Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

INTRODUCTION: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. METHODS: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. RESULTS: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. CONCLUSIONS: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Prothrombin 3'end Gene Variants in Patients With Sporadic Colon Adenocarcinoma.

BACKGROUND/AIM: Thrombin plays significant roles in various types of cancer. However, the expression levels of prothrombin, the thrombin precursor, in cancer remain unclear. Variants of the 3'end of the prothrombin gene lead to increased prothrombin expression. This study aimed to analyze prothrombin 3'end gene variants in colon tumor and adjacent normal tissue samples. MATERIALS AND METHODS: The study group consisted of 93 patients suffering from colon adenocarcinoma. The 3'end of the prothrombin gene was analyzed by DNA sequencing. RESULTS: Three variants, all previously associated with increased prothrombin expression were detected. Frequency of the FII 19911G allele was 46.77% and 47.85% in tumor and normal tissue, respectively. For the FII 20210A allele, the detected frequencies were 2.15% and 1.61%, respectively. The frequency of the FII c.1824T allele was 0.54% in both tissues. Four patients showed different genotypes in tumor and normal tissue. CONCLUSION: Prothrombin 3' end gene variants may play a role in colorectal cancer.