Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation

Background: Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. Objective and methods: To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. Results: Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. Conclusions: These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.</p

Hemostatic and protein C pathway dysfunction in the pathogenesis of experimental cerebral malaria

Accumulating data suggest that hemostatic dysfunction contributes to Plasmodium falciparum malaria pathogenesis. In addition, specific mechanisms through which the protein C pathway modulates P. falciparum pathogenesis have been described. We hypothesized that the anticoagulant and anti-inflammatory activities of recombinant activated protein C (APC) may possess therapeutic value in the setting of cerebral malaria (CM). In order to address this hypothesis, we assessed hemostatic parameters in an established murine model of experimental cerebral malaria (ECM), and using the same model, investigated the ability of recombinant APC to ameliorate ECM. In keeping with findings in patients with severe P. falciparum malaria, we observed that dysregulated thrombin generation and protein C pathway dysfunction were both late features of ECM. Furthermore, pretreatment with a monoclonal anti-EPCR antibody that blocks protein C/APC binding prior to P. berghei inoculation significantly reduced overall survival. Conversely, mice treated with recombinant APC exhibited a marked attenuation in clinical ECM progression and parasitemia, in parallel with a significant increase in overall survival. All together, these findings confirm that hemostatic and protein C pathway dysfunction are both consistent features in human and ECM, and demonstrate for the first time a role for recombinant APC in reducing clinical progression and mortality in ECM. </div