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

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 (PF 3 )  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 ExtrinsicIntrinsicCommon CofactorSubstrateEnzymeCofactorSubstrateEnzymeCofactorSubstrateEnzyme III VII VIII IX VVVV IIII II HMWK XI XXXX XII XIII PK ExtrinsicIntrinsicCommonCofactorMineral Carrier- Other CofactorMineral CofactorMineral III IV PF3 VIII IV PF3 VVVV IV PF3 vWF HMWK VI

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.http://www.sigmaaldrich.com/img/assets/27040/fibrinogen-cleave.jpg

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.http://www.sigmaaldrich.com/img/assets/23640/Prothrombin_Image.gif

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. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb75_1.html

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://www.clarian.org/ADAM/doc/graphics/images/en/18122.jpg. 2008.http://www.clarian.org/ADAM/doc/graphics/images/en/18122.jpg Graphic accessed at URL http://www1.istockphoto.com/file_thumbview_approve/1010778/2/istockphoto_1010778_calcium_suppleme nts.jpg, 2008. http://www1.istockphoto.com/file_thumbview_approve/1010778/2/istockphoto_1010778_calcium_suppleme nts.jpg

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  -granules Constitutional deficiency of the factor results in Owren’s disease also known as parahemophilia. Cofactor for prothrombinase complexprothrombinase complex Graphic accessed at URL http://blood.uvm.edu/lab/domains.jpghttp://blood.uvm.edu/lab/domains.jpg, 2008.http://blood.uvm.edu/lab/domains.jpghttp://blood.uvm.edu/lab/domains.jpg Graphic accessible at URL http://blood.uvm.edu/lab/domains.jpg. 2008.http://blood.uvm.edu/lab/domains.jpg Factor 5 gene

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

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

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 Travels in plasma as a heterodimer glycoprotein complexed to vWF Thrombin substrate Cofactor for TENASE complexTENASE Graphic accessed at URL http://www.uq.edu.au/vdu/VWF.gif, 2008.http://www.uq.edu.au/vdu/VWF.gif

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!!!!!!!! Vitamin K Dependent!!!!!!!! Vitamin K Dependent!!!!!!!! Vitamin K Dependent!!!!!!!! SERINE PROTEASE!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://dbb.urmc.rochester.edu/bcbp/members/images/fay2.gif, 2008.http://dbb.urmc.rochester.edu/bcbp/members/images/fay2.gif

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 complexTENASE Cofactor for prothrombinase complex Vitamin K Dependent!!!!!!!! Vitamin K Dependent!!!!!!!! SERINE PROTEASE!!!!!!!!!! Graphic accessed at URL http://en.wikipedia.org/wiki/Factor_X, 2008.http://en.wikipedia.org/wiki/Factor_X

15. Coagulation Factor XI Graphic accessed at URL http://www.haemtech.com/images2/XIa.jpg, 2008.http://www.haemtech.com/images2/XIa.jpg

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!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! Graphic accessed @ http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig17-1.jpg, 2008.http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig17-1.jpg

17. COAGULATION FACTOR XIII a.k.a. Fibrin Stabilizing Factor MWT = 320K (D) MWT = 320K (D) Half-life 3 - 6 days Half-life 3 - 6 days Mean plasma concentration = 1 - 2 mg/DL 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.http://www.haemtech.com/images2/Factor%20XIII.jpg

18. High Molecular Weight Kininogen a.k.a. Fitzgerald Factor MWT = 120K (D) MWT = 120K (D) Half-life 6.5 days Half-life 6.5 days Mean plasma concentration = 5 mg/DL 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.http://journals.prous.com/journals/dnp/20001304/html/dn130213/images/Heit_f1.gif

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!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! SERINE PROTEASE!!!!!!!!!! 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  -granules Circulate in plasma concentrations 7 – 10  g/mL Ligand for PLT receptor Ib/IX  See vWB/VIII complex 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  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. IIVII IXX

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 (α IIb β 3 ) 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: 1. Identify the name, Roman numeral designation, active form, site of production, and hemostatic role for each of the clotting factors. 2. Describe then classify the coagulation factors as subtrates, cofactors, or enzymes. 3. Evaluate the role of vitamin K in the production and function of vitamin K dependent plasma clotting protein. 4. Characterize the nature and hemostatic function of the tenase, the prothrombinase, and the vWB/VIII complexes. 5. Define these key terms: a) serine proteases, b) cofactor, and c) PF3. 6. Integrate the function of contact factors with ancillary systems of hemostasis (e.g., fibrinolysis, inflammation, etc.) 7. 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/ 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 http://medsci.indiana.edu/c602web/602/c602web/ coagtut/docs/intro1.html Laboratory Tests On-line at URL http://www.labtestsonline.org/ http://www.labtestsonline.org/