Haemostasis

Question 1

What is normal haemostasis?

Answer 1

The mechanisms for dealing with clotting and healing

Most of the time the body is in an anti-clotting (anticoagulant) state (but in pregnancy and postpartum women are in a pro-coagulant state to clot immediately after birth, thus at risk of DVT)

Haemostatic mechanisms:

1. maintain the fluidity of circulating blood under normal circumstances
2. Upon vessel injury limit and arrest bleeding by formation of a blood clot
   whilst at the same time maintaining blood flow through the damaged
   vessel
3. Removal of a blood clot upon completion of wound healing

Normal haemostasis requires the interaction of three compartments:
1. The blood vessels
- Blood vessels constrict and mediators tell us to stop
2. The platelets
- form a primary plug (primary heamostatic factors)
3. The coagulation factors (soluble plasma proteins)
- secondary haemostasis to form the clot
→Must be tightly controlled

Question 2

What is the mechanism of haemostasis?

Answer 2

Haemostatic mechanism is three distinct phases:

1. Primary haemostasis
   
   • interactions between blood vessels, platelets and von
     Willebrand factor
2. Secondary haemostasis
   
   • pathways of coagulation to generate a fibrin strand
3. Fibrinolysis
   
   • biochemical system that degrades the fibrin clot

Question 3

What are the features of veins and arteries?

Answer 3

Three distinct layers:

1. Intima is the inner layer of endothelial cells that rests on a basement membrane of microfibrils that are mainly collagen and elastin. Firmly in a resting anticoagulant state, releasing NO. In injury collagen is exposed.
2. The middle layer – Media- smooth muscle layer which allows contraction and relaxation.
3. Outer layers – Adventitia – comprised of collagen layers and fibroblasts that help protect and anchor the blood vessel

Centrifugal force means blood flow is faster in the centre of the blood vessel than at the edges near the vessel wall
Due to this, large cells such as WBC and RBC tend to flow towards the centre and platelets closer to the walls
• Under normal conditions the inner layer ‘intima’ is antithrombotic
• When damaged the sub-endothelium is exposed and is thrombogenic
• Sub-endothelium promotes both primary and secondary haemostasis
• Vascular injury results in the release of endothelin-1, a potent
vasoconstrictor from damaged endothelial cells

Question 4

What is primary haemostasis?

Answer 4

When the endothelium is injured, the pro-coagulant subendothelial matrix (collagen and tissue factors) is exposed and immediately initiates primary haemostasis:

1. Platelet adhesion: Platelets adhere to the exposed subendothelial matrix (directly or indirectly via vWf)
2. Upon tethering platelets are activated, then recruit (and activate) additional platelets to the injured site. Upon activation they become sticky and ‘spread out’
3. Platelet plug formation: Fibrinogen forms bridges between activated platelets to form the platelet plug

Platelets provide the cell base platform for haemostasis to occur

Question 5

What is Von Willebrand’s Factor?

Answer 5

A large glycoprotein synthesised by megakaryocytes and endothelial cells

VWF is the product of the VWF gene on the short arm of chromosome 12

It performs two major roles in haemostasis:

1. Mediates the adhesion of platelets to sites of vascular injury
   
   • primary haemostasis
2. Binds and stabilises the procoagulant protein factor VIII (FVIII)
   
   • secondary haemostasis

Mature protein is 2050-aa long

Released into the circulation through a constitutive pathway (always present in blood vessels) and also upon stimulation

The VWF monomer has multiple domains that deliver its unique
haemostatic abilities, including platelet-binding sites and FVIII-binding site

VWF can multimerize via disulphide bonds at cysteine residues allowing for a range of VWF complexes that span from dimers to multimers that contain >40 subunits

Question 6

How is VWF involved in primary haemostasis?

Answer 6

Circulating VWF is immobilized to the exposed collagen

Glycoproteins on platelets (GPIb) are complementary to VWF

Once tethered via the initial VWF-GPIb interaction, blood flow forces the platelet to roll over

This promotes further initial VWF-GPIb interactions

Question 7

How is resting endothelium maintained in an antithrombotic state?

Answer 7

Resting endothelium provides an environment that inhibits activation of haemostasis:

1. inhibit platelet activation (PGI2, NO, ADPase)
2. inhibit coagulation (heparan sulphate (HS) as a cofactor for antithrombin and thrombomodulin for activation of protein C, which inactivates activated FVa and FVIIIaEC = endothelial cells

When endothelium is damaged, it secretes substances that:
- activate platelets (TXA2 - Thromboxin A2 helps bind to vessels wall,
but Aspirin interferes interacting with COX inhibitors and forming an
irreversible bond., PAF, ET) and bind them to the vessel wall (VWF)
- activate coagulation: tissue factor initiates the formation of
fibrin

Question 8

What is secondary haemostasis?

Answer 8

Platelet plug alone is not enough to stem the blood loss - needs to be reinforced

Secondary haemostasis (coagulation pathway) is usually initiated 
simultaneously with primary haemostasis upon endothelial damage

Result of activation of coagulation pathways is that soluble fibrinogen is converted to insoluble fibrin that reinforces the platelet plug - happens through a series of reactions using clotting factors (procoagulants proteins)

Question 9

What is the coagulation cascade?

Answer 9

Circulating inactive zymogens are sequentially activated to the
active enzyme forms. Consists of two distinct pathways:

1. the extrinsic (tissue factor pathway): requires enzymes and
   factors present in the plasma as well as an activator
2. the intrinsic pathway: requires enzymes and factors all present
   in the plasma

• Both pathways converge on a common pathway to generate the
  fibrin clot

Under normal conditions intrinsic takes lead - contact is required, but everything is already there in the vessel. Whereas the extrinsic pathway requires vessel damage to expose tissue factor 3 (activator)

Question 10

What are the events of coagulation in the EXTRINSIC pathway?

Answer 10

1. Endothelial injury exposes tissue factor (TF) III on fibroblasts to
   the pro-enzyme FVII (circulating ligand in plasma)
2. Binding to TFII activates FVII and promotes its activity
3. The TF-FVIIa complex (with calcium is the extrinsic Xase complex)
   then activates the proenzyme FX to FXa (this is also activated by
   the intrinsic pathway)——> (COMMON PATHWAY)
4. FXa is able to cleave its substrate, prothrombin (FII), to
   thrombin
5. Only small amounts of FXa are generated by this reaction

     ((3 + 7 = 10  —>  10 cleaves prothrombin to thrombin in
                                  the common pathway))

Question 11

What are the coagulation factors involved in the INTRINSIC pathway?

Answer 11

12 -> 12a

12a acts on 11 to give 11a

11a plus Calcium can convert 9 to the intrinsic Xase complex (9a + 8a + phospholipids + calcium)

This complex acts on FX to give FXa (also activated by the extrinsic pathway)

(12 + 11 + 9 + 8 + 10 - COMMON)

Question 13

What are the events in the COMMON pathway?

Answer 13

1. FXa + FVa (via addition of phopspholids and Ca2+ ions) give
   the prothombinase complex
2. The prothrombinase complex is able to cleave its substrate
   prothrombin (FII) to thrombin

((FIBRINOLYSIS))

3. Thrombin can cleave FXIII to FXIIIa AND allows the conversion 
     of fibrinogen (factor 1) to fibrin

4. FXIIIa can then cross link the fibrin

(10a + 5 = prothrombinase complex, this converts prothrombin to thrombin. Thrombin leads to FXIIIa (from FXIII) and fibrin (from fibrinogen). FXIIIa acts on fibrin to give crosslinked fibrin)

Question 14

What is amplification?

Answer 14

The small amount of thrombin (FIIa) generated in the common pathway enters the amplification stage

Thrombin (FIIa) is able to activate FV (for the prothrombinase complex) and FVIII (intrinsic Xase complex) and also platelets

The increased availability of FVa and FVIIIa helps to generate more thrombin for the common pathway

Question 15

What occurs during fibrinolysis/fibrin generation?

Answer 15

Fibrinogen is a glycoprotein synthesised in hepatocytes (90% coag factors are made in the liver - patients with liver disease have acquired haemostatic disorders)

Fibrin is formed from fibrinogen via the action of thrombin (IIa)

Present in high concentration in plasma (3.0 g/L)

Platelets take up fibrinogen from the plasma by endocytosis and store it in their alpha granules

Fibrinogen has a and b and a chains and b chains, thrombin chops them up, then X111a puts them together by covalent links. Plasmin degrades polymers to fragments of diff sizes, including D dimers. We can assay for d dimers to see if there’s been a coagulation event eg DVT.

Question 16

What is fibrinolysis?

Answer 16

Fibrin clots are degraded through proteolysis

Fibrin degraded by the enzyme plasmin

                         PA                      Fibrin  Plasminogen ——> Plasmin      |
                                                      FDP

FDP = Fibrin degradation products, lots of types, specifically D DIMERS

Question 17

What are naturally occurring inhibitors

Answer 17

Naturally occurring inhibitors of coagulation that prevent the initiation or amplification of the coagulation cascade

Factors that promote fragment breakdown and inhibition of coag pathway = feedback mechanism

• alpha-1 antitrypsin
• tissue factor pathway inhibitor
• alpha-2 macroglobulin
• protein-z dependent protease inhibitor
• protein Z

Question 18

What is anti-thrombin?

Answer 18

Serpin protease inhibitor – serine protease inhibitors

Can inhibit all serine proteases - thrombin, IXa, Xa, XIa, XIIa, kallikrein and plasmin (most parts of the pathway but specifically a factor 10 and thrombin inhibitor)

Produced by hepatocytes

Forms stable 1:1 complexes with its target enzymes (coagulation factors its breaking down)

Inefficient in the absence of co-factors

Cofactor activity is provided by heparin sulphate (produced by mast cells. Results in activation, but inefficient alone, 1000x more active why bound. Why MI patients receive heparin to potentiate this pathway)

Question 19

What is the protein C pathway?

Answer 19

Activated protein C (APC) is a serine protease that inhibits Va and VIIIa

Protein C and its co-factor protein S are VITAMIN K dependent proteins

Synthesised in the liver

Exposed collagen in the inner vessel initiates the coagulation cascade. But don’t want full occlusion of the vessel - clotting is limited by APC, its cofactor protein S and vitamin K

Protein C and S are always circulating but inactive

1. Thrombin (FIIa) accumulates on the vessel wall at the site of injury
2. Thrombomodulin (TM) on the endothelial cell forms a complex with thrombin

3. This complex activates PC, which then in association with free 
protein S (PS) inactivates FVa (amplification and extrinsic pathway) and FVIIIa (intrinsic pathway)

Thrombin thus activates it’s own inhibition pathway = feedback loop

Question 20

What is tissue factor pathway inhibitor?

Answer 20

TFPI inhibits the FVIIa-TF complex in the extrinsic pathway

Protein S (PS) serves as a co-factor for TFPI’s inhibition of Xa

The microvascular endothelium is the major source of TFPI

Most TFPI binds to heparin on the surface of endothelial cells

Rest circulates in blood bound to low density lipoproteins

TFPI binds to FXa and neutralizes it, resulting in a conformational change of TFPI that promotes binding of the FXa/TFPI complex with the FVIIa/TF complex in a calcium-dependent reaction.