Abstract
Coagulation factor VIII (FVIII) comprises multiple domains that in a concerted manner mediate the role of FVIII as a cofactor for activated factor IX (FIXa) in the coagulation cascade. These domains of FVIII are grouped in a heavy chain (domains A1-A2-B) and a light chain (domains A3-C1-C2). At sites of vascular injury, FVIII is activated and assembles with FIXa on procoagulant membranes comprising phosphatidylserine (PS) in the outer leaflet of the bilayer. Defects therein are associated with the bleeding disorder hemophilia A, which can be treated with intravenous infusion with purified FVIII. Effective treatment, however, is severely hampered by the fact that FVIII is rapidly removed from the circulation. One relevant biological modifier of the FVIII half-life is von Willebrand factor (VWF), which forms a tight complex with FVIII in the circulation. In this complex, FVIII is protected from even faster clearance from plasma. The role of the A domains and the C2 domain in the interaction with phospholipid membranes, FIXa, VWF and the FVIII clearance receptors has been extensively studied. The potential function of the C1 domain in these molecular mechanisms has been hardly investigated and is therefore poorly understood. Yet, anti-C1 domain antibodies have been identified that effectively inhibit multiple FVIII functions.
We investigated the role of the C1 domain in the biological processes that involve FVIII by using single chain variable domain antibody fragment (scFV) KM33 that requires residues 2092-2093 in the C1 domain for binding FVIII. Furthermore, we utilised FVIII YFP constructs in which Lys-2092 and/or Phe-2093 were replaced by alanines.
The results show that this scFV is particularly effective in blocking the interaction of FVIII with phospholipid membranes and with activated platelets. We furthermore report that C1 domain residues 2092 and 2093 contribute to binding phospholipid membranes comprising a low percentage of PS. To assess the role of these residues in the interaction with physiological relevant procoagulant surfaces, we performed functional studies employing the FVIII YFP derivatives and activated platelets. The results show that residues 2092-2093 are also of major importance for the interaction of FVIII with the activated platelets.
Employing LRP-expressing U87MG cells, we furthermore demonstrated that this C1 domain region is also critical for the uptake of FVIII. An intriguing finding is that there is a cell surface binding step for FVIII that is independent of LRP. This binding step is of critical importance in the mechanism of LRP-dependent uptake of FVIII. We show that the C1 domain residues 2092-2093 contribute to both cell surface binding as well as receptor binding and found that the cell binding step of FVIII is not mediated by PS.
Furthermore, we determined the role of VWF in endocytosis of FVIII and investigated the inhibitory mechanism of KM33 and variants thereof on the biological processes where FVIII is involved. The results described in the present thesis contribute to the knowledge of the function of coagulation FVIII.
Original language | English |
---|---|
Qualification | Doctor of Philosophy |
Awarding Institution |
|
Supervisors/Advisors |
|
Award date | 13 Aug 2011 |
Publisher | |
Print ISBNs | 978-90-8570-778-3 |
Publication status | Published - 13 Aug 2011 |