To test the hypothesis that platelet activation contributes to tumor dissemination, we studied metastasis in mice lacking G␣q, a G protein critical for platelet activation. Loss of platelet activation resulted in a profound diminution in both experimental and spontaneous metastases. IntroductionA persuasive body of evidence has accumulated associating hemostatic factors with tumor growth, stroma formation, and tumor dissemination. [1][2][3] Clinical studies have shown that expression of procoagulants by cancer cells is prognostic of poor outcome. [4][5][6] Furthermore, the expression of tissue factor by tumor cells has been shown to promote metastatic disease in experimental animals, 7 whereas inhibitors of thrombin and other coagulation factors diminished metastatic potential. 1,8 The available data support the general hypothesis that local thrombin generation enhances tumor dissemination. However, it is presently not clear which specific thrombin substrates are important mediators of the metastatic process. Recent studies revealed that fibrinogen deficiency significantly diminishes metastatic potential, suggesting that fibrinogen is at least one thrombin substrate important in metastasis. 9,10 However, it is likely that other thrombin substrates also contribute to tumor cell metastasis based on the finding that pharmacologic inhibition of thrombin resulted in decreased metastatic potential even in the absence of fibrinogen. 10 Several studies support the view that thrombin-mediated platelet activation may play a role in tumor biology. The elimination of circulating platelets with antiplatelet antibodies was shown to result in a significant diminution in metastases using several transplantable murine tumor models. 11,12 Competitive inhibition of the key platelet integrin, ␣ IIb ␤ 3 , either pharmacologically or with antibodies to ␤ 3 , also diminished metastatic potential. 13,14 Similarly, pharmacologic inhibitors of platelet activation have been shown to decrease the metastatic potential of circulating tumor cells. 15,16 More recently, the genetic loss of the integrin ␤ 3 subunit in mice was shown to diminish metastasis. 17 Platelets could influence metastatic potential via several mechanisms. Platelet granules contain a variety of cellular growth factors (eg, platelet-derived growth factor [PDGF], vascular endothelial growth factor [VEGF]), matrix proteins (eg, vitronectin, fibronectin), and inflammatory mediators (eg, platelet factor-4, interleukin-8, macrophage inflammatory protein 1␣ , RANTES [regulated on activation, normal T expressed, and secreted], CCL17, CCXL1, CXCL5) that might influence tumor cell behavior and stroma formation. [18][19][20] Platelets may also contribute to the physical interaction between circulating tumor cells and vascular endothelial cells by supporting the stable adhesion to endothelium and/or transmigration of tumor cells out of the vasculature. Local platelet activation could promote the migration of inflammatory cells, enhancing tumor stroma formation. Alternatively, tumor c...

IntroductionTissue factor (TF) is the membrane-associated glycoprotein receptor for coagulation factors VIIa and X that serves as the primary physiologic initiator of blood coagulation. In addition to supporting proteolytic events that ultimately lead to local thrombin generation, TF is also proposed to directly contribute to intracellular signaling events through the TF cytoplasmic domain and TF/fVIIa/fXamediated activation of PAR-1 and PAR-2. 1-4 A significant body of evidence has accumulated linking tumor cell-associated procoagulant function to cancer biology. Multiple clinical studies have shown a correlation between TF expression by tumor cells and advanced disease stage and poor outcome. [5][6][7][8][9] Furthermore, experimental data generated using animal models of tumor metastasis strongly favor the view that TF expression by malignant cells supports metastatic success. [10][11][12][13][14][15] Similarly, thrombin-mediated proteolysis, 16-21 fibrin(ogen), 22,23 and PAR-mediated platelet activation 24 also appear to be significant determinants of metastatic potential. Both platelets and fibrinogen were shown to support metastatic potential by limiting the capacity of natural killer (NK) cells to clear newly established micrometastatic foci. 25,26 However, hemostatic factors are likely to influence tumor dissemination through multiple mechanisms and the precise pathways coupling TF to malignancy remain to be defined.The tandem importance of tumor cell-associated TF and circulating coagulation system components in malignancy is consistent with the hypothesis that TF supports metastasis by providing cancer cells a means of directing proteolytic events leading to local thrombin generation and the formation of tumor cell-associated microthrombi. However, an intriguing alternate possibility is that TF supports tumor cell dissemination by mechanism(s) uncoupled from "traditional" thrombin generation and subsequent thrombus formation. In this regard, significant attention has focused on potential intracellular signaling events coupled to the cytoplasmic portion of TF. This interest was driven in part by early studies indicating that tumor cells expressing a mutant form of TF lacking the cytoplasmic domain were far less metastatic than tumor cells expressing full-length TF. 11,12,14 However, interpretation of these early studies was made more complex by the use of nonmurine tumor lines in xenograft assays in mice, the use of tumor cells expressing human TF or human TF derivatives in a setting where all other factors were of murine origin, and the requisite use of immunocompromised mice. Nevertheless, many studies have provided provocative evidence for an important linkage between TF-mediated signaling events and several key cellular processes capable of influencing metastasis, including cytoskeletal organization, 27 cell adhesion/migration, 28-30 apoptosis, 31,32 and angiogenesis. 33,34 An important role for the TF cytoplasmic domain in cellular signaling is also supported by more recent studies of transgenic mic...

Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib -) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin α M β 2 binding motif (Fibγ 390-396A ) or the α IIb β 3 platelet integrin-binding motif (Fibγ Δ5 ), were challenged with collagen-induced arthritis (CIA). Fib -mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibγ 390-396A mice, which retain full clotting function. In contrast, arthritis in Fibγ Δ5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib -and Fibγ 390-396A mice with CIA displayed reduced local expression of TNF-α, IL-1β, and IL-6, which suggests that α M β 2 -mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-α expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to α M β 2 -mediated inflammatory processes.

A link between colitis and colon cancer is well established, but the mechanisms regulating inflammation in this context are not fully defined. Given substantial evidence that hemostatic system components are powerful modulators of both inflammation and tumor progression, we employed gene-targeted mice to directly test the hypothesis that the coagulation factor fibrinogen contributes to colitis-associated colon cancer in mice. This fundamental provisional matrix protein was found to be an important determinant of colon cancer. Fibrinogen deficiency resulted in a dramatic diminution in the number of colonic adenomas formed following azoxymethane/dextran sodium sulfate challenge. More detailed analyses in mice expressing a mutant form of fibrinogen that retains clotting function, but lacks the leukocyte integrin receptor αMβ2 binding motif (Fibγ390-396A) revealed αMβ2-mediated engagement of fibrin(ogen) is mechanistically coupled to local inflammatory processes (e.g., IL-6 elaboration) and epithelial alterations which contribute to adenoma formation. Consistent with these findings, the majority of Fibγ390-396A mice developed no discernable adenomas, whereas penetrance was 100% in controls. Furthermore, the adenomas harvested from Fibγ390-396A mice were significantly smaller than those from control mice and less proliferative based on quantitative analyses of mitotic indices, suggesting an additional role for fibrin(ogen) in the growth of established adenomas. These studies demonstrate, for the first time, a unique link between fibrin(ogen) and the development of inflammation-driven malignancy. Given the importance of antecedent inflammation in the progression of numerous cancers, these studies suggest that therapies targeting fibrin(ogen)-αMβ2 interactions may be useful in preventing and/or treating this important subset of malignancies.