Abnormal endothelium + Complement & thromb

Question 1

Causes of endothelial injury

Answer 1

Inflammation

      - infectious (bacteria, viruses)
      - non-infectious (immune-mediated)

Apoptosis and Necrosis
- toxins

Oxidative stress (hypoxia/reoxygenation)

Mechanical injury (shear stress, stretch)

Question 2

What is shear stress? What are the effects of shear stress?

Answer 2

Normal, physiological levels of shear stress are
required for endothelial production of NO, expression of genes encoding coag factors , GF, and adhesion molecules.

Shear stress, the frictional force exerted by laminar flow induces extensive changes in endothelial cell behaviour and has been implicated in vasculogenesis, re-endothelialisation of vascular grafts, atherosclerosis, and angiogenesis.

Question 3

Where are you most likely to get athersclerotic plaques? Why?

Answer 3

Endothelial cells in large vessels are subjected to high laminar flow and are well aligned, but in branching points, where shear stress is low or turbulent, endothelial cells lack alignment. These areas are prone to the formation of atherosclerotic plaques .

Question 4

What happens as a result of shear stress? (Physically)

Answer 4

Physically: In order to minimise forces acting on their surface, endothelial cells adapt to shear stress by alignment and migration within the direction of the flow. Failure to adapt to shear stress results in endothelial damage contributing to generation of atherosclerotic plaques or abnormal vessel repair.

Question 5

What happens as a result of shear stress? (Chemically)

Answer 5

Shear stress affects endothelium by activating signalling cascades via mechanosensors eg integrins, stress-activated cation channels, junctional proteins

Early: IP3, DAG = Ca influx = protein kinase activation
Later: TF/gene regulation; both pro-and anti-thrombotic genes are affected as well as some kinases and genes regulating the cytoskeleton. E.g. TF Nuclear factor B is @
Last: Cell alignment & migration and ECM synthesis\degradation.

Question 6

Consequences of shear stress

Answer 6

o Cause vascular remodelling: display a cobblestone morphology
o Cause vasoconstriction
o Cause inflammation and thrombosis
Stretch leads to junctional loss and monocyte adherence (role of HTN in atherosclerosis)

Question 7

Atherosclerosis recap

Answer 7

o Step 1 - fatty streak formation & adhesion of monocytes (reversible)
o Step 2 - intermediate lesion with layers of macrophages & SMCs in the wall
o Step 3 - fibrotic plaque covering a necrotic core (irreversible)
o Step 4 - plaque cap ruptures, exposing the thrombus to blood flow

Question 8

Fate of a thrombus after a plaque ruptures

Answer 8

• Completely/partially resolves
• Progresses and grows because of failure of normal anticoagulant/fibrinolytic mechanisms to cope. This can lead to vessel occlusion.
• Incorporation into vessel wall following fibroblast/ SMC migration
• Fragmentation and embolization

Question 9

What is the complement system?

Answer 9

• Innate immune system
• Was named ‘complement’ originally as is was a component of serum that complemented antibodies in killing of bacteria
• Now know it consists of 30 proteins that contributes to 3g/L to overall serum protein

Question 10

Complement system activation

Answer 10

Can be activated by 3 different pathways that terminate in activating C3 convertase. (Classical, alternative and MBL)

Question 11

Which antibodies can activate complement?

Answer 11

IgM and IgG

Question 12

How do the complement and coagulation system interact?

Answer 12

In vitro data suggest that thrombin, as well as coagulation factors (F) XIa, Xa, IXa, and plasmin, can activate C5 to C5a in the absence of C3

Question 13

How does complement complement the coag system?

Answer 13

Complement activation can amplify coagulation and inhibit fibrinolysis, through C5a, which induces expression of TF and plasminogen activator inhibitor 1 (PAI-1)

Question 14

What was described in 1929 by Ecker and Gross?

Answer 14

The anti-complement activation effect of heparin, first described in 1929 by Ecker and Gross, may be through its inhibitory effects on thrombin and FXa, which are known to cause activation of C3 and C5 producing C3a, C5a and subsequently SC5b-9.

Question 15

What role does complement play in thrombosis?

Answer 15

Disorders of complement activation or dysregulation can result in excessive complement generation and thrombosis, such as in paroxysmal nocturnal haemoglobinuria (PNH) , atypical haemolytic uremic syndrome (aHUS) thrombotic thrombocytopenic purpura (TTP) and antiphospholipid syndrome (APS)

Question 16

What is PNH?

Answer 16

fatal disease of the blood characterized by destruction of RBC by the complement system, a part of the body’s innate immune system. This destructive process occurs due to the presence of defective surface protein DAF on the RBC which normally function to inhibit such immune reactions.

Question 17

Why is DAF defective?

Answer 17

Due to a deficiency of PIG-A (somatic mutation) – required for synthesis of glycosyl-PI (GPI) phospholipid. A lack of GPI-linked proteins eg DAF (i.e CD55) which regulates formation of C3 convertase and CD 59 – restricts formation of MAC and does not allow RBC protection against terminal complement

Question 18

Clinical picture of PNH + epidemiology

Answer 18

Only a minority of affected people have the telltale red urine in the morning that originally gave the condition its name
chronic haemolysis, fatigue, pain, thrombotic events (not understood).
Median age – early 30s; median survival as low as 10-15 yrs

Question 19

Hypothetical mechanisms of PNH

Answer 19

• Release of free hemoglobin, which activates the endothelium and scavenges nitric oxide (NO)
• complement-mediated damage of glycosyl phosphatidylinositol (GPI)-deficient blood cells may result in the release of pro-coagulant microparticles into the circulation and platelet activation
  deficiency of GPI-anchored fibrinolytic factors such as urokinase plasminogen activator receptor (u-PAR), and anticoagulant factors such as tissue factor pathway inhibitor (TFPI) and, potentially, proteinase 3 (PR3),may further disturb the hemostatic balance.

Question 20

PNH treatment mechanism

Answer 20

The alternative pathway of complement is continually activated through a process called “tick-over.” Stressors, such as infections, can intensify this process.

1. DAF and CD59 are complement regulatory proteins that are linked to the surface of RBCs by GPI anchors and protect the RBCs from alternative pathway-mediated lysis.
2. These proteins are absent from some RBCs in patients with PNH.
3. @ of the alternative pathway causes extravascular hemolysis of RBCs by coating the RBC with the C3b fragment, and causes intravascular hemolysis through formation of C5b-9, or the MAC, on the RBC surface.

Eculizumab reduces hemolysis by preventing formation of C5b-9. Compstatin (Peptide inhibitors of C3 activation) prevents the formation of C3b and C5b-9 on PNH RBCs.

Question 21

mutations that have been mapped to aHUS

Answer 21

Factor H -Loss of complement regulation  - 24-28 
MCP (CD46) LCR 5-9
Factor I LCR 4-8 
C3 Gain of complement @ 2-8 6
Factor B Gain of complement @ 0-4

Question 22

Factor H, function

Answer 22

If the regulator factor H binds to C3b, the convertase enzyme is inactivated and no complement activation follows. The simultaneous interaction of factor H with
both C3b and cell surface sialic acids (or possibly glycosaminoglycans [GAGs]) is essential for proper regulation on self RBC, platelets, and EC. If this fails, disbalance between activation and regulation may lead to pathogenesis of atypical HUS.

Question 23

Factor I /MCP function

Answer 23

They inhibit C3bBbP C3 convertase and

C3bC3b BbP C5 convertase