Investigations into hemostasis in Sickle Cell Disease (SCD) suggests that thrombin and fibrin generation are increased during steady state, with conflicting data whether further activation occurs in vasocclusive crisis (VOC). Platelet activation during VOC occurs with variable findings during steady state. A selective, evaluation of hemostatic pathways i.e. intrinsic or extrinsic activation, fibrinolysis, and platelet-endothelial activation has not been reported. Neither has a longitudinal evaluation in infants during the unique transition when HbF levels fail from 80% to <10%. We propose to test the hypothesis that elevated HbF levels in the infant protects against coagulation activation seen in the older child and adult. Study design will allow us to ascertain if protection is due to inhibition of intraerythrocyte HbS polymerization with protection from in vivo sickling-desickling, the absence consequently of circulating red cell membrane pro-coagulant vesicle and thus no signs of hemostatic activation. Studies in older children will assess whether intrinsic activation (relevant to the origin of pain and acute inflammation) occurs only during VOC, and the role of monocyte TF expression in extrinsic. Studies will include intrinsic markers [high molecular (HK) and low molecular weight kininogen (LK), cleavages, western blotting, and kallikrein-alpha/2 macroglobulin]; extrinsic markers [TF assays on monocytes, TFPI and factor VIIa]; other activation and fibrinolytic markers [prothrombin F1.2, FPA, TAT, tPA, PAI-I, D-dimer and PAP] urokinase-type plasminogen activator receptor (uPAR) and soluble uPAR; platelet-endothelial markers [of activation dependent epitopes, in vivo platelet microparticles, circulating soluble adhesion molecules and urinary eicosanoids. Erythrocyte evaluation include quantitative HbF, F cells, adhesion, surface adhesion molecules, and erythrocyte procoagulant activity. Neutrophil evaluation include quantitative HbF, F cells, adhesion, surface adhesion molecule, and erythrocyte procoagulant activity. Neutrophil activation will be monitored via Mac-1 and plasma elastase-alpha1 protease complexes. Our use of the unique transition period when HbF levels fall from neonatal to adult levels should elucidate the contribution to coagulation activation of red cell procoagulent activity in vivo. This information is of pertinence since therapy targeted to reverse the Hb switch has gained ascendancy. A delineation of the roles of monocyte TF, fibrinolytic and cellular activation in the genesis of VOC will provide insights into SCD pathophysiology. Unequivocal demonstration of contact activation during VOC would provide a link between VOC and its accompanying phenomenon of pain, and inflammation. This could facilitate the design of treatment strategies (i.e. use of protease inhibitors, mab to FXII, etc). Finally our studies should provide a unique perspective on the continuum of hemostatic changes that unfold during the course of SCD, and those that develop as vascular insufficiencies supervene in the adult.
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