Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non-mucosal venous injury model in haemophilic rodents.

Essentials The efficacy of systemic antifibrinolytics for hemophilic non-mucosal bleeding is undetermined. The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored. Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis. The non-mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis. SUMMARY: Background Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. Objective To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy. Methods A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non-mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays. Results The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice. Conclusions The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non-mucosal tissues.

Balloon dilation and surgical valvotomy comparison in non-critical congenital aortic valve stenosis.

Percutaneous balloon aortic valvoplasty (BAV) and surgical aortic valvotomy (SAV) are palliative procedures in patients with non-critical congenital valve stenosis. The aim of the study was to evaluate long-term BAV and SAV results after up to 24 years of follow-up. From 1987 to 2013, 74 consecutive interventions were performed in patients with aortic stenosis, and 62 were included in the study (39 BAVs and 23 SAVs). Age of BAV patients was 1.3 months to 17 years, and of SAV patients 1.2 months to 15 years. Although BAV patients were older, there was no difference between groups according to sex, valve function/morphology, and early/late follow-up results, with exception to hospitalization period. Significant pressure gradient reduction and aortic regurgitation increment were registered after procedures. Three patients did not survive early period after surgery. Follow-up period was 7.0 ± 5.4 and 9.0 ± 8.0 years after BAV and SAV, respectively (p = 0.242). Follow-up pressure gradient rose only in the BAV group, and was emphasized after 10-year-follow-up (p = 0.020). Significant aortic insufficiency progression was registered after 15 years of follow-up in both groups (p = 0.007 and p = 0.009, respectively). Mean reintervention-free survival was 12.0 years in the BAV and 14.5 years in the SAV group (p = 0.733), and mean survival without aortic valve replacement was 15.2 and 17.4 years, respectively (p = 0.877). BAV and SAV in patients with congenital aortic stenosis are very comparable in both early and late follow-up results.

Pharmacokinetics of tranexamic acid in patients undergoing cardiac surgery with use of cardiopulmonary bypass.

We conducted a study to assess pharmacokinetics of high-dose tranexamic acid for 24 h after administration of the drug in patients undergoing cardiac surgery with cardiopulmonary bypass. High-dose tranexamic acid involved a bolus of 30 mg.kg(-1) infused over 15 min followed by a 16 mg.kg(-1) .h(-1) infusion until chest closure with a 2 mg.kg(-1) load within the pump prime. Tranexamic acid followed first-order kinetics best described using a two-compartment model, with a total body clearance that approximated the glomerular filtration rate. Mean plasma tranexamic acid concentrations during the intra-operative period and in the first 6 postoperative hours were consistently higher than the suggested threshold to achieve 100% inhibition and 80% inhibition of tissue plasminogen activator. With recent studies implicating high-dose tranexamic acid as a possible aetiology of postoperative seizures following cardiac surgery, the minimum effective yet safe dose of tranexamic acid in high-risk cardiac surgery needs to be refined.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study.

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases.

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ((1)HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants K(a) were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants K(aI) of PBZ-SA complex. Similarly, PBZ influences K(aI) of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of K(aII) values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of (1)HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex.

Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

Alterations of furosemide binding to serum albumin induced by increased level of fatty acid.

Localization of high and low affinity binding sites of furosemide in human serum albumin (HSA) as well as the influence of myristic acid on the drug binding to the albumin using fluorescence quenching method was investigated. Two independent classes of binding site in subdomain IIA of HSA structure were found. Alteration of protein affinity towards the drug and the participation of tryptophanyl and tyrosil residues in drug-albumin interaction for the determined binding sites were studied. It was concluded that association of myristic acid in its low affinity binding sites which corresponds to elevated fatty acid level in vivo, significantly decreases albumin affinity towards furosemide.

The influence of dietary habits and pathological conditions on the binding of theophylline to serum albumin.

The influence of fatty acids (FA) on theophylline (Th) binding to human serum albumin (HSA) in its high and low affinity binding sites was investigated. The content of studied FA solutions corresponds to the ones associating with different dietary habits and pathological states in vivo. Using fluorescence and (1)H NMR spectroscopy two high and two low affinity binding sites of Th in HSA structure were found. For each site several binding parameters in the absence and presence of FA were estimated. The results showed that the impact of FA on the affinity of HSA towards Th in high affinity binding sites is negligible whereas binding of the drug in low affinity sites decreases significantly in the presence of FA. It was observed that this effect is dependent on the number of fatty acid molecules bound to the protein while the chemical structure of fatty acids contained in the solution plays a minor role.

Binding of 6-propyl-2-thiouracil to human serum albumin destabilized by chemical denaturants.

We compared the binding affinity of 6-propyl-2-thiouracil (PTU) with native and destabilized human serum albumin (HSA) as a model to assess the binding ability of albumin in patients suffering from chronic liver or renal diseases. Urea (U) and guanidine hydrochloride (Gu.HCl) at a concentration of 3.0M were used as denaturation agents. Increasing the concentration of PTU from 0.8x10(-5) to 1.20x10(-4)M in the systems with HSA causes a decrease in fluorescence intensity of the protein excited with both 280 and 295nm wavelengths. The results indicate that urea and Gu.HCl bind to the carbonyl group and then to the NH-group. To determine binding constants we used the Scatchard plots. The presence of two classes of HSA-PTU binding sites was observed. The binding constants (K(b)) are equal to 1.99x10(4)M(-1) and 1.50x10(4)M(-1) at lambda(ex)=280nm, 5.20x10(4)M(-1) and 1.65x10(4)M(-1) at lambda(ex)=295nm. At lambda(ex)=280nm the number of drug molecules per protein molecule is a(I)=1.45 and a(II)=1.32 for I and II binding sites, respectively. At lambda(ex)=295nm they are a(I)=0.63 and a(II)=1.54 for the I and II binding sites. The estimation of the binding ability of changed albumin in the uremic and diabetic patients suffering from chronic liver or renal diseases is very important for safety and effective therapy.

Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study.

The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ((1)H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sulkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

Changes of serum albumin affinity for aspirin induced by fatty acid.

Saturated fatty acids such as myristic acid play an important role in the pathogenesis of cardiovascular disorders. Using the quenching fluorescence method we examined the influence of myristate on the changes of transporting protein affinity towards aspirin-the most popular anticoagulant. Our results showed that the presence of the myristic acid alters the stability of the anticoagulant-albumin complex. The ranges of [myristate]/[albumin] molar ratio at which the stability of drug-protein complex increases or decreases were determined. The differences in interaction between ligands and human or bovine serum albumins were identified. The competition in binding of ligands with these albumins was also described.