RESUMEN
OBJECTIVE: Introduction: Chronic kidney disease (CKD) is a critical medical and social problem. Every year 7& of such patients require renal replacement therapy associated with one of the most life threatening complications - disturbed hemostasis and development of thrombophilic conditions which are typical of complicated comorbid diseases. Prediction of potential thrombogenesis in that category of patients requires multidimensional approach, including comprehensive laboratory investigation of hemostasis on the one hand, and creation of mathematical model based on thrombophilia predictors on the other. The aim: To create the prognostic model of thrombophilia development in patients with stage VD CKD, treated by long term hemodialysis, using discriminant function analysis and biochemical variables of hemostasis. PATIENTS AND METHODS: Materials and methods: Of 12 hemostatic parameters (factors of influence), studied in 85 patients with stage VD CKD, two groups of variables were formed - pre-thrombosis (soluble fibrin (sF), D-dimer (D-d), protein C (PC)) and post-thrombosis (D-d, H (maximal end turbidity of plasma clot) and pC). RESULTS: Results: Linear discriminant functions (two models), based on two groups of variables, demonstrated rather high coincidence rate (92.2÷93.5&) in group distribution. The patients referred to those with thrombotic complications, 100& matched the prospective (for a year) study. CONCLUSION: Conclusions: By the results of correlation analysis, the most significant molecular biochemical hemostatic parameters (sF, D-d, pC, H) were determined. Using definite biochemical parameters, discriminant models complementing each other and having high prediction (Ñ <0.0001) of thrombophilic states in 92.2-93.5& of cases, were developed for patients with stage VD CKD, treated by long term hemodialysis.
Asunto(s)
Análisis Discriminante , Insuficiencia Renal Crónica/diagnóstico , Trombofilia/diagnóstico , Humanos , Pronóstico , Estudios Prospectivos , Insuficiencia Renal Crónica/complicaciones , Trombofilia/complicacionesRESUMEN
We have discovered that addition of monomeric desAB fibrin to prothrombin leads to appearance of the thrombin-like activity of prothrombin towards S2238 chromogenic substrate. DesA and desABß(15-42)2 fibrin forms did not cause any activation of prothrombin. From this observation we could suggested that amino acid residues of the 15-42 fragment of BßN-domain presented in desAB fibrin, cleaved in desABß(15-42)2 fibrin and protected in desA fibrin, play a crucial role in the non-enzymatic activation of prothrombin. To identify the Bß amino acid residues involved in the fibrin-prothrombin binding we used monoclonal antibodies 1-5G and 2d2a with epitopes in Bß26-42 and Bß12-26 fibrin fragments respectively. The thrombin-like activity in the mixture of prothrombin and desAB fibrin was monitored in the presence of each of these monoclonal antibodies. It was found that anti-Bß12-26 antibody does not exhibit any inhibitory effect on the thombin-like activity of the mixture. In contrast, adding of Bß26-42 antibody into the mixture of desAB fibrin with prothrombin diminished the thrombin-like activity by 70%. Recombinant dimeric peptides Bß(15-44)2 and Bß(15-66)2 that mimic amino acid residues in fibrin were also tested for their ability to activate prothrombin. It was found that both peptides were able to induce non-enzymatic activation of prothrombin. The activation was more evident in the case of Bß(15-44)2 peptide. From the data obtained we can conclude that desAB fibrin binds to prothrombin through the Bß26-42 amino acid residues and the formation of such a complex caused a non-enzymatic activation of prothrombin.
Asunto(s)
Fibrina/química , Protrombina/química , Aminoácidos/química , Anticuerpos Monoclonales/química , Catálisis , Coagulasa/química , Epítopos/química , Productos de Degradación de Fibrina-Fibrinógeno/química , Fibrinógeno/química , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10(-6) M (IC(50)). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC(50) was 1.26 × 10(-6) M at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC(50) = 1.31 × 10(-4) M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site 'A' (Aα17-19), which ordinarily initiates protofibril formation in a 'knob-hole' manner. This suggestion was confirmed by an HPLC assay, which showed a host-guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site 'A'. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10(-5) M and 1.10 × 10(-5) M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.