Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and localization of factor V activation fragments.
Clotting factor V and factor VIII are each represented by the domain structure A1-A2-B-A3-C1-C2 and share 40% sequence homology in the A and C domains. Rotary-shadowed samples of human factor V and factor VIII were examined in the electron microscope. Single-chain factor V molecules exhibited a globular "head" domain 12-14 nm in diameter. In addition, up to 25% of these molecules showed a rod-like "tail" of up to 50 nm. Glycerol-gradient centrifugation of factor V treated with thrombin partially resolved the factor Va heterodimer from a larger activation peptide of 150 kDa, as determined by gel electrophoresis. Electron microscopy of factor Va revealed globular molecules with several smaller appendicular structures but lacking the tails seen in factor V. Images of the 150-kDa activation peptide showed rod-like structures, similar in width to the tail of intact factor V and approximately 34 nm long. Rotary shadowing was also used to visualize factor VIII that had been fractionated into heterodimers containing heavy chains of distinct sizes. Each factor VIII preparation showed a globular structure approximately 14 nm in diameter, but the associated tails were observed much more frequently with factor VIII heterodimers containing the higher-molecular-weight heavy chains. These results, in conjunction with results of studies using other biophysical techniques, suggest a model in which the A and C domains of each cofactor constitute a globular head and the connecting B domain is contained in a two-stranded tail that is released by thrombin cleavage.
Fowler
W E
WE
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
Fay
P J
PJ
Arvan
D S
DS
Marder
V J
VJ
D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure*; D005169:Factor VIII / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure*; D015943:Factor Va / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008854:Microscopy, Electron; D008961:Models, Structural; D011487:Protein Conformation; D013917:Thrombin
D015943:Factor Va; D005165:Factor V; D005169:Factor VIII; D013917:Thrombin