The study examined how deficiencies in Factor V (FV) affect clot structure in a mouse model. Mice were genetically altered to have 15%, 50%, or 100% of normal FV plasma levels. Clot formation was induced in the carotid artery, then sections were stained and analyzed. When FV levels were 100%, occlusive clots commonly formed. At 50% FV, clots were partially occlusive. With 15% FV, arteries contained red blood cells but minimal clot formation. Lower FV concentrations decreased fibrin and fibrinogen production, resulting in less clot formation after injury. Therefore, FV deficiencies can lead to uncontrolled bleeding due to impaired coagulation.

1. The Efect of Reduced Plasma F V on Clot Structures 2009
The Efect of Reduced Plasma F V on Clot Structures
Michael Gomez-Melendez1, Kellie R. Machlus2, Jessica Cardenas2,
Hongmin Sun3 , Alisa Wolberg2
Department of Chemistry, University of Puerto Rico, Cayey, PR ;
Department of Pathology and Laboratory Medicine, University of North
Carolina, Chapel Hill, NC ; Department of Internal Medicine,
University of Missouri, Columbia, MS
Abstract
Background: 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.
Introduction
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2. The Efect of Reduced Plasma F V on Clot Structures 2009
When the Factor V is over the normal
Approximately one in one million of levels (100%) have no effect in the
persons will have a problem with the thrombin production2, therefore the clot
generations of Factor V. The first produced in a 200% Concentration of
disease related to Factor V was reported Factor V and 100% Concentration of
by the Norwegian hematologist Paul Factor V have the same capacity to
Owren and he proposed the existence of develop clots in the arteries . The
this factor and propose a new model of difference between the thrombin
the cascade for the coagulation process. produced is approximately 0% to 30% .
Bleedings disorders related with Factor Factor V produce activate the Factor
V as subdivided in two categories in the VIII and they share 40% of their
Type 1 the Factor V produced is so low sequences1 . Investigations that study
that is not measurable; the Type 2 is reduced concentrations Factor V present
only a deficiency, where the antigen don’t agree, presenting that low
level produced is below the normal concentrations don’t have big difference
amount but and is measurable 1. Factor 3
versus the ones that found that low
V is approximately 80% in blood concentrations of Factor V the thrombin
plasma and 20% in platelets6. Generally generation is reduced.
deficiency of Factor V cause abnormal
bleeding, but has been reported cases Materials and Methods
without bleeding complications 4. The Clot Induction
reduced levels of Factor V in humans Mice were modified genetically to have
can be congenital by gene mutations or reduced concentrations of Factor V in
acquired and the first symptoms can their blood plasma. The clots were
occur before the age of 6 5. The principal induced in the carotid artery with a patch
areas of bleeding are: mucosa and skin , of 10% ferric chloride in the carotid
joint and muscle, genitourinary tract, artery, until coagulation. Then the
Central Nervous System and carotid arteries were removed and
gastrointestinal tract 5. fixated with 4% paraformaldehyde,
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3. The Efect of Reduced Plasma F V on Clot Structures 2009
return the coplin jar for 10 seconds on
Histology high power, stir the solution and
The tissue was dehydrate with ethanol incubate the slides for 15 minutes. Rinse
80%. 95% and 100%. Then our samples with tap water (1 minute) and with
were embedded in paraffin and sectioned distilled water (1 minute) . Place the
in slices of 4 nm. slides in 25 mL of Ferric Ammonium
H&E Sulfate Solution and heat for 15 seconds
A protocol of Hematoxylin and Eosin on high power and incubate for 2
Staining (H & E) to identify the clot in minutes. Then heat 25 mL of PTAH
the slides. First we stained one of each Stain in the microwave for 20 seconds in
three slides (1, 4, 7, 10…). The process high power, stir and place the slides, and
started with the hydratation of the tissue incubate for 15 minutes. After the
with three changes with ethanol 100%, incubation time heat for 10 seconds and
95% 80% and D.I. water. The tissue was incubate again. for 15 minutes. Proceed
moved to Hematoxylin, rinsed in tap to a dehydratation process with one
water, dipped in 0.25% acid alcohol, changes of 95% alcohol, three changes
blued in 1.36% Lithium Carbonate , of 100% and 3 changes of Xylene. Then
briefly exposed to Eosin with alcohol + mount with Vectamount Mounting
phloxine. Then the samples were Solution.
differentiated with 80% EtOH , 95%
EtOH, 100% EtOH and Xylenes. Results
PTAH
The slides were processed through Factor V [100%]
Xylene to remove the paraffin and Factor V [100%] present groups of red
hydrated from alcohol 100% to tap cells coming together, then an artery and
water. Then the samples were exposed to finally we see a partial clot and then an
DI Water. In a coplin jar add 50 ml of occlusive clot. These control present
10% Zinc Chloride Solution and heat expected results on a normal coagulation
for 20 second on high power, remove pathway.
and stir (to balance the temperature) and
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4. The Efect of Reduced Plasma F V on Clot Structures 2009
In our samples
in the H & E
(rows labeled
with
characters)
the pale pink
is the collagen
outside the
artery; the
deep pink is
the muscle of
the artery ; the
cherry red
Factor V [50%]
inside the artery are the erythrocytes
In Factor V [50%] the red cells groups
and the pale pink inside the artery is the
are less and then we have a partial clot.
fibrin produced in the coagulation
In this case the clot formation activity is
cascade. The PTAH staining in the rows
reduced with a mix of red cells between
labeled with characters show us the
the disorganized clot. .
fibrin with the light blue-purple, the
nuclei are stained with deep blue-purple
Factor V [15%]
and the collagen with a light blue .
Following the same pattern in Factor V
[15%] have a big group of red cells but
Our result suggest that when we have
less fibrin, but in this case the fibrin is
normal levels of Factor V the
more organized. Also the occlusion time
coagulation is more uniform and have
was increasing while the concentrations
bigger clots present, but when we
were decreased. The results can be
reduced the Factor V to 15% the clot
appreciated in Figure 1.
starts a disorganization, making less
uniform the balance between red cells
Discussion
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5. The Efect of Reduced Plasma F V on Clot Structures 2009
and fibrin. This study was supported by grants
from: NIH K01 AR051021 (ASW) ,
The production of fibrin was affected University of North Carolina Institutes
because the Factor V activates less of Aging (ASW) , The Gustavus and
Factor X and les F II is converted to FIIa Louise Pfeiffer Research Foundation
and as a consequence less Fibrinogen is (ASW), and The National Hemophilia
converted into Fibrin. This deficiency Foundation (ASW) . Thanks to Vanessa
will cause hemophilia because the clot González. Pat Phelps, Paul Medina and
formation activity is completely affected Eileen Hayter for all the support. Thanks
in a negative way. to RISE Program (Grant GM59429) at
University of Puerto Rico at Cayey and
Conclusion BBSP Summer Research Program for
According to the results we can conclude make this possible.
that when the Factor V levels in the
plasma are below 100% , clot formation References
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to uncontrolled bleeding because the and Cell Biology 2004; 36: 1393-1399.
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or is slower. procoagulant concentration on rate,
peak, and total thrombin generation in a
Future Direction model system. Journal of Thrombosis
Immunohistochemistry will be and Haemostasis 2004; 2:402-413
performed to identify the fibrin present 3) Duchemin J, et al. Influence of
(not fibrinogen) in mouse clots and coagulation factors and tissue factor
compare amount of fibrin produced in concentration in the thrombin generation
plasma with different Factor V Levels. test in plasma. Journal of Thrombosis
and Haemostasis 2008; 99: 767-773
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Michael Gómez, Et. Al. Page 5

6. The Efect of Reduced Plasma F V on Clot Structures 2009
haemophilia A associated with transitory deficiency: a concise review .
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Michael Gómez, Et. Al. Page 6