UVM Theses and Dissertations
Format:
Print
Author:
Haynes, Laura M.
Dept./Program:
Biochemistry
Year:
2012
Degree:
PhD
Abstract:
The activation of the zymogen prothrombin to the serine protease [alpha]-thrombin is a critical step in the coagulation process as [alpha]-thrombin is at the crossroads of both the pro-and anti-coagulant pathways. The major activator of prothrombin is the prothrombinase complex, which consists of the serine protease factor Xa and its protein cofactor assembled on an appropriate phospholipid membrane in the presence of Ca². Although the activation of prothrombin by the prothrombinase complex has been studied for over forty years, much of our understanding of its function comes from closed system models that do not take into account the dynamics of flow within the vasculature. This dissertation presents the development of a model system for studying the activation of prothrombin by preassembled prothrombinase under physiologically relevant shear rates.
When prothrombin, at its mean plasma concentration, is activated by prothrombinase assembled on a supported synthetic phospholipid bilayer under flow the absolute levels thrombin generated were mediated by flow induced dilution effects-and no diffusion-mediated regulation due to substrate depletion near the catalytically active walls of the flow chamber was observed. This is in contrast to an analogous experiment in which the zymogen factor X at its physiologic plasma concentration is activated to factor Xa by the extrinsic tenase complex assembled on a supported phospholipid bilayer under flow that exhibits diffusion-mediated control in which significant substrate depletion occurs in the catalytically-active wall region. Furthermore, when there is competition for membrane binding sites between the zymogens prothrombin and factor X, as well as the protease factor Xa, the dilution-and diffusion-mediated effects observed when prothrombin and factor X, respectively, are activated under flow become diminished.
The activation of prothrombin under flow is also regulated by feedback proteolysis of prothrombin by active thrombin species, [alpha]-thrombin and the catalytically active intermediate meizothrombin, resulting in the loss of prothrombin's phospholipid binding domain and decreased zymogen activation. This effect is observed with prothrombinase assembled on both synthetic phospholipids and activated platelets-the primary site of assembly in vivo. This feedback cleavage may be a mechanism to prevent the thrombus spread downstream from the site of injury. However, the mechanism of prothrombin activation by prothrombinase assembled on a synthetic phospholipid bilayer and activated platelets are different. Prothrombinase assembled on synthetic phospholipids activates prothrombin via an inefficient reaction in which the two required cleavages may occur at distinct prothrombinase complexes. The mechanism of platelet prothrombinase, however, appears to be an efficient processive mechanism.
Finally, the inhibition of the prothrombinase complex with the novel anticoagulant rivaroxaban, a direct factor Xa inhibitor, was examined at a typical venous shear rate (100 sec⁻¹). It was determined that under a typical dosing regime of rivaroxaban there is dose-dependent inhibition of prothrombinase-associated factor Xa. Upon cessation of rivaroxaban delivery to inhibited prothrombinase a rapid dissociation is observed, as it washed downstream-potentially explaining the severe thrombotic events reported in patients who have discontinued rivaroxaban therapy.
When prothrombin, at its mean plasma concentration, is activated by prothrombinase assembled on a supported synthetic phospholipid bilayer under flow the absolute levels thrombin generated were mediated by flow induced dilution effects-and no diffusion-mediated regulation due to substrate depletion near the catalytically active walls of the flow chamber was observed. This is in contrast to an analogous experiment in which the zymogen factor X at its physiologic plasma concentration is activated to factor Xa by the extrinsic tenase complex assembled on a supported phospholipid bilayer under flow that exhibits diffusion-mediated control in which significant substrate depletion occurs in the catalytically-active wall region. Furthermore, when there is competition for membrane binding sites between the zymogens prothrombin and factor X, as well as the protease factor Xa, the dilution-and diffusion-mediated effects observed when prothrombin and factor X, respectively, are activated under flow become diminished.
The activation of prothrombin under flow is also regulated by feedback proteolysis of prothrombin by active thrombin species, [alpha]-thrombin and the catalytically active intermediate meizothrombin, resulting in the loss of prothrombin's phospholipid binding domain and decreased zymogen activation. This effect is observed with prothrombinase assembled on both synthetic phospholipids and activated platelets-the primary site of assembly in vivo. This feedback cleavage may be a mechanism to prevent the thrombus spread downstream from the site of injury. However, the mechanism of prothrombin activation by prothrombinase assembled on a synthetic phospholipid bilayer and activated platelets are different. Prothrombinase assembled on synthetic phospholipids activates prothrombin via an inefficient reaction in which the two required cleavages may occur at distinct prothrombinase complexes. The mechanism of platelet prothrombinase, however, appears to be an efficient processive mechanism.
Finally, the inhibition of the prothrombinase complex with the novel anticoagulant rivaroxaban, a direct factor Xa inhibitor, was examined at a typical venous shear rate (100 sec⁻¹). It was determined that under a typical dosing regime of rivaroxaban there is dose-dependent inhibition of prothrombinase-associated factor Xa. Upon cessation of rivaroxaban delivery to inhibited prothrombinase a rapid dissociation is observed, as it washed downstream-potentially explaining the severe thrombotic events reported in patients who have discontinued rivaroxaban therapy.