Proline racemase catalyzes the interconversion of <em>D</em>- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from <em>D</em>L-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other <em>D</em>-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per <em>dimer</em> become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-<em>2</em>-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.

BACKGROUND

It has been claimed that regional citrate anticoagulation (RCA) improves unfavorable calcium and magnesium dependent cellular and humoral events due to blood/dialyzer membrane interactions during hemodialysis (HD). This study aimed to verify whether the favorable effect of RCA on biocompatibility is independent from coagulation pathway modulation.

METHODS

A randomized controlled cross-over single blind trial comparing the activity of the coagulation pathway (thrombinantithrombin complexes (TAT), fibrinopeptide A (FPA), prothrombin fragments 1+<em>2</em> (F 1+<em>2</em>) and <em>D</em>-<em>dimer</em> (<em>D</em><em>D</em>)), complement activation (C3a) and interleukin-1 beta secretion (IL-1beta) in nine chronic H<em>D</em> patients treated with RCA or heparin. Blood samples were obtained from the arterial (C3a, IL-1beta, TAT, F 1+<em>2</em>, FPA and <em>D</em><em>D</em>) and venous (TAT, F 1+<em>2</em>, FPA) lines <em>2</em> min after starting treatment and repeatedly during the procedure after 15 min (C3a and IL-1beta), 30 min (C3a), 45 (C3a) and 180 min (TAT, F 1+<em>2</em>, FPA and <em>D</em><em>D</em>).

RESULTS

In both treatment protocols significant enhancement was observed in the coagulation activity during the dialysis session, documented by an increase in TAT (p<0.001), F 1+<em>2</em> (p<0.001) and FPA (p=0.001). Comparing the two anticoagulation modalities, no differences were noticed in the activity of the coagulation pathway, but a significantly higher complement activity (C3a=886 (83<em>2</em>-908) vs. 770 (645-857) ng/mL, p<0.05) and lower IL-1beta secretion (<em>2</em>35 (<em>2</em>06-<em>2</em>85) vs. 538 (346-974) pg/mL, p<0.05) was observed in RCA.

CONCLUSIONS

Due to an RCA protocol guaranteeing the same extent of anticoagulation activation as standard heparin, we demonstrated that the significantly lower IL-1beta secretion obtained with RCA is independent from the anticoagulation modulation and dissociated from the complement activity.

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.<em>2</em> and 3.0 A, are presented. The molecule crystallized in space group P41<em>2</em>1<em>2</em>, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of <em>2</em>1.1 and <em>2</em>1.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. <em>D</em>., Faber, H. R., Bystrom, C. E., Pettigrew, <em>D</em>. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues <em>2</em>34-<em>2</em>36) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic <em>2</em>-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg <em>2</em>36 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly <em>2</em>34 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg <em>2</em>36 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote <em>dimer</em>-tetramer assembly and to inactivate the tetramers.

CmtR from Mycobacterium tuberculosis is a winged helical <em>D</em>NA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a <em>dimer</em> with the metal bound to Cys-10<em>2</em> from the C-terminal region of one subunit and two Cys associated with helix alphaR from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the <em>dimer</em> could be solved at 31<em>2</em> K. The apoprotein at the same temperature is still a <em>dimer</em>, but it experiences a large conformational exchange at the <em>dimer</em> interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H(<em>2</em>)O-<em>D</em>(<em>2</em>)O exchange rates, especially at the <em>dimer</em>ic interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-10<em>2</em> as a metal-ligand. <em>D</em>NA binds to the apo <em>dimer</em> with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-<em>D</em>NA complex causes <em>D</em>NA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the <em>dimer</em> interface impairs <em>D</em>NA association by excluding the apo-conformers suited to bind <em>D</em>NA.

The worl<em>d</em> is ami<em>d</em> a pan<em>d</em>emic cause<em>d</em> by severe acute respiratory syn<em>d</em>rome-coronavirus <em>2</em>. Severe acute respiratory syn<em>d</em>rome-coronavirus causes serious respiratory tract infections that can lea<em>d</em> to viral pneumonia, acute respiratory <em>d</em>istress syn<em>d</em>rome, an<em>d</em> <em>d</em>eath. Some patients with coronavirus <em>d</em>isease <em>2</em>019 (COVI<em>D</em>-19) have an activate<em>d</em> coagulation system characterize<em>d</em> by elevate<em>d</em> plasma levels of <em>d</em>-<em>dimer</em>-a biomarker of fibrin <em>d</em>egra<em>d</em>ation. Importantly, high levels of <em>D</em>-<em>dimer</em> on hospital a<em>d</em>mission are associate<em>d</em> with increase<em>d</em> risk of mortality. Venous thromboembolism is more common than arterial thromboembolism in hospitalize<em>d</em> COVI<em>D</em>-19 patients. Pulmonary thrombosis an<em>d</em> microvascular thrombosis are observe<em>d</em> in autopsy stu<em>d</em>ies, an<em>d</em> this may contribute to the severe hypoxia observe<em>d</em> in COVI<em>D</em>-19 patients. It is likely that multiple systems contribute to thrombosis in COVI<em>D</em>-19 patients, such as activation of coagulation, platelet activation, hypofibrinolysis, en<em>d</em>othelial cell <em>d</em>ysfunction, inflammation, neutrophil extracellular traps, an<em>d</em> complement. Targeting these <em>d</em>ifferent pathways may re<em>d</em>uce thrombosis an<em>d</em> improve lung function in COVI<em>D</em>-19 patients.

Keywords: coronavirus; fibrin; orthomyxoviridae; pandemics; thrombosis.

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a <em>D</em>-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment <em>D</em>-<em>dimer</em>, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment <em>D</em>-<em>dimer</em> from plasmin degradation ( approximately 186 k<em>D</em>a). In contrast, fragment <em>D</em>-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment <em>D</em> from plasmin degradation of Fg ( approximately 94 k<em>D</em>a). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and <em>D</em>/<em>D</em>-<em>dimer</em> alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 10<em>2</em>-Asn 103. <em>D</em>egradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 1<em>2</em>3-Leu 1<em>2</em>4, beta Asn 137-Val 138, and beta Glu 141-Tyr 14<em>2</em>, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -<em>2</em>, and -9 and MT<em>2</em>-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.

CP1<em>2</em> is a protein of 8.7 k<em>D</em>a that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAP<em>D</em>H) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP1<em>2</em> (isoform <em>2</em>) of Arabidopsis thaliana show that CP1<em>2</em>-<em>2</em> is poorly structured. CP1<em>2</em>-<em>2</em> is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -3<em>2</em>6 and -35<em>2</em> mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP1<em>2</em>-<em>2</em> and GAP<em>D</em>H (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NA<em>D</em>, oxidized CP1<em>2</em>-<em>2</em> interacts with A(4)-GAP<em>D</em>H (K(<em>D</em>) = 0.18 microm) to form a binary complex of 170 k<em>D</em>a with (A(4)-GAP<em>D</em>H)-(CP1<em>2</em>-<em>2</em>)(<em>2</em>) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a <em>dimer</em> and by interacting with this binary complex (K(<em>D</em>) = 0.17 microm) leads to a 498-k<em>D</em>a ternary complex constituted by two binary complexes and two PRK <em>dimers</em>, i.e. ((A(4)-GAP<em>D</em>H)-(CP1<em>2</em>-<em>2</em>)(<em>2</em>)-(PRK))(<em>2</em>). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP1<em>2</em>-<em>2</em> upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP1<em>2</em> in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAP<em>D</em>H and PRK during darkness in photosynthetic tissues.

The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol <em>δ</em>-interacting protein <em>2</em> (PolDIP<em>2</em>) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP<em>2</em> in the TLS reaction. However, the consequences of PolDIP<em>2</em> interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP<em>2</em> on normal and TLS by five different human specialized Pols from three families: Pol <em>δ</em> (family B), Pol η and Pol ι (family Y), and Pol λ and Pol β (family X). Our results show that PolDIP<em>2</em> also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols β or ι. Additionally, we provide evidence that PolDIP<em>2</em> stimulates Pol <em>δ</em> without affecting its fidelity, facilitating the switch from Pol <em>δ</em> to Pol λ during 8-oxo-G TLS. PolDIP<em>2</em> stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine <em>dimers</em>. Finally, PolDIP<em>2</em> silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP<em>2</em> is an important mediator for TLS.

Abnormalities of haemorheology (plasma viscosity, fibrinogen), endothelial function [von Willebrand factor (vWf)], platelet activation (soluble P-selectin) and thrombogenesis [plasminogen activator inhibitor (PAI), and fibrin <em>D</em>-<em>dimer</em>] are common in cardiovascular disease. We investigated changes in these markers in 86 patients (58 males) presenting with acute stroke (all age < 75 years, with ictus < 1<em>2</em> h), and sequential changes at six time points (baseline on admission, 48 h, 1 week, <em>2</em> weeks, 3 months and 6 months following the onset of stroke). Baseline plasma viscosity, haematocrit, fibrinogen, vWf, PAI, soluble P-selectin and fibrin <em>D</em>-<em>dimer</em> levels were increased in the acute stroke patients compared with 35 age-matched and sex-matched controls. Following admission, there were significant increases in haematocrit at <em>2</em> weeks, vWf at 48 h and 1 week, fibrinogen at 1 week, PAI at 48 h and 1 and <em>2</em> weeks, soluble P-selectin at 48 h, and fibrin <em>D</em>-<em>dimer</em> at 48 h and 1 week following admission. Using univariate 'time to event' analysis, high >> median) mean age (log-rank test, P = 0.0<em>2</em>6<em>2</em>), diastolic blood pressure (P = 0.01), haematocrit (P = 0.0<em>2</em>34), PAI-1 (P = 0.0066) and fibrin <em>D</em>-<em>dimer</em> levels (P = 0.0356) were associated with a shortened event-free survival. Using a multivariate Cox survival analysis, only PAI-1 levels remained an independent predictor of survival (P = 0.0349). We conclude that acute stroke patients have marked baseline abnormalities of haemorheology, endothelial disturbance, thrombogenesis, platelet activation and abnormal fibrinolysis, with further changes over the subsequent follow-up period. Abnormal thrombogenesis and fibrinolysis may significantly influence survival in patients with acute stroke. These changes may have potential implications for the pathogenesis of stroke and its complications, although the possibility remains that we are documenting an acute phase response that previous studies, which included stroke patients with a wide time range since ictus onset, have neglected to consider.

OBJECTIVE

To clarify the clinical features of systemic lupus erythematosus (SLE) complicated with pulmonary hypertension (PH) and to evaluate the efficacy of immunosuppressive treatment.

METHODS

Case records were reviewed for 194 patients with SLE who were admitted to Aoyama Hospital of Tokyo Women's Medical University between 199<em>2</em> and 1999. There were 1<em>2</em> patients with PH [8 SLE and 4 SLE + systemic sclerosis (SSc) overlap syndrome]. These patients were compared with 59 age and sex matched patients with SLE for clinical characteristics and laboratory findings. The efficacy of treatments for PH was also evaluated.

RESULTS

In our cohort of 194 patients with SLE, 6.<em>2</em>% had PH. The plasma thrombin-antithrombin III complex and plasma <em>D</em>-<em>dimer</em> levels were significantly higher in patients with PH compared with those without PH. Eight patients with PH (4 SLE and 4 SLE + SSc) were treated with corticosteroids (CS) +/- cyclophosphamide (CYC). Right ventricular systolic pressure (RVSP) was improved in 7 of 8 patients. In 6 of 7 responders to the therapy, the treatment was started as soon as they were diagnosed with PH. PH relapsed in <em>2</em> patients treated with oral CS +/- CS pulse therapy, but their RVSP was decreased again by immunosuppressive treatment.

CONCLUSIONS

CS +/- CYC was effective for PH associated with SLE. Immunosuppressive treatment should be performed during the early stage of PH to improve prognosis.

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a <em>2</em>:1 ratio of the OAS-TL <em>dimer</em> to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of <em>dimers</em> of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(<em>D</em>) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(<em>D</em>)) and the regulation of cysteine sensitivity of SAT within the CSC.

Coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) is a prominent pandemic disease that emerged in China and hurriedly stretched worldwide. There are many reports on COVI<em>D</em>-19 associated with the amplified incidence of thrombotic events. In this review, we focused on COVI<em>D</em>-19 coupled with the coagulopathy contributes to severe outcome inclusive of comorbidities such as venous thromboembolism, stroke, diabetes, lung, heart attack, AKI, and liver injury. Initially, the COVI<em>D</em>-19 patient associated coagulation disorders show an elevated level of the <em>D</em>-<em>dimer</em>, fibrinogen, and less lymphocyte count such as lymphopenia. COVI<em>D</em>-19 associated with the Kawasaki disease has acute vasculitis in childhood which further affects the vessels found all over the body. COVI<em>D</em>-19 linked with the thrombotic microangiopathy triggers the multiple vasculitis along with the arterioles thrombosis, medium, large venous and arterial vessels mediates the disseminated intravascular coagulation (<em>D</em>IC). SARS-Co-V-<em>2</em> patients have reduced primary platelet production, increased destruction of the platelet, decreased circulating platelet leads to the condition of increased thrombocytopenia which contributes to the coagulation disorder. Endothelial dysfunction plays an important role in the coagulation disorders via increased generation of the thrombin and stops fibrinolysis further leads to hypercoagulopathy. Along with that endothelial dysfunction activates the complement system pathways and contributes to the acute and chronic inflammation via cytokine storm with the production of the cytokines and chemokines, coagulation in different organs such as lung, brain, liver, heart, kidney and further leads to multi-organ failure.

<strong class="sub-title"> Keywords: </strong> COVI<em>D</em>-19; Coagulation; Complement system; <em>D</em>isseminated intravascular coagulation; Hypercoagulability; Inflammation; SARS-CoV-<em>2</em>; Thromboembolism.

OBJECTIVE

The degree of oxidative stress and its association with a thrombophilic condition, if any, were investigated in alcoholics before the onset of severe liver disease.

METHODS

Reactive oxygen species and total antioxidant capacity were evaluated using two new kinetic spectrophotometric methods in a selected group of 45 consecutive chronic alcohol abusers and 4<em>2</em> apparently healthy moderate drinkers, used as controls. The hemostatic system was explored by detecting the plasma levels of prothrombin fragment 1+<em>2</em> (F1+<em>2</em>) and thrombin-antithrombin complexes (TAT) with enzyme-linked immunosorbent assays, while <em>D</em>-<em>dimer</em> plasma levels were measured with a turbidimetric immunoassay.

RESULTS

Reactive oxygen species were significantly higher (p<0.001) in heavy drinkers than in controls: 3<em>2</em>8.1 (143.4-847.<em>2</em>) U.CARR vs <em>2</em>50 (<em>2</em>00.7-366.8) U.CARR, respectively. The total antioxidant capacity was similar in chronic alcohol abusers and in moderate drinkers: 360.<em>2</em> (336.8-374.4) microMol HClO/mL vs 369 (36<em>2</em>-378.4) microMol HClO/mL, respectively. All molecular markers of hemostatic system activation were significantly increased in chronic alcohol abusers in comparison with those in moderate drinkers, as follows: TAT: <em>2</em>.5 (1.4-13) microg/L vs 1.5 (1-4.1) mocrog/L, respectively (p<0.001); F1+<em>2</em>: 1.7 (0.5-5.<em>2</em>) nMol/L vs 0.9 (0.4-1.1) nMol/L, respectively (p<0.01); <em>D</em>-<em>dimer</em>: <em>2</em>35.5 (<em>2</em>08-46<em>2</em>) ng/mL vs 163.5 (71-<em>2</em>33) ng/mL, respectively (p<0.001).

CONCLUSIONS

Our results suggest that oxidative stress and a thrombophilic condition can be observed in heavy drinkers without severe liver disease. The new test available for measuring reactive oxygen species in serum proved to be reliable and useful as an early marker of tissue damage.

OBJECTIVE

Recent clinical trials have established that adjusted-dose warfarin (international normalized ratio [INR] <em>2</em>.0 to 3.0) is highly effective in the reduction of ischemic stroke in patients with nonvalvular atrial fibrillation (AF). We hypothesized that the introduction of fixed low-dose warfarin alone or in combination with aspirin (300 mg) could normalize hemostatic markers, namely plasma fibrin <em>D</em>-<em>dimer</em> (an index of thrombogenesis), plasminogen activator inhibitor-1 (PAI-1, an index of fibrinolysis), fibrinogen, and von Willebrand factor (vWf, an index of endothelial dysfunction), in a manner comparable to adjusted-dose warfarin (target INR <em>2</em>.0 to 3.0). METJO<em>D</em>S: Sixty-one patients with AF (44 men, mean+/-S<em>D</em> age 64+/-19 years) who were not receiving any antithrombotic therapy were prospectively randomized into 1 of 3 treatment groups: warfarin (<em>2</em> mg) (n=<em>2</em>3; group 1), combination 1 mg warfarin plus 300 mg aspirin (n=<em>2</em>1; group <em>2</em>) or combination <em>2</em> mg warfarin plus 300 mg aspirin (n=17; group 3). Subjects from all 3 AF groups were matched for sex, age, and blood pressure. Blood samples were taken for sequential measurements for changes in plasma fibrin <em>D</em>-<em>dimer</em>, PAI-1, fibrinogen, and vWf before and at <em>2</em> and 8 weeks after randomization (phase 1). All patients were subsequently offered adjusted-dose warfarin therapy (phase <em>2</em>), and an additional blood sample was taken 6 weeks later.

RESULTS

When pretreatment results were compared with those from 60 age- and sex-matched healthy control subjects in sinus rhythm, there were significant elevations in levels of fibrinogen (P=0.0<em>2</em>5), vWf (P<0.0001), and fibrin <em>D</em>-<em>dimer</em> (P<0.0001) in patients with AF compared with control subjects. There were no significant changes in the levels of various indices measured after <em>2</em> and 8 weeks of therapy in all 3 groups, except for an increase in PAI-1 level (P=0.0<em>2</em>4) in group 3. After 6 weeks of therapy with dose-adjusted warfarin (INR <em>2</em>.0 to 3.0), there was a significant decrease in plasma fibrinogen (P=0.0<em>2</em>3) and fibrin <em>D</em>-<em>dimer</em> (P=0.0067) levels. There were no significant changes in the levels of PAI-1 (P=0.198) or vWf (P=0.33).

CONCLUSIONS

The present results confirmed that high levels of vWf, fibrinogen, and fibrin <em>D</em>-<em>dimer</em> levels were present in patients with AF compared with control subjects. Moreover, the introduction of 300 mg aspirin plus low-dose warfarin (1 mg/d), low-dose warfarin alone (<em>2</em> mg/d), or 300 mg aspirin plus low-dose warfarin (<em>2</em> mg/d) did not significantly reduce any of the hemostatic markers studied (except PAI-1 levels), whereas conventional full-dose warfarin (INR <em>2</em>.0 to 3.0) significantly reduced levels of fibrin <em>D</em>-<em>dimer</em> and fibrinogen. These results are in keeping with the disappointing ineffectiveness of low-intensity warfarin therapy, aspirin-warfarin combination, and ultralow-dose warfarin therapy in the recent prematurely terminated clinical trials and the established benefits of conventional adjusted-dose anticoagulation therapy.

BACKGROUND

Individuals with sleep-disordered breathing (SDB) are at increased cardiovascular risk, possibly due to SDB-related stresses contributing to atherosclerosis.

OBJECTIVE

We postulate that pathways associated with a prothrombotic potential are up-regulated in SDB.

METHODS

Morning and evening plasminogen activator inhibitor-1 (PAI-1), morning fibrinogen, and morning <em>D</em>-<em>dimer</em> were measured in 537 Cleveland Family Study adults. Piecewise multivariable linear mixed models estimated relative mean change or mean change in the biomarker per 5-unit increase in apnea-hypopnea index (AHI) in two groups: AHI less than 15 and AHI greater than or equal to 15, and hypoxia defined as percentage of sleep time with Sa(O(<em>2</em>)) less than 90% (< <em>2</em>%, ≥ <em>2</em>%).

RESULTS

Nonlinear associations were demonstrated: morning and evening PAI-1 increased by 1<em>2</em>% (95% confidence interval [CI], 5-<em>2</em>0%; P < 0.001) and 11% (95% CI, <em>2</em>-<em>2</em>0%; P = 0.01), respectively per 5-unit AHI increase until an AHI of 15, when no further increase in PAI-1 was demonstrated. The association between AHI and morning PAI-1 remained significant after adjusting for evening PAI-1 level (10%; 95% CI, 3-17%; P < 0.01). Morning fibrinogen increased on average by 8.4 mg/dl (95% CI, 3.1<em>2</em>-13.65; P = 0.00<em>2</em>) per five-unit AHI increase until an AHI of 15. There was no association between AHI and morning <em>D</em>-<em>dimer</em>. Hypoxia severity was not associated with thrombotic marker levels.

CONCLUSIONS

PAI-1 and fibrinogen levels increase monotonically with AHI at degrees of SDB considered mildly to moderately abnormal, suggesting that even mild SDB levels may increase prothrombotic processes. There may be a plateau in this effect, occurring at levels considered to reflect only moderate SDB severity. These relationships with mild-to-moderate SDB were not observed with D-dimer.

Particulate air pollution has been associated with increased risk of cardiopulmonary diseases. However, the underlying mechanisms are not fully understood. We have previously demonstrated that single dose exposure to diesel exhaust particle (<em>D</em>EP) causes lung inflammation and peripheral thrombotic events. Here, we exposed mice with repeated doses of <em>D</em>EP (15 µg/animal) every <em>2</em>(nd) day for 6 days (a total of 4 exposures), and measured several cardiopulmonary endpoints 48 h after the end of the treatments. Moreover, the potential protective effect of curcumin (the yellow pigment isolated from turmeric) on <em>D</em>EP-induced cardiopulmonary toxicity was assessed. <em>D</em>EP exposure increased macrophage and neutrophil numbers, tumor necrosis factor α (TNF α) in the bronchoalveolar lavage (BAL) fluid, and enhanced airway resistance to methacoline measured invasively using Flexivent. <em>D</em>EP also significantly increased plasma C-reactive protein (CRP) and TNF α concentrations, systolic blood pressure (SBP) as well as the pial arteriolar thrombosis. It also significantly enhanced the plasma <em>D</em>-<em>dimer</em> and plasminogen activator inhibitor-1 (PAI-1). Pretreatment with curcumin by oral gavage (45 mg/kg) 1 h before exposure to <em>D</em>EP significantly prevented the influx of inflammatory cells and the increase of TNF α in BAL, and the increased airway resistance caused by <em>D</em>EP. Likewise, curcumin prevented the increase of SBP, CRP, TNF α, <em>D</em>-<em>dimer</em> and PAI-1. The thrombosis was partially but significantly mitigated. In conclusion, repeated exposure to <em>D</em>EP induced lung and systemic inflammation characterized by TNFα release, increased SBP, and accelerated coagulation. Our findings indicate that curcumin is a potent anti-inflammatory agent that prevents the release of TNFα and protects against the pulmonary and cardiovascular effects of <em>D</em>EP.

Coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) is a recently described infectious disease caused by severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>). Since late <em>2</em>019, COVI<em>D</em>-19 has rapidly spread in virtually all countries, imposing the adoption of significant lockdown and social distancing measures. The activation of the coagulation cascade is a common feature of disseminated intravascular coagulation and adverse clinical outcomes in COVI<em>D</em>-19 patients. In this study, we conducted a meta-analysis aiming to investigate differences in serum <em>D</em>-<em>dimer</em> concentrations in patients with and without severe COVI<em>D</em>-19 disease. An electronic search in Medline (PubMed), Scopus and Web of Science was performed with no language restrictions, and 13 articles were reporting on 1,807 patients (585, 3<em>2</em>.4% with severe disease) were finally identified and included in the meta-analysis. The pooled results of all studies revealed that the <em>D</em>-<em>dimer</em> concentrations were significantly higher in patients with more severe COVI<em>D</em>-19 (SM<em>D</em>: 0.91 mg/L; 95% CI, 0.75 to 1.07 mg/L, <i>p</i> < 0.0001). The heterogeneity was moderate (<i>I</i> ^<em>2</em> = 46.5%; <i>p</i> = 0.033). Sensitivity analysis showed that the effect size was not modified when any single study was in turn removed (effect size range, 0.87 mg/L to 0.93 mg/L). The Begg's (<i>p</i> = 0.76) and Egger's tests (<i>p</i> = 0.38) showed no publication bias. In conclusion, our systematic review and meta-analysis showed that serum <em>D</em>-<em>dimer</em> concentrations in patients with severe COVI<em>D</em>-19 are significantly higher when compared to those with non-severe forms.

<strong class="sub-title"> Keywords: </strong> COVI<em>D</em>-19; <em>D</em>-<em>dimer</em>; SARS-CoV-<em>2</em>; coagulation; thrombosis.

Lipids are indispensable in the SARS-CoV-<em>2</em> infection process. The clinical significance of plasma lipid profile during COVI<em>D</em>-19 has not been rigorously evaluated. We aim to ascertain the association of the plasma lipid profile with SARS-CoV-<em>2</em> infection clinical evolution. Observational cross-sectional study including 1411 hospitalized patients with COVI<em>D</em>-19 and an available standard lipid profile prior (n: 1305) or during hospitalization (n: <em>2</em>97). The usefulness of serum total, L<em>D</em>L, non-H<em>D</em>L and H<em>D</em>L cholesterol to predict the COVI<em>D</em>-19 prognosis (severe vs mild) was analysed. Patients with severe COVI<em>D</em>-19 evolution had lower H<em>D</em>L cholesterol and higher triglyceride levels before the infection. The lipid profile measured during hospitalization also showed that a severe outcome was associated with lower H<em>D</em>L cholesterol levels and higher triglycerides. H<em>D</em>L cholesterol and triglyceride concentrations were correlated with ferritin and <em>D</em>-<em>dimer</em> levels but not with CRP levels. The presence of atherogenic dyslipidaemia during the infection was strongly and independently associated with a worse COVI<em>D</em>-19 infection prognosis. The low H<em>D</em>L cholesterol and high triglyceride concentrations measured before or during hospitalization are strong predictors of a severe course of the disease. The lipid profile should be considered as a sensitive marker of inflammation and should be measured in patients with COVI<em>D</em>-19.

IL-1 induces a significant number of metabolic and hematological changes. In experimental animals, IL-1 treatments cause hypotension due to rapid reduction of systemic blood pressure, reduced vascular resistance, increased heart rate and leukocyte aggregations. IL-1 causes endothelial dysfunction, the triggering factor of which may be of a different nature including pathogen infection. This dysfunction, which includes macrophage intervention and increased protein permeability, can be mediated by several factors including cytokines and arachidonic acid products. These effects are caused by the induction of IL-1 in various pathologies, including those caused by pathogenic viral infections, including SARS-CoV-<em>2</em> which provokes COVI<em>D</em>-19. Activation of macrophages by coronavirus-19 leads to the release of pro-inflammatory cytokines, metalloproteinases and other proteolytic enzymes that can cause thrombi formation and severe respiratory dysfunction. Patients with COVI<em>D</em>-19, seriously ill and hospitalized in intensive care, present systemic inflammation, intravascular coagulopathy with high risk of thrombotic complications, and venous thromboembolism, effects mostly mediated by IL-1. In these patients the lungs are the most critical target organ as it can present an increase in the degradation products of fibrin, fibrinogen and <em>D</em>-<em>dimer</em>, with organ lesions and respiratory failure. It is well known that IL-1 induces itself and another very important pro-inflammatory cytokine, TNF, which also participates in hemodynamic states, including shock syndrome in COVI<em>D</em>-19. Both IL-1 and TNF cause pulmonary edema, thrombosis and bleeding. In addition to hypotension and resistance of systemic blood pressure, IL-1 causes leukopenia and thrombocytopenia. The formation of thrombi is the main complication of the circulatory system and functionality of the organ, and represents an important cause of morbidity and mortality. IL-1 causes platelet vascular thrombogenicity also on non-endothelial cells by stimulating the formation of thromboxane A<em>2</em> which is released into the inflamed environment. IL-1 is the most important immune molecule in inducing fever, since it is involved in the metabolism of arachidonic acid which increases from vascular endothelial organs of the hypothalamus. The pathogenesis of thrombosis, vascular inflammation and angigenesis involves the mediation of the activation of the prostanoid thromboxane A<em>2</em> receptor. In 1986, in an interesting article (<i>Conti P, Reale M, Fiore S, Cancelli A, Angeletti PU, <em>D</em>inarello CA. In vitro enhanced thromboxane B<em>2</em> release by polymorphonuclear leukocytes and macrophages after treatment with human recombinant interleukin 1. Prostaglandins. 1986 Jul;3<em>2</em>(1):111-5</i>), we reported for the first time that IL-1 induces thromboxane B<em>2</em> (TxB<em>2</em>) releases in activated neutrophils and macrophages. An increase in thromboxane can induce leukocyte aggregation and systemic inflammation, which would account for the dramatic thrombi formation and organ dysfunction. Hence, IL-1 stimulates endothelial cell-leukocyte adhesion, and TxB<em>2</em> production. All these events are supported by the large increase in neutrophils that adhere to the lung and the decrease in lymphocytes. Therefore, ecosanoids such as TxA<em>2</em> (detected as TxB<em>2</em>) have a powerful action on vascular inflammation and platelet aggregation, mediating the formation of thrombi. The thrombogenesis that occurs in COVI<em>D</em>-19 includes platelet and cell aggregation with clotting abnormalities, and anti-clotting inhibitor agents are used in the prevention and therapy of thrombotic diseases. Prevention of or induction of TxA<em>2</em> avoids thrombi formation induced by IL-1. However, in some serious vascular events where TxA<em>2</em> increases significantly, it is difficult to inhibit, therefore, it would be much better to prevent its induction and generation by blocking its inductors including IL-1. The inhibition or lack of formation of IL-1 avoids all the above pathological events which can lead to death of the patient. The treatment of innate immune cells producing IL-1 with IL-1 receptor antagonist (IL-1Ra) can avoid hemodynamic changes, septic shock and organ inflammation by carrying out a new therapeutic efficacy on COVI<em>D</em>-19 induced by SARS-CoV-<em>2</em>.

<strong class="sub-title"> Keywords: </strong> COVI<em>D</em>-19; IL-1; SARS-CoV-<em>2</em>; inflammation; thrombosis; thromboxane.

Systemic sclerosis (SSc) is a disease characterized by progressive microvascular occlusion and fibrosis resulting in irreversible organ damage, the pathogenesis of which is felt to be of vascular origin. To gain a comprehensive view of the coagulation/fibrinolytic balance in SSc, a number of haemostatic and fibrinolytic variables were measured in <em>2</em>6 SSc patients (11 limited, 15 diffuse) and in <em>2</em><em>2</em> control subjects. Of the coagulation activation markers, the mean plasma level of prothrombin fragment 1 + <em>2</em> (F1 + <em>2</em>), but not of thrombin-antithrombin complexes (TAT), was higher in SSc patients than in controls (P < 0.001). Plasma levels of fibrin split product <em>D</em>-<em>dimer</em> (<em>D</em><em>D</em>), fibrinogen (FNG) and von Willebrand factor (vWF) were higher amongst patients than controls (P < 0.001). vWF and FNG levels were positively correlated (P < 0.001). Mean levels of <em>D</em><em>D</em> and vWF were more elevated in patients with diffuse than limited disease (P = 0.001 and P = 0.04, respectively). On the fibrinolytic side, defective tissue plasminogen activator (tPA) release (venous occlusion test, stimulated level < basal level) was noted in 46% (1<em>2</em>/<em>2</em>6) of SSc patients, but only in 4% (1/<em>2</em><em>2</em>) of controls. Patients had higher mean levels of tPA inhibitor (PAI) than controls (P < 0.001), levels being more elevated amongst patients with diffuse than limited disease (P = 0.01). An abnormally high lipoprotein (a) [Lp(a)] level was found in 9% (<em>2</em>/<em>2</em>0) of control subjects, but in 30% (8/<em>2</em>6) of SSc patients (P = 0.04) where it clustered with fibrinolytic defects. Altogether, these data suggest that patients with SSc are in a hypercoagulable state characterized by elevated plasma levels of FNG and vWF, by a dual hypofibrinolytic pattern (defective tPA release and elevated PAI), and by increased thrombin generation with enhanced fibrin formation. Higher levels of vWF, <em>D</em><em>D</em> and PAI in patients with diffuse disease are consistent with more extensive (micro)vascular involvement, although no causal relationship can be inferred. The lack of a parallel increase of TAT with F1 + <em>2</em>, in the presence of normal levels of antithrombin III (ATIII), indirectly suggests an impairment of the heparan sulphate-ATIII system which would favour thrombin generation. Since thrombin may act as a mitogen for fibroblasts, may upregulate vWF, PAI and endothelin production by endothelial cells, and may promote fibrin deposition on the vessel wall leading to worsening of microvascular occlusions, limitation of thrombin generation, besides fibrinolytic enhancement, could represent a possible coadjuvant interventional strategy.

The authors assessed hemostasis and fibrinolysis serially: on admission and on the 1st and 7th days after surgery for subarachnoid hemorrhage (SAH), examining the complications of aneurysm rupture and its surgical repair. Of 3<em>2</em> patients, <em>2</em>5 with SAH were compared with seven control patients who underwent surgery for an unruptured intracranial aneurysm. On admission, patients with SAH had higher thrombin-antithrombin III complex (TAT) levels (13.3 +/- 3.8 vs. 3.8 +/- 0.6 ng/ml, p = 0.01), fibrin degradation product, <em>D</em>-<em>dimer</em> levels (1310 +/- <em>2</em><em>2</em>0 vs. 556 +/- 89 ng/ml, p = 0.0001), and leukocyte counts (14.6 +/- 0.7 vs. 10.6 +/- 1.8 x 10(9) cells/L, p < 0.05) than did control patients. Postoperative <em>D</em>-<em>dimer</em> values (p = 0.007) remained higher in the SAH group. Furthermore, admission <em>D</em>-<em>dimer</em> levels were higher in the patients in poor clinical condition than in those in good condition (<em>2</em>017 +/- 377 vs. 934 +/- <em>2</em>08 ng/ml, p = 0.007), and <em>D</em>-<em>dimer</em> levels were associated with the outcome at 3 months after admission. Additionally, thrombin generation and fibrinolytic markers measured on admission were related to clinical grade, amount of subarachnoid blood seen on computerized tomography (CT) scanning, and patient fatality. Patients with hypodense lesions verified on follow-up CT scanning or with persistent neurological deficits at 3 months had higher prothrombin fragments 1 and <em>2</em>, TAT, <em>D</em>-<em>dimer</em>, and plasminogen activator inhibitor-1 values on the 1st day postoperatively than did patients without such lesions. In short, in patients with SAH, activation of coagulation and fibrinolysis was strongly associated with clinical state, patient fatality, and outcome at 3 months, and postoperatively this activation correlated with the development of brain infarction.

BACKGROUND

Abacavir (ABC) treatment has been associated with an increased incidence of myocardial infarction. The pathophysiological mechanism is unknown. In this study markers of inflammation and coagulation in HIV-infected patients using antiretroviral therapy with or without ABC were examined to pinpoint a pathogenic mechanism. Given the important role of high sensitivity C-reactive protein (hsCRP) levels in predicting cardiovascular risk, patient groups were also analyzed according to hsCRP levels.

METHODS

Patients treated with ABC and a matched control group treated without ABC were selected retrospectively. Vascular endothelial growth factor (VEGF) and markers of endothelial cell activation (von Willebrand factor (vWF), factor VIII), fibrin formation (fibrinogen, <em>D</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em> (F1+<em>2</em>), endogenous thrombin potential (ETP)), anticoagulation markers (protein C and S, activated protein C sensitivity ratio (APCsr)) and inflammation markers (IL-6, hsCRP) were measured in citrated plasma.

RESULTS

A total of 81 patients were included of whom <em>2</em>7 patients used an ABC-containing regimen and 54 used a non-ABC-containing regimen. Patient characteristics were not significantly different between the groups except for longer duration of use of the current antiretroviral regimen in the ABC group (p = 0.01). The median time on ABC was 68 months (interquartile range 59-80 months). No differences in coagulation and inflammation markers according to ABC use were observed. For the whole patient group elevated vWF and F1+<em>2</em> levels were observed in <em>2</em>3% and 37%, respectively. Compared to the reference ranges for the general population increased APCsr was found in 79% and lower protein C and VEGF levels in 40% and 43%, respectively. Patients in the high-risk category for cardiovascular disease with hsCRP levels>> 3 mg/L had significantly higher fibrinogen, <em>D</em>-<em>dimer</em>, F1+<em>2</em> and ETP levels compared to patients from the low-risk category with hsCRP levels < 1 mg/L.

CONCLUSIONS

HIV-infected patients using ABC showed no specific abnormalities in coagulation or inflammation markers that might explain the increased risk of myocardial infarction. For the whole group, regardless of ABC use, evidence of a prothrombotic state was observed. Thirty-three percent of patients with long-term use of antiretroviral treatment had hsCRP levels above 3 mg/L, which is strongly associated with cardiovascular disease in HIV-uninfected individuals.

We have recently reported that lipid structure regulates the interaction with membranes, recruitment to membranes, and distribution to membrane domains of heterotrimeric Galphabetagamma proteins, Galpha subunits, and Gbetagamma <em>dimers</em> (J Biol Chem <em>2</em>79:36540-36545, <em>2</em>004). Here, we demonstrate that modulation of the membrane structure not only determines G protein localization but also regulates the function of G proteins and related signaling proteins. In this context, the antitumor drug daunorubicin (daunomycin) and oleic acid changed the membrane structure and inhibited G protein activity in biological membranes. They also induced marked changes in the activity of the alpha(<em>2</em>A/<em>D</em>)-adrenergic receptor and adenylyl cyclase. In contrast, elaidic and stearic acid did not change the activity of the above-mentioned proteins. These fatty acids are chemical but not structural analogs of oleic acid, supporting the structural basis of the modulation of membrane lipid organization and subsequent regulation of G protein-coupled receptor signaling. In addition, oleic acid (and also daunorubicin) did not alter G protein activity in a membrane-free system, further demonstrating the involvement of membrane structure in this signal modulation. The present work also unravels in part the molecular bases involved in the antihypertensive (Hypertension 43:<em>2</em>49-<em>2</em>54, <em>2</em>004) and anticancer (Mol Pharmacol 67:531-540, <em>2</em>005) activities of synthetic oleic acid derivatives (e.g., <em>2</em>-hydroxyoleic acid) as well as the molecular bases of the effects of diet fats on human health.

A prominent model for the mechanism of transcription-coupled <em>D</em>NA repair proposes that an arrested RNA polymerase directs the nucleotide excision repair complex to the transcription-blocking lesion. The specific role for RNA polymerase II in this mechanism can be examined by comparing the extent of polymerase arrest with the extent of transcription-coupled repair for a specific <em>D</em>NA lesion. Previously we reported that a cyclobutane pyrimidine <em>dimer</em> that is repaired preferentially in transcribed genes is a strong block to transcript elongation by RNA pol II (<em>D</em>onahue, B.A., Yin, S., Taylor, J.-S., Reines, <em>D</em>., and Hanawalt, P. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 850<em>2</em>-8506). Here we report the extent of RNA polymerase II arrest by the C-8 guanine <em>D</em>NA adduct formed by N-<em>2</em>-aminofluorene, a lesion that does not appear to be preferentially repaired. Templates for an in vitro transcription assay were constructed with either an N-<em>2</em>-aminofluorene adduct or the helix-distorting N-<em>2</em>-acetylaminofluorene adduct situated at a specific site downstream from the major late promoter of adenovirus. Consistent with the model for transcription-coupled repair, an aminofluorene adduct located on the transcribed strand was a weak pause site for RNA polymerase II. An acetylaminofluorene adduct located on the transcribed strand was an absolute block to transcriptional elongation. Either adduct located on the nontranscribed strand enhanced polymerase arrest at a nearby sequence-specific pause site.