Secondary Hemostasis Coagulation Factors

1. Secondary HemostasisCoagulation Factors Ahmad Sh. Silmi Msc, FIBMS IUG MT department

2. Coagulation System Composed of 14 coagulation factors (serine proteases) which are interdependent (Factors I through XIII – there is no Factor VI – and PK and HMWK) Inactive form of each is an enzyme precursor which is usually designated by a Roman numeral but also given a name – Ex. Factor I fibrinogen. Numbers correspond to order of discovery NOT order in cascade. Active forms are usually designated by the letter “a” after the Roman numeral and may also have a different name – Ex. Ia Fibrin Cofactors are needed for many reactions in the cascade – Ex. Calcium, platelet factor 3 (PF3) Each molecule must be present in sufficient quantity as well as functioning normally Final product is fibrin mesh or clot which completely stops bleeding Secondary hemostasis Slow contraction and lysis of the clot occurs

3. Coagulation factors

4. Coagulation Factors

5. Coagulation Factor I • Fibrin Zymogen • Customarily called fibrinogen • MWT = 340K (D) • Half-life 4 – 6 days • Mean plasma concentration = 200 – 400 mg/DL • Glycoprotein • Found in PLT a-granules • Mirror image dimer: a, b, g polypeptides linked by di-sulfide bonds • Primary substrate for thrombin • Substrate for plasmin Graphic accessed at URL http://www.sigmaaldrich.com/img/assets/27040/fibrinogen-cleave.jpg, 2008.

6. Coagulation Factor II • Thrombin Zymogen • Customarily called thrombin • MWT = 71.6K (D) • Half-life 2 - 4 days • Mean plasma concentration = 10 mg/DL • Vitamin K Dependent!!!!!!!! • Glycoprotein • Found in PLT a-granules • Mirror image dimer: a, b, g polypeptides linked by di-sulfide bonds • Primary substrate for thrombin • Substrate for plasmin • SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://www.sigmaaldrich.com/img/assets/23640/Prothrombin_Image.gif, 2008.

7. Coagulation Factor III • a.k.a Tissue Factor • Synonymous with tissue thromboplastin, a crude mixture of TF and phospholipid • MWT = 44K (D) • Half-life – insoluble • Mean Plasma concentration = Non-circulating • Lipoprotein • Constitutive lipoprotein of subendothelium • Procoagulant Graphic accessed at URL http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb75_1.html, 2008.

8. Coagulation Factor IV • a.k.a. ionic calcium, an essential mineral • MWT = 40 (D) • Half-life – N/A • Mean plasma concentration 8 – 10 mg/dL Graphic accessed at URL http://www1.istockphoto.com/file_thumbview_approve/1010778/2/istockphoto_1010778_calcium_supplements.jpg, 2008. Graphic accessed at URL http://www.clarian.org/ADAM/doc/graphics/images/en/18122.jpg. 2008.

9. Coagulation Factor V • a.k.a. labile factor, a.k.a proaccelerin • At one time identified as factor VI • Subtrate for thrombin and Xa • MWT = 330K • Half-life = 0.5 – 1.5 days • Mean plasma concentration = 1 mg/dL • 25% of available concentration stored in PLT a-granules • Constitutional deficiency of the factor results in Owren’s disease also known as parahemophilia. • Cofactor for prothrombinase complex Graphic accessible at URL http://blood.uvm.edu/lab/domains.jpg. 2008. Graphic accessed at URL http://blood.uvm.edu/lab/domains.jpghttp://blood.uvm.edu/lab/domains.jpg, 2008. Factor 5 gene

10. Coagulation Factor VI • DOES NOT EXIST!!!!!!

11. Coagulation Factor VII • a.k.a Stable Factor • Customarily called Factor VII • MWT = 50K (D) • Half-life 5 – 8 hours • Mean plasma concentration = 0.05 mg/DL • Vitamin K dependent Glycoprotein • Circulates as a zymogen and in low quantities of active form • SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://path.upmc.edu/cases/case119/dx.html, 2008.

12. Coagulation Factor VIII • a.k.a Antihemophilic Factor (AHF) • MWT 330K (D) • Plasma half-life = 8 – 12 hours • Very labile unless bound to vWF • Individuals with von Willebrand disease have both diminished vWF and VIII activity levels • Individuals with hemophilia A have low VIII activity but normal vWF levels • Plasma concentration = 0.01 mg/dL • Travels in plasma as a heterodimer glycoprotein complexed to vWF • Thrombin substrate • Cofactor for TENASE complex Graphic accessed at URL http://www.uq.edu.au/vdu/VWF.gif, 2008.

13. Coagulation Factor IX • a.k.a. Christmas Factor • Deficiency results in Hemophilia B • MWT = 57K (D) • Half-life 1 - 3 days • Mean plasma concentration = 0.3 mg/DL • Activated by TF:VIIa complex and XIa • Vitamin K Dependent!!!!!!!! • SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://dbb.urmc.rochester.edu/bcbp/members/images/fay2.gif, 2008.

14. Coagulation Factor X • a.k.a. Stuart-Prower factor • MWT = 58.8K (D) • Half-life 2 – 2.5 days • Mean plasma concentration = 1 mg/dL • Activated by TENASE complex and TF:VIIa complex • Cofactor for prothrombinase complex • Vitamin K Dependent!!!!!!!! • SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://en.wikipedia.org/wiki/Factor_X, 2008.

15. Coagulation Factor XI • a.k.a. Plasma thromboplastin antecedent (PTA) • Deficiency results in Hemophilia C • MWT = 143K (D) • Half-life 2 – 3.5 days • Mean plasma concentration = 0.5 mg/DL • Activated by Thrombin and contact factor complex (XIIa, HMWK, and PK) • SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://www.haemtech.com/images2/XIa.jpg, 2008.

16. Coagulation Factor XII • a.k.a. Hageman Factor • MWT = 84K (D) • Half-life 2 – 3 days • Mean plasma concentration = 2 – 4 mg/DL • Activated in vitro by contact with negatively charged surfaces • SERINE PROTEASE!!!!!!!!!! Graphic accessed @ http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig17-1.jpg, 2008.

17. COAGULATION FACTOR XIII • a.k.a. Fibrin Stabilizing Factor • MWT = 320K (D) • Half-life 3 - 6 days • Mean plasma concentration = 1 - 2 mg/DL • Transglutaminase • “mortar” for fibrin strands/mesh • a unit manufactured by megakaryocytes; b unit manufactured by hepatocytes • Thrombin substrate Graphic accessed at URL http://www.haemtech.com/images2/Factor%20XIII.jpg, 2008.

18. High Molecular Weight Kininogen • a.k.a. Fitzgerald Factor • MWT = 120K (D) • Half-life 6.5 days • Mean plasma concentration = 5 mg/DL • Bound in circulation to PK • Monomeric a-globulin Graphic accessed at URL http://journals.prous.com/journals/dnp/20001304/html/dn130213/images/Heit_f1.gif, 2008.

19. Prekallikrein • Also known as Fletcher Factor • Half-life = 1.5 days • MWT = 85K (D) • Precursor of plasma kallikrein • PK activated by activated Hageman factor • Does not required ionized calcium • SERINE PROTEASE!!!!!!!!!!

20. Platelet Factor 3 (PF 3) • a.k.a. Phosphotidyl serine (phospholipid) • Functions as an assembly molecule for coagulation • Platelet derived

21. von Willebrand Factor (vWB) • Multimeric glycoprotein • Parts made in endothelial cells and mekaryocytes • Stored in endothelial cells (Weibel-Palade Bodies) and PLT a-granules • Circulate in plasma concentrations 7 – 10 mg/mL • Ligand for PLT receptor Ib/IX • See vWB/VIII complex

22. VITAMIN K DEPENDENCY • Lipophilic, hydrophobic vitamin needed for the posttranslational modification of coagulation factors II, VII, IX, AND X • Quinone found in green, leafy vegetables, fish, liver and produced by intestinal organisms B. fragilis and E. Coli. • Addition of second carboxyl group to the g carbon of glutamic acid residues near the terminal end of II, VII, IX, and X • Creates a pocket for Ca++ that promotes phospholipid binding • Vitamin K deficiency or presence of warfarin (vitamin K antagonist) renders II, VII, IX, X unable to participate in coagulation reactions. II VII IX X

23. SERINE PROTEASES • Endopeptidases which hydrolyze arginine or lysine peptide bonds of zymogens • Important amino acid in active site = serine • Proteolytic activity controlled by serpins • Clannish: chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans. • Participate in a wide range of functions in the body, including blood clotting, immunity, and inflammation, and digestion.

24. PROTHROMBINASE COMPLEX • Important coagulation cascade complex that cleaves (“activates”) prothrombin • Composed of Xa, Va, phospholipid (PLT-derived) and ionic calcium • The first step in the ‘common’ pathway of coagulation

25. vWF/FACTOR VIII COMPLEX • VWF binds platelet glycoprotein (GP) Ib/V/IX to provide platelet adhesion. • RGD (arginine-glycine-aspartic acid) sequences bind GP IIb/IIIa (αIIbβ3) to promote platelet aggregation. • GP IIb/IIIa also binds RGD sequences of fibrinogen. • A third VWF site binds collagen. • VWF also provides a binding site for coagulation factor VIII. VIII

26. TENASE COMPLEX • Important coagulation cascade complex that cleaves (“activates”) Factor X • Composed of VIIIa, IXa, phospholipid (PLT-derived) and ionic calcium • The end product of the ‘instrinsic ’ pathway of coagulation X

27. Learning Objectives – Upon completion of required reading, after careful study, and following this lecture, the student will be able to: Identify the name, Roman numeral designation, active form, site of production, and hemostatic role for each of the clotting factors. Describe then classify the coagulation factors as subtrates, cofactors, or enzymes. Evaluate the role of vitamin K in the production and function of vitamin K dependent plasma clotting protein. Characterize the nature and hemostatic function of the tenase, the prothrombinase, and the vWB/VIII complexes. Define these key terms: a) serine proteases, b) cofactor, and c) PF3. Integrate the function of contact factors with ancillary systems of hemostasis (e.g., fibrinolysis, inflammation, etc.) Apply knowledge of the coagulation factors to correctly answer clinical case study questions of hemostasis.

28. REFERENCES • McKenzie, Shirlyn B (2004). Clinical Laboratory Hematology. Pearson Prentice Hall. Chapter 35 • The Fritsma Factor at URL http://www.fritsmafactor.com/ • Coagulation Disorders Tutorial by Indiana University School of Medicine (2001) at URL http://medsci.indiana.edu/c602web/602/c602web/coagtut/docs/intro1.html • Laboratory Tests On-line at URL http://www.labtestsonline.org/