Structural Model of Porcine Factor Vil and Factor Villa 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 (Al-A2) to 166,000 (A,-A2-B). Proteolytic activation of fVIII by thrombin results in fVIlla heterotrimers lacking B domains (Al, A2, A3-Cl-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 Al-A2-B heavy chain (fVIII 166/76) or an Al-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STE;M) 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 Al-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 fingerlike 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 cshaped 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. MolecuThis work was presented in part at the 12t