Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin.

BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a ß-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both ß-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by ß-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

Anticoagulation and Bleeding during Veno-Venous Extracorporeal Membrane Oxygenation: Insights from the PROTECMO Study.

Rationale: Definitive guidelines for anticoagulation management during veno-venous extracorporeal membrane oxygenation (VV ECMO) are lacking, whereas bleeding complications continue to pose major challenges. Objectives: To describe anticoagulation modalities and bleeding events in adults receiving VV ECMO. Methods: This was an international prospective observational study in 41 centers, from December 2018 to February 2021. Anticoagulation was recorded daily in terms of type, dosage, and monitoring strategy. Bleeding events were reported according to site, severity, and impact on mortality. Measurements and Main Results: The study cohort included 652 patients, and 8,471 days on ECMO were analyzed. Unfractionated heparin was the initial anticoagulant in 77% of patients, and the most frequently used anticoagulant during the ECMO course (6,221 d; 73%). Activated partial thromboplastin time (aPTT) was the most common test for monitoring coagulation (86% of days): the median value was 52 seconds (interquartile range, 39 to 61 s) but dropped by 5.3 seconds after the first bleeding event (95% confidence interval, -7.4 to -3.2; P < 0.01). Bleeding occurred on 1,202 days (16.5%). Overall, 342 patients (52.5%) experienced at least one bleeding event (one episode every 215 h on ECMO), of which 10 (1.6%) were fatal. In a multiple penalized Cox proportional hazard model, higher aPTT was a potentially modifiable risk factor for the first episode of bleeding (for 20-s increase; hazard ratio, 1.07). Conclusions: Anticoagulation during VV ECMO was a dynamic process, with frequent stopping in cases of bleeding and restart according to the clinical picture. Future studies might explore lower aPTT targets to reduce the risk of bleeding.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-ß-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-ß-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity.

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells.

Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration, which is typically reliant on bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line and confirm that protease-activated receptor 1 (PAR1) activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we found that gentle contact compression, exerted using a custom built 'cell press' to mechanically stimulate cells, reduced thrombin-induced blebbing. Thrombin-induced blebbing was similarly attenuated using the small molecule Yoda1, an agonist of the mechanosensitive Ca2+ channel Piezo1, and this attenuation was impaired in Piezo1-depleted cells. Additionally, Piezo1 activation suppressed thrombin-induced phosphorylation of ezrin, radixin and moesin (ERM) proteins, which are implicated in the blebbing process. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.

New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Stoichiometry and architecture of the platelet membrane complex glycoprotein Ib-IX-V.

Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbß, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.

Low molecular weight heparin decreases pro-coagulant activity in clinical MSC products.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). METHODS: Clinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. RESULTS: Presence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DISCUSSION: All MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.

Sustainable inflammatory activation following spinal cord injury is driven by thrombin-mediated dynamic expression of astrocytic chemokines.

Acute spinal cord injury (SCI) always results in sustainable recruitment of inflammatory cells driven by sequentially generated chemokines, thereby eliciting excessive neuroinflammation. However, the underlying mechanism of temporally produced chemokines remains elusive. Reactive astrocytes are known to be the main sources of chemokines at the lesion site, which can be immediately activated by thrombin following SCI. In the present study, SCI was shown to induce a sequential production of chemokines CCL2 and CCL5 from astrocytes, which were associated with a persistent infiltration of macrophages/microglia. The rapidly induced CCL2 and later induced CCL5 from astrocytes were regulated by thrombin at the damaged tissues. Investigation of the regulatory mechanism revealed that thrombin facilitated astrocytic CCL2 production through activation of ERK/JNK/NFκB pathway, whereas promoted CCL5 production through PLCß3/NFκB and ERK/JNK/NFκB signal pathway. Inhibition of thrombin activity significantly decreased production of astrocytic CCL2 and CCL5, and reduced the accumulation of macrophages/microglia at the lesion site. Accordingly, the locomotor function of rats was remarkably improved. The present study has provided a new regulatory mechanism on thrombin-mediated sequential production of astrocytic chemokines, which might be beneficial for clinical therapy of CNS neuroinflammation.

Measurement of recombinant porcine factor VIII in patients with congenital haemophilia A and inhibitors in the presence of emicizumab.

INTRODUCTION: Recombinant porcine factor VIII (rpFVIII) is a treatment option for break-through bleeds in patients with congenital haemophilia A with inhibitors (CHAwI) on emicizumab. However, there are limited data about the measurement of rpFVIII in the presence of emicizumab. AIM: To analyse whether rpFVIII can be measured with a chromogenic assay with bovine component (bCSA) in plasma from CHAwI on emicizumab treatment. METHODS: In the first part of the study, FVIII deficient plasma was spiked with rpFVIII, in the second part, commercial plasma from CHAwI was spiked with emicizumab and rpFVIII, and in the third part, plasma from CHAwI on emicizumab treatment was spiked with rpFVIII. FVIII was then measured with bCSA and a chromogenic assay with human component (hCSA). Thrombin generation (TG) and clot-waveform analysis (CWA) were also carried out. RESULTS: The recovery of rpFVIII measured with bCSA is approximately 80% and is further influenced by the presence of an anti-porcine inhibitor. rpFVIII assessed with hCSA was influenced by emicizumab. CWA and TG showed a weak correlation with baseline emicizumab concentration, but peak thrombin and CWA correlated well with increasing emicizumab concentrations and rpFVIII activities. CONCLUSION: This study indicates that rpFVIII can be measured in the presence of emicizumab with a bCSA. A calibration curve for the measurement of rpFVIII with bCSA should be established.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

EUS-Guided Thrombin Injection and Coil Implantation for Gastric Varices: Feasibility, Safety, and Outcomes.

BACKGROUND AND AIMS: Gastric varices (GV) are reported in up to 20% of patients with portal hypertension, and bleeding is often more severe and challenging than oesophageal variceal bleeding. There is limited data on prophylaxis of GV bleeding or management in the acute setting, and different techniques are utilised. This study aims to evaluate outcomes following endoscopic ultrasound (EUS) guided placement of coils in combination with thrombin to manage GV. METHODS: We retrospectively reviewed all patients treated with combination EUS-guided therapy with coils and thrombin between October 2015 and February 2020. RESULTS: 20 patients underwent 33 procedures for GV therapy; 16/20 (80%) were type 1 Isolated GV (IGV1), and the remainder were type 2 Gastroesophageal Varices (GOV2). Median follow-up was 842 days (Interquartile range (IQR) 483-961). 17/20 (85%) had underlying cirrhosis, the most common aetiologies being alcohol-related liver disease and non-alcoholic steatohepatitis (NASH). The median Child-Pugh (CP) score was 6 (IQR 5-7). In 11/20 (55%) cases, the indication was secondary prophylaxis to prevent rebleeding; in 2/20 (10%), the bleeding was acute. Technical success was achieved in 19/20 (95%) of cases. During follow-up, the obliteration of flow within the varices was achieved in 17/20 (85%) cases. The 6-week survival was 100%, and 2 adverse events were reported: cases of rebleeding at day 5 and day 37; both rebleeds were successfully managed endoscopically. CONCLUSIONS: EUS-guided GV obliteration combining coil placement with thrombin, in our experience, is technically safe with good medium-term efficacy. A multicenter randomised controlled trial comparing different treatment strategies would be desirable to understand options better.

Serum Proteomics Identified TAFI as a Potential Molecule Facilitating the Migration of Peripheral Monocytes to Damaged White Matter During Chronic Cerebral Hypoperfusion.

Neuroinflammation is assumed as the critical pathophysiologic mechanism of white matter lesions (WMLs), and infiltrated peripheral monocyte-derived macrophages are implicated in the development of neuroinflammation. This study sought to explore the blood molecules that promote the migration of peripheral monocytes to the sites of WMLs. The serum protein expression profiles of patients and Sprague-Dawley rat models with WMLs were detected by data-independent acquisition (DIA) proteomics technique. Compared with corresponding control groups, we acquired 62 and 41 differentially expressed proteins (DEPs) in the serum of patients and model rats with WMLs respectively. Bioinformatics investigations demonstrated that these DEPs were linked to various Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms involved in neuroinflammation. Afterward, we identified thrombin-activatable fibrinolysis inhibitor (TAFI) as a shared and overexpressed protein in clinical and animal serum samples, which was further verified by enzyme-linked immunosorbent assay. Additionally, an upregulation of TAFI was also observed in the white matter of rat models, and the inhibition of TAFI impeded the migration of peripheral monocytes to the area of WMLs. In vitro experiments suggested that TAFI could enhance the migration ability of RAW264.7 cells and increase the expression of Ccr2. Our study demonstrates that neuroinflammatory signals can be detected in the peripheral blood of WMLs patients and model rats. TAFI may serve as a potential protein that promotes the migration of peripheral monocytes to WMLs regions, thereby providing a novel molecular target for further investigation into the interaction between the central and peripheral immune systems.