Rivaroxaban and apixaban induce clotting factor Xa fibrinolytic activity.

Essentials Activated clotting factor X (FXa) acquires fibrinolytic cofactor function after cleavage by plasmin. FXa-mediated plasma fibrinolysis is enabled by active site modification blocking a second cleavage. FXa-directed oral anticoagulants (DOACs) alter FXa cleavage by plasmin. DOACs enhance FX-dependent fibrinolysis and plasmin generation by tissue plasminogen activator. BACKGROUND: When bound to an anionic phospholipid-containing membrane, activated clotting factor X (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaß and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaß and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaß enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesized that they similarly modulate FXa fibrinolytic function. METHODS: DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots. RESULTS: The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaß. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaß, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma. CONCLUSION: The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaß in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications.

Muscle power in children, youth and young adults who acquired HIV perinatally.

OBJECTIVES: To compare muscle power between youth who acquired HIV perinatally and HIV unexposed uninfected (HUU) youth. METHODS: We assessed muscle power (relative to body mass, Pmax/mass), muscle force normalized to body weight (Fmax/BW), force efficiency, jump height (Hmax) and velocity (Vmax) during a single two-legged jump with hands on waist on a force platform (Leonardo) in HIV+ youth (n=35, 9-21 y). Thirty-three and 22 participants returned at 12- and 24-months, respectively. We compared age- and sex-specific z-scores in the HIV+ youth to those in HUU controls (n=716, 9-21 y) adjusting for height and muscle cross-sectional area (MCSA, by pQCT). RESULTS: At baseline, z-scores for Pmax/mass, Fmax/BW and Vmax were less than 1 standard deviation lower than HUU after adjusting for height and MCSA (p⟨0.05). Pmax/mass z-score was negatively associated with level of immunosuppression (p=0.013), but this relationship was not significant after adjusting for height and MCSA (p=0.07). Z-scores for all mechanography outcomes remained stable over time in HIV+ youth. CONCLUSION: Small deficits in muscle power were apparent in children and youth who acquired HIV perinatally, and the trajectory of muscle power did not change over two years. Further study is needed to identify effective strategies to improve dynamic muscle function in this population.

Bone geometry and strength are adapted to muscle force in children and adolescents perinatally infected with HIV.

OBJECTIVES: To determine if bone health is compromised in perinatally HIV-infected youth. METHODS: We assessed BMC at the proximal femur, lumbar spine and total body using DXA in perinatally HIV-infected youth (n=31; 9-18y). Using pQCT, we assessed muscle CSA, total and cortical bone area, cortical BMD and thickness and strength strain index at the tibial shaft. Thirty and 18 participants returned at 12- and 24-months, respectively. We calculated age- and sex-specific z-scores for the HIV-infected youth using data from a healthy cohort (n=883; 9-18y). RESULTS: At baseline, height and MCSA were reduced in HIV-infected youth (-0.79 to -0.23, p<0.05). BMC z-scores adjusted for height and lean mass were lower than controls at all sites except the lumbar spine (-0.57 to -0.27, p<0.05). Bone area and strength z-scores were not different from zero after adjusting for tibial length and MCSA. In contrast, cortical BMD z-scores were greater in HIV-infected youth (0.46, p=0.011). Z-scores for all bone outcomes showed positive trends over time in HIV-infected youth. CONCLUSION: Although HIV infection may be associated with bone mass deficits during growth, bone geometry and strength appear adapted to muscle force. Further, deficits in bone mass may dissipate over time in this population.

[The impact of lesions in the right hemisphere on linguistic skills: theoretical and clinical perspectives]. / Impacto de las lesiones del hemisferio derecho sobre las habilidades lingüísticas: perspectivas teórica y clínica.

INTRODUCTION: A lesion of the right hemisphere of right-handers can result in verbal communication impairments. The recent development of theoretical frameworks with regard to discourse and pragmatic abilities, among others, now allows us to recognize and describe these impairments. AIM: To offer an overview of the verbal communication deficits that can be found in right-hemisphere-damaged individuals. These deficits can interfere, at different levels, with prosody, the semantic processing of words and discourse and pragmatic abilities. DEVELOPMENT: Such impairments appear to be present in about half of right-hemisphere-damaged patients and, when present, can result in different clinical profiles. These deficits raise the question of their labeling and their relationship with aphasia. CONCLUSIONS: Given the evolution of the concept of language and the universal definition of aphasia, it is proposed that these deficits correspond to another manifestation of aphasia, thus challenging the idea that they are of a 'non-aphasic' nature.

Impacto de las lesiones del hemisferio derecho sobre las habilidades lingüísticas: perspectivas teórica y clínica / The impact of lesion in the right hemisphere on linguistic skills: theoretical and clinical perspectives

Una lesión en el hemisferio derecho (HD) en pacientes diestros puede provocar alteraciones de lacomunicación verbal. El reciente desarrollo de marcos teóricos centrados en las habilidades discursivas y pragmáticas ha permitido un mejor reconocimiento y descripción de estas alteraciones. Objetivo. Ofrecer un panorama sobre los déficit decomunicación verbal que pueden registrarse en pacientes con lesiones del HD. Desarrollo. Dichos déficit pueden interferir, de manera diferencial, en la prosodia, el procesamiento semántico de las palabras y/o en las habilidades discursivas y pragmáticas.Se calcula que las dificultades pueden estar presentes en la mitad de los lesionados derechos y, en estos casos, dan lugar a perfiles clínicos diferentes. Hablar de lesionados derechos conlleva una discusión no sólo acerca del etiquetado, sino que, además, pone en cuestión las relaciones de estas alteraciones con las afasias. Conclusiones. Dada la evolución del concepto de lenguaje y la definición universal de afasia, proponemos que los daños resultantes de una lesión en el HD pueden considerarse como parte de las afasias. Esto constituye un desafío a la idea de que son déficit de naturaleza no afásica

A lesion of the right hemisphere of right-handers can result in verbal communication impairments.The recent development of theoretical frameworks with regard to discourse and pragmatic abilities, among others, now allows us to recognize and describe these impairments. Aim. To offer an overview of the verbal communication deficits that can befound in right-hemisphere-damaged individuals. These deficits can interfere, at different levels, with prosody, the semantic processing of words and discourse and pragmatic abilities. Development. Such impairments appear to be present in about halfof right-hemisphere-damaged patients and, when present, can result in different clinical profiles. These deficits raise the question of their labeling and their relationship with aphasia. Conclusions. Given the evolution of the concept of languageand the universal definition of aphasia, it is proposed that these deficits correspond to another manifestation of aphasia, thus challenging the idea that they are of a 'non-aphasic' nature

Decreased skeletal muscle mitochondrial DNA in patients treated with high-dose simvastatin.

Statins are generally well tolerated, but can cause myopathy and have been associated with mitochondrial abnormalities. The aim of this study was to determine whether muscle mitochondrial DNA (mtDNA) levels are altered during statin therapy. We retrospectively quantified mtDNA in 86 skeletal muscle biopsy specimens collected as part of a previously published clinical trial of high-dose simvastatin or atorvastatin versus placebo.

Human immunodeficiency virus type 1 protease cleavage site mutations associated with protease inhibitor cross-resistance selected by indinavir, ritonavir, and/or saquinavir.

We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6(gag)) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A-->V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein.

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.

Fibrinogen bellingham: a gamma-chain R275C substitution and a beta-promoter polymorphism in a thrombotic member of an asymptomatic family.

Congenital dysfibrinogenemia is a rare cause of unexplained thrombosis. However, most individuals with dysfibrinogenemia are asymptomatic, suggesting that co-morbid factors contribute to thrombo-embolic events. The potential roles of additional genetic or acquired prothrombotic risk factors are poorly understood because detailed family studies are lacking. Herein, we describe a family whose propositus was a young Caucasian man with recurrent venous thrombo-emboli and dysfibrinogenemia due to heterozygosity for an Arg-->Cys substitution at residue 275 in the gamma-chain. The only additional thrombophilic abnormality found in the proband was heterozygosity for a G/A transition at position -455 in the fibrinogen beta-chain promoter; a genotype associated with high acute phase levels of fibrinogen. The proband's father, who died of a cerebral artery thrombosis, carried the gammaR275C substitution but not the beta-promoter -455 variant. Among 14 living relatives, eight were heterozygous for one or the other mutation and only one, a 21-year-old niece, was dually affected. None had suffered bleeding or thrombosis. In vitro studies of the proband's purified fibrinogen revealed markedly abnormal thrombin-catalyzed polymerization and delayed fibrin clot lysis by tPA-activated plasmin. We hypothesize that the gammaR275C substitution predisposes to thrombosis by generating clots that are relatively resistant to fibrinolysis. The clinical risk is low, however, in the absence of an additional thrombophilic mutation. The beta-promoter variant could, theoretically, contribute to this risk by augmenting expression of the dysfibrinogen under conditions of stress. Like the common hereditary thrombophilias, heterozygous familial dysfibrinogenemia induces thrombosis in the setting of multiple prothrombotic influences.

Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin.

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.

Clinical utility of testing human immunodeficiency virus for drug resistance.

Human immunodeficiency virus (HIV) type 1 drug-resistance testing is quickly moving from the research laboratory to the clinic as data defining its utility as a prognostic indicator of response to therapy become available. In July 1998, a panel of the International AIDS Society-USA did not recommend the widespread application of resistance testing, but by May 2000 this panel endorsed and recommended the incorporation of resistance testing in patient-care management. Considerable data supporting the use of drug-resistance testing have now been published or presented at international conferences. These data strongly suggest that drug-resistance testing is of considerable value in many clinical settings. Prospective trials of resistance testing as a clinical management tool are still ongoing, and the long-term benefits still need to be evaluated. Nevertheless, early results from several studies showed a significantly better virological response when treatment regimens were based on resistance-testing data, rather than on the standard of care. HIV drug-resistance testing is also useful as a tool for new antiretroviral drug design and development, as well as for monitoring the spread of primary HIV drug resistance.

Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.

Circadian variations in the pharmacokinetics of a new microemulsion formulation of cyclosporine in cardiac transplant recipients.

We attempted to characterize circadian variations in pharmacokinetic parameters of a new formulation of cyclosporine (CsA) in nine cardiac allograft recipients. A secondary objective was to determine the sampling time that correlated best with exposure of patients to the drug. This was a two-period study with each period lasting 12 hours. All patients received two equal doses of a new microemulsion of CsA 12 hours apart. Blood samples to measure drug levels were obtained at administration and 1, 2, 3, 4, 6, 9, and 12 hours after each dose. We found no statistically significant difference in pharmacokinetic parameters (areas under the curve, minimum blood concentration, oral clearance) measured during the day and during the night. However, maximum blood concentrations during the day were 30% higher than those at night (p<0.05). We found a good correlation between minimum concentrations in the morning and overall exposure of patients to CsA (r=0.89). This new microemulsion appears to have few circadian variations of blood concentrations in cardiac transplant recipients. The clinical significance of higher maximum blood concentration during daytime remains to be elucidated. Our results support the most widely accepted method for monitoring CsA, which is based on minimum concentrations in the morning.

Inhibition of meizothrombin and meizothrombin(desF1) by heparin cofactor II.

Meizothrombin and meizothrombin(desF1) are intermediates formed during the conversion of prothrombin to thrombin by factor Xa, factor Va, phospholipids, and Ca2+ (prothrombinase). These intermediates are active toward synthetic peptide substrates but have limited ability to interact with platelets or macromolecular substrates such as fibrinogen. Meizothrombin and meizothrombin(desF1) activate protein C, however, and may exert primarily an anticoagulant effect. In this study, we investigated the inhibition of meizothrombin and meizothrombin(desF1) by two glycosaminoglycan-dependent protease inhibitors, heparin cofactor II (HCII) and antithrombin (AT). Purified recombinant meizothrombin and meizothrombin(desF1) were inhibited by HCII in the presence of dermatan sulfate with maximal second-order rate constants of 8 x 10(6) M-1.min-1 and 1.8 x 10(7) M-1.min-1, respectively, but were inhibited less than one-tenth as fast by AT in the presence of heparin. Similarly, the products of the prothrombinase reaction were inhibited in situ more effectively by HCII than by AT. When HCII and dermatan sulfate were present continuously during the prothrombinase reaction, meizothrombin was trapped as a sodium dodecyl sulfate-stable complex with HCII and no amidolytic activity could be detected with a thrombin substrate. Our findings indicate that HCII is an effective inhibitor of meizothrombin and meizothrombin(desF1) and, therefore, might regulate the anticoagulant activity of these proteases.

The polymerization pocket "a" within the carboxyl-terminal region of the gamma chain of human fibrinogen is adjacent to but independent from the calcium-binding site.

The carboxyl-terminal region of the gamma chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. Protein fragments encoding amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbggammaC30 and gamma'C30 were protected from plasmin degradation by the presence of Ca2+ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca2+-binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.

The primary fibrin polymerization pocket: three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro.

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition.

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

A new method for characterization and epitope determination of a lupus anticoagulant-associated neutralizing antiprothrombin antibody.

A patient had both lupus anticoagulant hypoprothrombinemia syndrome and celiac disease. The presence of a neutralizing antiprothrombin antibody in the patient's serum was demonstrated by coagulation tests, immunoadsorption, and Western blot analysis. The probable cause for the severe hypoprothrombinemia was clearance of prothrombin-antibody complexes from the circulation. Studies showed the antiprothrombin antibody binding to human prothrombin was phospholipid- and Ca(++)-independent; the antibody did not bind to human thrombin. The target epitope of the antibody was studied by Western blot analysis of mutated recombinant human prothrombin molecules. The antibody reacted with the fragment 2-A region of prothrombin, spanning the second kringle domain and the thrombin A chain within prothrombin. Based on this new method, the proposed mechanism for the neutralizing action of the antibody is impairment of prothrombin activation by the prothrombinase complex, either by steric hindrance of the hydrolysis of prothrombin by factor Xa or by interference of the interaction of prothrombin with factor Va; both reactions are required for efficient conversion of prothrombin to thrombin.