The central focus of the project concerns the early events that lead to the initial appearance of procoagulant activity in sickle cells, and the mechanisms by which such cells may revert to a nonprocoagulant state. Procoagulant activity is defined as supplying the requirement of several reactions in clotting for negatively charge phospholipid, and it is caused by loss of asymmetry in the cell's phospholipid bilayer. Both sickled cells, and membrane fragments derived from them, are known to have such activity. But there is much less known about the earlier reversible stages. These include the following major questions, which will be addressed using highly sensitive assays for the phospholipid dependence of two major clotting reactions: the activation of factor X by the factor IXa-VIIIa complex, and of prothrombin by the factor Xa-Va complex. (1) Do reversibly sicklable cells (RSC) have procoagulant activity in the unsickled state; or is their reported activity due to the presence of irreversibly sickled cells (ISC), and perhaps other procoagulant peripheral cells like platelets? This will be approached by using gradient-fractionated sickle-cell preparations, sickle-trait cells, and potent platelet inhibitors. (2) Is the appearance of procoagulant activity in sickled RSC reversible upon reoxygenation? There is evidence that red cells can regain membrane asymmetry; we will measure this, and its rate, using measurements of procoagulant activity. (3) What is the role of the red cell's metabolism in the maintenance of the non-procoagulant state? There are two facets to this question. The first concerns maintenance of the cytoskeleton, which in turn is required for anchoring the inner membrane leaflet. The cytoskeleton is dependent on essential thiol groups, and these tend to be oxidized in sickled cells through the action of hemoglobin SS. In addition, however, thiols are maintained actively through the GSSH/GSH balance of the cell, which depends on active metabolism and generation of NADPH. The second metabolic role concerns an ATP-dependent translocase, which can relocate external phosphatidyiserine (PS) to the inner leaflet. In the absence of both the anchor and metabolism, it is likely that unregulated membrane flip-flop occurs, causing the appearance of procoagulant PS in the outer membrane leaflet. This will be studied, using procoagulant measurements of outer-leaflet PS, with RSC, ISC, and red-cell ghosts. We expect these studies to show that circulating procoagulant RSC are a significant feature in sickle-cell disease, and to delineate the causes of appearance of procoagulant activity.
| Martin, D W; Jesty, J (1995) Calcium stimulation of procoagulant activity in human erythrocytes. ATP dependence and the effects of modifiers of stimulation and recovery. J Biol Chem 270:10468-74 |
