low (MCF-7) TF-expressing human breast carcinoma cells were generated ex vivo in whole blood or in buffer under stirring conditions for 45 minutes. The numbers (MPs/ml) of total and TF-expressing tumor-derived particles (TMPs) in cell-free plasmas were measured by flow cytometry using FITC-labeled annexin V and a PE-labeled monoclonal anti-TF antibody respectively. The PCA of TMPs was measured by a one stage clotting assay and a highly sensitive fluorogenic thrombin generation assay. In order to evaluate the PCA of circulating TMPs, we injected 2x106 TF+ MPs derived from MDA-231 cells into mice via the tail vein. Human TF antigen and activity were measured in cell-free mouse plasmas at various intervals (5–420 min) after injections by ELISA and clotting assay, respectively. MPs less than 1μm in diameter were released from tumor cells in both buffer and whole blood by stirring. TMPs positive for TF consisted of approximately 40% of the annexin V+ MPs, and such particles derived from as low as 1x105 MDA-231 cells could be enumerated reliably (2.5x104 MPs/105 cells). By ultracentrifugation of cell-free plasmas, we confirmed that TF antigen was associated entirely with the MP fraction. TMPs derived from as few as 450 MDA-231 cells shortened plasma recalcification times from 525 ± 114 to 265 ± 148 (P<0.01), and significantly accelerated thrombin generation as evidenced by a 3 fold shortening in lag time, and a 2 fold increase in the rate of thrombin formation and thrombin concentration. No PCA was detected with MCF-7-derived TMPs. The PCA of TMPs was inhibited completely by a blocking anti-TF monoclonal antibody, but not by saturating concentrations of annexin V (an inhibitor of phospholipid PCA) or corn trypsin inhibitor (an inhibitor of the intrinsic pathway). Five minutes following the injection of TMPs into mice, appreciable levels of human TF antigen and activity were detected in cell-free plasmas. Both TF activity and antigen decreased over time and were detectable no longer than 30 minutes after injection, indicating a rapid clearance of circulating TMPs in vivo. In contrast, when TMPs were incubated in human whole blood ex vivo for various intervals, TF activity remained unchanged in cell-free plasmas for at least 5 hrs and TF antigen was not detected by flow cytometry on any blood corpuscles, including platelets, at all intervals. However, when whole blood containing TMPs was clotted by recalcification, no TF activity could be detected in the serum, indicating the incorporation of TMPs in formed clots. In summary, MPs bearing active TF are released readily from tumor cells and can significantly activate coagulation even at very low concentrations. These results provide new insights towards the potential contribution of TMPs to the pathogenesis of cancer-associated thrombosis.