Florian Langer

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.

Experimental metastasis and primary tumor growth in mice with hemophilia A

Summary.  During experimental lung metastasis, tumor cells adhere to the pulmonary microvasculature and activate coagulation via surface‐expressed tissue factor (TF), leading to local fibrin deposition and platelet aggregation. While interventional studies have demonstrated great efficacy of anticoagulants and antiplatelet agents in inhibiting metastasis, no information is available on how tumor biology may be affected by congenital bleeding disorders such as hemophilia A. We therefore used a syngeneic model to study experimental metastasis and primary tumor growth in factor VIII (FVIII)‐deficient mice. By conventional reverse transcription‐polymerase chain reaction, flow cytometry, and one‐stage clotting assays, we demonstrated constitutive expression of TF mRNA, antigen, and procoagulant activity in the murine B16F10 melanoma cell line. In hemophilic mice, B16F10 lung metastasis was significantly (P < 0.001) enhanced by a single dose of human FVIII (100 U kg−1), suggesting that FVIII played a critical role during the early blood‐borne phase of the metastatic cascade. In contrast, lung seeding was significantly (P < 0.05) reduced by lepirudin, a direct thrombin inhibitor, suggesting that thrombin generation contributed to pulmonary metastasis even in the absence of FVIII. Consistent with this finding, intravenous injection of B16F10 cell‐evoked laboratory changes of a hemolytic thrombotic microangiopathy and consumptive coagulopathy in both hemophilic and non‐hemophilic mice. Subcutaneous implantation of B16F10 cells into mice with hemophilia A gave rise to primary tumors in an exponential growth pattern similar to that observed in non‐hemophilic mice. Although TF expression by B16F10 cells may promote thrombin‐dependent metastasis in mice with hemophilia A, amplification of coagulation by host FVIII appears to be necessary for maximum lung seeding.