Molecular epidemiological analysis to assess the influence of pet-ownership in the biodiversity of Staphylococcus aureus and MRSA in dog- and non-dog-owning healthy households.

It has been suggested that zoonotic transmission of Staphylococcus aureus (SA) and methicillin-resistant S. aureus (MRSA) can occur between owners and their pets within the same household. However, the influence that pet-ownership could have in the biodiversity of SA/MRSA strains circulating among owners is not fully understood. The objective of this study was to perform a molecular epidemiological analysis to evaluate and compare the biodiversity of SA/MRSA strains in dog-owning and non-dog-owning healthy households within the same community. Antimicrobial resistance, SCCmec type, USA type and clonality were assessed. Overall, 33·1% (165/499) of human subjects carried SA and 2·8% (14/499) carried MRSA. Among dogs, 7·1% (8/113) carried SA but none were MRSA positive. No difference was detected in the diversity index of SA/MRSA pulsotypes between dog-owning and non-dog-owning households; but, a marked variation was still observed in the pulsotypes circulating in each type of household. Additionally, simultaneous carriage of the same SA pulsotype in owner(s) and dog was observed in 57% of households with positive humans and pets. These results demonstrate that dogs can indeed participate in the circulation of SA/MRSA pulsotypes within a home and that the presence of a pet does not seem to favour certain strains within their household.

The alphavbeta3 integrin regulates alpha5beta1-mediated cell migration toward fibronectin.

This study examines the interactions of alphavbeta3 and alpha5beta1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express alpha5beta1 endogenously were transfected with alphavbeta3 and beta3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An alphavbeta3 blocking antibody and the alphavbeta3 ligand cyclic G-Pen-GRGDSPC-A inhibited alpha5beta1-mediated migration toward Fn, but not attachment to Fn. This function was alphavbeta3-specific since alphavbeta5 transfection and alphavbeta5 blocking antibody did not produce this effect. Mutations introduced into the beta3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation beta3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation beta3-S752P into the cytoplasmic domain or its truncation (beta3-Delta717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by alphavbeta3 blocking antibody. alphavbeta3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (beta3-Delta693). These findings demonstrate alphavbeta3 regulation of alpha5beta1-mediated cell migration and suggest that the beta3 transmembrane domain is essential for this function.

Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alphaIIb beta3 in human embryonic kidney 293 cells.

The platelet-specific integrin alphaIIb beta3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of alphaIIb beta3, we expressed recombinant alphaIIb beta3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet alphaIIb beta3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alphaIIb beta3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alphaIIb beta3 is expressed in a "transitional" activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alphaIIb beta3 antibody PAC1 to cells expressing the full-length recombinant alphaIIb beta3. Deletion of the entire cytoplasmic domain of the beta subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of alphaIIb beta3 that is capable of mediating fibrinogen-dependent cell aggregation.

Comparison of binding to rapidly activating delayed rectifier K+ channel, IKr, and effects on myocardial refractoriness for class III antiarrhythmic agents.

Saturation binding studies in guinea pig ventricular myocytes with 3H-dofetilide, a radioligand for the cardiac rapidly activating delayed rectifier K+ IKr channel, indicated specific high-affinity binding with a Kd of 83 nM and a Bmax of 0.18 pmol/mg cellular protein (1.36 x 10(6) sites/cell). Using displacement of high-affinity 3H-dofetilide binding as a measure of interaction with the IKr channel, potencies (Ki values) for binding to the IKr channel in guinea pig myocytes for six class III antiarrhythmic agents were characterized and compared to indices of functional electrophysiologic activity in isolated guinea pig papillary muscles [EC25 values, concentration required to increase effective refractory period (ERP) 25% above baseline]. Dofetilide, E-4031, sematilide, and d-sotalol, which have been characterized previously as selective IKr blockers, displayed good agreement between Ki values for displacement of 3H-dofetilide binding (47 +/- 7 nM, 38 +/- 8 nM, 12 +/- 5 microM, and approximately 100 microM, respectively) and EC25 values for increasing ERP in papillary muscles (45.0 nM, 76.9 nM, 20.2 microM and 63.5 microM, respectively). Ibutilide and RP58866, which have been reported to act via mechanisms other than IKr block, had Ki values for displacement of 3H-dofetilide binding (16 +/- 7 nM and 17 +/- 2 nM, respectively) that were approximately 10-fold lower than EC25 values for increasing ERP in papillary muscles (185.8 nM and 223.5 nM, respectively). The potent displacement of high-affinity 3H-dofetilide binding by ibutilide and RP58866 strongly suggest a role for interaction with IKr in their actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy. A comparison of factor Xa versus thrombin inhibition.

OBJECTIVE: The aim was to examine the abilities of the direct thrombin inhibitor, recombinant hirudin (rHIR), and the coagulation factor Xa inhibitor, recombinant tick anticoagulant peptide (rTAP), given in combination with rt-PA as high dose front loading plus low dose maintenance infusions, to enhance reperfusion and maintain vessel patency in a canine model of left circumflex coronary artery stenosis and electrolytic lesion. METHODS: Occlusive coronary artery thrombosis was induced in anaesthetised dogs by electrical injury (150 microA) of the intimal surface of the vessel. Thirty minutes after occlusive thrombosis, high dose front loading infusions (45 min) of rTAP (200 micrograms.kg-1 x min-1) and rHIR (300 micrograms.kg-1 x min-1) were initiated concomitant with the start of a 90 min infusion of recombinant tissue-type plasminogen activator (rt-PA). Following the termination of front loading infusions, maintenance infusions of rTAP (10 or 20 micrograms.kg-1 x min-1) or rHIR (20 micrograms.kg-1 x min-1) were initiated and continued for the duration of the protocol (180 min after rt-PA termination). RESULTS: Reperfusion was incomplete in the rHIR group (7/9; 78%), whereas all rTAP-treated preparations reperfused (8/8 per group, aggregate 16/16; 100%). Following thrombolysis, the rHIR group had a high incidence of reocclusion, ranging from intermittent to long periods of occlusion, with only 2/7 (29%) of the preparations which initially recanalised remaining patent during the 180 min period following rt-PA termination. In contrast, 5/8 preparations in each of the two rTAP groups [aggregate 10/16; 63%] remained patent during the same period. The greater efficacy of rTAP v rHIR in maintaining vessel patency was also reflected in integrated coronary artery blood flows [91.0(SEM 5.8)% and 84.9(6.1)% of preocclusion flow in rTAP groups v 57.5(12.2)% of preocclusion flow in rHIR group], times to reocclusion [123.3(22.8) and 128.0(6.7) min in rTAP groups v 36.6(23.2) min in rHIR group; p < 0.05], and residual thrombus masses [1.8(0.3) and 2.0(0.3) mg in rTAP groups v 10.4(3.8) mg in rHIR group; p < 0.05]. CONCLUSIONS: With the present front loading plus low dose maintenance infusions designed to limit the duration of "high dose" conjunctive therapy, rTAP was more effective than rHIR at equimolar plasma concentrations in maintaining post-thrombolysis vessel patency, preserving coronary artery blood flow, and reducing residual thrombus mass. These findings further support the therapeutic potential of inhibiting factor Xa in the setting of coronary artery thrombolysis.

Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites.

Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.

Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site.

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.

Pathways of coagulation activation in situ in rheumatoid synovial tissue.

Immunohistochemical techniques were applied to rheumatoid synovium in order to detect components of coagulation and fibrinolysis pathways within these tissues. These techniques revealed an intact coagulation pathway and plasminogen activator inhibitor-2 associated with macrophage-like cells that were present throughout these tissues, especially in subsurface areas. Cell-associated thrombin generation appeared to account for conversion of abundant fibrinogen to fibrin. Occasional macrophage-like cells also stained for urokinase but tissue-type plasminogen activator and plasminogen activator inhibitor-1 were restricted to vascular endothelium. Intense synovial fibrin deposition (with the limited evidence for associated fibrinolysis) may contribute to local inflammation and explain certain clinical features of rheumatoid arthritis. These findings suggest novel treatment hypotheses for this disease.

Anticoagulant efficacy and immunogenicity of the selective factor Xa inhibitor antistasin following subcutaneous administration in the rhesus monkey.

The antithrombotic efficacy and duration of action of a single subcutaneous administration of the selective factor Xa inhibitor recombinant antistasin (rATS) was evaluated in a rhesus monkey model of mild disseminated intravascular coagulation. rATS (1 mg/kg) was shown to be fully effective and comparable to standard heparin (1,000 U/kg) in the suppression of thromboplastin-induced fibrinopeptide A generation for at least 5 h following a single subcutaneous administration. The absorption rate of rATS, as measured by ex vivo activated partial thromboplastin times (aPTT), mirrored that of standard heparin exhibiting peak anticoagulant activity between 1 and 2 h post administration. The anticoagulant effects of a single rATS dose lasted for longer than 30 h maintaining an aPTT value at least 2-fold higher than baseline. Repeated subcutaneous administrations of rATS resulted in the generation of fully neutralizing antibodies. These results suggest that specific factor Xa inhibition may be as effective as standard heparin in the treatment of venous thrombosis. Due to its antigenicity however, rATS is probably not suitable for chronic subcutaneous anticoagulant therapy.

Cellular localization of activated factor X by Xa-specific probes.

A probe, recombinant antistasin, that reacts specifically with the activated form of factor X (Xa) was used in immunohistochemical procedures to detect cellular sites of Xa generation within intact tissues. Factor Xa was detected on tumor cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma. Tumor-associated macrophages (but not tumor cells) expressed Xa in adenocarcinoma and squamous cell carcinoma of the lung, and Hodgkin's disease. Factor Xa in these locations corresponded to evidence reported previously for an intact coagulation pathway and thrombin formation associated with these tumor cells and macrophages. By contrast, only rare connective tissue cells stained for Xa in breast and colon cancer, tumor types shown previously to lack an intratumoral coagulation pathway and thrombin generation, and in normal liver, lung, breast, kidney, and placental tissues. Hepatocytes did not stain. These results suggest that such probes may be useful for studying the activation state of cell-associated factor X in situ within intact tissues.

Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis.

An in vivo thromboplastin (TP)-induced venous stasis thrombosis model in rabbits was used to compare the efficacy of standard heparin with the selective factor Xa inhibitors, recombinant tick anticoagulant peptide (rTAP) and recombinant antistasin (rATS), in prophylactic prevention of thrombus formation. Heparin significantly reduced TP-induced clot formation at doses of 55 and 100 U kg-1h-1 yielding clot weights of 9 +/- 4 and 6 +/- 2%, respectively. Clot formation was significantly decreased by i.v. infusions of rTAP at doses of 21, 37 and 64 micrograms kg-1 min-1 resulting in normalized clot weights of 13 +/- 3, 8 +/- 2 and 2 +/- 1%, respectively. rATS was approximately 10-fold more potent than rTAP, reducing normalized clot weights to 16 +/- 5, 2 +/- 1 and 1 +/- 0.8% at rATS doses of 1.25, 2.5 and 5.0 micrograms kg-1 min-1, respectively. These data suggest that factor Xa-mediated inhibition of coagulation with rTAP and rATS is as effective as conventional anticoagulant treatment with heparin in preventing venous thrombosis.

Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa.

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.

Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis.

Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM.

Isolation and structural characterization of a potent inhibitor of coagulation factor Xa from the leech Haementeria ghilianii.

The present work reports the discovery and characterization of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalis, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host's blood.

Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.

Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.

The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure.

Antistasin is a 15-kDa protein from the salivary glands of the Mexican leech, Haementeria officinalis, which manifests anticoagulant activity by inhibiting factor Xa. Previous work demonstrating the presence of this activity in salivary gland extracts and its partial purification has been reported (Tuszynski, G. P., Gasic, T. B, and Gasic, G.J. (1987) J. Biol. Chem. 262, 9718-9723). The present study includes further purification to homogeneity of antistasin and its subsequent fragmentation and complete amino acid sequence determination. The protein, which possesses 119 amino acid residues, is blocked at its amino terminus by the presence of a pyroglutamic acid residue and has an unusually high cysteine content, with 20 cysteine residues. The primary structure of antistasin shows no homology to hirudin, a 65-residue anticoagulant protein from the medicinal leech, Hirudo medicinalis. Of great interest is the finding of significant internal homology within antistasin where a 2-fold internal repeated structure is observed. At least four isoforms of antistasin have been identified in leech salivary gland extracts by high performance liquid chromatography analysis, and partial amino acid sequence analysis of these isoforms indicates they differ by 1 or 2 amino acid residues.

Identification of novel proteins synthesized in bone cells by antibody screening of a cDNA expression library.

Novel proteins synthesize predominantly in bone have been identified by antibody screening of bone cell cDNA expression libraries. Two unique cDNAs were identified whose structures do not match any known nucleic acid or protein sequence in the NIH computer bank. The first cDNA clone, BP-I, encoded a mRNA of 2300 bases in size which was expressed at high levels in 17/2.8 rat osteosarcoma cells, rat calvarial bone cells and placenta. A second clone, BP-II, encoded a mRNA of 1500 bases which was expressed at high levels in 17/2.8 osteosarcoma cells and in salivary gland. Expression of both mRNAs in osteosarcoma cells was modulated by the calciotropic hormone, vitamin D. Southern blot analyses indicated that the two cDNAs represented distinct, single copy genes in the rat genome. These novel gene products may serve as potential new markers to study bone turnover in metabolic bone disease.