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Written Report of The Effect of Reduced Plasma Factor V on Clot Structure
 

Written Report of The Effect of Reduced Plasma Factor V on Clot Structure

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    Written Report of The Effect of Reduced Plasma Factor V on Clot Structure Written Report of The Effect of Reduced Plasma Factor V on Clot Structure Document Transcript

    • 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 Michael Gómez, Et. Al. Page 1
    • 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, Michael Gómez, Et. Al. Page 2
    • 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 Michael Gómez, Et. Al. Page 3
    • 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 Michael Gómez, Et. Al. Page 4
    • 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 after the ferric chloride injury of a 1) Stefano D., et al. Molecules in mouse carotid artery is reduced. Focus Coagulation factor V. The Therefore Factor V deficiencies can lead International Journal of Biochemistry to uncontrolled bleeding because the and Cell Biology 2004; 36: 1393-1399. coagulation isn’t completely functional 2) Allen G., et al. Impact of 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 Acnowledgments 4) Ryman A. , et al. Acquired Michael Gómez, Et. Al. Page 5
    • The Efect of Reduced Plasma F V on Clot Structures 2009 haemophilia A associated with transitory deficiency: a concise review . and severe factor V deficiency during Haemophilia 2008; 14: 1164-1169 bullous pemphigoid: First Report . 6) Rosing J. , et al. Molecules in Focus Journal of Thrombosis and Hameostasis Factor V . The International Journal of 2009; 101:582-583 . Biochemistry and Cell Biology 1997; 29: 5) Huang J.N. , et al. Factor V 1123-1126 Michael Gómez, Et. Al. Page 6