Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

Background The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. Objective To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. Methods We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Conclusions Our data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function

Acute inflammation is exacerbated in mice genetically predisposed to a severe protein C deficiency

The anticoagulant, activated protein C (aPC), possesses antithrombotic, profibrinolytic, anti-inflammatory, and antiapoptotic properties, and the level of this protein is an important marker of acute inflammatory responses. Although infusion of aPC improves survival in a subset of patients with severe sepsis, evidence as to how aPC decreases mortality in these cases is limited. Because a total deficiency of PC shows complete neonatal lethality, no animal model currently exists to address the mechanistic relationships between very low endogenous aPC levels and inflammatory diseases. Here, we show for the first time that novel genetic dosing of PC strongly correlates with survival outcomes following endotoxin (LPS) challenge in mice. The data provide evidence that very low endogenous levels of PC predispose mice to early-onset disseminated intravascular coagulation, thrombocytopenia, hypotension, organ damage, and reduced survival after LPS challenge. Furthermore, evidence of an exacerbated inflammatory response is observed in very low PC mice but is greatly reduced in wild-type cohorts. Reconstitution of low-PC mice with recombinant human aPC improves hypotension and extends survival after LPS challenge. This study directly links host endogenous levels of PC with various coagulation, inflammation, and hemodynamic end points following a severe acute inflammatory challenge

Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27

Please see erratum at DOI: 10.1182/blood-2014-09-604066 The errors have been corrected in the online version, which now differs from the print version.Cellular junctions are essential to the normal functioning of the endothelium and control angiogenesis, tissue leak, and inflammation. From a screen of micro RNAs (miRNAs) altered in in vitro angiogenesis, we selected a subset predicted to target junctional molecules. MiR-27a was rapidly downregulated upon stimulation of in vitro angiogenesis, and its level of expression is reduced in neovessels in vivo. The downregulation of miR-27a was essential for angiogenesis because ectopic expression of miR-27a blocked capillary tube formation and angiogenesis. MiR-27a targets the junctional, endothelial-specific cadherin, VE-cadherin. Consistent with this, vascular permeability to vascular endothelial growth factor in mice is reduced by administration of a general miR-27 inhibitor. To determine that VE-cadherin was the dominant target of miR-27a function, we used a novel technology with “Blockmirs,” inhibitors that bind to the miR-27 binding site in VE-cadherin. The Blockmir CD5-2 demonstrated specificity for VE-cadherin and inhibited vascular leak in vitro and in vivo. Furthermore, CD5-2 reduced edema, increased capillary density, and potently enhanced recovery from ischemic limb injury in mice. The Blockmir technology offers a refinement in the use of miRNAs, especially for therapy. Further, targeting of endothelial junctional molecules by miRNAs has clinical potential, especially in diseases associated with vascular leak.Jennifer A. Young, Ka Ka Ting, Jia Li, Thorleif Moller, Louise Dunn, Ying Lu, Joshua Moses, Leonel Prado-Lourenço, Levon M. Khachigian, Martin Ng, Philip A. Gregory, Gregory J. Goodall, Anna Tsykin, Ilana Lichtenstein, Christopher N. Hahn, Nham Tran, Nicholas Shackel, James G. Kench, Geoffrey McCaughan, Mathew A. Vadas, and Jennifer R. Gambl