This study examined how reduced levels of Factor V (FV) in plasma affect clot structure in a mouse model. Mice with genetically altered levels of 15%, 50%, or 100% normal FV were used. Lower FV levels resulted in less occlusive clots after injury to the carotid artery. With 15% FV, arteries showed minimal clotting and were full of red blood cells. Reduced FV decreases fibrin and fibrinogen production and the amount of clot formed after injury.

1. The Effect of Reduced Plasma Factor V on Clot Structure
Michael Gómez-Meléndez 1 , Kellie R. Machlus 2, Jessica Cardenas 2 , Hongmin Sun 3, and Alisa S. Wolberg 2
1
Department of Chemistry, University of Puerto Rico , Cayey, PR , 2 Department of Pathology and Laboratory Medicine,
University of North Carolina, Chapel Hill, NC , 3 Department of Internal Medicine, University of Missouri, Columbia, MS
ABSTRACT MATERIALS AND METHODS CONCLUSION
PTAH
Background: In the coagulation cascade, Factor V (FV) acts as a . When the FV levels in the plasma are below 100%, clot formation
cofactor to factor X, which together form the prothrombinase Clot Fixation in Paraffin Staining
Sectioning Hematoxylin and
complex and convert prothrombin to thrombin.Thrombin then cleaves Induction Paraformaldehyde Embedding after ferric chloride injury of a mouse carotid artery is reduced.
Eosin Staining
fibrinogen, forming the fibrin necessary to create a stable clot.
Deficiencies in Factor V result in a hemorrhagic phenotype.1
FUTURE DIRECTIONS
Objectives: We examined how deficiencies in FV affect clot
structure in a mouse model of thrombosis.
Methods: Clot formation was induced by administration of a ferric RESULTS •Purpose
chloride patch for two minutes to the carotid in the artery of mice
genetically altered to have 15, 50 or 100% of normal FV plasma •Specifically identify fibrin (not fibrinogen) in mouse clots
levels. Vessel sections containing the clots were then fixed in and compare the amount of fibrin produced in plasma with
paraformaldehyde, dehydrated and embedded in paraffin for
different FV levels .
sectioning. Individual sections were stained with both Hematoxylin
= H & E Stained
and Eosin (H&E) and Phosphotungstic Acid Hematoxylin (PTAH) for •Protocol
= PTAH Stained
histological analysis. • For immunohistochemistry Antibody 59D8 will be used.
Results: In wild-type mice (100% FV), the presence of an occlusive
clot following ferric chloride injury was common. When the level of Day 1 Day 2
plasma FV was reduced to 50%,however, the clot was only partially
occlusive. Additionally, when plasma levels of FV were 15%, arteries Factor V [100%] • Xylene • Rinse in PBS
• Methanol • Incubate in DAKO
were full of red cells and had only minimal clot formation. Envision-HRP for mice
• Hidratation in dilutions of DAKO
Conclusions: Lower concentrations of FV in the plasma of mice H&E Deparaffinization
Ethanol Envision-HRP • Rinse in PBS
decreased the production of fibrin and fibrinogen and ultimately, the
amount of clot formed after ferric chloride injury. • Incubate slides in Vector
• Boil Sections in Citrate Novared.
A B C D E Buffer Vector
• Rinse in MilliQ
INTRODUCTION Antigen • Cool on ice Novared • Staing with Hematoxyline
Retrieval
Approximately one in one million people have a problem generating PTAH
FV.1 The first disease related to FV was reported by the Norwegian
hematologist Paul Owren, who proposed the existence of this factor • Dilute 1:1000 • Rinse in water.
• Incubate overnight with • Dehydrate.
and a new model of the coagulation process.1 Generally, deficiencies 59D8 antibody and PBS/BSA Mounting • Mount with Pertex
in FV cause abnormal bleeding, but some cases without bleeding 1 2 3 4 5
Monoclonal
complications have been reported.2 The reduced levels of FV in
humans can be congenital or acquired.3 The principal areas of Factor V [50%]
bleeding are mucosa, skin, joints, muscles, genitourinary tract,
central nervous system and the gastrointestinal tract. 4
H&E
ACKNOWLEDGEMENTS
• This study was supported by grants from: NIH K01 AR051021 (ASW),
University of North Carolina Institute on Aging (ASW), The Gustavus
HYPOTHESIS A B C D E
and Louise Pfeiffer Research Foundation (ASW), and The National
PTAH
Hemophilia Foundation (ASW).
• Thanks to Vanessa González, Pat Phelps, Paul Medina and Eileen
Hayter for all the support.
1 2 3 4 5 • Thanks to RISE Program (Grant GM59429) at University of Puerto
Rico at Cayey and BBSP Summer Research Program for making this
Factor V [15%] possible.
H&E
REFERENCES
1) Stefano D., et al. Molecules in Focus Coagulation factor V. The International
Journal of Biochemistry and Cell Biology 2004; 36: 1393-1399.
A B C D E
2) Ryman A. , et al. Acquired haemophilia A associated with transitory and
PTAH severe factor V deficiency during bullous pemphigoid: First Report . Journal of
Thrombosis and Hameostasis 2009; 101:582-583 .
When the concentration of FV in plasma is
reduced, the product of the coagulation 3) Huang J.N. , et al. Factor V deficiency: a concise review . Haemophilia 2008;
14: 1164-1169
cascade (fibrin) will be less. Therefore, the
size of clots will be reduced. 1 2 3 4 5 4) Rosing J. , et al. Molecules in Focus Factor V . The International Journal of
Biochemistry and Cell Biology 1997; 29: 1123-1126