Structure and function of coagulation factors

The coagulation cascade comprises several serine proteases that act in combination with a non-enzymatic cofactor on phospholipid-containing membranes. During the past decade we have been focusing on the mechanism by which activated factor IX assembles with its cofactor factor VIII. These proteins are indispensable for proper functioning of the coagulation cascade as functional absence of factor VIII (FVIII) and factor IX is associated with the bleeding disorder hemophilia A and hemophilia B. FVIII is composed of a series of repeated domains which appear in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A-domains of FVIII mediate the binding to activated factor IX and factor X whereas the C2 domain has been implicated in binding to phospholipids.

We recently identified a human antibody (designated KM33) that inhibits FVIII cofactor activity. We further established that this antibody blocked endocytosis of FVIII by various cellular systems which may contribute to the regulation of the FVIII plasma level. Binding studies revealed that KM33 is directed towards the C1-domain of FVIII and that KM33 blocked the binding of FVIII to phospholipid membranes suggesting involvement of C1 domain in this process. We next established that FVIII-YFP variants in which the residues Lys2092 and Phe2093 at the tip of the C1 domain are replaced by alanines exhibit a reduced binding to KM33. FVIII-YFP-2092A/2093A also did not interact with phospholipid membranes containing a low percentage of phosphatidyl-L-serine. The binding of this variant was, however, indistinguishable from the corresponding wild type FVIII when membranes containing higher concentrations of phosphatidyl-L-serine were used. In agreement with these findings phospholipid membranes containing relatively high concentrations of phosphatidyl-L-serine were needed to develop full cofactor activity of FVIII-YFP-2092A/2093A. Together these findings provide evidence for the involvement of Lys2092/Phe2093 in binding to phosphatidyl- L-serine containing phospholipids.

We next investigated the role of region 2992/2093 on the endocytic uptake of FVIII by cells that express FVIII’s catabolic receptor LDL receptor-related protein (LRP). Confocal microscopy studies and flow cytometry studies showed that there was a marked defect in the uptake of FVIII-YFP-2092A/2093A relative to FVIII-YFP. These studies further demonstrated that there is a cell surface binding event that precedes the LRP dependent uptake of FVIII. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. This finding implies that the amino acid residues of region 2092-2093 comprise a binding site for the cell surface. Surface plasmon resonance analysis showed that these substitutions affect the direct binding of FVIII to LRP as well. This dual role of region 2092-2093 for both cell surface and LRP binding explained the major reduction of endocytic uptake of the FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092-2093 are of major importance for FVIII function. This region not only contributes to cofactor function but is also involved in the cellular uptake of FVIII.

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