Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug. IntroductionUnfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs) are well-established anticoagulants commonly used to prevent or treat thromboembolic disorders and to prevent clotting of extracorporeal blood during hemodialysis and cardiac surgery. Besides the continuous monitoring required for UFH, both UFH and LMWHs have serious side effects, such as hemorrhage, heparin-induced thrombocytopenia, 1 or adverse reactions related to a contaminating product in heparins derived from animal tissues. 2 Fondaparinux is a synthetic pentasaccharide whose structure and mechanism of action present several homologies with UFH and LMWH. It will thus be designated as a heparin derivative in the present work. Fondaparinux is the first factor Xa (FXa) selective inhibitor that has received approval for clinical use. It has been evaluated in different clinical trials and appears at least as effective as enoxaparin (a LMWH) for several indications. It also appears safer compared with UFH or LMWHs because of its synthetic origin and its favorable pharmacokinetic profile, which allows a daily injection without continuous monitoring. Furthermore, only few rare cases of thrombocytopenia under fondaparinux treatment were reported, with controversy on a direct causal relationship between the drug and the symptoms in one case. [3][4][5] As with every anticoagulant, heparin derivatives are associated with a bleeding risk, and with fondaparinux, this risk is potentiated by its long half-life of approximately 17 hours. 6 UFH and LMWHs can be totally or partially reversed by the administration of protamine sulfate, a nonspecific heparin inhibitor, but prota...

Background Prolonged recovery is common after SARs‐CoV‐2 infection, however the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF‐ADAMTS13 imbalance, dysregulated angiogenesis and immuno‐thrombosis are hallmarks of acute COVID‐19. We hypothesized that VWF‐ADAMTS13 imbalance persists in convalescence together with EC activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. Patients and Methods Fifty patients were reviewed at a minimum of 6 weeks following acute COVID‐19. ADAMTS13, WPB proteins and angiogenesis‐related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n=20) and acute COVID‐19 patients (n=36). Results ADAMTS13 levels were reduced (p=0.009) and the VWF/ADAMTS13 ratio was increased in convalescence (p=0.0004). Levels of Platelet Factor 4 (PF4), a putative protector of VWF, were also elevated (p=0.0001). A non‐significant increase in WPB proteins Angiopoietin‐2 (Ang‐2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis‐related proteins was observed in convalescent COVID‐19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. Conclusion Our data provide insights into sustained EC activation, dysregulated angiogenesis and VWF/ADAMTS13 axis imbalance in convalescent COVID‐19. In keeping with the pivotal role of immuno‐thrombosis in acute COVID‐19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.

To cite this article: Fazavana J, Bianchini EP, Saller F, Smadja C, Picard V, Taverna M, Borgel D. A chemically-modified inactive antithrombin as a potent antagonist of fondaparinux and heparin anticoagulant activity. J Thromb Haemost 2013; 11: 1128-36.Summary. Background: Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. Objectives: To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs. Methods: Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. Results: AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. Conclusions: AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.

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.

Terminal sialylation determines plasma VWF half-life. A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we show that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF:Ag levels (p

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 µg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.

Essentials• Cytoadhesion of infected erythrocytes is critical in cerebral malaria (CM) pathobiology.• Markedly elevated plasma VWF levels represents an early hallmark feature of CM.• Platelets directly impact malaria pathogenesis through multiple mechanisms.• VWF-ADAMTS-13 axis targeting in CM may offer novel therapeutic opportunities.