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.

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.

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.

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.

The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) relies on the demonstration of antibodies to the heparin-platelet factor 4 (H-PF4) complex. Assays are based on the functional ability of H-PF4 antibodies to activate platelets, or detect the antibody directly by immunological methods. Multiple assays in each category are currently in clinical use and newer, rapid immunological assays are becoming available. The aim of this study was to compare available methods for detecting H-PF4 antibodies in a prospective study of patients with clinically suspected HIT. Functional assessment included serotonin release assay (SRA) and lumi-aggregometry (LA). Immunological assessment included ELISA (GTI), and particle gel immunoassay (PGIA; Diamed and Akers). Circulating platelet microparticles (PMP) were assessed by flow cytometry. Patients were also assessed for the pre-test probability of HIT using the Warkentin 4-T scoring system. 151 patients were enrolled. 54/151 patients (35.8%) had a positive GTI ELISA, while 53/151 (35.1%) and 39/151 (25.8%), respectively, had positive Akers and Diamed PGAI tests. Only 15/149 (10.1%) patients had a positive SRA, while only 5/150 (3.3%) gave a positive result by lumi-aggregometry. There was a strong correlation between the ELISA OD values obtained in serum and plasma using both fresh (r=0.98) and frozen (r=0.99) samples, although slightly more positive results were obtained using serum. Differences were only seen with OD values around the cut-off of 0.4. The majority (77.8%) of H-PF4 antibodies detected by ELISA were neutralized by heparin in the ‘confirmatory’ procedure. Weak antibodies (OD 0.4–0.5) were more likely to be non-neutralizable (5/12; 42%) than strong antibodies (OD>1.0; 4/23; 17%). 47 patients positive by ELISA were retested to determine the predominant immunoglobulin subclass. 15/47 (32%) were positive (OD>0.4) for IgG; 27/47 (57%) for IgM, and 12/47 (25%) for IgA. The Diamed assay more closely correlated with the GTI ELISA than the Akers test (82.1% vs. 56.7%, respectively). The PGIAs were only moderately correlated with each other (64%) with the Akers assay giving more “false positive” results relative to the ELISA. PMP were higher in patients with a positive ELISA (6.2 vs 4.7 × 106/ml) or positive SRA (5.5 vs. 5.1 ×106/ml) but this was not statistically significant due to the wide range of results. Of 119 patients assessed, 87 had a low pre-test probability of HIT (4-T score 0–3), 27 had an intermediate probability (4–5), and 5 had a high probability (6–8). The GTI ELISA was positive in 24, 56 and 80% of low, intermediate and high probability cases. The Akers PGIA was positive in 39, 41 and 40% respectively; the Diamed assay in 21, 33 and 40%, and the SRA in 7, 11 and 40%, respectively. This study was conducted in a patient population biased towards cardiovascular surgery, and confirms previously reported observations that immunoassays are more frequently positive than functional assays. The ELISA correlated better than the PGIA tests with the pre-test probability of HIT, although the Diamed test showed acceptable correlation with the ELISA. In contrast, the Akers assay correlated poorly with the ELISA, often producing positive results when the latter test was negative. We conclude that while the PGIA tests are rapid and convenient, further studies are needed to determine the basis for disparate results relative to the widely used ELISA.