Hina Desai

Tissue factor‐bearing microparticles derived from tumor cells: impact on coagulation activation

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 vitro‐generated MP or in cell‐free plasmas from nude mice bearing orthotopically injected human cancer cells. Results: Tumor cell‐derived 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.

The presence and release of tissue factor from human platelets

Tissue factor (TF) is a transmembrane receptor for FVII that triggers blood coagulation. It is not normally exposed to circulating blood, but may be produced by endothelium and monocytes under pathological conditions. Platelets take up TF-positive microparticles from leukocytes and TF appears on platelets adhering to leukocytes following collagen stimulation of blood. However, the presence of TF in circulating platelets has not been directly demonstrated. In this study, flow cytometric analysis of washed platelets from five healthy adult volunteers demonstrated TF-antigen on both resting platelets and platelets activated by thrombin (0.1 U/ml), collagen (5 w g/ml) or ADP (5 w M). TF released by platelets was demonstrated in the supernatants of non-activated and activated washed platelets by dot-immunoblotting and Western blotting. The amount of TF released from non-activated and activated platelets was quantitated using an enzyme-linked immunosorbent assay (ELISA). Washed non-activated and platelets activated by thrombin, collagen or ADP released 27-35 pg TF per mg protein. TF associated with the platelet surface was biologically inactive, although released TF was functionally active as determined by a two-stage factor X activation assay. We conclude that platelets contain an inactive form of TF that may develop functional activity following its release. However, the role of platelet TF in health and disease remains to be determined.

Bevacizumab immune complexes activate platelets and induce thrombosis in FCGR2A transgenic mice

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 FcγRIIa (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 FcγRIIa activation. Methods and results: We found that Bev immune complexes (IC) activate platelets via FcγRIIa, and therefore attempted to reproduce this finding in vivo using FcγRIIa (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 Bev’s 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 FcγRIIa receptor, and that this provides a better explanation for the thrombotic events observed in vivo.

Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454

Platelets are known to play a role in blood borne metastasis. Previous experimental studies have suggested that platelet GpIIb/IIIa may be a therapeutic target. However, the need for intravenous administration limits the potential application of current GpIIb/IIIa inhibitors to cancer therapy. The aim of the present study was to assess the efficacy of a novel, non-peptide oral GpIIb/IIIa antagonist (XV454) on tumor cell-induced platelet aggregation in vivo and on experimental metastasis. A Lewis lung carcinoma (LL2) mouse model of experimental metastasis was used in this study. XV454 (100 micro g) was administered intravenously (via tail vein) or orally (gavages) to 20 g mice. To determine the effect of XV454 on platelet aggregation, blood samples were collected by cardiac puncture 10 minutes after intravenous and 1-24 hrs after oral XV454, and platelet function was assessed by aggregometry, thrombelastography and the Platelet Function Analyzer (PFA100). The effect of XV454 on tumor cell-induced thrombocytopenia was determined 10 minutes after intravenous and 3 hrs after oral XV454 administration. Tumor cells (2 x 10(6)) were injected intravenously and 15 minutes after cell injection, platelet count was measured and compared to baseline (pre-injection) counts. To assess the effect on metastasis, XV454 was administered intravenous or orally 10 minutes and 3 hrs before tumor cell injection, respectively. Eighteen days later, surface lung tumor nodules were counted and the total lung tumor burden assessed. In a fourth group, in addition to the initial oral dose (before tumor cell injection), oral XV454 was given daily for the first week and three times in the second week. Administration of XV454 (5 mg/kg) completely inhibited platelet aggregation and this effect persisted for at least 24 hrs after oral delivery. Both intravenous and oral XV454 significantly inhibited tumor cell-induced thrombocytopenia (P < 0.01), the number of surface lung tumor nodules (80-85%; P < 0.001) and total tumor burden (83% for intravenous group; 50% oral [single treatment] group; 91% oral [multiple treatment] group, P < 0.001). Overall, these data provide further evidence for the effect of oral and intravenous GpIIb/IIIa antagonism on tumor cell-platelet interaction and metastasis.

Anti-CD40L Immune Complexes Potently Activate Platelets In Vitro and Cause Thrombosis in FCGR2A Transgenic Mice

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.

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

Human platelets contain and release TWEAK

The multifunctional cytokine, TWEAK (TNF-like weak inducer of apoptosis), is a member of the TNFα superfamily. TWEAK is found in a broad range of cell types and has been linked to cell growth and survival, angiogenesis and other inflammatory processes. These functions and their importance in inflammatory diseases have made TWEAK an attractive pharmaceutical target, particularly for immunotherapy with monoclonal antibodies (mAbs). Immunotherapy targeting another TNFα family member, CD154, was associated with thrombosis in clinical trials. Subsequent studies identified platelets, which contain CD154, as a possible contributing factor to thrombosis in these trials. Since clinical trials with anti-TWEAK mAbs have already begun, we considered it important to determine whether platelets contain TWEAK. Using a variety of immunologic methods we found that, upon activation, human platelets expose TWEAK antigen and release it in soluble form (sTWEAK). By flow cytometry we determined that human platelets activated by TRAP (Thrombin Receptor Agonist Peptide) and other agonists expose TWEAK antigen (22% median positivity) and release TWEAK positive microparticles. The presence of TWEAK on platelets was confirmed by confocal microscopy. By ELISA, we found that sTWEAK is released by activated platelets. Finally, western blot analysis revealed TWEAK protein (∼34 kDa) in washed platelet lysates. The finding that human platelets contain TWEAK raises important questions about its possible functions in normal physiology, as well as in inflammatory diseases and their treatment.

Purification and properties of human melanoma cell tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that acts as a receptor for nonactivated and activated factor VII (FVII) and triggers the coagulation cascade. TF plays an important role in hemostasis, but may also have noncoagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered by the lack of purified TF. In this study, TF antigen was identified on human A375 malignant melanoma cells using flow cytometry. We further purified TF apoprotein 2.000-fold to homogeneity from A375 melanoma cells using immunoaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under reduction, purified TF apoprotein gave two major protein bands corresponding to molecular weights of 53 and 34 to 36 KD. The identity of these forms of TF was confirmed by Western blotting using a polyclonal antibody against human brain TF. Under reduction, the TF antibody bound with a monomeric form of TF (53 KD), and without reduction, to several forms of TF (34 to 128 KD). Preliminary carbohydrate analysis suggested that TF is a glycoprotein and contains about 22% total carbohydrates. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipids. The effects of varying concentrations (0 to 8 μg) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibited more than 70% of the procoagulant activity of TF in an FX activation assay. The complex formation between purified TF apoprotein and FVlla was demonstrated by using an enzyme-linked immunosorbent assay. TF formed a complex with FVIIa in a concentration-dependent and saturable manner. We conclude that in human melanoma cells, TF occurs in monomeric and heterodimeric forms and appears to have similar properties as reported for TF from other sources.

Deficiencies in the CD40 and CD154 receptor-ligand system reduce experimental lung metastasis

It is established that experimental metastasis requires platelet activity. CD154 expressed on and released from activated platelets induces an inflammatory response in endothelial cells and monocytes, including tissue factor production. CD154 has also been shown to activate platelets in vitro and promote thrombus stability in vivo. These CD154 effects may be mediated, at least in part, by CD40 signaling on platelets and vascular endothelial cells. We have previously demonstrated prolonged bleeding and PFA-100 closure times in mice deficient for Cd154 or its receptor Cd40. In the present study, we hypothesized that Cd40 and Cd154 promote lung tumor formation in experimental metastasis in mice. We created mice doubly deficient in Cd40 and Cd154 (Dbl KO) and found them to be both fertile and viable. Injected tumor cells seeded poorly in mice deficient in Cd40 or Cd154, as well as Dbl KO, compared to wild-type mice. We sought to determine whether blood-borne Cd40 versus endothelial Cd40 contribute differentially to reduced experimental lung metastasis, as observed in Cd40 deficient mice. By bone marrow transplantation, we created mice deficient for Cd40 either in the blood compartment but not in the endothelium, or vice versa. We found that mice deficient in blood compartment Cd40 had fewer lung nodules compared to wild-type mice and mice deficient in endothelial Cd40. Our findings suggest an important contribution of the Cd40-Cd154 pathway to experimental lung metastasis. Furthermore, the data points to a selective role for peripheral blood cell Cd40 in this process.