ACTIVATION OF PROTHROMBIN IN WHOLE HUMAN BLOOD

资助期内在政府资源中心进行的调查(12-1-96至 11-30-97)专注于血友病A的凝血(凝血因子VIII缺乏)， 血友病C(凝血因子XI缺乏)和正常血液。在鲜血中 含有接触途径的抑制物(玉米胰蛋白酶抑制物， 特定于激活的Hageman因子)，凝血是由组织启动的 因子(25 PM)使生物相关的组织因子凝集 对途径进行了研究。我们早期的研究表明，凝血酶 在正常血液中的生成有三个阶段。跟随 在组织因子的启动下，血液在一段时间内保持液体 哪一种凝血酶原活性保持在低水平(起始期)。在.期间 随后的传播阶段，凝血酶被爆炸性地产生，并且一个 可见的血块形态。爆炸性凝血酶的产生一直持续到 终止阶段，该阶段的反应通过耗尽 底物或内源抑制物的抑制。我们最新的 血友病A型血研究证实因子缺乏导致 到略微延长的启动阶段并基本完成 在繁殖阶段抑制爆炸性凝血酶的产生， 凝血酶生成率(TAT)是 正常汇率。在这个阶段，凝血酶的生成没有这种抑制作用 观察为血友病C(凝血因子XI缺乏)。一段漫长的时间 检测到起始期，但仅在极低的组织因子下 浓度(下午5点左右)。与一株单克隆人的初步结果 正常血液中凝血因子XI的抗体抑制物表明 在低Tf的全正常血液中可以模拟凝血因子XI缺乏的情况， 血友病C支持上述结论。此外， 重组组织因子途径抑制物(3-5 nM TFPI)的加入 正常血液显著延长凝血酶的起始期 世代分布，较高的浓度会有效地抑制 凝血酶生成。这些研究表明，TFPI可能会 诱导严重出血怎么说都不为过，这表明它在 仅限临床会场，极其谨慎。中获得的其他结果 这些研究已经提出了一种对自然界差异的解释 正常和血友病受试者血液中形成的凝块。相比较 在凝血正常的情况下，血友病A的凝血会有轻微的延迟 但显著延迟和减少了血小板的活化 纤维蛋白肽A在相同引发剂水平上的释放(下午25 Tf)，这些观察在血友病C血中并不明显，其中 血小板活化和纤维蛋白形成在运动结束时达到最大 做实验。这些数据表明，凝血酶在 稳定血栓生长的传播阶段，特别是在 关于纤维蛋白的形成，并与其他研究一致 这表明血友病患者最初的血小板堵塞是 到裂解[J.J.Sixma和A.van den Berg(1984)BrJ Haematol 58：741].一个 给出了这些结果(摘要编号2067)的初步报告 在第38届美国血液学会年会上(#年12月 1996年6月10日，佛罗里达州奥兰多)，手稿已提交给 血液杂志详细描述了这些研究。目前我们正在延长这些 研究可能影响凝血的其他因素 血友病的稳定性。我们把研究的范围扩大到正常的整体 以检查其他对凝血反应的影响。跟随 1995~1996年抗血小板药物的初步研究 研究年，我们开始确定阿司匹林是否可测量地影响 在我们的模型中是凝固的。阿司匹林对健康的影响问题 凝血酶的生成已经提高，但还没有得到完全的回答 例如，见A.Szczeklik(1994)在Thromb Haemost 72：988中的信]。 每12小时服用一次阿司匹林(3倍，325毫克) 在实验前一小时最后一次给药，没有显著效果。 用25 PM Tf检测凝血酶生成或血小板活化 发起人。阿司匹林在较低的转铁蛋白浓度下的作用尚未见报道。 被调查过了。在添加血小板凝血酶受体的研究中 活化肽(序列SFLLRN)结合全血，我们观察到 血小板活化速度显著加快(在 反应的第一分钟)，而凝血酶生成情况是 只是稍微加速了一下。这些结果强烈地表明， 在我们的模型中，全血凝血的限制步骤不是血小板 激活，而是促凝血蛋白的激活和组装 导致凝血酶生成的复合体。最后，初步研究表明 在S部分蛋白缺乏症患者的血液中进行了检测 (约30%正常)，表明凝血速度略快于正常 缓慢的反应(凝血和凝血酶-抗凝血酶复合体) 加入纯化蛋白S约1-2分钟(不含C4b- 结合蛋白)。相信这些研究将会给我们带来一些启示。 S蛋白在限制正常人和正常人血栓形成潜能中的作用 S蛋白虚血。

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.