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