Project Description:
Platelet- and plasma-derived factor Va serve essential roles in thrombin generation catalyzed by the prothrombinase complex assembled on activated platelets. Several observations indicate that the platelet-derived cofactor is more important in maintaining normal hemostasis, and is physically and structurally distinct than its plasma counterpart. Despite these differences, it has been demonstrated unequivocally that the entire platelet-derived factor V pool originates via a specific, receptor-mediated, clathrin-dependent endocytosis of plasma-derived factor V by megakaryocytes, platelet precursor cells.

Recent studies by our laboratory have demonstrated that factor V endocytosis by megakaryocytes is mediated by a two receptor system. In this model, factor V initially binds to a specific factor V receptor expressed only on megakaryocytes able to endocytose factor V. This binding event facilitates an interaction between a second factor V molecule and LDL receptor-related protein-1 (LRP-1), an endocytic receptor, which subsequently mediates the endocytosis of its bound factor V molecule. These combined observations represent a unique role for LRP-1 in endocytosis of a coagulation protein not destined for lysosomal degradation.

The overall goal of our laboratory is to identify and characterize the proteins that form the megakaryocyte two receptor system involved in the binding and endocytosis of plasma-derived factor V.

Some specific projects include: (1) Identification of the specific, factor V receptor expressed on human megakaryocytes using a proteomics approach; (2) Determination of the fates of the factor V receptor and LRP-1 subsequent to factor V endocytosis, and during megakaryocyte development and platelet formation by immunofluorescence; (3) Identification of the minimum factor V amino acid sequence(s) involved in its interactions with the factor V receptor, as well as LRP-1 expressed on megakaryocytes; and (4) Definition of the intracellular signaling events that regulate factor V binding and endocytosis by megakaryocytes by determining the effects of kinase inhibitors and Ser/Thr phosphatases on factor V endocytosis, and binding to LRP-1 expressed on megakaryocytes.

Bouchard, B.A., Meisler, N.T., Nesheim, M.E., Liu, C.X., Strickland, D.K., and Tracy, P.B. A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes. J Thromb Haemosta 6(4):638-44, 2008.

Bouchard, B.A., Butenas, S., Mann K.G. and Tracy, P.B. Interactions between platelets and the coagulation system. In: Platelets, Michelson AD (2nd Ed), Academic Press, San Diego, CA, pp. 377-402, 2007.

Bouchard, B.A., Williams, J.L., Meisler, N.T., Long, M.W. and Tracy, P.B. Endocytosis of plasma factor V by megakaryocytes occurs via a clathrin-dependent, specific receptor-mediated event. J Thromb Haemosta 3:541-551, 2005

Damron, D.P., Bouchard, B.A., Shapiro, R.E., Schonberg, A.L. and Bernstein, I.M. Platelet activation, sympathetic tone, and plasma volume in nulligravid women of reproductive age. Obstet Gynecol 103:931-936, 2004