UVM Theses and Dissertations
Format:
Print
Author:
Wood, Jeremy Paul
Title:
Dept./Program:
Biochemistry
Year:
2010
Degree:
PhD
Abstract:
Thrombin is generated from its precursor protein, prothrombin, through the action of prothrombinase, an enzymatic complex composed of the serine protease factor Xa and its non-enzymatic cofactor factor Va (consisting of a heavy and light chain) assembled in a Ca⁺-dependent manner on the surface of activated platelets. In addition to providing the membrane for prothrombinase assembly, platelets also contain and release about 20-25% of the total factor VNa found in blood, while the remaining 75-80% circulates within the plasma. As such, this dissertation focuses on both the unique features of the factor VNa released· from activated platelets, and the pathway through which prothrombin activation occurs on the activated platelet surface.
Studies of factor VIVa, isolated from pooled plasma or platelets, have demonstrated that the platelet-derived factor VNa pool is both physically and functionally distinct from its plasma-derived counterpart. Techniques have now been developed to isolate sufficient quantities of both plasma-and platelet-derived factor Va from a single donor, in order to determine which structural differences are consistently present between the two pools. Using this purification procedure, and subsequent mass spectrometric analyses, a novel post-translational modification was identified which serves to further differentiate the two cofactor pools: Tyr²¹⁹⁶ was determined to be phosphorylated in plasma-derived, but not platelet-derived, factor Va.
In addition to the soluble pool of factor VN a that is released by platelets and binds to the platelet surface, there also exists a non-dissociable, membrane-bound form of the cofactor. This factor Va accounts for approximately 35% of the total factor Va activity expressed on the surface of activated platelets, and analyses now reveal that it is attached to the platelet membrane through a GPI anchor modification on the C-terminus of the heavy chain. As GPI anchors are prototypically added to proteins immediately following translation, this represents a novel mechanism by which factor V is endocytosed by megakaryocytes, proteolytically-processed to free the heavy chain, trafficked to the endoplasmic reticulum, and modified by addition of a GPI anchor.
Not only is the structure of platelet-derived factor Va unique, but the activity of prothrombinase assembled on the platelet surface is unique as well. The activation of prothrombin has been studied extensively using model systems containing phospholipid vesicles of defined content. In these systems, activation occurs through a sequential, ordered cleavage, generating the active intermediate meizothrombin before ultimately producing thrombin. However, on the physiological surface of activated platelets, the order of cleavage is reversed, and the inactive intermediate prethrombin-2 is produced rather than meizothrombin. The prethrombin-2 is then rapidly cleaved to produce thrombin. As meizothrombin has significant anticoagulant activity, and prethrombin-2 does not, we hypothesize that the pathway employed on the platelet surface optimizes the expression of procoagulant activity when thrombin is produced at sites of vascular injury.
Studies of factor VIVa, isolated from pooled plasma or platelets, have demonstrated that the platelet-derived factor VNa pool is both physically and functionally distinct from its plasma-derived counterpart. Techniques have now been developed to isolate sufficient quantities of both plasma-and platelet-derived factor Va from a single donor, in order to determine which structural differences are consistently present between the two pools. Using this purification procedure, and subsequent mass spectrometric analyses, a novel post-translational modification was identified which serves to further differentiate the two cofactor pools: Tyr²¹⁹⁶ was determined to be phosphorylated in plasma-derived, but not platelet-derived, factor Va.
In addition to the soluble pool of factor VN a that is released by platelets and binds to the platelet surface, there also exists a non-dissociable, membrane-bound form of the cofactor. This factor Va accounts for approximately 35% of the total factor Va activity expressed on the surface of activated platelets, and analyses now reveal that it is attached to the platelet membrane through a GPI anchor modification on the C-terminus of the heavy chain. As GPI anchors are prototypically added to proteins immediately following translation, this represents a novel mechanism by which factor V is endocytosed by megakaryocytes, proteolytically-processed to free the heavy chain, trafficked to the endoplasmic reticulum, and modified by addition of a GPI anchor.
Not only is the structure of platelet-derived factor Va unique, but the activity of prothrombinase assembled on the platelet surface is unique as well. The activation of prothrombin has been studied extensively using model systems containing phospholipid vesicles of defined content. In these systems, activation occurs through a sequential, ordered cleavage, generating the active intermediate meizothrombin before ultimately producing thrombin. However, on the physiological surface of activated platelets, the order of cleavage is reversed, and the inactive intermediate prethrombin-2 is produced rather than meizothrombin. The prethrombin-2 is then rapidly cleaved to produce thrombin. As meizothrombin has significant anticoagulant activity, and prethrombin-2 does not, we hypothesize that the pathway employed on the platelet surface optimizes the expression of procoagulant activity when thrombin is produced at sites of vascular injury.