Turoctocog alfa pegol (N8-GP) is a novel glycoPEGylated extended half-life recombinant factor VIII (FVIII) product developed for prophylaxis and treatment of bleeds in patients with haemophilia A, to enable higher activity levels with less frequent injections compared with standard FVIII products. This phase III (NCT01480180), multinational, open-label, non-randomised trial evaluated the safety and clinical efficacy of N8-GP when administered for treatment of bleeds and for prophylaxis, in previously treated patients aged ≥12 years with severe haemophilia A. Patients were allocated to receive N8-GP for prophylaxis or on-demand treatment for up to 1.8 years. Patients on prophylaxis were administered one dose of 50 IU/kg of N8-GP every fourth day. Bleeds were treated with doses of 20-75 IU/kg. Total exposure to N8-GP in the trial was 14,114 exposure days (159 patient-years). For the prophylaxis arm (n=175), the median annualised bleeding rate (ABR) was 1.33 (interquartile range, 0.00-4.61), the mean ABR was 3.70 (95 % confidence interval 2.94-4.66) and 70 (40 %) patients had no bleeds during the trial. Across treatment arms, 83.6 % of bleeds resolved with one injection and 95.5 % with up to two injections. N8-GP had a favourable safety profile and was well tolerated. The frequency and types of adverse events reported were as expected in this population. One patient developed inhibitory antibodies against FVIII (≥0.6 Bethesda units [BU]) after 93 N8-GP exposure days. No clinically significant safety concerns were identified and N8-GP was effective for prophylaxis and treatment of bleeds in previously treated patients.

CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36 -mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl 3 -induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36 -donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring α 2b β 3 activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36 -mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.

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The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte, platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2 hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R = 0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with APS and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies.

Antiphospholipid antibodies (aPL), the majority of which are directed against β2-glycoprotein I (β2GPI), are associated with an increased incidence of venous and arterial thrombosis. The pathogenesis of antiphospholipid/anti-β2GPI-associated thrombosis has not been defined, and is likely multifactorial. However, accumulating evidence suggests an important role for endothelial cell activation with the acquisition of a procoagulant phenotype by the activated endothelial cell. Previous work demonstrated that endothelial activation by antiphospholipid/anti-β2GPI antibodies is β2GPI-dependent. We extended these observations by defining annexin A2 as an endothelial β2GPI binding site. We also observed that annexin A2 plays a critical role in endothelial cell activation induced by anti-β2GPI antibodies, and others have described direct endothelial activation by anti-annexin A2 antibodies in patients with aPL. Similar findings have been reported using human monocytes, which also express annexin A2. Because annexin A2 is not a transmembrane protein, how binding of β2GPI/anti-β2GPI antibodies, or anti-annexin A2 antibodies, to endothelial annexin A2 causes cellular activation is unknown. Recent studies, however, suggest an important role for the Toll-like receptor family, particularly TLR4. In this article, we review the role of these interactions in the activation of endothelial cells by aPL. The influence of these antibodies on the ability of annexin A2 to enhance t-PA-mediated plasminogen activation is also discussed.