1. ENZYME CASCADES: BLOOD CLOTTING
Medical Biochemistry, Lecture 27

2. Lecture 27, Outline The extrinsic and intrinsic clotting pathways
Specific reactions in the coagulation pathway: a. thrombin-fibrinogen-fibrin; b. Factor XIIIa; c. Hemophilia; d. anti-protease system; e. auto-regulation of thrombin; f. fibrinolysis

3. Blood Clotting
Because of the importance of blood in regulating pH and the transport of oxygen, nutrients, carbon dioxide, and wastes, maintaining the integrity of the process is crucial to life. When ruptures in the system do occur, the process of blood clotting is initiated as an emergency measure to halt the loss of blood. Biochemically, blood clotting is an example of signal amplification caused by the simultaneous activation and inhibition of many enzymes. A basic overview of the process and how the enzymes are regulated will be the focus of this lecture.

4. Red blood cells enmeshed in
the insoluble strands of a fibrin
clot
Electron micrograph
of a fibrin fiber

6. PLEASE REMEMBER
Do not worry about memorizing the whole pathway and all of the factors involved - you will be tested on specific examples, not the entire pathway - the focus will be on using this pathway to illustrate all of the interactions between proteins, enzymes and regulatory mechanisms described in the preceding lectures.

7. Blood Vessel Damage
When injury to a blood vessel occurs, three major events happen to rapidly stop the loss of blood:
1) clumping of blood platelets at the site of injury to create a physical plug.
2) vasoconstriction occurs to reduce blood flow through the area.
3) aggregation of fibrin into an insoluble clot that covers the rupture and stops loss of blood.
The clot is dissolved after actual repair of the blood vessel.

8. Platelet Aggregation
The initial phase of platelet aggregation is a complex formed between the platelets and underlying collagen fibrils exposed in the ruptured vessel. An additional circulating protein, von Willebrand factor (vWF), mediates binding of platelets to collagen and each other resulting in activation of the platelets and release of various activators. The next slide schematic illustrates the complex biochemical effects that binding of platelets, collagen and vWF have on activation of the blood clotting pathway

10. Blood Vessel Damage (cont)
In the normal, undamaged vascular endothelium, platelet aggregation does not occur since collagen fibrils are not exposed and other activating factors (like ADP) are not present in sufficient amounts. Besides exposure of the collagen fibrils in the underlying matrix of the vessel, other membrane proteins in this matrix are exposed to the circulating blood. It is these matrix and membrane proteins that serve as receptors for the various zymogens and protein co-factors that are released by the activated platelets (or that were already present in the blood).

11. Blood Vessel Damage (cont)
Ultimately, the final blood clot is formed by the conversion of fibrinogen to fibrin eventually resulting in insoluble, cross-linked fibrin polymers. Two activation pathways (initiated by complexes with exposed membrane matrix proteins), historically termed the extrinsic and intrinsic pathways, supply the protease (Factor Xa) that activates the thrombin catalyzed production of fibrin

12. Intrinsic and Extrinsic
Pathways Schematic

13. General Characteristics of the Coagulation Cascade
The predominant mechanism illustrated in the activation pathway is that of an enzyme amplification cascade. One enzyme at the start of the activation pathway can activate many molecules of a given substrate as illustrated:

14. Amplification Cascade Model
This preceding model can be interpreted as follows: If each of 100 individual Factor VIIa enzymes catalyzed the activation of 100 Factor Xa enzymes, and each one of these 10,000 Factor Xa enzymes catalyzed 100 thrombin activations, and so on - there is a resulting million-fold activation from the start of the pathway to fibrinogen cleavage. It is this amplification process that allows a clot to form rapidly and also illustrates the magnitude that one defect in these steps could have on the whole process (an example to be discussed will be hemophilia).

15. General Characteristics (cont)
Another characteristic of this process is the regulation of zymogen enzymes. In both pathways, Factors XII, XI, IX, VII, X and II are zymogens of serine proteases. Cleavage of one zymogen leads to an active protease that activates another zymogen protease and so on. These zymogens are themselves regulated by a series of non-protease protein co-factors that also must be activated by proteases (like thrombin activation of Factor VIII to VIIIa and Factor V to Va).

16. General Characteristics (cont)
These protease and protein cofactor interactions lead to large multi-protein complexes, yet they can be thought of just like any small molecule substrate to product enzymatic reaction that we have discussed previously. It is the specificity of the protein sequence and conformation in combination with various metal, lipid or protein co-factors that determine whether a factor is a substrate for an activated protease (For instance, Factor XIIa will not cleave prothrombin to thrombin, only Factor XI to XIa).

18. Intrinsic Pathway and
Protein Complex

19. Extrinsic Pathway and
Protein Complex

20. Thrombin-Fibrinogen-Fibrin
Thrombin is generated from a prothrombinase complex consisting of prothrombin, factor Va, factor Xa, calcium and anionic phospholipids. Prothrombin is synthesized in the liver and contains 10 Gla residues in its amino-terminal domain. Factor Xa cleaves prothrombin into its activated form (thrombin). Thrombin acts on fibrinogen, a large fibrous protein consisting of two tripeptide units. These subunits form three globular domains - two on the ends and one in the middle where the two tripeptides associate. Thrombin cleaves four peptides termed either fibropeptide A (from the a-subunit) or fibropeptide B (from the b-subunit). These cleaved fibropeptides are unique in that they contain many negatively charged Asp and Glu residues, plus unique sulfated tyrosine residues. Loss of this net negative charge on the fibrin monomer induces conformational changes in the middle globular domains that create new sites with high affinities for the terminal globular domains of other fibrin monomers.

21. Prothombinase Pathway
and Protein Complex

22. Fibrinogen Activation
Peptides cleaved by thrombin contain many Asp, Glu and sulfated-Tyr
residues, thus very highly negatively charged

23. Factor XIIIa (transglutaminase)
The "soft clot" of fibrin monomers is stabilized and converted into the final clot by covalent cross-linkages between specific glutamine residues on one monomer and lysine residues on another monomer. This peptide (amide) bond is catalyzed by activated Factor XIIIa, a transglutaminase:

24. Factor XIIIa - Fibrin Cross-linking

26. Roles of Calcium, Carboxyglutamate, and Vitamin K
Some of these factors (thrombin, VII, IX and X) contain a unique modified glutamate residue, called carboxyglutamate (Gla). This amino acid is a natural high affinity binder (or chelator) of calcium ions, hence the designation of calcium as a co-factor in the schematic. It is this complex of calcium with the Gla-factors that allow specific interactions with acidic membrane lipids that ultimately lead to the correct tertiary and quaternary protein stuctures recognized by other proteins in the pathway. Synthesis of Gla residues result from post-translational modifications of the newly synthesized factors in the liver endoplasmic reticulum by a vitamin K-dependent carboxylase

27. Vitamin K - Carboxyglutamate

28. Dicoumarol / Warfarin
Two analogs of Vitamin K, dicoumarol (I) and warfarin (II), have been shown to inhibit formation of the Gla residues of prothrombin and Factors VII, IX and X. The lack of Gla residues, and hence binding of calcium, was shown to inhibit the participation of these proteins in the blood coagulation process. This is the basis for using coumarin drugs clinically as anti-coagulants.

29. Structures of the Coumarin Drugs (Vitamin K analogs) VITAMIN K
DICOUMAROL
Structures of the
Coumarin Drugs
(Vitamin K analogs)
WARFARIN

30. Physiological Effect of Inhibiting Gla Production with Coumarin Drugs
The presence of Gla peptides
released during the clotting
activation cascade signals new
synthesis of Gla-proteases in
the liver. Blocking of the Gla
modification by drugs prevents
secretion of the newly
synthesized proteases and thus
limits their numbers in
circulating blood. In the
absence of drugs, this pathway
replenishes the circulating
pool of zymogen factors

31. Hemophilia
Hemophilia is a genetically inherited disease characterized by an inability to form proper blood clots. This deficiency has been attributed to defective production of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Hemophilia A is the most common (80%) form of the disease.

32. Hemophilia - Mechanistic Defect
It may not be readily apparent why defects in Factor VIII and Factor IX lead to poorly formed clots. Both of these factors are in the intrinsic pathway. In hemophiliacs, the extrinsic pathway still functions normally. As will be discussed, the whole clotting pathway is tightly regulated and interdependent. When an injury occurs in a patient with hemophilia, a disproportionate clotting factor response to the wound occurs (i.e., defective intrinsic pathway activation), and regulation of the entire process becomes impaired.

33. Anti-protease system
Critical features of the blood coagulation pathway are the mechanisms of inactivation. If activated thrombin was not regulated, then the fibrin clot could continue to form and eventually block circulation. Also present in the blood are protein protease inhibitors that specifically bind to the different activated serine proteases. In normal blood circulation scenarios, the antiproteases proteins are present in sufficient concentrations to act as security against random thrombin activation (or other proteases) and the resulting fibrin clots.

34. Anti-protease system (cont)
An example is antithrombin III, which tightly binds and inactivates thrombin - this complex is later cleared from circulation in the liver. The clinical administration of heparin promotes the association between antithrombin III and thrombin. This is the basis for use of heparin as an anti-coagulant.

35. Auto-regulation of thrombin
Besides cleaving fibrinogen, thrombin has forms complexes with an endothelial cell protein receptor termed thrombomodulin. Thrombomodulin and calcium act as co-factors of thrombin activation of Protein C, a Gla containing protease. When activated, Protein C in conjunction with another protein co-factor, Protein S, will proteolyze and inactivate Factors Va and VIIIa.

37. Regulation of Thrombin - Summary
Activation of the blood coagulation process involves the site-specific, rapid amplification and activation of factors that quickly form the fibrin clot. Since this activation is localized to the site of injury, formation of fibrin clots should not be occuring throughout the entire circulatory system. The combination of the antiproteases and Protein C ensures this. Since thrombin is near the end of the amplification cascade, there will be a high localized concentration of thrombin at the site of injury. This high level of thrombin will correspondingly activate a high level of Protein C.

38. Thrombin Summary (cont)
Protein C inactivation of Factor Va leads to an inhibition of Factor Xa activation of prothrombin (and thus more thrombin) while inactivation of Factor VIIIa effectively inhibits formation of additional stable fibrin clots. Circulating anti-proteases further down-regulate the activated clotting factors. Thus the activation-inactivation systems of blood coagulation exist in a dynamic equilibrium that can be rapidly mobilized and just as quickly turned off.

39. Mechanism of Clotting Summary
INITIATE
AMPLIFY
INHIBIT

40. Dissolving of Blood Clots: Fibrinolysis
Similar mechanisms of activation and inactivation described for clot formation apply to this pathway, too. Plasmin, which exists in circulating blood as plasminogen, is the protease responsible for degrading the fibrin clots. The activator of plasminogen, another protease termed tissue plasminogen activator (tPA), binds with high affinity to the fibrin clot along with plasminogen. The tPA-plasminogen-fibrin complex results in proteolytic activation of plasminogen to plasmin, which then begins digesting the fibrin.

41. Clot Dissolving Pathway

42. Fibrinolysis (cont)
To keep one plasmin molecule from degrading the whole clot, after plasmin degrades a region of the clot, the resulting digested peptides dissociate from the clot and take the plasmin-tPA complex with them. Again similar to the antiprotease factors described above, anti-plasmin and anti-tPA proteins have been described that perform the same function as the anti-coagulation proteins

43. Coagulation Complexes