CONTROL MECHANISMS IN HEMOSTASIS

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The aims of this proposal cover three major objectives: 1. To determine the mechanism(s) of action of lupus anticoagulants. (LAC's) and the hemostatic significance of circulating antibodies to anionic phospholipids: We shall investigate binding of LAC's to activated platelets and to endothelial cells (EC) exposed to IL-1, endotoxin or tumor necrosis factor, and correlate binding with exposure of phosphatidylserine. We shall determine binding-induced alterations in 'prothrombinase' capacity of platelets and EC, effects on cytokine-induced synthesis of PGI2 by EC, and alterations in EC-thrombomodulin-thrombin- inducec activation of protein C. We shall investigate LAC binding to other IL-1- or TNF-sensitive cells, as well as exposure of PS. We shall investigate the mechanism of hypoprothrombinemia, seen in many patients with LAC's, by doing 125 I-prothrombin turnover studies. 2. To study the role of endothelial cell GpIb in EC function: We shall examine binding of vWF and asialo-vWF to resting EC and to EC stimulated with IL-1, TNF or endotoxin. We shall measure GpIb expression in resting and stimulated EC, using monoclonal antibodies to GpIb, and determine whether these antibodies, or polyclonal antibodies produced in our laboratory, inhibit vWF or AS-vWF binding. We shall examine the relationship between the EC cytoskeleton and EC GpIb in resting cells and cells exposed to cytokines. We shall study modulation of EC GpIb-alpha mRNA by cytokines using a 5'-1.1 Kb probe and a 3'-1.3 Kb probe and correlate changes with membrane GpIb expression. We shall examine GpIb distribution by electron microscopy before and after EC stimulation by cytokines and other agents. We shall investigate the binding of platelets to stimulated EC and the possible role of vWF, AS-vWF, fibrinogen and, possibly, other adhesive proteins in such interactions. 3. To investigate possible structural abnormalities of GpIb or of cytoskeletal components in platelets from patients with pseudo- vWd: Using the polymerase chain reaction, we shall amplify mRNA from patient platelets and shall search for structural abnormalities using platelet GpIb-alpha probes. We shall examine cytoskeletal components of patient platelets, resting and after stimulation with aggregating agents, as well as the interaction between membrane GpIb and the cytoskeleton. We shall determine the effect on cytoskeletal components of exposure to conditions activating platelet calpain, such as calcium ionophore, thrombin + collagen, and the anesthetics dibucaine and tetracaine.