Lisa K. Jennings - US grants

Human platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) are proteins on the membrane surface which mediate the aggregation of platelets in response to vascular injury. These glycoproteins are mainly involved in what is considered conditional receptor function in that the aggregation sites on these proteins are exposed only after platelet stimulation. The studies proposed in this application are designed to continue to define the structure/function relationship of GPIIb and GPIIIa as aggregation receptors on the platelet membrane. Monoclonal antibodies prepared by this laboratory will be used as specialized probes for epitope mapping. Two techniques will be used primarily: 1) the western blot technique with purified intact glycoproteins and of proteolytic digests 2) flow cytometry of intact or permeabilized platelets. Information gained by these techniques will be used in correlating the functional importance of these identified polypeptide regions. Functional parameters to be examined are platelet aggregation and fibrinogen receptor expression. Some antibodies will clearly not have an effect on platelet function. These probes will be useful in topographical mapping. It is anticipated that these studies will contribute to further characterization of platelet GPIIb and GPIIIa and will lead to defining the molecular changes occurring during platelet response to stimuli. Recent evidence has implicated that glycoproteins similar to GPIIb and GPIIIa are present in other cells which have a role in cell adhesion. Preliminary studies in this laboratory suggest that neutrophils and neutrophil-like cell lines have GPIIb and GPIIIa- like molecules. The studies proposed are to further characterize these proteins on both human neutrophils and identified cell lines. One such cell line is the HL-60 cell line. Western blots, immunoprecipitation experiments and flow cytometry will be used to identify these proteins. A time course will be done to determine if the expression of these platelet-like glycoproteins on the HL-60 cells is a function of differentiation. Such studies will lead to an examination of the altered expression of GPIIb and GPIIIa-like molecules on neutrophil membrane surface. It is possible that the expression of polypeptide regions on the neutrophil which are responsible for adherence to endothelial cells change upon inflammation much like the conditional receptor function in human platelets. This study should provide information on the relationship of platelet antigens to the function of normal and abnormal leukocytes.

Surface plasmon resonance biosensors have become important tools for measuring the binding affinities and kinetic constants of reversible interactions between molecules. BIACORE, a novel analytical system, uses surface plasmon resonance detection to rapidly determine the affinity and kinetics of ligand-receptor interactions, macromolecular complex formations, DNA protein binding and other interactions between biological macromolecules. A group of NIH-supported investigators at the University of Tennessee, Memphis campus have active research programs in the areas of cell adhesion, drug discovery, signal transduction, DNA protein binding and structure-function analysis. A BIACORE surface plasmon resonance biosensor is needed to study the reversible interactions between biological molecules where increased resolution in kinetic analysis is essential, increased sensitivity is required and for mufti-sample analysis through automation that will facilitate experimentation and save time. We are requesting a BIACORE 3000 to enhance our research activity and productivity and to assist us in accomplishing the goals of our funded NIH grants.