The influence of age on the regulation of thrombin - Relevance to the development of thromboembolic disease and its treatment in children

During child-hood the regulation of thrombin is enhanced physio-logically and the optimal regulation of thrombin by anticoagulants, which is critically important for the treatment of TEs, differs in the young compared to adults. Further, clots made from newborn plasma have decreased thrombin activity compared to clots pro-duced from adults, which suggests that lower concen-trations of anti-coagulants may effec-tively inhibit clot growth. It is thus hypothesized that the regu-lation of thrombin by newly available anticoagulants (hirudin, agatroban, and orgaran) will be age dependent, and that the treatment of TEs in the young might require de-creased doses of either direct or indirect thrombin inhibitors compared to treatment of TEs in adults. These hypo-theses will be tested in an in-vitro and in-vivo setting. First, thrombin generation will be measured in the presence of umbilical cord vein cell surfaces by amidolysis of a chromo-genic substrate after defibination, contact activation, and recalci-fication of the test plasma. Each plasma which will be supplemented with hirudin (10-1000ng/mL plasma (Behring)), arga-troban (0.1-500M (SmithKline Beecham)) or orgaran (0.1-20 anti-FXaU/mL (Org 10172, Organon). Thrombin generations with anticoagulant containing plasmas will be compared to results from control ex-peri-ments with untreated plasma. Differences in thrombin generation, prothrombin consumption or throm-bin inhibition for each agent will deter-mined by ANOVA, as will differences between anticoagulants in the same plasma type. Secondly, the benefit-to-risk profile of currently used, as well as new, anticoa-gulants will be investigated in an animal model of DVT and bleeding in the young. Central venous lines (CVL) will be introduced into adult and newborn pigs. Clot-formation will be induced by intra-CVL injec-tion of bovine thrombin. Six hours after clot-induction, animals will be injected with 1 Ci/kg of 125I-fibri-no-gen, and will then receive 5 mL intravenous bolus (IV) / 5 mL subcuta-neous (SQ) of: 0.15 M NaCl (con-trol), 100 U/kg IV/400 U/kg SQ unfractioned heparin, 100 U /kg IV/400 U/kg SQ low-molecular weight heparin, 0.1 - 0.5 mg/kg IV/0.25 - 5 mg/kg SQ hirudin, 0.1 - 1 mg/kg IV/0.1 - 1 mg/kg SQ argatroban, or 20 -500 anti-FXa U/kg IV orgaran. At 2, 5 and 10 h post treatment, CVL + clot will be dissected out. Ana-lysis of the effectiveness of each agent will be carried out by assessment of clot size and by clot-accretion of 125I-fibrin. In order to test the safety of each anticoagulant, animals will be anaesthetized, injected with anticoagulant (same doses as in the DVT experiments) and 5 small incisions made by scalpel in an ear. Blood loss will be determined by im-mersing the ear in a 37oC H2O bath and analyzing subsamples taken from the bath over time for red cell hemo-globin. The ratio of treatment efficacy (either decrease in clot weight or 125I-fibrin accretion compared to saline con-trol) to bleeding risk (blood loss in L) will be calcu-lated for each treatment in each age group. Data will be analyzed by 2-way ANOVA to determine if any significant differences exist in efficacy to bleeding risk ratios between the different anticoagulants and/or age groups.