The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

The specificity of interactions between proteins and sulfated polysaccharides

Os polissacarídeos sulfatados são capazes de se ligar às proteínas com diferentes níveis de especificidade. São macromoléculas altamente ácidas que podem se ligar de forma inespecífica a qualquer domínio básico da superfície de uma proteína em soluções com baixa força iônica, contudo tais interações não parecem ser fisiologicamente significativas. Por outro lado, foram identificados vários sistemas nos quais componentes estruturais muito específicos dos polissacarídeos sulfatados conferem alta afinidade para algumas proteínas. O exemplo mais conhecido é o pentassacarídeo da heparina com alta afinidade pela antitrombina. Outros exemplos podem ser observados no estudo de invertebrados marinhos, tais como a importância da estrutura fina do dermatam sulfato para sua interação com o cofator II da heparina e o envolvimento defucanas sulfatadas encontradas no gel que envolve osóvulos dos ouriços-do-mar na espécie especificidade da fertilização. Um terceiro exemplo de interação específica é aquele descrito para o glicosaminoglicano heparam sulfato encontrado na superfície celular. Neste caso, o padrão de sulfatação pode determinar diferentes afinidades do carboidrato por citoquinas, fatores de crescimento e outras proteínas encontradas na superfície celular e na matriz extracelular. Estas interações complexas entre proteínas e carboidratos são capazes de influenciar a difusão das proteínas através dos tecidos, assim como modelar a resposta celular a estas moléculas.

Laboratory approach in Thai patients with venous thrombosis.

This retrospective study aimed to analyze laboratory findings in Thai patients with venous thrombosis in Phramongkutklao Hospital from August 1997 to October 2004. Blood samples obtained from 166 patients with ages ranging from 10 months to 87 years were tested for protein S (PS), protein C (PC), antithrombin (AT), factor V Leiden (FVL) and prothrombin G20210A. It was found that low levels of PS, PC, and AT were observed in 23 patients (13.9%), 21 patients (12.7%) and 11 patients (6.6%), respectively. The incidence of combined low levels of anticoagulant factors occurred in 23 patients (13.9%). Three patients (1.8%) were positive for FVL. All patients were negative for prothrombin G20210A. Additionally, 85 patients (51.2%) were negative for all tests. In conclusion, it is recommended that the screening tests for anticoagulant factors PS, PC and AT be used to investigate the causes of thrombosis in Asian populations due to their cost-effectiveness. However, the detection of gene mutations inducing thrombosis should be considered.