MECHANISMS OF THROMBOSIS IN SICKLE DISEASE

在镰状细胞中充分描述了止血途径的体内活化 疾病（SCD），无论是在无症状稳态疾病和疼痛期间 危机然而，目前尚不清楚血栓形成是否起到直接的致病作用， 在SCD血管闭塞现象的病理生理学中的作用。我们有 开发了一种凝血试验来测量全血组织的水平， 因子（TF）促凝血活性（PCA）。该测定的优点是 其灵敏度，避免单核细胞（Mo）分离的需要，以及 样本可以在远离采集的时间进行分析。 出乎意料的是，我们在正常人中发现了可检测到的TF PCA， 这是一个新发现全血TF PCA水平显著高于 在SCD患者中升高。Mo可能是全血TF的来源 PCA。在我们的第一个具体目标中，我们将确定TF PCA是否真正 可在Mo表面上通过以下方式引发体内凝血： 证明是否存在增加的复合物形成， 组织因子途径抑制物（TFPI），以及是否存在预期的 第七因子的周转率增加。根据我们最近的观察， 急性时相蛋白，C反应蛋白（CRP），诱导Mo合成 TF，我们假设有充分记录的血清中 SCD患者的CRP水平是导致全血中CRP水平升高的原因。 TF PCA。在我们的第二个具体目标中，我们将测量血清CRP（以及 TNF-α、IL-1和IL-6）水平，并检查是否 这些血清诱导Mo中TF的重新合成与CRP成比例 浓度.将寻求CRP重要作用的进一步证据 通过CRP免疫耗竭后重新检测血清TF诱导。在 我们的第三个具体目标，我们将测试的假设，而损失的 镰状红细胞膜的不对称性使得表面 凝血酶原酶复合物的组装，它并不同样支持 活化蛋白C（APC）介导的因子Va的失活，因此进一步 与正常细胞相比， 红细胞（RBC）。镰状红细胞亚群和无血影蛋白 膜针状体也将被测试其支持这些的能力。 前体和抗凝血凝固酶复合物。凝血活性 将与膜外瓣叶内容相关， 磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）。最后，在我们的 第四个具体目标，我们将研究是否上述 SCD患者血浆中蛋白S缺乏是由于 存在抗蛋白S的自身抗体。我们记录了 这种抗体在其他临床情况下， 与抗磷脂抗体相关（如 SCD）。

In vivo activation of hemostatic pathways is well described in sickle cell disease (SCD), both in asymptomatic steady-state disease and during pain crisis. However, it is unclear whether thrombosis plays a direct causative role in the pathophysiology of the vasoocclusive phenomena of SCD. We have developed a coagulation assay to measure levels of whole blood tissue factor (TF) procoagulant activity (PCA). The advantages of this assay are its sensitivity, avoidance of the need for monocyte (Mo) isolation, and the fact that samples may be assayed at a time remote from collection. Unexpectedly, we find detectable TF PCA in normal individuals, which represents a novel finding. Whole blood TF PCA levels are significantly elevated in patients with SCD. Mo are likely the source of whole blood TF PCA. In our first specific aim, we will determine whether TF PCA is truly available on the surface of Mo to initiate coagulation in vivo, by demonstrating whether or not there is increased complex formation with tissue factor pathway inhibitor (TFPI), and whether there is the expected increased turnover of factor VII. Based on our recent observation that the acute-phase protein, C-reactive protein (CRP), induces Mo to synthesize TF, we hypothesize that the well-documented periodic elevations in serum CRP levels in SCD patients are responsible for the increase in whole blood TF PCA. In our second specific aim, we will measure serum CRP (as well as TNF-alpha, IL-1, and IL-6) levels in SCD patients and examine whether these sera induce de novo synthesis of TF in Mo in proportion to CRP concentration. Further proof of the important role of CRP will be sought by re-testing sera for TF induction following immunodepletion of CRP. In our third specific aim, we will test the hypothesis that while the loss of membrane asymmetry in sickle erythrocytes allows efficient surface assembly of the prothrombinase complex, it does not equally support activated protein C (APC)-mediated inactivation of factor Va, thus further exaggerating the net procoagulant effect of these cells compared to normal red blood cells (RBCs). Sickle RBC subpopulations and spectrin-free membrane spicules will also be tested for their ability to support these pro and anticoagulant coagulation enzyme complexes. Clotting activities will be correlated with membrane outer leaflet content of phosphatidylserine (PS) and phosphatidylethanolamine (PE). Finally, in our fourth specific aim, we will examine whether the previously described deficiency of protein S in the plasma of patients with SCD is due to the presence of auto-antibodies to protein S. We have documented the existence of such antibodies in other clinical situations where protein S deficiency occurs in association with anti-phospholipid antibodies (as is true for SCD).