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Updated: Jul 31, 2026
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and
W E Fowler1, P J Fay, D S Arvan
1Department of Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
Insights
Clotting factors V and VIII share structural similarities. Electron microscopy reveals their globular heads and distinct tails, with thrombin cleavage releasing a rod-like activation peptide from factor V.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Clotting factors V and VIII are essential for hemostasis.
- Both factors share a conserved domain structure (A1-A2-B-A3-C1-C2) and sequence homology in A and C domains.
Purpose of the Study:
- To investigate the three-dimensional structure of human factors V and VIII.
- To elucidate the structural changes upon activation of factor V by thrombin.
Main Methods:
- Rotary shadowing electron microscopy was used to visualize purified human factors V and VIII.
- Glycerol-gradient centrifugation and gel electrophoresis were employed to analyze thrombin-treated factor V.
Main Results:
- Single-chain factor V exhibited a globular head (12-14 nm) and a rod-like tail (up to 50 nm).
- Thrombin cleavage of factor V yielded factor Va and a 150 kDa activation peptide; factor Va lacked the prominent tail.
- Factor VIII preparations showed globular heads, with tails observed more frequently in heterodimers containing higher-molecular-weight heavy chains.
Conclusions:
- A structural model is proposed where A and C domains form the globular head, and the B domain forms a two-stranded tail.
- Thrombin cleavage releases the B domain as an activation peptide, altering the overall molecular structure.
Abstract:
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.
