Online

Gertz, Jacqueline Michelle

Biochemistry

2015

PhD

Platelet- and plasma-derived factor Va are absolutely essential for thrombin generation catalyzed by the prothrombinase complex, a 1:1 stoichiometric complex of the serine protease factor Xa and the nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of calcium ions. Two whole blood pools of the procofactor, factor V, exist: approximately 75% circulates in the plasma as a single chain inactive molecule, while the other 25% resides in platelet α-granules in a partially proteolytically-activated state. Our laboratory demonstrated that the platelet-derived cofactor originates following endocytosis of plasma-derived factor V by megakaryocytes, the platelet precursor cells, via a two receptor system including an uncharacterized, specific factor V receptor and low density lipoprotein receptor related protein-1. Following endocytosis factor V is physically and functionally modified and trafficked to the platelet α-granule from where it is released upon platelet activation at sites of vascular injury. The first goal of this dissertation was to define how factor V endocytosis changes over the course of megakaryocyte development. Hematopoietic multipotential stem cells were isolated from human umbilical cord blood and subjected to ex vivo differentiation into megakaryocytes. Megakaryocyte differentiation was assessed by flow cytometry using fluorescently-labeled antibodies against megakaryocyte- and platelet-specific markers and factor V directly conjugated to a fluorophore over 12 days. Differentiation was confirmed by a decrease in a stem cell marker (CD34) and an increase in a mature megakaryocyte marker (CD42) and coincident with factor V endocytosis. Live cell imaging verified differentiation and permitted the observation of proplatelet formation, the precursor to circulating platelets. Analogous experiments verified the trafficking of factor V into proplatelet extensions. Factor V is a highly glycosylated protein: potential roles of these glycans may be endocytosis and trafficking by megakaryocytes. We previously demonstrated that factor V endocytosis is mediated by the light chain region of the procofactor. This region of factor V contains three glycans - one high mannose and two complex N-linked glycans. In the second part of this dissertation, a role for the complex N-linked glycans at Asn1675 and Asn2181 of the factor V light chain in factor V endocytosis by megakaryocytes was assessed. Exoglycosidases were used to selectively trim the complex N-linked glycans on human factor V under native conditions. Treatment with neuraminidase removed 100% of the sialic acid residues on the factor V light chain as demonstrated by gel electrophoresis and mass spectrometry. Treatment with β-1,4-galactosidase removed 69% of the galactose residues at Asn1675 and 100% at Asn2181. Glycosidase-treated factor Va behaves similarly to untreated factor Va in thrombin generation assays suggesting that cofactor activity is unaltered by glycan trimming. In addition, glycan removal had no effect on factor V endocytosis by megakaryocyte-like cells. These observations suggest that complex N-linked glycans on the factor V light chain are not important for factor Va cofactor activity or factor V endocytosis by megakaryocyte-like cells, which strongly suggests that they have a role in trafficking.