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.

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Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G 2 /M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasisassociated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).LIM kinase 1 (LIMK1) 1 belongs to a novel dual specificity (serine/threonine and tyrosine) kinase family that contain two amino-terminal LIM domains (1). LIMK1 gene is expressed predominantly in brain and in developing neural tissues (2), and its deletion (microdeletion of chromosome 7q11.23) is typical for Williams syndrome (3). Cofilin, one of the actin-binding proteins, considered to be a potent regulator of the actin dynamics (4) by means of its activity in F-actin depolymerization, is the only known substrate of LIMK1. The function of cofilin is inhibited by phosphorylation at the Ser-3 residue (5) by LIMK1, which leads to accumulation of F-actin. The catalytic activity of LIMK1 is regulated by distinct members of the Rho subfamily of small GTPases (Rho, Rac, and Cdc42), which controls actin filament dynamics and focal adhesions assembly in response to extra-and intracellular stimuli. Rho, Rac, and Cdc42 induce formation of stress fibers, assembly of lamellipodia and membrane ruffles, and regulation of filopodial protrusions, respectively (6). LIMK1 has been shown to mediate specifically Rac-induced actin cytoskeleton reorganization and focal adhesion complexes (5, 7, 8). Rac-induced activation of LIMK1 is mediated by PAK1, which phosphorylates LIMK1 on its Thr 508 residue (9). Other studies also proposed that Rhoand Cdc42-induced cytoskeletal changes are mediated through phosphorylation of LIMK1 by Rho-dependent protein kinase ROCK (10) and Cdc42-regulated protein kinase PAK4 (11) and MRCK␣ (12).The non-catalytic domain of LIMK1 contains two tandem repeats of a LIM motif, a putative zinc binding motif, and a PDZ domain, which contains two tandem nuclear exit signal sequence...

Clotting activation occurs frequently in cancer. Tissue factor (TF), the most potent initiator of coagulation, is expressed aberrantly in many types of malignancy and is involved not only in tumor-associated hypercoagulability but also in promoting tumor angiogenesis and metastasis via coagulation-dependent and coagulation-independent (signaling) mechanisms. Tissue factor pathway inhibitor (TFPI) is the natural inhibitor of TF coagulant and signaling activities. Studies have shown that TFPI exhibits antiangiogenic and antimetastatic effects in vitro and in vivo. In animal models of experimental metastasis, both circulating and tumor cell-associated TFPI are shown to significantly reduce tumor cell-induced coagulation activation and lung metastasis. Heparins and heparin derivatives, which induce the release of TFPI from the vascular endothelium, also exhibit antitumor effects, and TFPI may contribute significantly to those effects. Indeed, a non-anticoagulant low-molecular-weight heparin with intact TFPI-releasing capacity has been shown to have significant antimetastatic effect in a similar experimental mouse model. The evidence supporting the dual inhibitory functions on TF-driven coagulation and signaling strengthen the rationale for considering TFPI as a potential anticancer agent. This article primarily summarizes the evidence for antiangiogenic and antimetastatic effects of TFPI and describes its potential mechanisms of action. The possible application of TFPI and other inhibitors of TF as potential anticancer agents is described, and information regarding potential antitumor properties of TFPI-2 (which has structural similarities to TFPI) is also included.

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44-and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is ϳ5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial ϳ2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.Giardia lamblia, an evolutionary primitive eukaryotic protozoan parasite and an intestinal pathogen of humans and animals, is one of the major causes of water-borne diseases worldwide (1). This flagellated protozoan undergoes complex life cycle stages while inside the host. Exposure to the highly acidic condition in stomach and proteases in the upper small intestine triggers excystation of trophozoites from the ingested cysts. Newly emerged trophozoites swim freely in the intestinal fluid and colonize the upper small intestine to replicate (2, 3). As enterocytes migrate to the tip of the villus and get sloughed off into the intestinal lumen, the attached trophozoites either reattach to new enterocytes to remain in the intestine or differentiate into infective cysts. Although the life cycle of this primitive eukaryote and physiological signals that regulate induction of excystation and encystation have been studied extensively, the molecular mechanisms by which trophozoites sense and respond quickly to the environmental signals in the intestine to initiate encystation remain largely unknown.Encystation is an adaptive process to cope with the depletion of nutrients, specifically cholester...