Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity

Abstract

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33 scFv , and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 3 Ala/ Ala (fVIII YFP 2092/93) and 2 single mutants Lys2092 3 Ala and Phe2093 3 Ala. KM33 scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII YFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33 scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33 scFv inhibited binding to platelets and fVIII YFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIII YFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII YFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII YFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain. (Blood. 2009;114:3938-3946) Introduction Factor VIII functions as a cofactor in the membrane-bound intrinsic factor Xase complex. Together with the enzyme factor IXa, activated factor VIII binds to phosphatidyl-L-serine (Ptd-L-Ser)-containing membranes 1,2 to form an enzyme complex that cleaves the zymogen factor X to factor Xa. 3,4 Factor Xa is thereafter responsible for catalyzing prothrombin cleavage to thrombin. 5 The importance of the factor Xase complex is illustrated by the disease hemophilia, in which a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Despite the central importance of membrane binding, this aspect of factor VIII function remains poorly understood. Factor VIII is synthesized as a single polypeptide chain containing 2351 amino acids (molecular weight, 280 kDa) and shows a domain structure of A1-a1-A2-a2-B-a3-A3-C1-C2, where a1, a2, and a3 are spacer regions that separate the domains from each other. 6 Factor VIII is homologous to factor V in amino acid sequence and domain structure. 7 The A domains are homologous with ceruloplasmin, the C domains with discoidin I, and with lactadherin, 8,9 and the B domain is unique to each protein. 10 The A domains mediate the dominant interactions with factor IXa and factor X in the factor Xase complex, whereas binding to Ptd-L-Ser-containing membranes is mediated predominantly by the C2 domain. 11-15 The structure-function relationships of factor V resemble those of factor VIII in that the A domains mediate the dominant interactions with the enzyme and substrate and the C2 domain mediates the dominant membrane-binding inter...

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

“…Within each of the LRP clusters II and IV, approximately four adjacent CRs form a binding site for FVIII (31,33), whereas the matching epitope of FVIII involves multiple lysines on all three domains of its light chain (65)(66)(67)(68). Consistent with this, the anti-FVIII ScFv KM33, which recognizes the C1-domain of the light chain, interferes with FVIII binding to both LDLR and LRP (46,48,69). Our present study shows that similarly to LRP, four adjacent CRs of LDLR form a site for FVIII, and on the FVIII side, the contact interface involves the C1-domain.…”

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…The activated factor X substrate S-2765 containing the thrombin inhibitor I-2581 was from Chromogenix (Milan, Italy). All other chemicals were from Merck (Darmstadt, Germany).Proteins-Monoclonal antibodies CLB-EL14 (EL14), CLB-KM33 (KM33), CLB-CAg12, CLB-CAg9, and CLB-CAg117 have been described before (14,21, 22). ESH4 was obtained from American Diagnostica (Greenwich, CT).…”

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

“…For the FVIIIa stability analysis, the FVIII variants were first incubated at varying time intervals with 2 nM thrombin and 1.5 mM CaCl 2 in the presence and absence of increasing concentrations of FIXa (0 -16 nM) at 25°C in 40 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.2% (w/v) BSA, and 25 M phospholipid vesicles. The generated amount of FXa was assessed as described (30). Residual FVIII activity was derived from the initial rate of FXa formation as a function of the FIXa concentration.…”

“…The reaction was started with the addition of 1.5 mM CaCl 2 and 1 nM thrombin. The amount of FXa generated per minute was subsequently assessed as described (30). For the FVIIIa stability analysis, the FVIII variants were first incubated at varying time intervals with 2 nM thrombin and 1.5 mM CaCl 2 in the presence and absence of increasing concentrations of FIXa (0 -16 nM) at 25°C in 40 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.2% (w/v) BSA, and 25 M phospholipid vesicles.…”

“…FXa-generating Assay-Factor Xase activity measurement and the preparation of phospholipid vesicles were performed as described before (30). Briefly, 25 M sonicated phospholipid vesicles comprising 15% phosphatidylserine, 20% phosphatidylethanolamine, and 65% phosphatidylcholine were mixed with 0.3 nM FVIII, 200 nM FX, and 0 -16 nM FIXa in a buffer containing 40 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.2% (w/v) bovine serum albumin (BSA) (Merck).…”