Liza Robles-Carrillo

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.

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.

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.

CalDAG-GEFI deficiency protects mice from FcγRIIa-mediated thrombotic thrombocytopenia induced by CD40L and β2GPI immune complexes.

Platelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)‐mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or β2 Glycoprotein I (β2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild‐type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG‐GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs.

Assessment of protein C anticoagulant pathway by thrombin generation assay in the presence of endothelial cells

The generation of thrombin is the pivotal event in the process of blood coagulation. In vivo, thrombin generation is regulated by cooperation between the vascular endothelium and the procoagulant and anticoagulant systems in blood, such as the thrombin/endothelial cell– dependent activation of the protein C anticoagulant pathway that ultimately leads to factor Va (FVa) and FVIIIa inactivation [1,2]. In vitro, thrombin generation is assessed most commonly in plasma by use of a fluorogenic substrate–based assay (TGA) in microtiter plates [3]. While this assay can accurately measure the kinetics of thrombin generation in plasma (including lag time [LT], peak thrombin [PT], and endogenous thrombin potential [ETP]), it does not assess the influence of the endothelial cell–dependent protein C pathway on thrombin generation. Therefore, to make the assay sensitive to the inactivation of FVa and FVIIIa by the activated protein C (APC) system in a physiologically relevant manner, we introduced a surrogate endothelial component to the TGA. Wells of flat-bottomed microtiter plates were coated with quiescent EA.hy926 cells (hybrid of human umbilical vein endothelial cells [HUVECs] and A549 lung carcinoma cells; ATCC, Manassas, VA, USA), which consistently express thrombomodulin (TM) as well as the endothelial protein C receptor (EPCR) [4]. This cell line was chosen to provide endothelial cell–like APC function without the reproducibility and stability problems associated with endothelial cells from natural sources (e.g., HUVECs) [5,6]. EA.hy926 cells were cultured to 80% confluence in 75-cm flasks in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. To establish quiescence, cells were washed in PBS and further incubated in serum-free medium for 24 h. Cells were then washed, harvested, and seeded (4 9 10 cells in 200 lL of complete growth medium) in half of the wells (48 wells) of a tissue culture grade 96-well microtiter plate (Costar 3599, Corning, NY, USA) and incubated for 12–18 h to allow optimum attachment, while avoiding further cell proliferation. Finally, EA.hy926 monolayers were washed and incubated for 24 h in serum-free medium to reestablish quiescence. Before assay, monolayers were washed once with PBS. Thrombin generation was evaluated according to the method of Hemker et al. [3] in normal pooled plasma (NP; Precision Biologics, Dartmouth, Canada) and in protein C–deficient (PCd; Affinity Biologicals, Ancaster, Canada), protein S–deficient (PSd; Affinity Biologicals), and heterozygous FV Leiden (fVL; from individual subjects) plasmas, in the presence or absence of EA.hy926 monolayers. After the addition of 5 pmol L (final concentration [f.c.]) recombinant relipidated human tissue factor (TF; Innovin, Dade Behring, Newark, DE, USA) and 15 lmol L (f.c.) phospholipids (PL; DOPS/DOPC/ DOPE, Avanti Polar Lipids, Alabaster, AL, USA) to plasmas, thrombin generation was initiated by a mixture of 16.7 mmol L (f.c.) CaCl2 and the fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-AMC.HCl, 417 lmol L [f.c.]; Bachem, Torrance, CA, USA) and monitored for 90 min. Assay calibrator was run for each sample (with or without cells) by adding a2-macroglobulin–thrombin complex (Diagnostica Stago, Parsippany, NJ, USA) instead of TF/PL. By using a chromogenic assay for measuring APC activity [7], we found that membrane-bound TM from 4 9 10 intact EA.hy926 cells (per well) had an activity equivalent to ~ 0.5 nmol L of soluble thrombomodulin (Haematologic Technologies, Essex Junction, VT, USA). Compared with bare microtiter wells, thrombin generation in NP was suppressed by EA.hy926 cells as evidenced by a 56% reduction in PT, 39% reduction in Correspondence: Enriqueta Coll, Center for Thrombosis Research, Florida Hospital, 2501 N. Orange Ave, Suite 786, Orlando, FL 32804, USA. Tel.: +1 407 3032440; fax: +1 407 3032441 E-mail: Enriqueta.coll@flhosp.org