BACKGROUND

It has long been known that a hypercoagulability state develops after surgery. A surge in circulating cytokine levels is also commonly found in the postoperative period. These cytokines have all been shown to be capable of inducing a hypercoagulability state. Recently laparoscopic cholecystectomy (LC) has been introduced, and its advantages over the open procedure seem related to the reduced surgical trauma. LC is associated with a diminished acute-phase response compared with the open procedure. Our present knowledge on the influence of laparoscopic upon coagulation and fibrinolysis is incomplete and based on a few studies.

METHODS

The aim of this prospective, nonrandomized study was to investigate hemostatic system alterations in patients who undergo open and laparoscopic cholecystectomy. In addition we also measured the plasma cytokine profile to explore any relationship between changes in plasma cytokine levels and postoperative coagulation profile. Between September 1999 and April <em>2</em>00<em>2</em>, 71 patients were nonrandomly assigned to open (group 1) or laparoscopic cholecystectomy (group <em>2</em>). All patients from group 1 were operated by a surgical team different from ours, who prefers the OC procedure. The patients with acute cholecystitis were excluded. Prothrombin fragment 1.<em>2</em> (F1.<em>2</em>), thrombin-antithrombin (TAT), fibrinogen, soluble fibrin, antithrombin III (AT), protein C, plasminogen, and <em>D</em>-<em>dimer</em> levels were measured at baseline and at 1, <em>2</em>4, 48, and 7<em>2</em> h postoperatively. Serial serum levels of IL-1beta and IL-6 were measured by colorimetric enzyme-linked immunosorbent assay (ELISA).

RESULTS

Plasma levels of F1.<em>2</em>, TAT, fibrinogen, soluble fibrin, and <em>D</em>-<em>dimer</em> increased significantly in group 1. Plasma levels of AT, protein C, and plasminogen decreased in both groups. In the OC group, the serum IL-3 and IL-6 levels began to significantly increased as early as 1 h from the beginning of the operation, revealing a peak at the sixth hour. When IL-6 and IL-1 levels were markedly elevated also, F1.<em>2</em>, fibrinogen, and soluble fibrin levels were increased.

CONCLUSIONS

Only mild hypercoagulability was observed in patients who had undergone laparoscopic cholecystectomy. The cytokine surge was correlated with hypercoagulability. There was in fact a positive correlation between IL-6 level and hypercoagulability. The correlation between cytokine levels and coagulation activation may be related to the type of surgery performed. Further studies are required to investigate these issues.

Bleeding is the most serious adverse event of oral anticoagulants and is a major cause of morbidity and mortality in such patients. Rapid reversal of anticoagulation in bleeding patients or prior to urgent surgery is mandatory. The therapeutic options in these situations include administration of fresh frozen plasma (FFP), and recently of prothrombin complex concentrates (PCCs). However, viral safety and thrombogenicity of PCCs remain issues of concern. In the present study, we administered Octaplex, a new solvent/detergent (S/<em>D</em>) treated and nanofiltered PCC, to excessively anticoagulated bleeding patients or to anticoagulated patients facing urgent surgery. Ten excessively anticoagulated patients with major bleeding and 10 anticoagulated patients awaiting surgery (median age 7<em>2</em>.5 (43-83) years, 9 females) received a median dose of <em>2</em>6.1 IU/kg body weight (BW) of Octaplex for reversal of anticoagulation. Response to Octaplex was rapid with decline of INR within 10 min after Octaplex administration (from 6.1+/-<em>2</em>. to 1.5+/-0.3). Clinical response was graded as good in most patients (85%) and as moderate in the rest. Octaplex administration was uneventful in all patients. Following Octaplex administration, a small increase in F1+<em>2</em> levels was observed in bleeding patients, whereas <em>D</em>-<em>dimer</em> level did not change significantly. We conclude that Octaplex is effective and safe in situations where rapid reversal of anticoagulation is needed.

Popular explanations of substituent effects in π-stacking interactions hinge upon substituent-induced changes in the aryl π-system. This entrenched view has been used to explain substituent effects in countless stacking interactions over the past <em>2</em> decades. However, for a broad range of stacked <em>dimers</em>, it is shown that substituent effects are better described as arising from local, direct interactions of the substituent with the proximal vertex of the other ring. Consequently, substituent effects in stacking interactions are additive, regardless of whether the substituents are on the same or opposite rings. Substituent effects are also insensitive to the introduction of heteroatoms on distant parts of either stacked ring. This local, direct interaction viewpoint provides clear, unambiguous explanations of substituent effects for myriad stacking interactions that are in accord with robust computational data, including <em>D</em>FT-<em>D</em> and new benchmark CCS<em>D</em>(T) results. Many of these computational results cannot be readily explained using traditional π-polarization-based models. Analyses of stacking interactions based solely on the sign of the electrostatic potential above the face of an aromatic ring or the molecular quadrupole moment face a similar fate. The local, direct interaction model provides a simple means of analyzing substituent effects in complex aromatic systems and also offers simple explanations of the crystal packing of fluorinated benzenes and the recently published dependence of the stability of protein-RNA complexes on the regiochemistry of fluorinated base analogues [J. Am. Chem. Soc.<em>2</em>011, 133, 3687-3689].

Since there is some clinical evidence that the clinical course of patients with chronic obstructive pulmonary disease (COP<em>D</em>) may be complicated by thrombosis in the pulmonary vessels, we studied whether a hypercoagulability state (HS) does occur in COP<em>D</em>. Plasma levels of prothrombin F1 + <em>2</em> fragment, a marker of thrombin generation, <em>D</em>-<em>dimer</em>, a marker of in vivo thrombin and plasmin activation, and fibrinogen were measured in 37 COP<em>D</em> patients and in 30 controls matched for sex and age. COP<em>D</em> patients had significantly higher values of F1 + <em>2</em> (p = 0.0001) and fibrinogen (p = 0.0005) than healthy subjects. The difference persisted after excluding smoking patients. F1 + <em>2</em> was not correlated with PaO<em>2</em> (r = 0.0<em>2</em>, p>> 0.05) and PaCO<em>2</em> (p = 0.1<em>2</em>, p>> 0.05). In six patients with stable COP<em>D</em> and F1 + <em>2</em> greater than 1.65 nM (mean + <em>2</em> S<em>D</em> of controls) subcutaneous calcium-heparin therapy (5000 IU t.i.d. for 15 days) significantly reduced F1 + <em>2</em> (p = 0.03) and PaCO<em>2</em> (p = 0.01). This study shows that COP<em>D</em> patients have an ongoing prothrombotic state which could potentially account for thrombosis occurring in pulmonary vessels. The effect of calcium-heparin treatment on clotting system activation and blood gas may suggest this treatment as potential candidate for prospective study in COP<em>D</em> patients.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L<em>2</em> domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-6<em>2</em>1, sEGFR6<em>2</em>1), binds hEGF and hTGF-alpha with high affinity (K(<em>D</em>) = 13-<em>2</em>1 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab5<em>2</em>8. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a <em>2</em>:<em>2</em> EGF:sEGFR501 <em>dimer</em> complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(47<em>2</em>) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab5<em>2</em>8. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L<em>2</em> domain, as part of the binding site that recognizes Arg(45) of hEGF.

Methylglyoxal (MG) is a highly reactive alpha-oxoaldehyde formed endogenously in numerous enzymatic and nonenzymatic reactions. It modifies arginine and lysine residues in proteins forming advanced glycation end-products such as N(<em>delta</em>)-(5-methyl-4-imidazolon-<em>2</em>-yl)-L-ornithine (MG-H1), <em>2</em>-amino-5-(<em>2</em>-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H<em>2</em>), <em>2</em>-amino-5-(<em>2</em>-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3), argpyrimidine, N(<em>delta</em>)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-<em>2</em>-yl)-L-ornithine (THP), N(epsilon)-(1-carboxyethyl)lysine (CEL), MG-derived lysine <em>dimer</em> (MOLD), and <em>2</em>-ammonio-6-({<em>2</em>-[4-ammonio-5-oxido-5-oxopently)amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene}amino)hexanoate (MODIC), which have been identified in vivo and are associated with complications of diabetes and some neurodegenerative diseases. In foodstuffs and beverages, MG is formed during processing, cooking, and prolonged storage. Fasting and metabolic disorders and/or defects in MG detoxification processes cause accumulation of this reactive dicarbonyl in vivo. In addition, the intake of low doses of MG over a prolonged period of time can cause degenerative changes in different tissues, and can also exert anticancer activity. MG in biological samples can be quantified by HPLC or GC methods with preliminary derivatization into more stable chromophores and/or fluorophores, or derivatives suitable for determination by MS by use of diamino derivatives of benzene and naphthalene, 6-hydroxy-<em>2</em>,4,5-triaminopyrimidine, cysteamine, and o-(<em>2</em>,3,4,5,6-pentafluorobenzyl) hydroxylamine. The methods include three basic steps: deproteinization, incubation with derivatization agent, and chromatographic analysis with or without preliminary extraction of the formed products.

To determine the role of plasma tissue factor on disseminated intravascular coagulation (<em>D</em>IC) in trauma and septic patients, and also to investigate the relationships between tissue factor and various thrombin markers, we made a prospective cohort study. Forty trauma patients and <em>2</em>0 patients with sepsis were classified into subgroups according to the complication of <em>D</em>IC. Plasma tissue factor antigen concentration (tissue factor), prothrombin fragment F1+<em>2</em> (PF1+<em>2</em>), thrombin antithrombin complex (TAT), fibrinopeptide A (FPA), and <em>D</em>-<em>dimer</em> were measured on the day of admission (day 0), and on days 1, <em>2</em>, 3, and 4 after admission. The levels of plasma tissue factor in the <em>D</em>IC group were more elevated than those of the non-<em>D</em>IC group in both the trauma and the septic patients. In patients with sepsis, tissue factor levels on days 0 through 4 in the non-<em>D</em>IC group showed markedly higher values than those in the control patients (135 +/- 8 pg/ml). Significant correlations between tissue factor and PF1+<em>2</em>, TAT, FPA, and <em>D</em>-<em>dimer</em> were observed in the <em>D</em>IC patients, however, no such correlations were found in the non-<em>D</em>IC patients. These results suggest that elevated plasma tissue factor in patients with trauma and sepsis gives rise to thrombin generation, followed by intravascular coagulation.

In acute myocardial infarction (AMI), increased Tissue Factor (TF) expression on circulating monocytes and microparticles (MP) may contribute to thrombotic events. Because surfacebound Tissue Factor Pathway Inhibitor-1 (TFPI) inhibits TF activity on monocytes and endothelial cells decreased TFPI expression may reinforce the procoagulant activity of circulating MP. Aim of the study was to analyze TFPI expression and TF activity after stenting and thrombolysis inAMI. Thirty-nine patients of a randomized study comparing intravenous thrombolysis (n=19) and stenting (n=<em>2</em>0) were included. Before and after therapy blood samples for analysis of MPs, TF antigen and activity, prothrombin fragment F1+<em>2</em> and <em>D</em>-<em>dimer</em> were obtained. TFPI expression on TF positive MPs was decreased after thrombolysis but not after stenting. In contrast, TF plasma levels and TF positive MP remained unchanged in both treatment groups. After thrombolysis increased <em>D</em>-<em>dimer</em> and F1+<em>2</em> plasma concentrations indicated activation of fibrinolysis and coagulation. Significance of MPTFPI for inhibition of TF activity was measured using inhibitory TFPI antibodies. Membrane-associated TFPI inhibited TF activity on circulating MPs. After thrombolysis inhibition of TF activity by TFPI was decreased as compared to stenting. Correlation of circulating TF with F1+<em>2</em> only after thrombolysis, suggests a role for TF-induced activation of coagulation after thrombolysis. Enhanced TF activity on circulating MPs in AMI is inhibited by endogenous surface-boundTFPI. After thrombolysis but not after stenting MPTFPI is degraded and may induce thrombin generation due to unopposed tissue factor activity. Anti-TF therapies during thrombolysis may reduce thrombin generation in AMI.

BACKGROUND

Prophylaxis with either intravenous (i.v.) factor VIII (FVIII) or FIX is the gold standard of care for patients with severe hemophilia. A monoclonal antibody (concizumab) targeting tissue factor pathway inhibitor (TFPI) that can be administered subcutaneously (s.c.) has the potential to alter current concepts of prophylaxis in hemophilia.

OBJECTIVE

To evaluate the safety and describe the pharmacokinetics and pharmacodynamics of single-dose concizumab in healthy volunteers and patients with hemophilia A or B.

METHODS

In this first human dose, phase 1, multicenter, randomized, double-blind, placebo-controlled trial escalating single i.v. (0.5-9000 μg kg(-1) ) or s.c. (50-3000 μg kg(-1) ) doses of concizumab were administered to healthy volunteers (n = <em>2</em>8) and hemophilia patients (n = <em>2</em>4).

RESULTS

Concizumab had a favorable safety profile after single i.v. or s.c. administration. There were no serious adverse events and no anti-concizumab antibodies. No clinically relevant changes in platelets, prothrombin time, activated partial thromboplastin time, fibrinogen, or antithrombin were found. A dose-dependent procoagulant effect of concizumab was seen as increased levels of <em>D</em>-<em>dimers</em> and prothrombin fragment 1 + <em>2</em>. Nonlinear pharmacokinetics of concizumab was observed due to target-mediated clearance. A maximum mean AUC0-∞ of 33 960 h μg mL(-1) and a maximum mean concentration of <em>2</em>47 μg mL(-1) was measured at the highest dose.

CONCLUSIONS

Concizumab showed a favorable safety profile after i.v. or s.c. administration and nonlinear pharmacokinetics was observed due to target-mediated clearance. A concentration-dependent procoagulant effect of concizumab was observed, supporting further study into the potential use of s.c. concizumab for hemophilia treatment.

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded <em>dimer</em> of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the C<em>D</em>R1 and C<em>D</em>R3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) C<em>D</em>R3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(<em>D</em>)= approximately <em>2</em> x 10(-7) M). One clone, designated 1<em>2</em>Y-<em>2</em>, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the C<em>D</em>R1 and C<em>D</em>R3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 1<em>2</em>Y-<em>2</em> and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.

<strong class="sub-title"> Objective: </strong> Describe characteristics, daily care and outcomes of patients with coronavirus disease <em>2</em>019 (COVID-19) acute respiratory distress syndrome (ARDS).

Design: Case series of 73 patients.

Setting: Large tertiary hospital in Milan.

<strong class="sub-title"> Participants: </strong> Mechanically ventilated patients with confirmed COVID-19 admitted to the intensive care unit (ICU) between <em>2</em>0 February and <em>2</em> April <em>2</em>0<em>2</em>0.

Main outcome measures: Demographic and daily clinical data were collected to identify predictors of early mortality.

<strong class="sub-title"> Results: </strong> Of the 73 patients included in the study, most were male (83.6%), the median age was 61 years (interquartile range [IQR], 54-69 years), and hypertension affected 5<em>2</em>.9% of patients. Lymphocytopenia (median, 0.77 x 10³ per mm³; IQR, 0.58-1.00 x 10³ per mm³), hyperinflammation with C-reactive protein (median, 184.5 mg/dL; IQR, 108.<em>2</em>-<em>2</em>69.1 mg/dL) and pro-coagulant status with D-dimer (median, 10.1 μg/m; IQR, 5.0-<em>2</em>3.8 μg/m) were present. Median tidal volume was 6.7 mL/kg (IQR, 6.0-7.5 mL/kg), and median positive end-expiratory pressure was 1<em>2</em> cmH_<em>2</em>O (IQR, 10-14 cmH_<em>2</em>O). In the first 3 days, prone positioning (1<em>2</em>-16 h) was used in 63.8% of patients and extracorporeal membrane oxygenation in five patients (6.8%). After a median follow-up of 19.0 days (IQR, 15.0-<em>2</em>7.0 days), 17 patients (<em>2</em>3.3%) had died, <em>2</em>3 (31.5%) had been discharged from the ICU, and 33 (45.<em>2</em>%) were receiving invasive mechanical ventilation in the ICU. Older age (odds ratio [OR], 1.1<em>2</em>; 95% CI, 1.04-1.<em>2</em><em>2</em>; <i>P</i> = 0.004) and hypertension (OR, 6.15; 95% CI, 1.75-<em>2</em>9.11; <i>P</i> = 0.009) were associated with mortality, while early improvement in arterial partial pressure of oxygen (PaO_<em>2</em>) to fraction of inspired oxygen (FiO_<em>2</em>) ratio was associated with being discharged alive from the ICU (<i>P</i> = 0.00<em>2</em> for interaction).

Conclusions: Despite multiple advanced critical care interventions, COVID-19 ARDS was associated with prolonged ventilation and high short term mortality. Older age and pre-admission hypertension were key mortality risk factors.

Trial registration: ClinicalTrials.gov identifier: NCT04318366.

<strong class="sub-title"> Background: </strong> To systematically review clinical and biochemical characteristics associated with the severity of the novel severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>)-related disease (COVID-19).

Materials and methods: Systematic review of observational studies from PubMed, ISI Web of Science, SCOPUS and Cochrane databases including people affected by COVID-19 and reporting data according to the severity of the disease. Data were combined with odds ratio (OR) and metanalysed. Severe COVID-19 was defined by acute respiratory distress syndrome, intensive care unit admission and death.

<strong class="sub-title"> Results: </strong> We included 1<em>2</em> studies with <em>2</em>794 patients, of whom 596 (<em>2</em>1.33%) had severe disease. A slightly higher age was found in severe vs non-severe disease. We found that prevalent cerebrovascular disease (odds ratio [OR] 3.66, 95% confidence interval [CI] 1.73-7.7<em>2</em>), chronic obstructive pulmonary disease (OR: <em>2</em>.39, 95% CI 1.10-5.19), prevalent cardiovascular disease (OR: <em>2</em>.84, 95% CI 1.59-5.10), diabetes (OR: <em>2</em>.78, 95% CI <em>2</em>.09-3.7<em>2</em>), hypertension (OR: <em>2</em>.<em>2</em>4, 95% CI 1.63-3.08), smoking (OR: 1.54, 95% CI 1.07-<em>2</em>.<em>2</em><em>2</em>) and male sex (OR: 1.<em>2</em><em>2</em>, 95% CI 1.01-1.49) were associated with severe disease. Furthermore, increased procalcitonin (OR: 8.<em>2</em>1, 95% CI 4.48-15.07), increased D-Dimer (OR: 5.67, 95% CI 1.45-<em>2</em><em>2</em>.16) and thrombocytopenia (OR: 3.61, 95% CI <em>2</em>.6<em>2</em>-4.97) predicted severe infection.

<strong class="sub-title"> Conclusion: </strong> Characteristics associated with the severity of SARS-CoV-<em>2</em> infection may allow an early identification and management of patients with poor outcomes.

<strong class="sub-title"> Keywords: </strong> SARS-CoV-<em>2</em>; d-dimer; infection; procalcitonin; severity; sex; thrombocytopenia.

Prothrombin complex concentrates (PCCs) are widely administered for emergency oral anticoagulation reversal and for coagulation defects in liver disease. Pharmacokinetic data may help to optimize treatment. The objective of this study was to characterize the pharmacokinetics of a PCC (Beriplex P/N) containing coagulation factors II (FII), VII (FVII), IX (FIX) and X (FX) and anticoagulant proteins C and S. Fifteen healthy volunteers received a single rapid 50 IU/kg infusion of PCC and underwent frequent blood sampling until 144 hours (h) after infusion. Coagulation factors and anticoagulant protein pharmacokinetic parameters were estimated by non-linear regression. The mean infusion rate of PCC was 7.9 ml/min, equivalent to 196.4 IU/min. By the earliest post-infusion sampling point at 5 minutes (min), plasma FIX concentration increased by a median of 73%. Median increases in FII, FVII and FX at 5 min were 1<em>2</em><em>2</em>%, 6<em>2</em>% and 158%, respectively. Proteins C and S also increased rapidly. The median terminal half-life of FIX was 16.7 h, FII 59.7 h, FVII 4.<em>2</em> h and FX 30.7 h. The median in-vivo recovery of FIX was 1.57 %/IU/kg and that of the other three coagulation factors>> <em>2</em> %/IU/kg. Plasma concentration of thrombogenicity marker <em>D</em>-<em>dimer</em> did not increase, and there was no clinical evidence of thrombosis. Through up to 1<em>2</em> weeks follow-up there were no laboratory findings indicating PCC-related viral exposure. Rapid PCC infusion produced prompt sustained increases in coagulation factors and anticoagulant proteins with no clinical evidence of thrombosis or viral transmission.

Introduction: Emerging evidence points to an association between severe clinical presentation of COVID-19 and increased risk of thromboembolism. One-third of patients hospitalized due to severe COVID-19 develops macrovascular thrombotic complications, including venous thromboembolism, myocardial injury/infarction and stroke. Concurrently, the autopsy series indicate multiorgan damage pattern consistent with microvascular injury.

Prophylaxis, diagnosis and treatment: COVID-19 associated coagulopathy has distinct features, including markedly elevated D-dimers concentration with nearly normal activated partial thromboplastin time, prothrombin time and platelet count. The diagnosis may be challenging due to overlapping features between pulmonary embolism and severe COVID-19 disease, such as dyspnoea, high concentration of D-dimers, right ventricle with dysfunction or enlargement, and acute respiratory distress syndrome. Both macro- and microvascular complications are associated with an increased risk of in-hospital mortality. Therefore, early recognition of coagulation abnormalities among hospitalized COVID-19 patients are critical measures to identify patients with poor prognosis, guide antithrombotic prophylaxis or treatment, and improve patients' clinical outcomes.

Recommendations for clinicians: Most of the guidelines and consensus documents published on behalf of professional societies focused on thrombosis and hemostasis advocate the use of anticoagulants in all patients hospitalized with COVID-19, as well as 2-6 weeks post hospital discharge in the absence of contraindications. However, since there is no guidance for deciding the intensity and duration of anticoagulation, the decision-making process should be made in individual-case basis.

Conclusions: Here, we review the mechanistic relationships between inflammation and thrombosis, discuss the macrovascular and microvascular complications and summarize the prophylaxis, diagnosis and treatment of thromboembolism in patients affected by COVID-19.

Keywords: COVID-19; Inflammation; Prophylaxis; SARS-CoV-2; Thrombosis; Venous thromboembolism.

Angiotensin-converting enzyme-<em>2</em> (ACE<em>2</em>) expression may increase due to upregulation in patients using angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARBs). Because renin-angiotensin system blockers increase levels of ACE<em>2</em>, a protein that facilitates coronavirus entry into cells, there is concern that these drugs could increase the risk of developing a severe and fatal form of COVI<em>D</em>-19. The impact of discontinuing ACEI and ARBs in patients with COVI<em>D</em>-19 remains uncertain. <em>D</em>ESIGN: BRACE CORONA is a pragmatic, multicenter, randomized, phase IV, clinical trial that aims to enroll around 500 participants at 34 sites in Brazil. Participants will be identified from an ongoing national registry of suspected and confirmed cases of COVI<em>D</em>-19. Eligible patients using renin-angiotensin system blockers (ACEI/ARBs) with a confirmed diagnosis of COVI<em>D</em>-19 will be randomized to a strategy of continued ACEI/ARB treatment versus temporary discontinuation for 30 days. The primary outcome is the median days alive and out of the hospital at 30 days. Secondary outcomes include progression of COVI<em>D</em>-19 disease, all-cause mortality, death from cardiovascular causes, myocardial infarction, stroke, transient ischemic attack, new or worsening heart failure, myocarditis, pericarditis, arrhythmias, thromboembolic events, hypertensive crisis, respiratory failure, hemodynamic decompensation, sepsis, renal failure, and troponin, B-type natriuretic peptide (BNP), N-terminal-proBNP, and <em>D</em>-<em>dimer</em> levels. SUMMARY: BRACE CORONA will evaluate whether the strategy of continued ACEI/ARB therapy compared with temporary discontinuation of these drugs impacts clinical outcomes among patients with COVI<em>D</em>-19.

The Ser/Thr kinase CK<em>2</em> (previously called casein kinase <em>2</em>) is composed of two catalytic chains (CK<em>2</em> alpha) attached to a <em>dimer</em> of noncatalytic subunits (CK<em>2</em> beta). CK<em>2</em> is involved in suppression of apoptosis, cell survival, and tumorigenesis. To investigate these activities and possibly affect them, selective CK<em>2</em> inhibitors are required. An often-used CK<em>2</em> inhibitor is 5,6-dichloro-1-beta-<em>D</em>-ribofuranosylbenzimidazole (<em>D</em>RB). In a complex structure with human CK<em>2</em> alpha, <em>D</em>RB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol. Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK<em>2</em> alpha around both <em>D</em>RB binding sites. After local rearrangement, the allosteric site serves as a CK<em>2</em> beta interface. This opens the potential to construct molecules interfering with the CK<em>2</em> alpha/CK<em>2</em> beta interaction.

A mutation in the <em>D</em>-loop of the second zinc finger of the <em>D</em>NA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR(dim)), has been suggested to be essential for dimerization and <em>D</em>NA binding of the GR, and genetically altered GR(dim) mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR(dim) mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGR(dim) (A458T) or the hGR(dim4) (A458T, R460<em>D</em>, <em>D</em>46<em>2</em>C, and N454<em>D</em>) mutant receptors in human osteosarcoma (U-<em>2</em> OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-<em>2</em> OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB repression was unaffected by either mutation. Interestingly, both the hGR(dim) and hGR(dim4) receptors readily formed <em>dimers</em> as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR(dim) cells were only partially resistant to apoptosis, whereas hGR(dim4) cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR(dim4) cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.

BACKGROUND

Inflammation has been linked to the genesis of stroke in atrial fibrillation (AF) and is implicated in early recurrent arrhythmia after AF ablation. We aimed to define the time course of inflammation, myocardial injury, and prothrombotic markers after radiofrequency ablation for AF and its relation to AF recurrence.

RESULTS

Ninety consecutive AF patients (53% paroxysmal) undergoing radiofrequency ablation were recruited. High-sensitivity C-reactive protein (hs-CRP), Troponin-T, creatine kinase-MB, fibrinogen, and <em>D</em>-<em>Dimer</em> concentrations were measured at baseline, at 1, <em>2</em>, 3, 7 days, and at 1 month after ablation. AF recurrence was documented at 3 days and at 1, 3, and 6 months follow-up. Troponin-T and creatine kinase-MB peaked at day 1 after procedure (both P<0.05). Hs-CRP peaked at day 3 after procedure (P<0.05). Fibrinogen (P<0.05) and <em>D</em>-<em>Dimer</em> (P<0.05) concentrations were significantly elevated at 1 week after procedure. Ln hs-CRP elevation correlated with Ln Troponin-T and fibrinogen elevation. The extent of Ln hs-CRP, Ln Troponin-T, and fibrinogen elevation predicted early AF recurrence within 3 days after procedure (P<0.05, respectively), but not at 3 and 6 months.

CONCLUSIONS

Patients undergoing radiofrequency ablation for AF exhibit an inflammatory response within 3 days. The extent of inflammatory response predicts early AF recurrence but not late recurrence. Prothrombotic markers are elevated at 1 week after ablation and may contribute to increased risk of early thrombotic events after AF ablation.

Essentials Neutrophil extracellular traps (NETs) might play a role in cancer-related coagulopathy. We determined NET biomarkers and followed cancer patients for venous thromboembolism (VTE). We found a constant association with VTE for citrullinated histone H3. Biomarkers of NET formation could reflect a novel pathomechanism of cancer-related VTE.

CONCLUSIONS

Background Neutrophil extracellular traps (NETs) are decondensed chromatin fibers that might play a role in the prothrombotic state of cancer patients. Objectives To investigate whether the levels of citrullinated histone H3 (H3Cit), a biomarker for NET formation, cell-free <em>D</em>NA (cf<em>D</em>NA) and nucleosomes predict venous thromboembolism (VTE) in cancer patients. Patients/Methods Nine-hundred and forty-six patients with newly diagnosed cancer or progression after remission were enrolled in this prospective observational cohort study. H3Cit, cf<em>D</em>NA and nucleosome levels were determined at study inclusion, and patients were followed for <em>2</em> years. VTE occurred in 89 patients; the cumulative 3-month, 6-month, 1<em>2</em>-month and <em>2</em>4-month incidence rates of VTE were 3.7%, 6.0%, 8.1%, and 10.0%, respectively. Results Patients with elevated H3Cit levels >> 75th percentile of its distribution, n = <em>2</em>36) experienced a higher cumulative incidence of VTE (<em>2</em>-year risk of 14.5%) than patients with levels below this cut-off (<em>2</em>-year risk of 8.5%, n = 710). In a competing-risk regression analysis, a 100 ng mL-1 increase in H3Cit level was associated with a 13% relative increase in VTE risk (subdistribution hazard ratio [SHR] 1.13, 95% confidence interval [CI] 1.04-1.<em>2</em><em>2</em>). This association remained after adjustment for high VTE risk and very high VTE risk tumor sites, <em>D</em>-<em>dimer</em> level, and soluble P-selectin level (SHR 1.13, 95% CI 1.04-1.<em>2</em><em>2</em>). The association of elevated nucleosome and cf<em>D</em>NA levels with VTE risk was time-dependent, with associations with a higher risk of VTE only during the first 3-6 months. Conclusion These data suggest that biomarkers of NET formation are associated with the occurrence of VTE in cancer patients, indicating a role of NETs in the pathogenesis of cancer-associated thrombosis.

One Von Hippel-Lindau (VHL) tumor suppressor gene is lost in most renal cell carcinomas while the nondeleted allele exhibits hypermethylation-induced inactivation or inactivating somatic mutations. As a result of these genetic modifications, there is an increased production of VEGF-A and pro-angiogenic growth factors in this disorder. The important role of angiogenesis in the pathogenesis of renal cell carcinomas and other tumors has focused the attention of investigators on the biology of VEGFs and VEGFR1-3 and to the development of inhibitors of the intricate and multifaceted angiogenic pathways. VEGFR1-3 contain an extracellular segment with seven immunoglobulin-like domains, a transmembrane segment, a juxtamembrane segment, a protein kinase domain with an insert of about 70 amino acid residues, and a C-terminal tail. VEGF-A stimulates the activation of preformed VEGFR<em>2</em> <em>dimers</em> by the auto-phosphorylation of activation segment tyrosines followed by the phosphorylation of additional protein-tyrosines that recruit phosphotyrosine binding proteins thereby leading to signalling by the ERK1/<em>2</em>, AKT, Src, and p38 MAP kinase pathways. VEGFR1 modulates the activity of VEGFR<em>2</em>, which is the chief pathway in vasculogenesis and angiogenesis. VEGFR3 and its ligands (VEGF-C and VEGF-<em>D</em>) are involved primarily in lymphangiogenesis. Small molecule VEGFR1/<em>2</em>/3 inhibitors including axitinib, cabozantinib, lenvatinib, sorafenib, sunitinib, and pazopanib are approved by the F<em>D</em>A for the treatment of renal cell carcinomas. Most of these agents are type II inhibitors of VEGFR<em>2</em> and inhibit the so-called <em>D</em>FG-Aspout inactive enzyme conformation. These drugs are steady-state competitive inhibitors with respect to ATP and like ATP they form hydrogen bonds with the hinge residues that connect the small and large protein kinase lobes. Bevacizumab, a monoclonal antibody that binds to VEGF-A, is also approved for the treatment of renal cell carcinomas. Resistance to these agents invariably occurs within one year of treatment and clinical studies are underway to determine the optimal sequence of treatment with these anti-angiogenic agents. The nivolumab immune checkpoint inhibitor is also approved for the second-line treatment of renal cell carcinomas. Owing to the resistance of renal cell carcinomas to cytotoxic drugs and radiation therapy, the development of these agents has greatly improved the therapeutic options in the treatment of these malignancies.

BACKGROUND

Perturbed sleep might contribute to cardiovascular disease by accelerating atherosclerosis. Sleep is poor in Alzheimer caregivers who are also a group at increased cardiovascular risk.

OBJECTIVE

To test the hypothesis that impaired sleep relates to elevated levels of biomarkers of atherosclerosis in community-dwelling elderly and that this association would possibly be stronger in caregivers than in non-caregiving controls.

METHODS

We studied 97 Alzheimer caregivers and 48 non-caregiving controls (mean age 71 +/- 8 years, 7<em>2</em>% women) who underwent wrist actigraphy at their homes. Measures of objective sleep were averaged across 3 consecutive nights. The Pittsburgh Sleep Quality Index was administered by an interviewer to rate subjective sleep quality. Morning fasting blood samples were collected to determine measures of inflammation, coagulation and endothelial dysfunction.

RESULTS

There were independent associations between decreased subjective sleep quality and increased levels of fibrin <em>D</em>-<em>dimer</em> (p = 0.0<em>2</em><em>2</em>, <em>D</em>eltaR(<em>2</em>) = 0.0<em>2</em>9) and von Willebrand factor antigen (p = 0.0<em>2</em>9, <em>D</em>eltaR(<em>2</em>) = 0.034) in all participants. Percent sleep (p = 0.0<em>2</em>5) and subjective sleep quality (p = 0.017) were lower in caregivers than in controls. In caregivers, the correlation between decreased percent sleep and elevated levels of interleukin-6 (p = 0.04<em>2</em>, <em>D</em>eltaR(<em>2</em>) = 0.039) and C-reactive protein (p < 0.10, <em>D</em>eltaR(<em>2</em>) = 0.0<em>2</em>7) was significantly stronger than in controls.

CONCLUSIONS

Perceived impairment in sleep related to increased coagulation activity and endothelial dysfunction in all participants, whereas objectively impaired sleep related to inflammation activity in caregivers. The findings provide one explanation for the increased cardiovascular risk in elderly poor sleepers and dementia caregivers in particular.

BACKGROUND

Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE.

METHODS

19 hospital-treated patients were involved. Plasma levels of <em>D</em>-<em>dimer</em>, prothrombin fragments 1+<em>2</em> (F1+<em>2</em>), activated partial thromboplastin time (APTT), prothrombin time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 3<em>2</em>-54 days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (<em>D</em>IC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH).

RESULTS

APTT was prolonged and <em>D</em>-<em>dimer</em> and F1+<em>2</em> increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F1+<em>2</em> was 3.4-fold and <em>D</em>-<em>dimer</em> even <em>2</em>4-fold higher compared with the recovery phase (median 7<em>2</em>6 vs <em>2</em>13 pmol/l, and median 4.8 vs 0.<em>2</em>mg/l, respectively, p<0.001 for both). Platelet count correlated with AT, PC, and PS (r=0.73, r=0.81, and r=0.71, respectively, p<0.001 for all) as well as with fibrinogen (r=0.7<em>2</em>, p<0.001). Only five patients fulfilled the ISTH diagnosis of <em>D</em>IC.

CONCLUSIONS

During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx hetero<em>dimer</em> and Prx glutathionylation. In contrast, 1-Cys <em>D</em>-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys <em>D</em>-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys <em>D</em>-Prx induces the dissociation of the Prx non-covalent homo<em>dimer</em>, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent <em>dimer</em>-monomer switch in the Prx family, similar to the decamer-<em>dimer</em> switch for the <em>2</em>-Cys Prxs.

Inflammatory and procoagulant host responses are closely related in sepsis. The protein C pathway serves as a regulatory pathway with anti-inflammatory and anticoagulant properties. Recently, recombinant human activated protein C (rhAPC) was shown to reduce mortality in severe sepsis. Nevertheless, the effects of rhAPC in humans are still ill defined. The infusion of low endotoxin doses into humans provides a standardized model to study inflammatory and hemostatic mechanisms. Thus, we investigated whether rhAPC acts as an anticoagulant or anti-inflammatory drug in human endotoxemia. There were <em>2</em>4 volunteers randomized to receive either <em>2</em>4 microg/kg per hour rhAPC or placebo intravenously for 8 hours. Lipopolysaccharide (LPS, <em>2</em> ng/kg) was administered <em>2</em> hours after starting the infusions. rhAPC decreased basal tissue factor (TF)-mRNA expression, and thrombin formation and action. In contrast, rhAPC did not significantly blunt LPS-induced thrombin generation. Consistently, rhAPC did not reduce LPS-induced levels of TF-mRNA or <em>D</em>-<em>dimer</em> and had no effect on fibrinolytic activity or inflammation. Finally, endogenous APC formation was enhanced during endotoxemia and appeared to be associated with inflammation rather than thrombin formation. In conclusion, even low-grade endotoxemia induces significant protein C activation. Infusion of rhAPC decreases "spontaneous" activation of coagulation but does not blunt LPS-induced, TF-mediated coagulation in healthy volunteers, which is in contrast to a number of anticoagulants.