Poster of The Effect of Reduced Plasma Factor V on Clot Structure
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Transcript of Poster of The Effect of Reduced Plasma Factor V on Clot Structure
The Effect of Reduced Plasma Factor V on Clot StructureMichael Gómez-Meléndez1 , Kellie R. Machlus2, Jessica Cardenas2 , Hongmin Sun3 , and Alisa S. Wolberg2
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
ABSTRACTBackground: In the coagulation cascade, Factor V (FV) acts as a
cofactor to factor X, which together form the prothrombinase
complex and convert prothrombin to thrombin.Thrombin then cleaves
fibrinogen, forming the fibrin necessary to create a stable clot.
Deficiencies in Factor V result in a hemorrhagic phenotype.1
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
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
levels. Vessel sections containing the clots were then fixed in
paraformaldehyde, dehydrated and embedded in paraffin for
sectioning. Individual sections were stained with both Hematoxylin
and Eosin (H&E) and Phosphotungstic Acid Hematoxylin (PTAH) for
histological analysis.
Results: In wild-type mice (100% FV), the presence of an occlusive
clot following ferric chloride injury was common. When the level of
plasma FV was reduced to 50%,however, the clot was only partially
occlusive. Additionally, when plasma levels of FV were 15%, arteries
were full of red cells and had only minimal clot formation.
Conclusions: Lower concentrations of FV in the plasma of mice
decreased the production of fibrin and fibrinogen and ultimately, the
amount of clot formed after ferric chloride injury.
INTRODUCTIONApproximately one in one million people have a problem generating
FV.1 The first disease related to FV was reported by the Norwegian
hematologist Paul Owren, who proposed the existence of this factor
and a new model of the coagulation process.1 Generally, deficiencies
in FV cause abnormal bleeding, but some cases without bleeding
complications have been reported.2 The reduced levels of FV in
humans can be congenital or acquired.3 The principal areas of
bleeding are mucosa, skin, joints, muscles, genitourinary tract,
central nervous system and the gastrointestinal tract. 4
ACKNOWLEDGEMENTS
• This study was supported by grants from: NIH K01 AR051021 (ASW),
University of North Carolina Institute on Aging (ASW), The Gustavus
and Louise Pfeiffer Research Foundation (ASW), and The National
Hemophilia Foundation (ASW).
• Thanks to Vanessa González, Pat Phelps, Paul Medina and Eileen
Hayter for all the support.
• Thanks to RISE Program (Grant GM59429) at University of Puerto
Rico at Cayey and BBSP Summer Research Program for making this
possible.
REFERENCES
1) Stefano D., et al. Molecules in Focus Coagulation factor V. The International
Journal of Biochemistry and Cell Biology 2004; 36: 1393-1399.
2) Ryman A. , et al. Acquired haemophilia A associated with transitory and
severe factor V deficiency during bullous pemphigoid: First Report . Journal of
Thrombosis and Hameostasis 2009; 101:582-583 .
3) Huang J.N. , et al. Factor V deficiency: a concise review . Haemophilia 2008;
14: 1164-1169
4) Rosing J. , et al. Molecules in Focus Factor V . The International Journal of
Biochemistry and Cell Biology 1997; 29: 1123-1126
FUTURE DIRECTIONS
•Purpose
•Specifically identify fibrin (not fibrinogen) in mouse clots
and compare the amount of fibrin produced in plasma with
different FV levels .
•Protocol
• For immunohistochemistry Antibody 59D8 will be used.
MATERIALS AND METHODS
Clot
Induction
Fixation in
Paraformaldehyde
Paraffin
Embedding
Sectioning Hematoxylin and
Eosin Staining
PTAH
Staining
CONCLUSION
. When the FV levels in the plasma are below 100%, clot formation
after ferric chloride injury of a mouse carotid artery is reduced.
Deparaffinization
• Xylene
• Methanol
• Hidratation in dilutions of Ethanol
AntigenRetrieval
• Boil Sections in CitrateBuffer
• Cool on ice
59D8 Monoclonal
• Dilute 1:1000
• Incubate overnight withantibody and PBS/BSA
DAKO Envision-HRP
• Rinse in PBS
• Incubate in DAKO Envision-HRP for mice
• Rinse in PBS
Vector Novared
• Incubate slides in Vector Novared.
• Rinse in MilliQ
• Staing with Hematoxyline
Mounting
• Rinse in water.
• Dehydrate.
• Mount with Pertex
HYPOTHESIS
When the concentration of FV in plasma is
reduced, the product of the coagulation
cascade (fibrin) will be less. Therefore, the
size of clots will be reduced.
RESULTS
= H & E Stained
= PTAH Stained
Day 1 Day 2
Factor V [50%]
Factor V [15%]
H & E
H & E
PTAH
PTAH
Factor V [100%]
A B C D E
A B C D E
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
A B C D E
H & E
PTAH