Investigations performed in the GCRC during the grant period (12-1-96 to 11-30-97) focused on coagulation in hemophilia A (factor VIII deficient), hemophilia C (factor XI deficient), and normal blood. In blood which contains an inhibitor of the contact pathway (corn trypsin inhibitor, specific for activated Hageman factor), coagulation is initiated by tissue factor (25 pM) so that the biologically relevant tissue factor coagulation pathway is examined. Our earlier studies had demonstrated that thrombin generation in normal blood is characterized by three phases. Following initiation by tissue factor, the blood remains fluid for a period during which prothrombin activation remains low (initiation phase). During the subsequent propagation phase, thrombin is generated explosively and a visible clot forms. Explosive thrombin generation continues until the termination phase where the reaction is attenuated by depletion of the substrates or inhibition by endogenous inhibitors. Our most recent studies in hemophilia A blood confirm that deficiency of factor VIII leads to a slightly prolonged initiation phase and virtually complete suppression of explosive thrombin generation during the propagation phase, evidenced by a rate of thrombin generation (TAT) that is 1/29th of the normal rate. No such suppression of thrombin generation during this phase was observed for hemophilia C (factor XI deficiency). A prolonged initiation phase was detected, but only at very low tissue factor concentrations (about 5 pM). Preliminary results with a monoclonal antibody inhibitor of factor XI in normal blood suggest that the condition of factor XI deficiency may be mimicked in whole normal blood at low TF, supporting the above conclusions for hemophilia C. Furthermore, the addition of recombinant tissue factor pathway inhibitor (3-5 nM TFPI) to normal blood significantly prolongs the initiation phase of the thrombin generation profile, with higher concentrations potently suppressing thrombin generation. These studies suggest that the potential of TFPI to induce severe hemorrhage cannot be overstated, indicating its use in the clinical venue only with extreme caution. Additional results obtained in these studies have suggested an explanation for differences in the nature of clots formed in blood from normal and hemophilic subjects. Compared with normal clotting, clotting in hemophilia A occurs with a modest delay in platelet activation but a significant delay and reduction in the liberation of fibrinopeptide A. At the same level of initiator (25 pM TF), these observations are not evident in hemophilia C blood, where platelet activation and fibrin formation are maximal by the end of the experiment. These data suggest a role for thrombin generated during the propagation phase in stabilizing the growing thrombus, particularly with respect to fibrin formation, and are in agreement with other research which has shown that the initial platelet plug of hemophiliacs is subject to lysis [J.J.Sixma and A. van den Berg (1984) Br J Haematol 58:741]. A preliminary report of these results (abstract number 2067) has been given at the 38th Annual Meeting of the American Society of Hematology (December 6-10, 1996, Orlando, FL), and a manuscript has been submitted to the journal BLOOD detailing these studies. At present we are extending these studies to examine the additional factors which may influence clot stability in hemophilia. We have extended the studies in normal whole blood to examine other influences on the coagulation reaction. Following preliminary studies with anti-platelet reagents during the 1995-96 research year, we set out to determine whether aspirin measurably affects coagulation in our model. The question of the influence of aspirin on thrombin generation has been raised, yet has not been fully answered [for instance, see the letter by A. Szczeklik (1994) in Thromb Haemost 72:988]. Using a regimen of aspirin administration every 12 hours (3x, 325 mg each) with the final dose one hour before the experiment, no significant effect on thrombin generation or platelet activation was detected with 25 pM TF initiator. The role of aspirin at lower TF concentrations has not yet been investigated. In studies adding the platelet thrombin receptor activation peptide (sequence SFLLRN) to whole blood, we observed that platelet activation was significantly accelerated (complete within the first minute of the reaction), while the thrombin generation profile was only slightly accelerated. These results strongly suggest that the limiting step in coagulation of whole blood in our model is not platelet activation, but is activation and assembly of the procoagulant protein complexes leading to thrombin generation. Finally, initial studies have been carried out in blood from a patient with partial protein S deficiency (about 30% normal), indicating a somewhat faster-than-normal coagulation reaction (clotting and thrombin-antithrombin complex) which was slowed approximately 1-2 minutes by addition of purified protein S (without C4b- binding protein). It is believed that these studies will shed some light on the role of protein S in limiting thrombotic potential in normal and protein S deficient blood.
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