5.2. Hemostasis and the role of thrombocytes. Blood coagulation. Fibrinolysis. Physiological anticoagulant mechanisms.

Question 1

I. Hemostasis
1. Definition of hemostasis

Answer 1

prevention of bleeding from a damaged vessel (prevention of hemorrhage)

Question 2

I. Hemostasis
2. What are the stages of hemostasis?

Answer 2

PRIMARY HEMOSTASIS
1. Vasoconstriction
2. Increased tissue pressure
3. Formation of a platelet plug

SECONDARY HEMOSTASIS
Coagulation (formation of a blood clot)

Question 3

I. Hemostasis
3A. What are the mechanism and role of vasoconstriction (primary hemostasis)?

Answer 3

1/ Mechanism:
- Upon injury of the blood vessel wall, there is an immediate response called vasoconstriction
- Vasoconstriction will decrease blood flow towards injured area, leading to decrease in blood loss

2/ Role: to give more time for more effective hemostatic processes to take place

Question 4

I. Hemostasis
3B. What are the causes of vasoconstriction (primary hemostasis)?

Answer 4

1/ Neurogenic reflex
2/ Endothelin release
3/ Release of vasoconstrictor substances

Question 5

I. Hemostasis
3B1. How does neurogenic reflex participate in vasoconstriction (primary hemostasis)?

Answer 5

produces vasoconstriction

Question 6

I. Hemostasis
3B2. How does Endothelin release participate in vasoconstriction (primary hemostasis)?

Answer 6

• Endothelin Binds to to ET-receptor on SMC (smooth muscle cell), leading to vasoconstriction

Question 7

I. Hemostasis
3B3. What are the vasoconstrictor substances that are released by thrombocytes? Consequences?

Answer 7

Chemical released by thrombocytes

• Thromboxane A2 (TXA2, synthesized by platelets)
  => Ca2+ signal
  => Vasoconstriction
• Serotonin (synthesized by platelets)
• Epinephrine (not synthesized, taken up by platelets) => ⍺1 adrenergic receptor acting on smooth muscle contraction

Question 8

I. Hemostasis
3C. Mechanism and role of Increased tissue pressure?

Answer 8

Increased tissue pressure contributes to hemostasis because:
- It decreases radius, which causes significant decrease in blood flow; decrease r by a factor of 2 would diminish flow by a factor of 16 according to - Poiseuille law.
E.g, We do this naturally by pressing a finger against a small cut to stop the bleeding.

Question 9

I. Hemostasis
3D. What is the role of Platelet plug formation (step 3 in primary hemostasis)?

Answer 9

platelets plug small ruptures in the endothelium

Question 10

I. Hemostasis
3E. What are the stages of Platelet plug formation (step 3 in primary hemostasis)?

Answer 10

1/ Adhesion
2/ Activation
3/ Aggregation

Question 11

I. Hemostasis
3F. What are platelets?

Answer 11

platelets are fragments of megakaryocytes

Question 12

I. Hemostasis
3G. What are characteristics (structure) with corresponding functions of platelets

Answer 12

1/ Have a dense tubular system
-> Acts as a Ca2+ storage

2/ Have dense granules containing ADP, serotonin and polyphosphates to activate platelets

3/ Have alpha granules which contain clotting factors

4/ Have actin + myosin for later contraction to reduce the size of the clot

5/ Have adhesion glycoproteins found on their surface
ex: GPIa/IIa, GPVI

Question 13

I. Hemostasis - Stages of platelet formation
3H1. Why is platelet adhesion necessary? When will platelet adhesion occur?

Answer 13

1/ Platelets do not adhere to endothelium directly because platelets have negative surface charge and so do the endothelium (proteoglycans – heparan sulfate).
2/ Platelet adhesion occurs in response to ↑ shear stress at platelet surface or endothelial cells, and in response to vessel injury or humoral signals.

Question 14

I. Hemostasis - Stages of platelet formation
3H2. What are the steps af platelet adhesion?

Answer 14

1/ Platelets first adhere to the injured space via glycoprotein-collagen binding with glycoprotein GP VI and GP Ia – IIa
=> This is a Direct platelet interaction

2/ Endothelial cells also release the von Willebrand factor (vWF) which also binds to exposed collagen and then create an indirect interaction between GP Ib – IX – B

Question 15

I. Hemostasis - Stages of platelet formation
3I1. How are platelets activated?

Answer 15

Binding of GP VI to collagen initiates tyrosine phosphorylation cascade -> Phospholipase C (PLC) activation -> IP3 -> Ca2+-release = platelet is activated
*Note: Inositol trisphosphate or inositol 1,4,5-trisphosphate abbreviated InsP3 or Ins3P or IP3

Question 16

I. Hemostasis - Stages of platelet formation
3I2. What are the 5 consequences of increased concentration of intracellular Ca2+?

Answer 16

1. Exocytosis of granules
   → ADP, 5-HT, epinephrine, Ca2+, procoagulant factors, fibrinogen…
   → Positive feedback
2. Phospholipase A2 (PL2) activation
   → TXA2-production → positive feedback → platelet activation increased
3. Cytoskeletal rearrangement
   → platelets undergo shape change, increased surface
4. Exposure of GPIIb-IIIa integrin complex
   → fibrinogen binding, platelet aggregation
5. Phospholipid externalization
   → coagulation cascade

Question 17

I. Hemostasis - Stages of platelet formation
3I3. The 5 consequences of increased contraction of intracellular Ca2+
-> What are the consequences of Exocytosis of granules?

Answer 17

→ ADP, 5-HT receptors (serotonin receptors), epinephrine, Ca2+, procoagulant factors, fibrinogen…
→ Positive feedback

Question 18

I. Hemostasis - Stages of platelet formation
3I4. The 5 consequences of increased contraction of intracellular Ca2+
-> What are the consequences of PLA2 activation?

Answer 18

→ TXA2-production → positive feedback → platelet activation increased

Question 19

I. Hemostasis - Stages of platelet formation
3I5. The 5 consequences of increased contraction of intracellular Ca2+
-> What are the consequences of 3. Cytoskeletal rearrangement?

Answer 19

→ platelets undergo shape change, increased surface

Question 20

I. Hemostasis - Stages of platelet formation
3I6. The 5 consequences of increased contraction of intracellular Ca2+
-> What are the consequences of 4. Exposure of GPIIb-IIIa integrin complex?

Answer 20

→ fibrinogen binding, platelet aggregation

Question 21

I. Hemostasis - Stages of platelet formation
3I7. The 5 consequences of increased contraction of intracellular Ca2+
-> What are the consequences of 5. Phospholipid externalization?

Answer 21

→ coagulation cascade

Question 22

I. Hemostasis - Stages platelet formation – 2. Platelet activation
3j. How is platelet activation inhibited?

Answer 22

By synthesis of PGI2 and NO in endothelial cells, it will inhibit the increase of intracellular Ca2+ concentration which subsequently inhibit platelet activation and aggregation
- PGI2 -> PGI2 – R (GS) -> Cause increase in cAMP
- NO -> GC -> Increase in cGMP

*Note:
- Prostaglandin I2 (PGI)

Question 23

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 3. Platelet plug formation
3K. What are the Steps of platelet aggregation?

Answer 23

1/ Thrombus initially develops through the stabilization of discoid platelet aggregate

2/ Shedding of microparticles from the platelet plasma membrane (caused by a high shear stress) also contributes to aggregation

3/ ADP + TXA2 released from platelets attract more platelets to the affected area

4/ Exposed GP IIb-IIIa binds to RGD sequence and RGD proteins
- RGD sequence is a tripeptide Arg-Gly-Asp motif in a subunit of fibrinogen
- RGD proteins are vWF, fibrinogen, fibronectin and vitronectin
=> Platelets cross-bind to each other

Question 24

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation a
3L. What are Characteristics of blood coagulation?

Answer 24

1/ Occurs primarily because of the conversion of fibrinogen to fibrin (via thrombin)

2/ Thrombin itself must first be activated and have the necessary cofactors to perform this function
- Thrombin activation occurs via intrinsic and extrinsic pathway
- Intrinsic (contact activation) and extrinsic (tissue factor) converge at the activation of factor X

Question 25

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation a 3M. How does Intrinsic pathway work?

Answer 25

1/ Intrinsic pathway: activated by factors that are found within blood. 2/ Factors XII gets activated = XIIa -> XIIa converts factor XI to factor XIa -> XIa activates factor IX to factor IXa -> IXa forms the “intrinsic tenase complex” with VIIIa, phosphotidylserine (PS) and Ca2+ = (Ixa – VIIIa – PS – Ca2+) -> Intrinsic tenase converts factor X to factor Xa -> Factor Xa is generated on the platelet surface

Question 26

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation a 3N. How does Extrinsic pathway work?

Answer 26

1/ Extrinsic pathway: activated by tissue factor found outside of the blood (VSM) 2/ With injury to a blood vessel, the factor VIIa is exposed to tissue factor -> form a complex together with Ca2+ called the ‘’extrinsic tenase complex’’ (TF-VIIa-Ca2+) 3/ Extrinsic tenase complex activates factor X -> factor Xa (common pathway) and factor IX -> factor IXa (intrinsic pathway) -> Factor Xa is generated by extrinsic tenase complex on the surface of fibroblasts

Question 27

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation a 3L. How does Final common pathway work?

Answer 27

1/ Factor Xa (gotten from both intrinsic + extrinsic pathway) forms a complex with factor Va, that is called ‘’prothrombinase complex’’ 2/ Prothrombinase complex is used to activate prothrombin (II) into thrombin (IIa) -> Thrombin cleaves fibrinogen to fibrin (fibrin mesh network)

Question 28

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation 3O. What are the 4 4 pro-coagulative effects by thrombin (IIa)?

Answer 28

1/ Activate platelets directly via PAR-2 2/ Catalyze the proteolysis of fibrinogen (I) into fibrin (Ia). 3/ Activate factor XIII (stabilizing factor) for cross links 4/ Activate factor V, VIII (positive feedback)

Question 29

I. Hemostasis - Stages of hemostasisPrimary hemostasis – 4. Blood coagulation a 3P. Compare Extrinsic vs Intrinsic pathway

Answer 29

1/ Extrinsic - Starts early - Explosive (within seconds) 2/ Intrinsic - Starts later - Slower (within minutes) - Accelerates fibrin formation - Stabilizes the blood clot

Question 30

I. Hemostasis - Stages of hemostasis - Secondary hemostasis – 4. Blood coagulation 3Q1. What is the structure of Gla proteins?

Answer 30

A complex consist of - Procoagulants: prothrombin, factor VII, IX, X - Anticoagulants: protein C & S

Question 31

I. Hemostasis - Stages of hemostasis - Secondary hemostasis – 4. Blood coagulation 3Q2. What does the synthesis of Gla proteins require?

Answer 31

1/ Requires vitamin K 2/ Occur in liver

Question 32

I. Hemostasis - Stages of hemostasis - Secondary hemostasis – 4. Blood coagulation 3R. How does Vitamin K-dependent carboxylation occur?

Answer 32

1/ γ-glutamyl carboxylase uses a vitamin K cofactor to convert glutamic acid into γ-carboxyglutamic acid, Gla (by adding carboxylic acid). 2/ In the process of serving as a cofactor (donate ē), vitamin K is converted to vitamin K epoxide. Vitamin K epoxide gets converted back into vitamin K quinone by epoxide reductase = reusable!

Question 33

I. Hemostasis - Stages of hemostasis - Secondary hemostasis – 4. Blood coagulation 3S. Importance of vitamin K in blood coagulation?

Answer 33

1/ Gla structure allows Ca++ to bind, which induces factor II, VII, IX, X to activate. 2/ Warfarin inhibits epoxide reductase, blocking vitamin K from getting recycled = coagulation cascade would not work properly.

Question 34

I. Hemostasis - Stages of hemostasis - Secondary hemostasis – 4. Blood coagulation 3T. What are the Steps of fibrin formation?

Answer 34

1/ The formation of fibrin takes place when thrombin converts fibrinogen dimers to fibrin monomers 2/ To stabilize the fibrin mesh network, factor XIIIa (transglutaminase) (activated by thrombin) converts the fibrin monomers to a network of fibrin polymers => The fibrin network is then insoluble and much more difficult to break => When plenty of thrombin is available, a dense fibrin network is formed and traps blood cells and plasma, preventing further bleeding

Question 35

II. Anticoagulation mechanisms 1. What are the factors involved in anticoagulation?

Answer 35

1/ Paracrine factors 2/ Anticoagulant factors – inhibition of the clotting cascade - Tissue factor pathway inhibitor - Antithrombin - Thrombomodulin - Protein C and S 3/ Clearance of activated clotting factors

Question 36

II. Anticoagulation mechanisms 1A. How are paracrine factors involved in anticoagulation?

Answer 36

Endothelial cells generate prostacyclin, which promotes vasodilation and inhibits platelet activation. They also generate NO (stimulated by thrombin), which inhibits platelet adhesion and aggregation through cGMP.

Question 37

II. Anticoagulation mechanisms 1B. How are Tissue factor pathway inhibitor (TFPI) and Antithrombin involved in anticoagulation?

Answer 37

1/ Tissue factor pathway inhibitor (TFPI) => Glycosylphosphatidylinositol (GPI) – anchored to endothelial cell membrane inhibits VIIa 2/ Antithrombin III binds and inhibits thrombin & Xa => Heparins (mast cells, basophils) & Heparin-sulfate (endothelial surface) promotes this, inhibiting coagulation

Question 38

II. Anticoagulation mechanisms 1C. How is Thrombomodulin involved in anticoagulation?

Answer 38

Thrombomodulin is the Glycosaminoglycan on endothelial cells -> Binds and removes thrombin from circulation -> Binds to protein C

Question 39

II. Anticoagulation mechanisms 1D. How is protein C involved in anticoagulation?

Answer 39

Protein C binds to thrombin-thrombomodulin complex -> Activated by thrombin -> Protein C inactivates Va & VIIa

Question 40

II. Anticoagulation mechanisms 1E. How is protein S involved in anticoagulation?

Answer 40

Protein S is a cofactor of protein C

Question 41

III. Procoagulant effects of thrombin 1. What are the 4 Procoagulant effects of thrombin?

Answer 41

1/ Activation of platelets (through PAR-1) 2/ Activation of downstream components in the clotting cascade 3/ Positive feedback at several upstream levels of the cascade 4/ Inhibition of early fibrinolysis

Question 42

III. Procoagulant effects of thrombin 1A. What are consequences of Activation of downstream components in the clotting cascade?

Answer 42

catalyze the proteolysis of fibrinogen -> activates XIII factor

Question 43

III. Procoagulant effects of thrombin 1B. What are consequences of Positive feedback at several upstream levels of the cascade?

Answer 43

- catalyze the proteolysis of prothrombin -> catalyze the activation of XI factor -> catalyze the formation of the cofactors Va and VIIIa

Question 44

III. Procoagulant effects of thrombin 1C. What are consequences of Inhibition of early fibrinolysis?

Answer 44

high local concentration → activation of fibrinolysis-inhibitor

Question 45

IV. What are Anticoagulant effect(s) of thrombin?

Answer 45

Activation of Protein C (through thrombomodulin)

Question 46

V. Fibrinolysis 1. What are definition and role of Fibrinolysis?

Answer 46

1/ Definition: Breakdown of blood clot 2/ Role - Prevents accumulatio of non-aquedate blood clot - Removes already unnecessary blood clot

Question 47

V. Fibrinolysis 2. What is the mechanism of Fibrinolysis?

Answer 47

1/ When a clot is formed, a large amount of plasminogen is trapped in the clot along with other plasma protein 2/ The injured tissues and vascular endothelium slowly release t-PA and u-PA , which coverts plasminogen to plasmin a Ser protease) 3/ Plasmin causes degradation of fibrin

Question 48

V. Fibrinolysis 3. How is fibrinolysis inhibited?

Answer 48

1/ Process: synthesis of plasminogen activator inhibitor such as ⍺2-antiplasmin and ⍺2-macroglobulin - Function: restriction of fibrinolysis 2/ TAFI (thrombin-activatable fibrinolysis inhibitor) - Function: inhibit the conversion of plasminogen into plasmin

Question 49

VI. What are some anticoagulants medications?

Answer 49

1/ vitamin K antagonists (e.g, warfarin) which inhibit FIX, FX, FII, FVII 2/ Direct thrombin inhibitors (e.g, dabigatran) which inhibit FIIa 3/ Anticoagulant outside the body - Heparin - Ca2+ chelators