Interaction of heparin with two synthetic peptides that neutralize the anticoagulant activity of heparin.

Two synthetic analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (Ac-SRGKAKVKAKVKDQTK-NH2) and the all-d-amino acid version of the same peptide (l-HIPAP and d-HIPAP, respectively) were synthesized, and their efficacy as agents for neutralization of the anticoagulant activity of heparin was assayed. The two analogue peptides were found to be equally effective for neutralization of the anticoagulant activity of heparin, as measured by restoration of the activity of serine protease factor Xa by the Coatest heparin method. The finding that l-HIPAP and d-HIPAP are equally effective suggests that d-amino acid peptides show promise as proteolytically stable therapeutic agents for neutralization of the anticoagulant activity of heparin. The interaction of l-HIPAP and d-HIPAP with heparin was characterized by 1H NMR, isothermal titration calorimetry (ITC), and heparin affinity chromatography. The two peptides were found to interact identically with heparin. Analysis of the dependence of heparin-peptide binding constants on Na+ concentration by counterion condensation theory indicates that, on average, 2.35 Na+ ions are displaced from heparin per peptide molecule bound and one peptide molecule binds per hexasaccharide segment of heparin. The analysis also indicates that both ionic and nonionic interactions contribute to the binding constant, with the ionic contribution decreasing as the Na+ concentration increases.

Efficacy of viral clearance methods used in the manufacture of activated prothrombin complex concentrates: focus on AUTOPLEX T.

Various methods are described for the elimination of infectious viruses from activated prothrombin complex concentrates (aPCCs) and for the analysis of the final products (AUTOPLEX T and FEIBA VH). Viruses of concern in human plasma-derived products are enveloped (hepatitis B and C, cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus [HIV]) and nonenveloped (hepatitis A and parvovirus B19). Donated blood used for AUTOPLEX T is screened for antihepatitis C, HBsAg, anti-HIV types 1 and 2, and p24 antigen. Plasma pools utilized for raw materials are also tested by PCR for HIV and hepatitis C virus. Partial virus inactivation and partitioning are achieved by purification of the aPCC. Further reduction of virus infectivity is accomplished by lyophilization and dry-heat treatment. Each step undergoes virus elimination validation studies in which a relevant sample is 'spiked' with the appropriate virus or model virus. The total reduction in virus from raw material to final product can then be calculated. For AUTOPLEX T the cumulative log10 reduction factors for several viruses vary from 4.2 to 14.3. This ensures an exceptionally high margin of safety. Definitive evidence for product safety was obtained by clinical observation of treated patients. The viral inactivation process of AUTOPLEX T involves a four-tier viral safety program, including Cohn alcohol fractionation and dry-heat treatment, in place of the two-stage vapour-heating process for FEIBA.

Solution-phase and solid-phase synthesis of novel transition state inhibitors of coagulation enzymes incorporating a piperidinyl moiety.

2-Amino-3-piperidin-4-yl-propionic acid containing peptidomimetics are potent protease inhibitors when combined with an appropriate keto-thiazole or keto-carboxylic acid moiety. A novel P1 residue in factor Xa and thrombin inhibitors has been found resulting in IC50 values as low as 0.048 microM, a factor of ten more potent than Argatroban. Starting with non-chiral synthetic routes, a new stereospecific route was developed as well as a new solid-phase method.

La vitamina K: bioquímica, función y deficiencia: revisión / The vitamin K: biochemistry, function and deficiency: riview

La vitamina K es un cofactor que actúa en la síntesis de los factores de coagulación II, VII, IX y X, de los inhibidores de la coagulación, proteínas C y S, y de proteínas de la matriz ósea. Su forma activa actúa como coenzima en la carboxilación del ácido glutámico de dichas proteínas. La función de los factores dependientes de la vitamina K se realizan mediante la formación de complejos enzimaticos, en los cuales estas proteínas se unen sobre la membrana fosfolipídica a un cofactor proteíco, en presencia de calcio. La insuficiencia de los mecanismos responsables de la gamma-carboxilación del ácido glutámico altera la función hemostática. La deficiencia hereditaria de factores dependientes de vitamina K, los antibióticos y los anticoagulantes orales disminuyen la capacidad de formación de los complejos enzimaticos, por lo que se produce síndrome hemorrágico o trombóticos y alteraciones de la masa ósea que son fácilmente tratadas con administración de preparados de vitamina K. Las causas principales de deficiencia son: falta de reservas hepáticas en recién nacidos, insuficiencia hepática, falta de ingesta, malabsorción, antibioticoterapia y admisnistración de cumarínicos. Para el estudio de la vitamina K se usan métodos indirectos, que miden las proteínas dependientes de su acción, y métodos directos que miden específicamente las quinonas