UVM Theses and Dissertations
Format:
Print
Author:
Whelihan, Matthew F.
Dept./Program:
Biochemistry
Year:
2009
Degree:
MS
Abstract:
During tissue factor-initiated blood coagulation, the essential procofactors factor V (FV) and factor VIII (FVIII) are activated by thrombin. Exposure of circulating factor VIla to tissue factor on the surface of extravascular cells gives rise to the extrinsic Xase. The extrinsic Xase produces sufficient factor Xa to support thrombin generation; adequate for activation of the procofactors, which leads to the amplification of the coagulation response. In the contact pathway, the sources of the FVIII and FV procofactor activation are unknown. In this pathway, factor XII is activated on a negatively charged surface and proceeds to activate factor XI (FXI), which in-turn activates factor IX (FIX).
FIXa feeds into the main stream of thrombin generation through activation ofFX. FIXa is very poor catalyst in the absence of its FVIIIa cofactor which increases FXa generation by 109. It is possible that an other enzyme upstream from FIXa, is capable of activating the FVIII and FV procofactors. We tested the hypothesis that FXla can activate FVIII and FV. Plasma derived FV and recombinant albumin-free FVIII were subjected to FXla proteolysis. During the reaction the relative activity and integrity of each procofactor were measured at selected time points using a clotting assay and SDS-PAGE.
FXla rapidly activates FVIII to a level of 14% compared to thrombin derived FVIIla. The addition of high molecular weight kininogen (HMWK) and zinc doubled both the rate and level ofthis activation. NHz-terminal sequencing reveals two heavy chain cleavages comparable to those seen with thrombin at R748 and R372 as well as three sequential proteolyses ofthe A2 COOH-terminus. Two light chain cleavages at R1652 and R1721 were also observed. FXI aactivated FV at a significantly slower rate and the addition of HMWK and zinc appeared to drive an inactivating proteolysis. NHz-terminal sequencing revealed a more complicated series ofproteolytic events than what was seen with FVIII. FXla cleaves the thrombin sites at R1545 and R709. Proteolysis at R510 in the heavy chain is preferred, followed by a cleavage COOH-terminal to R306 that has yet to be identified. A single cleavage at R1765 is observed in the light chain.
The activation of FVIII by FXla occurs rapidly and is most likely the more important of the two pathways. FXla's activation ofFV does not appear to be paramount in this context and FXla's ability to inactivate FVa is probably a more important role. This study suggests that FXla plays a role in early procofactor activation; primarily of FVIII that leads to the observed acceleration in thrombin generation.
FIXa feeds into the main stream of thrombin generation through activation ofFX. FIXa is very poor catalyst in the absence of its FVIIIa cofactor which increases FXa generation by 109. It is possible that an other enzyme upstream from FIXa, is capable of activating the FVIII and FV procofactors. We tested the hypothesis that FXla can activate FVIII and FV. Plasma derived FV and recombinant albumin-free FVIII were subjected to FXla proteolysis. During the reaction the relative activity and integrity of each procofactor were measured at selected time points using a clotting assay and SDS-PAGE.
FXla rapidly activates FVIII to a level of 14% compared to thrombin derived FVIIla. The addition of high molecular weight kininogen (HMWK) and zinc doubled both the rate and level ofthis activation. NHz-terminal sequencing reveals two heavy chain cleavages comparable to those seen with thrombin at R748 and R372 as well as three sequential proteolyses ofthe A2 COOH-terminus. Two light chain cleavages at R1652 and R1721 were also observed. FXI aactivated FV at a significantly slower rate and the addition of HMWK and zinc appeared to drive an inactivating proteolysis. NHz-terminal sequencing revealed a more complicated series ofproteolytic events than what was seen with FVIII. FXla cleaves the thrombin sites at R1545 and R709. Proteolysis at R510 in the heavy chain is preferred, followed by a cleavage COOH-terminal to R306 that has yet to be identified. A single cleavage at R1765 is observed in the light chain.
The activation of FVIII by FXla occurs rapidly and is most likely the more important of the two pathways. FXla's activation ofFV does not appear to be paramount in this context and FXla's ability to inactivate FVa is probably a more important role. This study suggests that FXla plays a role in early procofactor activation; primarily of FVIII that leads to the observed acceleration in thrombin generation.