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Project Description:
The principle objective of our laboratory is to develop a fundamental understanding of how platelets participate in and regulate the formation of the important bioregulatory effector molecule, thrombin. Thrombin generation is effected by a Ca2+-dependent, membrane-bound complex of the cofactor protein, factor Va, and the serine protease, factor Xa. One specific goal is to provide a quantitative understanding of the integrally related kinetic and binding events regulating the functional interactions of factors Va and Xa with the platelet membrane surface. The membrane receptors, the intracellular signalling pathways and the enzymatic processes controlling these events are being identified using biochemical and molecular biological approaches. A second major goal is to define how megakaryocytes, platelet progenitor cells, developmentally regulate the endocytosis and possible synthesis of the required cofactor factor V(a) and, to determine the cellular events regulating its endocytosis, its intracellular trafficking to storage granules and the phenotypic changes in the factor V molecule resulting from these interactions. Since the platelet-derived factor Va pool is essential for normal blood clotting, defining how platelets acquire this essential protein, process it and express it at their membrane surface is key in regulating thrombin generation. The formation of thrombin at the surface of platelets is pivotal to the physiological and pathophysiological functions they provide as they localize to vascular and extravascular tissue sites.
Student Opportunities:
Laboratory members become proficient in protein purification and several techniques used for protein characterization (SDS-PAGE, Western blotting, fluorography, autoradiography, amino acid analyses, HPLC, FPLC and MALDI-TOF mass spectrometry). Proficiency in the performance and analysis of chromogenic and fluoroscopic methods for assessing enzyme kinetic reactions can also be learned. Sterile technique is practiced as several cell lines are maintained in continuous culture. Platelets are isolated from whole blood donations on a daily basis. Platelet progenitor cells, megakaryocytes, are isolated from bone marrow aspirates as that primary mature cell population, or can be expanded and differentiated ex vivo from CD34+ bone marrow precursors. Other methods can include kinase assays, fluorescence-activated flow cytometric analyses, fluorescence-activated cell sorting, fluorescence microscopy and confocal microscopy.
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