Mechanisms regulating factor V endocytosis by megakaryocytes
The blood coagulation protein, factor Va, is a critical cofactor in blood clot formation via the formation of thrombin by the prothrombinase complex assembled on the surface of activated platelets adhered at sites of vascular injury. The procofactor, factor V, exists in two whole blood pools: 75-80% circulates in plasma (liquid portion of blood) while the remainder is stored in alpha-granules of blood cells called platelets from where it is released upon blood vessel injury and platelet activation. While both plasma and platelet factor Va function efficiently in blood clotting, the platelet molecule forms the more physiologically relevant cofactor pool in the normal blood clotting response as its local concentration at sites of blood vessel injury is >100-fold higher than plasma factor The entire platelet-derived factor Va pool originates via endocytosis of the procofactor, factor V, from plasma by megakaryocytes, the platelet precursor cells. Factor V endocytosis by megakaryocytes is specific, clathrin-dependent, and appears to be 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 bound factor V. These combined observations represent a unique role for LRP-1 in endocytosis of a coagulation protein not destined for lysosomal degradation. Rather, subsequent to its endocytosis, factor V is functionally modified, trafficked to, and stored in alpha-granules.
The overall goal of these studies is to further define how plasma-derived factor V is acquired by megakaryocytes using megakaryocyte derived ex vivo from human bone marrow or umbilical cord blood, and how its intracellular transport and modifications yield a physically and functionally distinct platelet protein. Delineation of the cellular processes regulating platelet acquisition of this unique factor V/Va pool is essential for understanding how fibrin formation is regulated at the activated platelet surface.
Assessment of procoagulant enzyme complex assembly and function by flow cytometry
Our laboratory is also involved in large studies of various patient populations. These studies, being performed in collaboration with Dr. Kathleen Brummel-Ziedins, use a flow cytometry-based assay of prothrombinase complex assembly on activated platelets in whole blood developed by our laboratory, which assesses prothrombinase complex assembly, and can be used to predict an individual’s propensity to bleed or clot.