MECHANISMS OF THROMBOSIS IN SICKLE DISEASE

点击翻译按钮获取中文摘要

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).