Mildred Amaya

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.

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin.

Summary.  The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low‐molecular‐weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non‐coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparins anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg−1) 4 h before intravenous administration of melanoma cells (2.0 × 105) markedly (89%) reduced lung tumor formation (3 ± 2) compared with controls (31 ± 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg−1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin‐treated animals, despite a 4‐fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg−1). Administration of tumor cells (2 × 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 ± 37 vs. 498 ± 94 × 106 mL−1, P < 0.01), but this was prevented by tinzaparin treatment (921 ± 104 × 106 mL−1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.

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.

Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins αIIbβ3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28± platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

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.

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet–tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell–platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)–CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.

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.

Assessment of Anti-Metastatic Effects of Anticoagulant and Antiplatelet Agents Using Animal Models of Experimental Lung Metastasis

It is well established that the blood coagulation system is activated in cancer. In addition, there is considerable evidence to suggest that clotting activation plays an important role in the biology of malignant tumors, including the process of blood-borne metastasis. For many years our laboratory has used experimental models of lung metastasis to study the events that follow the introduction of procoagulant-bearing tumor cells into circulating blood. This chapter focuses on the basic methods involved in assessing the anti-metastatic effects of anticoagulants and anti-platelet agents using rodent models of experimental metastasis. In addition, it summarizes our experience with these models, which collectively suggests that intravascular coagulation and platelet activation are a necessary prelude to lung tumor formation and that interruption of coagulation pathways or platelet aggregation may be an effective anti-metastatic strategy.