Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role.

DNA Mediated Blood Coagulation: Extracellular DNA Accelerates Fibrinogen Polymerization By Thrombin Independent of Contact Pathway

Introduction- Several in vivo studies and clinical studies have demonstrated extracellular DNA as a mediator of coagulation and this effect was reversed by the administration of DNA degrading enzyme DNase I. However, there is no clear understanding of the mechanism by which extracellular DNA activates coagulation in vitro. Conventionally, it was thought to be the activator of the contact pathway. But recent studies have shown that extracellular DNA isolated without contaminants like silica particles is a weak activator of the contact pathway. In this study, we have investigated the mechanism by which extracellular DNA is contributing to coagulation. Corroborating with recent results, we show that extracellular genomic DNA is a weak activator contact pathway. We determined that extracellular DNA accelerate fibrinogen polymerization by thrombin via a possible template mechanism. Our biophysical studies corroborate the interaction of DNA, thrombin, and fibrinogen. Understanding the mechanism of DNA induced blood coagulation will help address the gaps in the literature as well as develop inhibitors against DNA- mediated thrombosis. Methods- Silica-free extracellular DNA was purified with the PAXgene™ Blood DNA Kit. Contact activation in plasma was measured by monitoring the cleavage of the substrate S2302. To study the contact independent activation of plasma clotting by extracellular DNA, 1.5 µM Corn Trypsin Inhibitor (CTI) was applied to the plasma. Next, acceleration of fibrinogen polymerization by thrombin in presence of extracellular DNA was measured by monitoring the absorbance of 350 nm. Interaction of DNA with fibrinogen and thrombin in phosphate buffer was determined by CD spectroscopy. Results- Our results show that silica-free extracellular genomic DNA is a weak activator of the contact pathway of coagulation [Fig-A]. Moreover, genomic DNA accelerated the plasma clotting even when the contact pathway was inhibited with CTI indicating a contact independent mechanism of the procoagulant activity of extracellular DNA. Interestingly, the presence of extracellular DNA accelerated the polymerization of fibrinogen in presence of thrombin [Fig-B]. A bell-shaped dose-response curve for extracellular DNA indicates a likely template mechanism in which both thrombin and fibrinogen could assemble on the DNA molecule. These results are supported by the results from the CD spectroscopy studies where an alteration of the structure of fibrinogen and thrombin can be noticed in presence of extracellular DNA. Confocal studies further corroborate this observation. Our results also show different nucleic acids activate coagulation via different pathways. Significance- Procoagulant activity of extracellular DNA is demonstrated in several mouse models. However, a clear understanding of the mechanism of procoagulant activity of DNA in vitro has been challenging due to the caveats in the isolation of extracellular DNA where it is often contaminated with silica particles. Here we show a novel procoagulant mechanism of cell- free DNA where it augments the polymerization of fibrinogen by thrombin. These results provide insights into the mechanism of procoagulant activity of DNA which is key to develop therapeutics against procoagulant DNA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Disseminated intravascular coagulation and its immune mechanisms

Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and non-infectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of contact pathway in response to pathogen-associated or host derived damage-associated molecular patterns. The process is further amplified through inflammatory and immuno-thrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients associates with worsening morbidities and increased mortality regardless of the underlying pathology, therefore timely recognition of DIC is critical to reduce the pathologic burden. Due to diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia and fibrin/ogen conversion. Since current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of non-overt DIC and/or pre-DIC states. Therapeutic strategies for DIC patients involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in DIC patients remains high and new strategies, tailored to the underlying pathologic mechanisms, are needed.

Focal Laser Stimulation of Fly Nociceptors Activates Distinct Axonal and Dendritic Ca2+ Signals

Drosophila Class IV neurons are polymodal nociceptors that detect noxious mechanical, thermal, optical and chemical stimuli. Escape behaviors in response to attacks by parasitoid wasps are dependent on Class IV cells, whose highly branched dendritic arbors form a fine meshwork that is thought to enable detection of the wasp's needle-like ovipositor barb. To understand how mechanical stimuli trigger cellular responses, we used a focused 405-nm laser to create highly local lesions to probe the precise position needed in evoke responses. By imaging calcium signals in dendrites, axons, and soma in response to stimuli of varying positions, intensities and spatial profiles, we discovered that there are two distinct nociceptive pathways. Direct stimulation to dendrites (the contact pathway) produces calcium responses in axons, dendrites and the cell body whereas stimulation adjacent to the dendrite (the non-contact pathway) produces calcium responses in the axons only. We interpret the non-contact pathway as damage to adjacent cells releasing diffusible molecules that act on the dendrites. Axonal responses have higher sensitivities and shorter latencies. In contrast, dendritic responses have lower sensitivities and longer latencies. Stimulation of finer, distal dendrites leads to smaller responses than stimulation of coarser, proximal dendrites, as expected if the contact response depends on the geometric overlap of the laser profile and the dendrite diameter. Because the axon signals to the CNS to trigger escape behaviors, we propose that the density of the dendritic meshwork is high not only to enable direct contact with the ovipositor, but also to enable neuronal activation via diffusing signals from damaged surrounding cells. Dendritic contact evokes responses throughout the dendritic arbor, even to regions distant and distal from the stimulus. These dendrite-wide calcium signals may facilitate hyperalgesia or cellular morphological changes following dendritic damage.

Polyanions in Coagulation and Thrombosis: Focus on Polyphosphate and NETs

Neutrophils extracellular traps (NETs) and polyphosphates (polyP) have been recognized as procoagulant polyanions. The review summarizes the activities and regulation of two procoagulant mediators and compares their functions. NETs are composed of DNA and share a phosphate backbone with polyP that is built of phosphate units linked by high energy phospho-anhydride bonds. Both NETs and polyP form insoluble particulate surfaces composed of a DNA/histone meshwork or Ca2+-rich nanoparticles, respectively. The polyanions modulate coagulation involving an array of mechanisms and trigger thrombosis via activation of the factor XII (FXII)-driven procoagulant and proinflammatory contact pathway. This review outlines the current knowledge on NETs and polyP with respect to their procoagulant and prothrombotic nature, strategies for interference of their activities in circulation, as well as the crosstalk between these two molecules. A better understanding of these underlying, cellular mechanisms will shed light on the therapeutic potential of targeting NETs and polyP in coagulation and thrombosis.

A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study

Background The peak of the global COVID-19 pandemic has not yet been reached, and many countries face the prospect of a second wave of infections before effective vaccinations will be available. After an initial phase of viral replication, some patients develop a second illness phase in which the host thrombotic and inflammatory responses seem to drive complications. Severe COVID-19 disease is linked to high mortality, hyperinflammation, and a remarkably high incidence of thrombotic events. We hypothesize a crucial pathophysiological role for the contact pathway of coagulation and the kallikrein-bradykinin pathway. Therefore, drugs that modulate this excessive thromboinflammatory response should be investigated in severe COVID-19. Methods In this adaptive, open-label multicenter randomized clinical trial, we compare low molecular weight heparins at 50 IU anti-Xa/kg twice daily—or 75 IU anti-Xa twice daily for intensive care (ICU) patients—in combination with aprotinin to standard thromboprophylaxis in hospitalized COVID-19 patients. In the case of hyperinflammation, the interleukin-1 receptor antagonist anakinra will be added on top of the drugs in the interventional arm. In a pilot phase, the effect of the intervention on thrombotic markers (D-dimer) will be assessed. In the full trial, the primary outcome is defined as the effect of the interventional drugs on clinical status as defined by the WHO ordinal scale for clinical improvement. Discussion In this trial, we target the thromboinflammatory response at multiple levels. We intensify the dose of low molecular weight heparins to reduce thrombotic complications. Aprotinin is a potent kallikrein pathway inhibitor that reduces fibrinolysis, activation of the contact pathway of coagulation, and local inflammatory response. Additionally, aprotinin has shown in vitro inhibitory effects on SARS-CoV-2 cellular entry. Because the excessive thromboinflammatory response is one of the most adverse prognostic factors in COVID-19, we will add anakinra, a recombinant interleukin-1 receptor antagonist, to the regimen in case of severely increased inflammatory parameters. This way, we hope to modulate the systemic response to SARS-CoV-2 and avoid disease progressions with a potentially fatal outcome. Trial registration The EU Clinical Trials Register 2020-001739-28. Registered on April 10, 2020.

A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study

Background The peak of the global COVID-19 pandemic has not yet been reached and many countries face the prospect of a second wave of infections before effective vaccinations will be available. After an initial phase of viral replication, some patients develop a second illness phase in which the host thrombotic and inflammatory responses seems to drive complications. Severe COVID-19 disease is linked to high mortality, hyperinflammation, and a remarkably high incidence of thrombotic events. We hypothesize a crucial pathophysiological role for the contact pathway of coagulation and the kallikrein-bradykinin pathway. Therefore, drugs that modulate this excessive thromboinflammatory response should be investigated in severe COVID-19.Methods In this adaptive, open-label multicenter randomized clinical trial we compare low molecular weight heparins at 50 IU anti-Xa/kg twice daily - or 75 IU anti-Xa twice daily for intensive care (ICU) patients - in combination with aprotinin to standard thromboprophylaxis in hospitalized COVID-19 patients. In the case of hyperinflammation, the interleukin-1-receptor antagonist anakinra will be added on top of the drugs in the interventional arm. In a pilot phase, the effect of the intervention on thrombotic markers (D-dimer) will be assessed. In the full trial, the primary outcome is defined as the effect of the interventional drugs on clinical status as defined by the WHO ordinal scale for clinical improvement. Discussion In this trial we target the thromboinflammatory response at multiple levels. We intensify the dose of low molecular weight heparins to reduce thrombotic complications. Aprotinin is a potent kallikrein pathway inhibitor that reduces fibrinolysis, activation of the contact pathway of coagulation, and local inflammatory response. Additionally, aprotinin has shown in vitro inhibitory effects on SARS-CoV-2 cellular entry. Because the excessive thromboinflammatory response is one of the most adverse prognostic factors in COVID-19, we will add anakinra, a recombinant interleukin-1 receptor antagonist, to the regimen in case of severely increased inflammatory parameters. This way, we hope to modulate the systemic response to SARS-CoV-2 and avoid disease progressions with a potentially fatal outcome. Trial registration This trial is registered in the EU Clinical Trials Register. Registration number: 2020-001739-28. Registered on 2020-04-10.