title: Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.
abstract: Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the same core features as fVIII molecules. Molecular species corresponding to heterodimers (mass, 128 +/- 13 kD) and unassociated subunit chains (40-100 kD) were also observed in fVIIIa preparations, suggesting that heterotrimers have an appreciable tendency to dissociate, a phenomenon that could explain the decay of fVIIIa activity after thrombin activation of fVIII.
authors.affiliation: University of Wisconsin Medical School, Milwaukee 53233.
authors.forename: M W
authors.initials: MW
authors.lastname: Mosesson
authors.forename: D N
authors.initials: DN
authors.lastname: Fass
authors.forename: P
authors.initials: P
authors.lastname: Lollar
authors.forename: J P
authors.initials: JP
authors.lastname: DiOrio
authors.forename: C G
authors.initials: CG
authors.lastname: Parker
authors.forename: G J
authors.initials: GJ
authors.lastname: Knutson
authors.forename: J F
authors.initials: JF
authors.lastname: Hainfeld
authors.forename: J S
authors.initials: JS
authors.lastname: Wall
mesh_terms: D000818:Animals; D005169:Factor VIII / Q000648:ultrastructure*; D015944:Factor VIIIa / Q000648:ultrastructure*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D011487:Protein Conformation; D013552:Swine
chemical_list: D046911:Macromolecular Substances; D015944:Factor VIIIa; D005169:Factor VIII