Meems Henriët, Meijer Alexander B, Cullinan David B, Mertens Koen, Gilbert Gary E
Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands.
Blood. 2009 Oct 29;114(18):3938-46. doi: 10.1182/blood-2009-01-197707. Epub 2009 Aug 17.
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 --> Ala/Ala (fVIII(YFP 2092/93)) and 2 single mutants Lys2092 --> Ala and Phe2093 --> 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.
凝血因子VIII与含有磷脂酰-L-丝氨酸（Ptd-L-Ser）的膜的结合，部分是由定位于C2结构域的一个基序介导的。我们使用抗C1抗体片段KM33（单链抗体片段）和具有改变的KM33表位的凝血因子VIII突变体，评估了C1结构域假定的膜结合作用。我们制备了双突变体Lys2092/Phe2093→Ala/Ala（fVIII(YFP 2092/93)）以及两个单突变体Lys2092→Ala和Phe2093→Ala。KM33（单链抗体片段）抑制了荧光素标记的凝血因子VIII与合成膜的结合，并抑制了至少95%的因子X酶活性。fVIII(YFP 2092/93)对含有15% Ptd-L-Ser的膜的亲和力低3倍，但对含有4% Ptd-L-Ser的膜的亲和力降低超过10倍。在微量滴定板中，KM33（单链抗体片段）与抗C2抗体在阻断与15% Ptd-L-Ser囊泡的结合方面具有相加作用，而任何一种抗体均可阻断与4% Ptd-L-Ser囊泡的结合。KM33（单链抗体片段）抑制了与血小板的结合，而fVIII(YFP 2092/93)与A23187刺激的血小板的结合减少。fVIII(YFP 2092)在各种Ptd-L-Ser浓度下均表现出正常活性，而fVIII(YFP 2093)在Ptd-L-Ser低于12%时活性降低。fVIII(YFP 2092/93)的活性降低幅度比任何一个单突变体都更大。这些结果表明，Lys 2092和Phe 2093是凝血因子VIII C1结构域上膜结合基序的组成部分。
