The biophysical and biochemical basis for the 1 degree wave of platelet aggregation induced by ristocetin (von Willebrand factor (vWf) dependent) and ADP, epinephrine and thrombin (fibrinogen dependent) will be studied. A general hypothesis is proposed based on colloidal stability considerations and supported by my previous studies which suggest that (after the platelet surface is altered by the appropriate inducer) the vWf and fibrinogen may bind to the platelet at specific receptors and act as bridging molecules between platelets. In addition to a potential bridging function, I have shown that the binding of vWf may facilitate agglutination by reducing the platelet's surface charge and this may also be true for fibrinogen. The objectives of this grant are to test the above hypothesis and to begin a systematic analysis of the proteins which contribute charged groups to the platelet surface. The interaction of vWf and fibrinogen with their platelet receptors will be investigated by newly developed agglutination techniques utilizing solid beads containing the proteins covalently bound to their surface. Both the platelet and the protein can be selectively altered biochemically to define the molecular basis for these interactions. Attempts will be made to isolate the platelet receptors for these proteins by selective solubilization and affinity chromatography. The different forms of vWf and biochemically altered, purified vWf will be tested quantitatively for their ability to enhance the reduction in electrophoretic mobility of platelets produced by ristocetin. The electrophoretic mobility changes accompanying fibrinogen-dependent 1 degree aggregation will be tested in platelet-rich plasma and reconstituted systems to determine which agents are responsible for the changes. Comparisions between normal platelets and those from patients with vascular disease will be performed. The molecular organization and functional significance of platelet surface charge groups will be investigated by reacting platelets with labeled probes which are selective for different charge groups and assessing changes in electrophoretic mobility, platelet function and protein labelling (as judged by SDS polyacrylamide gel electrophoresis and fluorography). Finally, electron-spin resonance spectroscopy of fat soluble membrane probes will be performed to assess its usefulness in investigating changes in platelets during stimulation.
NIH ExPorter
NIH ExPorter