Introduction: Neutrophil extracellular trap (NET) formation was recently identified as a novel mechanism to kill pathogens. However, excessive NET formation in sepsis can injure host tissues. We have recently shown that parenteral vitamin C (VitC) is protective in sepsis. Whether VitC alters NETosis is unknown. Methods: We used Gulo−/− mice as they lack the ability to synthesize VitC. Sepsis was induced by intraperitoneal infusion of a fecal stem solution (abdominal peritonitis, FIP). Some VitC deficient Gulo−/− mice received an infusion of ascorbic acid (AscA, 200 mg/kg) 30 min after induction of FIP. NETosis was assessed histologically and by quantification for circulating free DNA (cf-DNA) in serum. Autophagy, histone citrullination, endoplasmic reticulum (ER) stress, NFκB activation and apoptosis were investigated in peritoneal PMNs. Results: Sepsis produced significant NETs in the lungs of VitC deficient Gulo−/− mice and increased circulating cf-DNA. This was attenuated in the VitC sufficient Gulo−/− mice and in VitC deficient Gulo−/− mice infused with AscA. Polymorphonuclear neutrophils (PMNs) from VitC deficient Gulo−/− mice demonstrated increased activation of ER stress, autophagy, histone citrullination, and NFκB activation, while apoptosis was inhibited. VitC also significantly attenuated PMA induced NETosis in PMNs from healthy human volunteers. Conclusions: Our in vitro and in vivo findings identify VitC as a novel regulator of NET formation in sepsis. This study complements the notion that VitC is protective in sepsis settings.

Factor XI (FXI) is the zymogen of a plasma protease, factor XIa (FXIa), that contributes to thrombin generation during blood coagulation by proteolytic activation of several coagulation factors, most notably factor IX (FIX). FXI is a homolog of prekallikrein (PK), a component of the plasma kallikrein-kinin system. While sharing structural and functional features with PK, FXI has undergone adaptive changes that allow it to contribute to blood coagulation. Here we review current understanding of the biology and enzymology of FXI, with an emphasis on structural features of the protein as they relate to protease function.

Vitamin C (VitC) or ascorbic acid (AscA), a cofactor for collagen synthesis and a primary antioxidant, is rapidly consumed post-wounding. Parenteral VitC administration suppresses pro-inflammatory responses while promoting anti-inflammatory and pro-resolution effects in human/murine sepsis. We hypothesised that VitC could promote wound healing by altering the inflammatory, proliferative and remodelling phases of wound healing. Mice unable to synthesise VitC (Gulo(-/-) ) were used in this study. VitC was provided in the water (sufficient), withheld from another group (deficient) and supplemented by daily intra-peritoneal infusion (200 mg/kg, deficient + AscA) in a third group. Full thickness excisional wounds (6 mm) were created and tissue collected on days 7 and 14 for histology, quantitative polymerase chain reaction (qPCR) and Western blotting. Human neonatal dermal fibroblasts (HnDFs) were used to assess effects of In conclusion, VitC favorably on proliferation. Histological analysis showed improved wound matrix deposition and organisation in sufficient and deficient +AscA mice. Wounds from VitC sufficient and deficient + AscA mice had reduced expression of pro-inflammatory mediators and higher expression of wound healing mediators. Supplementation of HnDF with AscA induced the expression of self-renewal genes and promoted fibroblast proliferation. VitC favourably impacts the spatiotemporal expression of transcripts associated with early resolution of inflammation and tissue remodelling.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Factor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged "surfaces" such as polymeric orthophosphate. FXII is comprised of non-catalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation, but restricts activation when FXII is not surface-bound. From the N-terminus, the heavy chain contains fibronectin type II (FN2), epidermal growth factor-1 (EGF1), fibronectin type I (FN1), EGF2, and kringle (KNG) domains, and a proline-rich region (PRR). It shares this organization with its homolog, pro-hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains to FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains, and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog Ɛ-aminocaproic acid. A model is proposed in which an Ɛ-aminocaproic acid-sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

Background Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long‐standing hypothesis has been that allosterism originating from heparin‐binding site(s) on coagulation enzymes is a promising approach to yield safer agents. Objectives To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk. Methods We employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work. Results Sulfated chiro‐inositol (SCI), a non‐saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin‐binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway‐based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum‐dose‐tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl3‐induced arterial and thromboplastin‐induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 μg/animal, which matched enoxaparin at 2500 μg/animal. Conclusions Overall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.

Background Allosteric inhibition is a promising approach to developing a new group of anticoagulants with potentially reduced bleeding consequences. Recently, we designed sulfated β-O4 lignin (SbO4L) as an allosteric inhibitor that targets exosite 2 of thrombin to reduce fibrinogen cleavage through allostery and compete with GPIbα to reduce platelet activation. Objective To assess 1) the antithrombotic potential of a novel approach of simultaneous exosite 2-dependent allosteric inhibition of thrombin and competitive inhibition of platelet activation and 2) the promise of SbO4L as the first-in-class antithrombotic agent. Methods A combination of whole blood thromboelastography, hemostasis analysis, mouse arterial thrombosis models and mouse tail bleeding studies were used to assess antithrombotic potential. Results and Conclusions SbO4L extended clot initiation time, reduced maximal clot formation, platelet contractile force and clot elastic modulus suggesting dual anticoagulant and antiplatelet effects. These effects were comparable to those measured for enoxaparin. A dose of 1 mg SbO4L per mouse prevented occlusion in 100% of arteries, while lower doses exhibited proportionally reduced response. Likewise, the time to occlusion increased ~70% with 0.5 mg dose in mouse Rose Bengal thrombosis model. Finally, tail bleeding studies demonstrated that SbO4L does not increase bleeding propensity. In comparison, a 0.3 mg dose of enoxaparin increased bleeding time and blood volume loss. Overall, this work highlights the promise of allosteric inhibition approach and presents SbO4L as a novel anticoagulant with potentially reduced bleeding side effects.

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