Hemostasis

Question 1

Steps to Hemostasis

Answer 1

1. Arteriolar vasoconstriction
2. Primary hemostasis
3. Secondary hemostasis
4. Regulation of thromus formation

Question 2

Steps in Local Vasoconstriction

Answer 2

1. Subendothelial basement membrane is exposed upon injury
2. Platelets recognize and bind Von Willebrand factor that is adhering to exposed collagen
   
   1. VWF Made in endothelial cells and platelets
3. Platelets change shape and become more flat
4. Platelets’ silent receptors are activated and that allows them to bind better
5. Platelets release granules containing Von Willebrand Factor (positive feedback)
6. Even more platelets aggregate at site of injury forming hemostatic plug

Question 3

Platelet formation and properties

Answer 3

1. Formed in bone marrow from megakaryocytes
2. Normal platelet count is 150-350K
3. Have 7-10 day lifespan
4. Platelets are normally NOT ACTIVE in circulation and are activated upon injury
5. Platelet size correlates with reactivity such that larger platelets are prothrombotic.
6. Platelets release trophic factors which maintain cell to cell contacts
   
   1. Therefore, low platelets leads to petichiae caused by small amounts of blood that has leaked out of the blood vessel.

Question 4

Extrinsic pathway of secondary hemostasis

Answer 4

1. Additional tissue factor (TF) in subendothelium is exposed and activated due to injury
   
   1. Tissue factor is a transmembrane protein that is not normally accessible
2. Tissue factor(TF) activates Factor VII in circulating blood
3. Interaction of TF-Factor VII results in recruitment of Factor IX ]- All three for initiation complex
4. Initation complex activates Factor X ]-common pathway
5. Factor X converts prothrombin to Factor IIa also known as Thrombin
6. Thrombin cleaves fibrinogen to fibrin

Amplification 1 (positive feedback loop of thrombin or factor IIa)

1. Thrombin or Factor IIa activates:
   
   1. Factor IX
   2. Factor VIII (cofactor for factor IX) which is circulating in blood in a complex with vWF
      
      1. vWF stabilizes Factor 8 and extends its half life from 8 min to 8 hours
   3. Factor IX and Factor VIII form a complex
   4. Factor IX and Factor VIII activate Factor X so that large amounts of it are made
   5. More Thrombin or Factor IIa is made
   6. More fibrinogen is cleaved to fibrin

Amplification 2 (Thrombin positive feedback loop)

1. Thrombin or Factor IIa activates:
   
   1. Factor V cofactor
   2. Factor V cofactor binds Factor X increasing its activity
   3. Factor X cleaves more prothrombin to Thrombin or Factor IIa 300,000 times faster
   4. Thrombin casuses more cleavage of Fibrinogen to Fibrin

Question 5

Secondary Hemostasis

Why is it needed?

Answer 5

1. Initial platelet plug formed through arteriole vasocontriction is not strong enough
2. The point of secondary hemostasis is to form cross-linked fibrin which forms a strong selanat around the platelet plug

Question 6

Vitamin K dependent Zymogens

Answer 6

1. Procoagulant factors
   
   1. Factors II, VII, IX, x
2. Anticoagulants
   
   1. Protein C and Protein S

Question 7

What does vitamin K do?

Answer 7

1. Factors II, VII, IX, X all have glutamates at N-terminus
2. In order for them to function properly, they must gain additional negative charge and they get this through carboxylation
3. Carboxylation if Vitamin K dependent.
4. Vitamin K creates a gamma-carboxyglutamate on each one of these vitamin K dependent factors.
5. Reason
   
   1. When platelets are activated after binding to endothelium, their membranes expose negative charge.
   2. Ca2+ binds to negative charge.
   3. Factors II, VII, IX, and X which now have negative gamma-carboxylgulamate can bind to positively charged Ca2+
6. Note: Warfarrin prevents coagulation by blocking bindng of Ca2+
7. You can prevent blood from clotting by chelating Ca2+

Question 8

Secondary hemostasis intrinsic pathway

Answer 8

1. Negatively charged surface (usually from phosphate residues) activates circulating Factor XII
2. Factor XII recruits HMGK and activates it
3. HMGK domain flips outward and acts as a landing pad for Factor XI
4. Factor XI activates Factor IX
5. Factor XI activates Factor X (common pathway)
6. Factor X cleaves prothrombin to thrombin of Factor IIa
7. Factor IIa converts fibrinogen to fibrin

Amplification

1. Thrombin can feedback to activate Factor XI

Question 9

Conversion of Fibrin to Fibrinogen

Answer 9

1. Fibrinogen is a multimer with 2 alpha and 2 beta chains circulating in blood stream
2. Ends of fibrinogen have fibrinopeptides that are cleaved by thrombin to make fibrin
3. Fibrin self-assembles into multimers and then fibers spontaenously
4. Factor XIII is a transglutaminase that is activated by thrombin and acts on fibrin to crosslink fibrin increasing its structural integrity.

*Factor XIII deficiency will result in:

1. Clot forms
2. Bleeding stops
3. Bleeding resumes again due to lack of structural integrity of fibrin

Question 10

Regulation of Thrombus Formation

Answer 10

1. Thrombomodulin
   
   1. Thrombomodulin is a receptor on surface of endothelial cells
   2. As production of thrombin is ramped up, thrombomodulin acts as a thrombin sink and prevents further thrombogenic activity so that fibrin is no longer made.
   3. When Thrombomodulin and thrombin bind, they robustly activate Protein C (serine protease) and its cofactor Protein S
      
      1. Note: Thrombin alone can activate Protein C but Thrombin/Thrombomodulin increase Protein C heavily
   4. Protein C and Protein S inactivate Factor V and Factor VIII to shut down clotting amplification.

Note: as soon as Thormbin is activated, not only does it turn on its own positive feedback loops in amplification 1 and 2, but it also experiences negative feedback.

1. Fibrinolysis- activated as soon as clot is formed and is used to keep clot within boundary
   
   1. Plasminogen is a protein circulating in blood
   2. Incorporated into clot and is activated by
      
      1. Thrombin
      2. Tissue plasminogen activator (used in MI)
   3. Plasmin is formed
   4. Plasmin chews up fibrin clot

Question 11

Leiden mutation

Answer 11

1. A mutation in Factor V can prevent it from being cleaved and inactivated by Protein C and S.
2. This results in increased clotting.

Question 12

Heparin

Answer 12

1. Heparin upregulates antithrombin
2. Antithrombin inhibits thrombin and factor V
3. Heparin is an anticoagulant.