Blood

Ursula Mariani, Blood Forum

Email: [email protected]

1. Structure and Function of Blood, Red Blood Cells and Haemoglobin

• Physiological function of blood in homeostasis
• Structure of blood – plasma contents and cell types
• Synthesis of blood cells
• Regulation of erythropoiesis (synthesis of red blood cells)
• Special features of red blood cells
• Synthesis and breakdown of haemoglobin
• Haemoglobin-oxygen binding
2. Anaemia
3. Blood groups, transfusion and haemolytic disease of the newborn
4. Haemostasis (clotting)
5. Anticoagulation, Fibrinolysis and Pathophysiology of Haemostasis
References
Fundamentals of Physiology, 4th Ed, L. Sherwood
Medical Biochemistry, 2nd Edition, John W. Baynes, Marek H. Dominiczak
 Function of Blood - Why do we need blood?
Blood releases CO2 in the lungs Lungs
Pulmonary Blood takes up O2 in the lungs
capillaries
Arterioles Venules

Pulmonary PULMONARY
CIRCULATION Pulmonary
artery
veins
Systemic
arteries
Systemic
veins Blood takes up
nutrients from
the GIT & storage
heart
SYSTEMIC
tissues
CIRCULATION
Systemic
Tissues capillaries
Blood takes up CO2 & Blood delivers O2 and
wastes in the tissues Venules Arterioles nutrients to all body tissues
Smaller arteries
Tissues convert O2 & nutrients branching off to
Modified from
Sherwood
to ATP, CO2, H2O & wastes supply various tissues Fig. 10-4, p. 264
 Summary: Function of Blood
Blood maintains homeostasis by:

• taking up oxygen from the lungs.

• taking up nutrients from the GIT and from tissues that store nutrients.

• transporting oxygen and nutrients to all cells of the body for metabolism.
ATP
• taking up carbon dioxide and other waste products produced by metabolism.

• releasing carbon dioxide into the lungs.

• releasing other metabolic waste products into the kidneys or liver for excretion.

• transporting many other chemicals, e.g. hormones, between different parts of the
body

These lectures focus on the synthesis, structure and function of red blood cells
which transport oxygen and how we prevent loss of blood (clotting).
 What is blood?
• Fluid
• circulates in vessels
• consists of:
• Plasma
• Water, electrolytes (HCO3-)
• Nutrients (glucose, lipids,
micronutrients)
• Proteins (albumin, inactive
clotting factors & anticoagulants)
• Hormones,
• Waste products (urea, uric acid,
bilirubin)
• “Cells” red blood cells
platelets
white blood cells
 Cellular components of Blood
3 different functions of “cells” lymphocyte
RBC Red blood cell
- Red blood cells (RBC): transport O2 (erythrocyte) Platelet
- Platelets: form clots and prevent
bleeding
- White cells (leucocytes): immunity=
defense of self against external
organisms (parasites, virus, bacteria) or
abnormal self cells (tumors and old or
damaged cells) (discussed in
immunology)
- Polynuclear: neutrophil, basophil,
eosinophil
- Mononuclear: lymphocytes and
monocytes
Origins of different blood cells
 Blood Production site(s)
• Embryo and Foetus
• Liver and spleen

• Adult life: bone marrow

• Bone marrow: in all bones, but in
particular
• flat bones (iliac bones, sternum,
ribs, vertebrae)
• proximal portions of long bones
(tibia, humerus, femur)
 Haematopoiesis (blood cell synthesis)

Pluripotent
haematopoietic
stem cell

In bone
marrow Common myeloid progenitor Common lymphoid progenitor

Megakaryocyte Granulocyte Erythrocyte Monocyte

precursors precursors precursors precursors

Lymphocytes in
lymphoid tissues

In blood Platelets Granulocytes Erythrocytes Monocytes Lymphocytes

Basophil Neutrophil B lymphocyte

Eosinophil T lymphocyte

Monocyte/macrophage

Modified from Sherwood Fundamentals of Physiology Fig. 11-13, p. 312

 Production of Different Blood Cell Types in Words
• All blood cells are made in red bone marrow.
• All blood cells are derived from pluripotent haematopoietic stem cells, also called
haemocytoblasts.
• In addition to replicating themselves, pluripotent haematopoietic stems cells
produce partially differentiated daughter cells that are common myeloid progenitor
cells or common lymphoid progenitor cells.
• Common lymphoid progenitor cells divide and differentiate to ultimately generate
T lymphocytes and B lymphocytes, which are important in the “adaptive” immune
system.
• B lymphocytes make antibodies, which are proteins that bind to pathogens, foreign
proteins and damaged cells and proteins as part of the immune response.
• Common myeloid progenitor cells divide and differentiate to produce precursor
cells for generating red blood cells (erythrocytes), granulocytes, monocytes and
megakaryocytes.
• Granulocytes include neutrophils, basophils and eosinophils, which are important for
fighting infections by different types of pathogens.
• Monocytes mature to become macrophages, which phagocytose pathogens and
damaged or old cells of the body, including erythrocytes.
• Megakaryocytes give off cell particles, called platelets, which are important for
blood coagulation (clotting) in combination with clotting factors in the plasma.
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Regulation of Erythropoiesis
(red blood cell synthesis)
 Haematopoiesis (blood cell synthesis)

Pluripotent
haematopoietic
stem cell

In bone
marrow Common myeloid progenitor Common lymphoid progenitor

Megakaryocyte Granulocyte Erythrocyte Monocyte

precursors precursors precursors precursors

Lymphocytes in
lymphoid tissues

In blood Platelets Granulocytes Erythrocytes Monocytes Lymphocytes

Basophil Neutrophil B lymphocyte

Eosinophil T lymphocyte

Monocyte/macrophage

Modified from Sherwood Fundamentals of Physiology Fig. 11-13, p. 312

 Erythropoiesis - Development and differentiation of red
blood cells
Cell division Death after
DNA must be made Haemoglobin is made ~120 days
Folate & vitamin B12 required Iron is required
Enucleation
RNA

Haematopoietic Common Unipotent Proerythroblasts Erythroblasts Reticulocytes Erythrocytes

stem cells myeloid erythroid stem
progenitors cells

Final maturation
in blood vessels
Self renewal
Haemoglobin Cell division
starts to be continues Reticulocytes
made move into
blood vessels
= ~1% of red
cells
 Notes: Erythrocyte Synthesis
• Haematopoietic stem cells divide to replicate and they to form partially
differentiated common myeloid precursor stem cells.
• Common myeloid stem cells replicate and differentiate to form unipotent stem cells
that start to make haemoglobin and can give rise only to erythrocytes.
• Unipotent stem cells replicate and form proerythroblasts, which continue to make
haemoglobin.
• Proerythroblasts replicate and form erythroblasts, which also make haemoglobin.
• Erythroblasts then expel their nucleus and organelles (mitochondria, endoplasmic
reticulum, etc.) to form reticulocytes, which still contain some RNA.
• Reticulocytes are released from the bone marrow into the blood, where they
mature into erythrocytes (mature red blood cells), which are disk-shaped and have
no RNA or DNA.
• Cell replication requires DNA replication.
• DNA replication requires nitrogenous bases.
• The micronutrients, folate (folic acid) and vitamin B12, are required for
biosynthesis of the nitrogenous bases used to make DNA, especially thymidine
(dTTP). (What happens to blood cells if a person has a deficiency of folate or
vitamin B12?)
• Synthesis of haemoglobin requires the micronutrient, iron. (What happens to blood
cells if a person has a deficiency of iron?)
• Erythrocytes die after ~120 days in the blood.
Clinical Case
• A 50-year old man has had kidney disease for years of years and it
getting worse.
• When he comes to the clinic, his skin is pale, he is out of breath and
tires very easily.
• How could his kidney disease explain his symptoms?
 Regulation of Erythropoiesis (red blood cell synthesis)

If there are too few RBCs:

- Too little oxygen delivery
- Decreased metabolism
- Tissue cell death

If there are too many RBCs (polycythaemia):

- Enough oxygen present, but
- Increased blood viscosity
- Decreased flow (blood clots and other problems)

How do we tell the bone marrow to increase or decrease

synthesis of RBCs?
RBC oxygen and erythropoietin
 Regulation of red blood cell synthesis

2 EPO secreted
Kidney

Erythropoietin
1
low oxygen
detected
3 EPO
Red bone
stimulates
5 More oxygen marrow
RBC
decreases EPO synthesis
secretion

4 More RBCs carry

more oxygen

Erythrocytes
Modified from Sherwood Fundamentals of Physiology Fig. 11-3, p. 301
 Erythropoietin (EPO)
• Erythropoietin is the main hormone that stimulates erythrocyte
(red blood cell) production.
• Made in the KIDNEY (90%) and liver (10%).
• Too little oxygen in the blood leads to low tissue oxygenation
(hypoxia).
• Hypoxia of the kidneys stimulates erythropoietin production.
• EPO is produced in cells bordering renal arterioles that are
immediately sensitive to O2 changes in RBCs.
• Release of EPO from kidney stops once tissue oxygenation is back
to normal.
• Increased EPO release can be elevated for long periods of time to
sustain long periods of exposure to hypoxia (e.g. in high altitude).
• If erythropoietin cannot be produced: lower stimulation of
erythrocyte production  anaemia.
 Regulation of Erythropoiesis by Erythropoietin

DNA synthesis
Enucleation

Haematopoietic Common Unipotent Proerythroblasts Erythroblasts Reticulocytes Erythrocytes

stem cells myeloid stem cells
progenitors

Other growth factors Erythropoietin

Increased release
stimulate cell (EPO)
of reticulocytes
division/replication stimulates cell
into blood (~5
to increase all blood division/replication
days)
cells of RBC precursors
only
Insufficient erythropoiesis
results in anaemia
Factors that decrease tissue oxygen (cause hypoxia)
and stimulate production of Erythropoietin (EPO)

• Anaemia = below-normal O2-carrying capacity of

the blood, characterized by a low haematocrit
and/or low haemoglobin.
• Poor blood flow: e.g. heart failure, low blood
pressure, haemorrhage.
• Pulmonary disease: decreased O2 entry from lung
to blood.
• High altitude: O2 in the air is decreased.
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Function of Red Blood Cells and

Haemoglobin Synthesis & Metabolism
 Red blood cells (erythrocytes)
• Whole blood allowed to stand or
centrifuged separates into:
• cellular components (at the bottom of the tube)
• leukocytes on top of erythrocytes
• plasma (on top of the tube).
• Cellular component (mostly red blood cells)
= 45-55% (in Johannesburg) of the whole
blood sample volume
 Percentage volume of RBCs in blood =
Haematocrit.

= ~5 x 1012 RBCs/litre of Blood (~5 million

million/l) (more in men than women) Essentials of physiology
4th edition– L Sherwood
(compare with ~5 x 109/litre WBCs= ~5
thousand million/l)
 Erythrocytes
• No nucleus (cannot replicate)
• No organelles (no protein synthesis, no oxidative
metabolism)
• Enzymes are present for anaerobic glycolysis, pentose
phosphate pathway (anti-oxidant), converting CO2 to
H2CO3 (carbonic acid, carbonic anhydrase)
• Disk-like biconcave shape
 Erythrocytes: important properties
• Biconcave disk shape provides a large surface area for O2 to
diffuse into the cell.
• Shape enables O2 to diffuse rapidly to the whole cell.
• Very flexible “loose” membrane allows RBCs to deform and
squeeze through vessels less than 3 μm in diameter (RBC
diameter = 7.8 μm)
• RBCs contain haemoglobin.
• 12 – 16 g/decilitre of blood (1/10 litre)
= 120 to 160 g/l

Why does haemoglobin need to be

“packaged” inside cells?
 Haemoglobin Synthesis

Succinyl
-CoA Fe2+
pyrrole
protoporphyrin
Haem (porphyrin + Fe2+)
1 Haem + 1 globin polypeptide chain (alpha or beta) = 1 subunit
of haemoglobin
1 molecule of haemoglobin contains 4 haemoglobin subunits
1 molecule of haemoglobin contains :
-4 haems
-4 polypeptide chains
-4 Fe++
2 alpha chains + 2 beta chains  haemoglobin A
 Haemoglobin
Α-globin chains (red)
What happens
in iron
deficiency?

β-globin chains (blue)

Iron (orange) What happens
to haemoglobin
Haem rings (green) when RBCs die?
https://commons.wikimedia.org/wiki/File:1GZX_Haemoglobin.png#/media/File:1GZX_Haemoglobin.png
 Erythrocyte and Haemoglobin Breakdown
• Lifespan of an erythrocyte ~120 days
• Membranes become fragile and break when erythrocytes go
through small vessels in the spleen (3 μm vs. 8 μm of diameter).
• When the cells break, free haemoglobin is bound by a plasma
protein, haptoglobin.
• Macrophages phagocytose haptoglobin-haemoglobin complex.
Fe2+ is released from haemoglobin & stored for reuse.
Porphyrin is converted to BILIRUBIN (yellow pigment).

Fe2+
Picture By Stefcho2 - Own work, Public Domain,
https://commons.wikimedia.org/w/index.php?curid=9872836

Haem = red/bluish-red Bilirubin = yellow

 Erythrocyte and Haemoglobin Breakdown
• Bilirubin is released into the blood
• Bilirubin is then transported to- and metabolized by- the liver and
intestines.
What happens if there is excessive breakdown of RBCs?
to:
plasma haptoglobin
plasma bilirubin

• Plasma haptoglobin concentration decreases.

• Liver’s capacity to metabolise bilirubin is exceeded and bilirubin
accumulates in blood and tissues  skin and mucosa and eye
conjunctivae become yellow (icteric) jaundice
http://cp4x3a.xara.hosting/jaundice.htm

https://www.healthunbox.com/en/jaundice/
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Function of Haemoglobin
 How does Haemoglobin (Hb) Bind Oxygen?
• 98% of O2 in blood is bound to Hb
• 1 molecular oxygen (O2) binds to Fe2+ in each HAEM
haem group.
• 4 O2 + Hb --> Hb(O2)4
• Binding is non-covalent and reversible
• Fe2+ is not oxidised (to Fe3+) (methaemoglobin).
• O2 binds to Hb easily (tightly) in the pulmonary
capillaries (high [O2]).
• O2 is released easily in the capillaries of the
peripheral tissues (low [O2]).
• When O2 is bound to Hb  blood appears red
(arteries)
• When O2 dissociates from (leaves) Hb  blood
appears blueish (veins)
How does Hb bind O2 tightly in the lungs, then release it easily in the tissues?
Cooperative Binding of Oxygen to Haemoglobin (Hb)
• Each subunit of Hb can exist in two different protein conformations, one with
low affinity for (looser binding of) O2 and 1 with higher affinity for (tighter
binding of) O2.
• The low affinity conformation is favoured (stabilised) when O2 is not bound and
the high affinity conformation is favoured when O2 is bound.
• I.e. binding of O2 changes the conformation of the subunit from the low affinity
conformation to the high affinity conformation.
• Binding of O2 to one or two subunits of the four subunits of Hb favours the
conversion of all four subunits to the high affinity conformation.
• This increases the binding affinity of the “empty” subunits for O2 and more O2
binds. (So the Hb molecule is “full”.)
O2 O2 O2 O2 O2 O2 O2
O2 O2

No O2 bound, O2 binding O2 binding O2 binds more

all subunits changes to favours high to high affinity
low affinity high affinity affinity conformation
conformation conformation conformation of subunits
empty subunits

• Cooperative binding allows Hb to bind lots of O2 in the lungs (high [O2], high
affinity) and release it in the tissues (low [O ], lower affinity, easier to “let go”).
Cooperative Binding of Oxygen to Haemoglobin (Hb)

% Hb subunits with oxygen bound

O2 concentration
in tissues O2 concentration
in lungs

Concentration of oxygen

P50

Figure modified from https://commons.wikimedia.org/wiki/File:Hb_saturation_curve.png

 Other things that bind to Haemoglobin (Hb)
also affect Oxygen Binding Affinity
• CO2 binds to the globin (not haem) and stabilises the low affinity
conformation of Hb  increased CO2 in tissues increases release of O2.
• H+ (hydrogen ions/protons) bind to negatively-charged amino acids of the
globins and stabilise the low affinity conformation of Hb  tissues
produce lactic acid during anaerobic respiration and RBCs convert CO2
to carbonic acid. Binding of H+ from both acids increases release of O2
from Hb in the tissues.
• 2,3-bisphosphoglycerate (2,3-BPG) binds to the globins and stabilises
the low affinity conformation of Hb  2,3-BPG is produced by RBCs in
hypoxia and increases release of O2 from Hb in the tissues.
 ↑ pH (↓H+ ions)
↓ CO2 Allosteric effects on
↓ 2,3BPG
↓ body temperature
Haemoglobin Oxygen Binding
Favour high affinity Hb
conformation,
increase Hb-O2 affinity,
% Hb subunits with oxygen bound left-shift curve,
O2 stays bound.

↓ pH (↑H+ ions, Bohr effect)

↑ CO2 (Haldane effect)
↑ 2,3-bisphosphoglycerate (2,3BPG,
low oxygen, anaemia, high
altitude)
↑ body temperature
Favour low affinity Hb conformation,
decrease Hb-O2 affinity,
right-shift curve,
O2 comes off. diagram modified from
https://derangedphysiology.com/mai
n/core-topics-intensive-care/arterial-
blood-gas-
interpretation/Chapter%204.0.5/facto
Concentration of oxygen rs-which-influence-affinity-
haemoglobin-oxygen
Allosteric effects of Carbon Monoxide on Haemoglobin Oxygen Binding

400 500 600

P50

Normal air 100% O2

High pH, low CO2, low 2,3-BPG, low ~20% O2
temperature
Low pH, high CO2, high 2,3-BPG, high
Figure modified from http://what-when-
temperature how.com/acp-medicine/lung-function-
assessment-and-thoracic-diagnostic-
techniques-part-1/
 Other things that bind to Haemoglobin (Hb)
affect Oxygen Binding Affinity
• CO2 binds to the globin (not haem) and stabilises the low affinity
conformation of Hb  increased CO2 in tissues increases release of O2.
• H+ (hydrogen ions/protons) bind to negatively-charged amino acids of the
globins and stabilise the low affinity conformation of Hb  tissues produce
lactic acid during anaerobic respiration and RBCs convert CO2 to carbonic
acid. Binding of H+ from both acids increases release of O2 from Hb in the
tissues.
• 2,3-bisphosphoglycerate (2,3-BPG) binds to the globins and stabilises the
low affinity conformation of Hb  2,3-BPG is produced by RBCs in
hypoxia and increases release of O2 from Hb in the tissues.
• CO (carbon monoxide) binds to haem iron with higher affinity (more tightly)
than O2 , preventing O2 binding and stabilises the high affinity conformation
preventing O2 release  cell death due to lack of O2 and ATP.
• NO (Nitric oxide): binds to haemoglobin in the lungs, then dissociates in
peripheral tissues  dilates the arterioles  more O2 gets to tissues.
 Summary Erythrocytes and Haemoglobin
• Erythrocytes make up ~50% of blood volume.
• Their biconcave disk shape is optimised for transfer of O2, with no organelles,
only a small number of enzymes (glycolysis, antioxidant & carbonic anhydrase)
and a large amount of haemoglobin.
• Haemoglobin consists of four subunits, each made up of a globin polypeptide
(alpha or beta) and a haem that consists of a porphyrin ring and ferrous (Fe2+)
iron.
• One molecule of oxygen (O2) binds non-covalently to each Fe2+ ion in the haem.
• Binding of O2 to one subunit of haemoglobin allosterically increases O2 binding
by the other subunits in the molecule.
• Binding of CO2, H+ and 2,3-bisphosphoglycerate and increased temperature
allosterically decrease haemoglobin binding affinity for O2, stimulating release of
O2 in peripheral tissues, whereas higher pH and lower CO2 concentrations in the
lungs increase affinity for O2 and increase haemoglobin binding of O2.
• Most CO2 produced by peripheral tissues is converted carbonic acid (H2CO3) and
transported as HCO3- in the plasma, while only a small amount is transported
bound to haem.
• CO is toxic because it binds to haemoglobin more tightly than O2, preventing O2
binding and stabilises the high affinity conformation preventing O2 release.
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]
1. Structure and Function of Blood, Red Blood Cells and Haemoglobin
2. Anaemia
• Definition, signs and symptoms
• Primary mechanisms
• Analysis of anaemia and calculations
• Decreased erythropoiesis – vitamin B12 and folate deficiency,
leukaemias and toxins
• Decrease haemoglobin synthesis – iron deficiencies and globin
mutations
• Loss or destruction of red blood cells.
3. Blood groups, transfusion and haemolytic disease of the newborn
4. Haemostasis
5. Anticoagulation and Fibrinolysis
References
Chapter 3, Overview of Anemias in Pathphysiology of Blood disorders Bunn & Furie, available through the Wits
health sciences library at https://0-
accessmedicine.mhmedical.com.innopac.wits.ac.za/content.aspx?sectionid=137394689&bookid=1900&jumpsection
ID=137394734&Resultclick=2#1150585730
 Definition and Signs & Symptoms of Anaemia
Definition
Anaemia is a lower than normal O2-carrying capacity of the
blood that is characterized by a low haematocrit and/or low
haemoglobin concentration.

Signs and symptoms

• Pallor, discoloration of skin and mucosa = pale skin, pale
conjunctivas, pale gums, pale nail beds. (shunting of blood)

• Shortness of breath (dyspnea) even with very small effort,

intense exercise usually not tolerated (heart attack symptoms
such as chest pains) (low oxygen/hypoxia)

• Fatigue (decreased ATP synthesis).

https://www.memorangapp.com/flashcar
ds/136011/CS+9%3A+Geriatric+Anemia/
 Three Major Mechanisms of Anaemia

• Decreased red blood cell (erythrocyte) replication

• Decreased haemoglobin synthesis

• Excessive loss of red blood cells (erythrocytes)

 How do we Measure Anaemia?
Haemoglobin concentration
• grams of haemoglobin per dL (1/10 litre) of Anaemia
whole blood (cells + plasma)  LOW (definition)

Haematocrit
• red blood cell volume as % of volume of whole
blood  LOW
Mean Cell Volume (MCV)
• = haematocrit (%) x 10/RBC count (x1012/litre) Normocytic=Normal MCV
Microcytic=Low MCV
• = size (volume) of the red blood cell (in Macrocytic=High MCV
femtolitres (fl), 1 fl = 10-15 litres)
• Normally 80 – 100 fl per cell.
• varies according to the type of anaemia
 How do we Measure Anaemia?
Mean Cell Haemoglobin (MCH)
• Amount of haemoglobin in each cell
• = Haemoglobin (g/dL) x 10/RBC count
(x1012/l)
• = Hb/cell (in pg = 10-12 grams)
• varies according to the type of anaemia
Normochromic=
normal MCH or MCHC
Mean Corpuscular Haemoglobin Hypochromic=
Concentration (MCHC) Low MCH or MCHC
• Haemoglobin concentration inside RBCs
• = Haemoglobin (g/dl) x 100/haematocrit (%)
• = haemoglobin concentration per dL of red
blood cells (in g/dL)
• varies according to the type of anaemia
 Erythrocyte indices (Hemoglobin,
Hematocrit, MCV, MCH & MCHC)

https://www.youtube.com/watch?v=QUHqYVK
-Nhg
 How do we Measure Anaemia?
Reticulocyte count (regenerative vs non-regenerative)
• Anaemia causes hypoxia. Hypoxia stimulates erythropoietin secretion.
Erythropoietin stimulates erythropoiesis. If kidneys and bone marrow
function correctly  erythropoiesis will be hyperstimulated
• Hyperstimulation increases reticulocytes circulating in the blood (>1-2%)
• This is called a regenerative anaemia
reticulocytes

• When bone marrow cannot increase reticulocyte synthesis (i.e. when renal
function, erythropoiesis or Hb synthesis is decreased) reticulocyte counts are
normal or low.
• This is called a non-regenerative anaemia
 Anaemia caused by decreased
erythrocyte production
Peripheral: kidneys produce insufficient erythropoietin
(EPO) (chronic renal disease)
Central: the bone marrow cannot produce cells even
when erythropoietin (EPO) is high
= Non-regenerative: decreased cell replication causes
decreased reticulocytes
 Decreased Red Cell Production
• Lifespan of erythrocytes: 120 days
• Lifespan of platelets: 11- 20 days
• Lifespan of white cells: 1- 4 days

• Blood cells need to be replenished constantly

• Cells must reproduce VERY rapidly
• Nucleotides (with bases A, C, G & T) are needed
• Nutrients are needed to synthesise nucleotides
(vitamin B12 and folate)
Peripheral causes of Decreased Red Cell Production
• Kidney disease  Kidney Decreased EPO
failure secretion
decreased ability to
synthesise and secrete
erythropoietin (EPO)
• Lack of erythropoietin 
decreased erythropoiesis low oxygen Decreased RBC
detected
• Decreased erythropoiesis synthesis

 fewer reticulocytes Red bone

marrow
Fewer RBCs carry
• Fewer reticulocytes  less oxygen
fewer RBCs
• RBCs have normal size
(MCV) and normal Hb
content (MCH and MCHC).

•  Anaemia is normocytic, normochromic and non-regenerative (low

reticulocyte %).
Decreased Erythropoiesis due to B12/folate deficiency
• Vitamin B12 and folates (folic acid) are essential for DNA synthesis
(thymidine triphosphate, T of the A, G, C, T bases of DNA)
• Too little folate or B12 too little new DNA  erythrocyte precursor cells
cannot divide to form erythroblasts 
•VOLUME (SIZE) of the erythrocyte increases  increased MCV =
macrocytic
•Fewer reticulocytes released  decreased reticulocyte % = non-
regenerative
•erythrocyte number decreases
•cells become oval shaped and red blood cell membrane is fragile 
haemolysis  fewer cells and jaundice
•More cells need to be made, but B12 and folate deficient  leads to
decreased haematocrit and decreased haemoglobin  anaemia
So even though there is more haemoglobin per erythrocyte, the decreased
number of erythrocytes/litre of blood  decreased haemoglobin/dl of
blood and decreased haematocrit (= anaemia)
Vitamin B12 Deficiency & Pernicious Anaemia

• Vitamin B12 must be obtained from food (mostly

animal-based foods and bacteria)
• Vitamin B12 is absorbed bound to protein called
Intrinsic Factor which is made by the stomach.
• In pernicious anaemia, patients develop ANTIBODIES
against Intrinsic Factor  too little Intrinsic Factor in
the gastric glands too little absorption of Vitamin
B12
• Takes up to 5 years to deplete large stores of vitamin
B12.
= Non-regenerative macrocytic anaemia
 Folate/Folic acid Deficiency Anaemia

• Folate (vitamin B9) in green vegetables, fruits, liver

and meats (but destroyed by HEAT).

• Folate deficiency can be caused by malabsorption e.g.

alcoholism, GIT disease, pregnancy, cancer or
chemotherapy

= non-regenerative macrocytic anaemia

Decreased Cell Division and Replication due to
Leukaemia or Toxins
• Leukaemia cells divide very rapidly and invade bone marrow
• Toxins damage bone marrow cells and cause fibrosis
• Cell invasion and fibrosis decrease the number of haematopoietic
stem cells
• Fewer erythroblasts are produced fewer reticulocytes  fewer
erythrocytes  normocytic normochromic anaemia
• decreased reticulocytes = non-regenerative anaemia
• Usually, decreased production of all blood cells:
• fewer normal white cells
• fewer platelets
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Anaemia Caused by
Decreased Haemoglobin
Synthesis
Iron deficiency
Anaemia of chronic disease
Genetic: Thalassemia
= Non-regenerative
 Decreased Haemoglobin Synthesis

Iron is required for

haemoglobin synthesis
Enucleation
RNA

Haematopoietic Common Unipotent Proerythroblasts Erythroblasts Reticulocytes Erythrocytes

stem cells myeloid erythroid stem
progenitors cells

Final maturation
in blood vessels

Haemoglobin
starts to be Reticulocytes
made move into
blood vessels
= ~1% of red
cells
 Decreased Haemoglobin Synthesis
• If haemoglobin cannot be made, each erythrocyte will be
smaller. (microcytic anaemia)
• This can happen in 3 circumstances:
• Genetic: mutation in the alpha or beta globin chain leading to
lower levels of globin  decreased haemoglobin/cell =
thalassemia
• Iron deficiency
• Anaemia of chronic inflammatory diseases
Iron Deficiency & Anaemia of Chronic Disease
• Both anaemias are due to iron not getting to the bone
marrow (for different reasons)
• Insufficient iron in bone marrow  decreased
haemoglobin synthesis
• Decreased haemoglobin in each red blood cell
 decreased ‘colour’ of the cell = hypochromic
(decreased MCH, MCHC)
decreased size of cells = microcytic (decreased MCV)
Decreased haemoglobin concentration and haematocrit
(= anaemia)
Iron Deficiency & Anaemia of Chronic Disease
• Iron, released by macrophages when they breakdown
haemoglobin or absorbed from food, is transported in the
blood as transferrin. Iron is then transferred to bone marrow
or stored in the liver as ferritin.
• In iron deficiency there is not enough iron stored in the body
• Ferritin (iron storage protein) is low
• In anaemia of chronic inflammatory disorders, a protein
made during chronic inflammation (hepcidin) prevents
release of iron from macrophages and liver  iron is
sequestered (stuck) in macrophages and liver as ferritin 
stored iron cannot be used by bone marrow to synthesize
haemoglobin.
• Ferritin is high.
 Iron Deficiency Anaemia
Insufficient intake
global malnutrition or special diets (vegetarian/vegan).

Excess loss
• chronic bleeding in the intestines (ulcer, cancer)
• heavy menses due to fibroids/coagulation disorders.

Decreased haemoglobin
Decreased MCV, MCH and MCHC
Decreased haematocrit and haemoglobin concentration
Microcytic, hypochromic anaemia
pallor of skin + mucosa, breathlessness, fatigue
 Summary of Non-Regenerative Anaemias
1. Lack of erythropoietin, e.g. due to renal disease, causes
decreased erythropoiesis, which results in a decreased numbers
of reticulocytes and RBCs, with normal size and Hb content. 
Anaemia is normocytic, normochromic and non-regenerative.
2. Decreased ability to synthesise DNA due to folate or vitamin
B12 deficiency  Anaemia is macrocytic, hypo/normochromic
and non-regenerative.
3. Decreased pluripotent haematopoietic stem cells due to
leukaemia or fibrosis  Anaemia is normocytic, normochromic
and non-regenerative.
4. Decreased haemoglobin synthesis from decreased availability
of iron or decreased globin protein synthesis  Anaemia is
microcytic, hypochromic and non-regenerative.
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Regenerative anaemias
Excessive loss of RBCs
• Haemorrhage (acute)
• Haemolysis

Increased EPO stimulates increased reticulocytes

Excessive Blood Loss: Haemorrhage & Bleeding
• Blood loss  fewer RBCs in blood  lower O2 carrying capacity
 tissue hypoxia
• Hypoxia in kidneys stimulates erythropoietin production
• Erythropoietin stimulates erythropoiesis  ↗ reticulocytes in the
blood
• Acute heavy blood loss (haemorrhage)  increased reticulocytes
appear in the blood after 2-3 days
Excessive Blood Loss: Haemorrhage & Bleeding

However
• Chronic blood loss (intestinal bleeding, eg, ulcers or
cancer)
• Initially (the first 2-3 months) high erythropoiesis 
>2% reticulocytes in blood
• Prolonged loss of RBCs  loss of iron
• Chronic loss of iron (over several months)  depletes
iron stores  iron deficiency
• Iron deficiency  decreased ability to make RBCs 
iron deficiency anaemia (non-regenerative)
 Excessive RBC Destruction: Haemolysis
• Red blood cell destruction  release of haemoglobin (Hb) and
enzymes (lactate dehydrogenase, LDH) into plasma
• increased LDH in plasma

• Increased Hb outside of erythrocyte  binds haptoglobin

• Macrophages phagocytose Hb-haptoglobin  decreased haptoglobin
in plasma (very specific test for haemolysis)

• Increased macrophage Hb  increased bilirubin production and

release
• Excess bilirubin in the plasma  yellow skin, mucosae and sclerae
(jaundice) and dark urine Size and colour of erythrocytes?
• Fewer RBCs  hypoxia  erythropoietin  erythropoiesis 
excess release of more immature RBCs, i.e. reticulocytes (>2%).
Causes of Haemolysis & Haemolytic Anaemia
Genetic Disorders
• Sickle cell anaemia
Mutation of β-globin gene causes Hb to form crystals inside the
RBC (at low O2)
erythrocyte becomes “sickle” shaped
RBCs cannot pass through narrow capillaries
crystals damage the erythrocyte membrane
many red blood cells break = haemolysis
• Glucose-6-phosphate dehydrogenase (G6PD) deficiency
Mutations of G6PD decrease ability of anti-oxidant enzyme to
protect against drugs that oxidise Hb and the RBC membrane.
 Using oxidising drugs makes RBCs fragile  haemolysis
Infections
• Malaria
Malaria parasites infect RBCs and lyse the cells  haemolysis
Antibodies
• Blood transfusion reactions & haemolytic disease of the newborn
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

1. Structure and Function of Blood, Red Blood Cells and

Haemoglobin
2. Anaemia
3. Blood groups, transfusion and haemolytic disease of the newborn
• Blood group antigens
• ABO blood groups, antibodies and transfusion reactions
• Rhesus blood groups, antibodies and erythroblastosis foetalis
4. Haemostasis
5. Anticoagulation, Fibrinolysis and Pathophysiology of Haemostasis

References
Fundamentals of Physiology, 4th Ed, L. Sherwood
 What are blood groups?
• Blood groups are “antigens” on the surface of red blood cells.

• Antigens are molecules that are bound by antibodies.

• Antibodies are proteins that are made by B-lymphocytes as part of

the immune response. They bind to specific “foreign” molecules,
called antigens, and stimulate removal of the antigen by the immune
system.

• Blood group antigens are modified sugar molecules (α-N –

acetylgalactosamine or α-D-galactose) that are attached or NOT to
the surfaces of red blood cells (and other cells), depending on the
ABO genes that the person inherits.
• OR
• Blood group antigens are proteins that are present or NOT in the
membrane of red blood cells (not other cells) depending on rhesus
genes.
 ABO Blood Types

A antigens B antigens A & B antigens

A A A A BAB A
A A A B B
B B B
A A A B B
A B ABA
B B A
Group O Group A Group B Group AB
erythrocyte erythrocyte erythrocyte erythrocyte

• Erythrocytes of blood type O have neither A nor B antigens.

• Erythrocytes of blood type A have A antigen

• Erythrocytes of blood Type B have B antigen

• Erythrocytes of blood type AB have both A and B antigens

• Presence of antigens is determined genetically.

 Genetic Determination of A, B and O Blood
Groups
• The ABO gene (chromosome 9) is an enzyme (glycosyltransferase)
that attaches a sugar molecule to the cell surface.

• 3 variants of the ABO gene.

• A variant/allele makes an enzyme that attaches one sugar (α-N
–acetylgalactosamine) to the cell surface  the A antigen.

• B variant/allele makes an enzyme that attaches a different

sugar (α-D-galactose) to the cell surface  the B antigen.

• O variant/allele makes an enzyme with NO activity.  no

sugar  no antigen attached.

How does blood group AB happen?

Genetic Determination of A, B and O Blood Groups
Two copies/alleles of the ABO gene
Genotype ABO Blood Group
AA A
AO A
AB AB
BB B
BO B
OO O
What will
Group O, most common~ 50%
Group A, 30-40% happen if AO
Group B, varies with ethnicity (8-25%) and BO have a
Group AB : 3-5% baby?
 Anti-Blood Group Antibodies
• We make antibodies against antigens NOT PRESENT
on our own erythrocytes.
IgM
• Shortly after birth.
• Antibodies are Immunoglobulin M (IgM) – bind ten
antigen molecules.
Group O people make antibodies against A and B Anti-A antibodies Anti-A antibodies
antigens = anti-A and anti-B antibodies A A A B B
A A A B B
B
A A A B B
Anti-A antibodies
Group A people make antibodies against B antigens B B
A A A B B
A A A = anti-B antibodies B
A A A B B

NO antibodies
A
A BAB B Group AB people make NO ABO blood group antibodies
A BAB B
A
A B B ABA A B B ABA
A A
 Peculiarity of Anti-ABO Blood Group Antibodies
• Usually antibodies are made only after exposure to antigen.

• But, anti-A and anti-B antibodies are made in the first months of life.

• Symbiotic intestinal bacteria have (sugar) molecules similar to A and

B antigens.

• Bacteria stimulate development of B-lymphocytes that make anti-A

and/or anti-B antibodies.

• If antigens are present on own cells = self, B-lymphocytes making the

matching antibodies are destroyed.

• If antigens are NOT present on cells = NON-self, B-lymphocytes

making the matching antibodies are retained and antibodies continue
to be made.
What does this mean for blood transfusions?
• Group O - both anti-A and anti-B antibodies  attack cells with
A and/or B antigens  can receive only Group O. No antigens 
not attacked by anti-A or anti-B antibodies  “universal” donor.
• Group A - anti-B antibodies  attack cells with B antigens 
can receive Group A or Group O. A antigens are attacked by anti-
A antibodies  can donate to Group A and AB.
• Group B - anti-A antibodies  attack cells with A antigens 
can receive Group B or Group O. B antigens  can donate to
Group B and AB.
• Group AB - no antibodies  Universal transfusion acceptors (can
take transfusions of packed red cells from groups A, B, AB and
O). A and B antigens  Can only give to group AB.
What does this mean for blood transfusions?
 What happens in a mismatched transfusion?
• E.g. Group A person transfused with AB blood
• Group A person’s anti-B Antibodies will recognize the B
antigen on the AB erythrocyte as foreign/non-self.
• The Anti-B Antibodies will coat the B antigens of the AB
erythrocytes Receiver’s Anti-B
antibodies

A A
A
A A
A A
Group A people make anti-B antibodies AB BABB B
B B
A A A A AB
BAB B A
Recipient’s Group A Transfused Group AB
erythrocyte erythrocyte

• Antibody coating signals the immune system – destroy

and remove.
What happens in a mismatched transfusion?
• Recipient's antibody recognition of foreign antigen causes a
transfusion reaction:
1. Erythrocytes agglutinate (join together) – IgM has 10
binding sites.
2. Destruction of antibody-coated erythrocyte by immune
system (lysis by complement system = haemolysis, and
phagocytosis)

Agglutinated red cells

 What happens in a mismatched transfusion?
• Clumping of erythrocytes leads to mini
‘strokes’ (blocked capillaries) 
disseminated intra-vascular coagulation
• Massive destruction of erythrocytes 
massive release of intracellular
haemoglobin  renal toxicity  acute
renal failure
• Antigen-Antibody reaction stimulates
cascades of pro-inflammatory
messengers  “cytokine storm”  fever
•  death or severe damage (stroke, renal
failure).
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Rhesus Blood Group System

• The D antigen is a membrane protein in erythrocytes
• People who have the D antigen are Rhesus positive (Rh+)
• People who don’t have it are Rh negative (Rh-)
• Anti-D antibodies are only produced in Rh- people after
exposure to Rh+ blood
• 1st exposure  sensitization
• 2nd exposure  antibodies bind the Rh+ red blood cells
 haemolysis of Rh+ cells
Why is the Rhesus blood group important?
 Rhesus Blood Group System

Mother Rh- and fetus Rh+

 Rhesus Blood Group System
Mother Rh- and foetus Rh+
• Foeto-placental barrier prevents foetal red blood cells from entering
maternal blood
• BUT maternal antibodies can enter the foetus.
• 1st pregnancy of Rh- mother  maternal immune system does not
“see” foetus’s blood before the birth
• Birth  maternal blood comes into contact with foetal Rh+ red cells
• Mother develops Anti-Rh antibodies
• Next pregnancy with Rh+ foetus  mother’s anti-Rh antibodies
react will foetal RBCs during pregnancy  haemolysis 
erythroblastosis foetalis or haemolytic disease of the newborn
•  haemolytic anaemia and severe jaundice in the second (and any
subsequent) baby  terminal heart failure or mental damage
 Genetics of Rhesus Blood Groups
• 2 variants of the Rhesus D gene.
• D variant/allele makes a protein that is expressed in the RBC
membrane  the D antigen  Rh+.
• d variant/allele makes a protein that is NOT expressed  NO
antigen  Rh-.

Genotype Rhesus Blood Group

DD + (positive)
Dd + (positive)
dd - (negative)

• D is dominant.
• Mother dd and father Dd  50% chance foetus is Rh+
• 55% of Rh+ fathers are Dd, only 45% are DD = 100% chance foetus is Rh+
 Testing before Transfusion
• Blood typing
• red blood cells are diluted with saline
• one portion is mixed with Anti-A antibodies
• one portion with Anti-B antibodies
• one portion with Anti-D antibodies
• look for clumping agglutination
• No clumping  antigen is not present.
• Verify compatibility
• Mixing red blood cells from potential donor with plasma from the
recipient.
• If no clumping  donor blood can be given to the recipient.

• Many other blood group systems

 tested before transfusions
 tested especially before organ and bone marrow
transplants to avoid rejection
Example
Agglutination reactions in Patient and a potential Donor
+ : agglutination occurred when antiserum was added to the blood
- : agglutination did not occur when antiserum was added to the blood

PATIENT DONOR
Anti-A + +
Anti-B - +
Anti D - +

1- does this picture belong to the donor or the patient?

2- Determine the blood group of the patient and the blood group of donor

3- Can you safely transfuse the blood of the donor into this patient? Explain why.
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

1. Structure and Function of Blood, Red Blood Cells and

Haemoglobin
2. Anaemia
3. Blood groups, transfusion and haemolytic disease of the newborn
4. Haemostasis – clot formation
• Vascular spasm
• Platelets
• Cell-based coagulation and fibrin clot
• Clot retraction and fibrinolysis
5. Haemostasis – laboratory analysis, anti-coagulation systems and
pathophysiology

References
Fundamentals of Physiology, 4th Ed, L. Sherwood
Pathophysiology of Blood Disorders, 2nd Ed, H.F. Bunn & B. Furie, Chapter 13, Overview of Hemostasis
https://0-accessmedicine.mhmedical.com.innopac.wits.ac.za/content.aspx?bookid=1900&sectionid=137395249
 Haemostasis
1. Define haemostasis in terms of vasculature, platelets and coagulation.
2. Describe vascular spasm.
3. Describe platelets in terms of their contents and membrane structure.
4. Explain platelet activation and function.
5. Explain the effects of endogenous platelet limiting factors and anti-platelet
therapeutics.
6. Explain platelet pathophysiology
7. Describe coagulation.
8. Describe the major clotting factors in terms of their tissue source, vitamin K
dependence, function, activation and co-factors.
9. Explain the cell-based coagulation pathway.
10. Describe fibrin polymerisation and cross-linking.
11. Describe clot retraction, fibrinolysis and production of D-dimers.
12. Explain the effects of the major anticoagulation systems.
13. Describe the biochemical measurement of (intrinsic and extrinsic) coagulation
pathways.
14. Explain the effects of the major therapeutic anticoagulants.
15. Explain the pathophysiology of hypercoagulabity.
16. Explain the pathophysiology of hypocoagulabity.
 Haemostasis
• Definition: prevention of blood loss/stopping
blood
• If vessel is severed, haemostasis is achieved by
1- vascular spasm Primary
2- formation of platelet plug Haemostasis

3- fibrin clot formation (coagulation) Secondary

Haemostasis
4- eventual growth of fibrous tissue into
the blood clot to permanently close the hole in
the vessel and removal of the clot

Clotting must be rapid, but limited & reversible

 Vascular Spasm
• Rupture of vessel  wall of vessel contracts, sticks
together
• Contraction results from
• nervous reflexes initiated by pain
• local smooth muscle spasm (main factor)
• local humoral factors from traumatized tissues and blood
platelets (Thromboxane A2)
• Smaller vessels: platelets mainly responsible for
vasoconstriction by releasing Thromboxane A2 and
forming a “plug”
• Spasm lasts several hours  time to form platelet plug
and fibrin clot.
Formation of platelet plug - Platelets
• Fragments from big megakaryocyte cells in the bone marrow
• Also called thrombocytes, but no nucleus

• Contain
1. Actin and myosin (molecules for shape change, secretion and
platelet contraction)
2. Residual Golgi apparatus which synthesizes enzymes
3. Mitochondria for energy metabolism
4. Enzymes that synthesize prostaglandins (thromboxane A2)
5. Granules containing ADP & serotonin
6. Growth factors for endothelial cell growth
Formation of platelet plug - Platelets
• Platelet membrane
1. Glycoproteins
• Complex I binds to injured endothelium (exposed collagen
& injured endothelial cells), but not normal endothelium.
• GPIIb/IIIa binds fibrinogen (fibrin clot formation)
2. Phospholipids activate clotting factors
3. Receptors for activator molecules
• Dead platelets are removed by macrophages, particularly
in the spleen.

What do platelets do when we bleed?

 Platelets Vessel
wall

Collagen

Von Willebrand factor

Formation of the platelet plug - Activation
• Von Willebrand factor (vWF) is a long soluble
plasma protein that is secreted by endothelial cells
of blood vessels.
• Vessel injury exposes collagen in interstitial tissue.
• von Willebrand factor in the plasma binds to
exposed collagen and changes conformation.
• Platelets bind to directly to collagen and to
collagen-bound von Willebrand factor.
• Binding to collagen and von Willebrand factor
activates the platelets.
• Platelets are also activated by:
• thromboxane A2
• ADP Platelets Vessel
wall

• serotonin
• thrombin
• Where do these activators come from?
 Platelets Vessel
wall

Collagen

Von Willebrand factor

TxA2, ADP,
serotonin
Formation of the platelet plug - Activation
Activated platelets:
• change shape – many filopodia (sticky spikes) Platelet Activated platelet
– not activated
• “catch” other platelets to form a loose “plug”.
• produce and secrete thromboxane A2, which
activates more platelets.
• release ADP and serotonin, which activate more
platelets.
• change the conformation of glycoprotein IIb/IIIa,
enabling it to bind fibrinogen, which joins platelets
together.
• display membrane phospholipid (phosphatidylserine),
which activates clotting factors IX and X.
fibrin
Formation of the platelet plug - Activation
On the surface of activated platelets:

• Factor X (bound to phospholipid and factor V) converts

prothrombin to thrombin, which
• activates more platelets
• converts fibrinogen to fibrin, a mesh that traps cells, including
RBCs.
• Platelets become tightly bound to each other
through fibrin and von Willebrand factor and
fibrin
then contract to form a tight/firm plug = clot.

Platelet activation and aggregation is a chain reaction (positive

feedback).
How do we stop it?
 Platelet-limiting factors and anti-platelet
pharmaceuticals
• Smooth surface and glycocalix layer of healthy endothelial cells
prevent platelet binding
• Undamaged endothelial cells secrete prostacyclin and nitric
oxide (NO).
• Both inhibit platelet activation and aggregation  the clot does
not extend beyond the injured zone.
• Both stimulate vasodilation  counteracts vasospasm,
increases blood flow.
• ASPIRIN inhibits synthesis of thromboxane A2  decreased
platelet activation
• Clopidogrel (Plavix®) blocks ADP binding to platelets 
decreased platelet activation
Formation of platelet plug - Pathophysiology
Multiple injuries of micro-vessels
Platelets plug small breaks  sufficient
to stop blood loss.
• Lack of platelets  micro-injuries are not
plugged micro-bleeds are visible as red
spots (purpura) in the skin.
• Lack of Von Willebrand factor  bleeding
gums and nosebleeds

Platelets form a loose “wall” (only bricks), sufficient for

small breaks.
For bigger breaks, fibrin is needed to “cement” the bricks.
 Physiological (cell-based) Coagulation

Pathophysiology of Blood Disorders, 2nd Ed, H.F. Bunn & B. Furie, Chapter 13, Overview of Hemostasis
https://0-
accessmedicine.mhmedical.com.innopac.wits.ac.za/content.aspx?bookid=1900&sectionid=137395249

How do we make the fibrin part of the clot?

 Blood coagulation = Secondary Haemostasis
• Coagulation = transformation of blood
from a liquid to a semi-solid, gel state =
clot.
• Initiated by:
• extravascular cells
• platelets
Serum
• damaged vessel walls
• Ultimate effect:
conversion of fibrinogen (soluble plasma Clot
protein) to fibrin (insoluble fibres) = clot

Fibrin clot adheres to the damaged vessel Red

surface and platelet plug, traps blood cells, blood
fills the breach and blocks the vessel (~20 cells
minutes).
What happens to make the fibrin?
 Blood coagulation – the clotting factors
Factor Name Tissue Source Function Activated by Co-factor
FII  FIIa Liver, requires Proteolysis of FXa/FVa
Prothrombin vitamin K fibrinogen, FV,
Thrombin FVIII, FXI, FXIII
FVII  FVIIa Liver, requires Proteolysis of FX, FXa/FVa, Tissue factor (TF),
vitamin K FIX thrombin, ++ Ca2+
FIX  FIXa Liver, requires Proteolysis of FX FXIa, FVIIa/TF Ca2+, FVIIIa,
vitamin K platelet
phospholipid
FX  FXa Liver, requires Proteolysis of TF/FVIIa FVa, Ca2+, platelet
vitamin K prothrombin (FII) FIXa/FVIIIa phospholipid
to thrombin (FIIa)
FXI FXIa Liver Proteolysis of FIX FXIIa, thrombin Ca2+
(FIIa)
FXII  FXIIa Liver Proteolysis of FXI Exposed collagen,
glass etc.
FXIII  FXIIIa Liver, platelets & Covalently cross- thrombin Ca2+
WBCs links fibrin
Tissue Factor (TF, Membranes of non- Co-factor of FVII, Exposed by
FIII) endothelial cells “start” signal broken vessel
Thromboplastin
FV  FVa Liver, also platelets Co-factor of FXa Thrombin
FVIII  FVIIIa Liver & endothelial Co-factor of FIXa Thrombin
tissues
Fibrinogen (FI)  Liver, also platelets Forms the clot Thrombin
Fibrin
 Blood coagulation – the clotting factors
• All clotting factors, except tissue factor, are produced by the
liver and are present in the blood as inactive precursors
(zymogens).
• When activated, factors IIa (thrombin), VIIa, IXa, Xa, XIa and
XIIa are proteolytic enzymes that activate other clotting
factors, by cutting the proteins.
• Four factors, II, VII, IX and X, can only be produced if
vitamin K is available in the liver
• Factor XIIIa is also an enzyme, but it cross-links fibrin.
• Fibrinogen (FI) is not an enzyme or a co-factor. When the
fibrinogen protein is cut by thrombin (FIIa) it becomes
insoluble and forms fibres, called fibrin, which is the clot.
 Blood coagulation – the clotting factors
• Five clotting factors VIIa, IXa, Xa, XIa and XIIIa
require Ca2+ as a cofactor. (Blocking Ca2+ prevents
coagulation in blood collection tubes.)
• Two protease clotting factors, IXa and Xa require
platelet phospholipid for full activity.
• Three protease clotting factors VIIa, Xa and IXa require
clotting “co-factor” proteins.
• The cofactor proteins are, respectively, tissue factor (TF,
FIII  TF/FVIIa), FVa (FVa/FXa) and FVIIIa
(FVIIIa/FIXa).
 Coagulation pathways
• Clotting proteins are inactive in plasma to avoid spontaneous clots and
tissue factor is absent.
• Vessel trauma  exposure of tissue factor and sequential activation
of the pro-coagulation factors  coagulation pathways  fibrinogen
becomes fibrin
• 3 different coagulation pathways form fibrin, 1 physiological, 2
“biochemical”.
• Cell-based coagulation pathway occurs in healthy, living people.
• Extrinsic coagulation pathway: started by addition of tissue factor
from outside the blood vessel, measured by prothrombin time
(PT).
• Intrinsic coagulation pathway: requires only factors normally
present inside the blood vessel, started by addition of negative
charge, measured by partial thromboplastin time (PTT).
 Cell-based Coagulation

Tissue factor

thrombin
prothrombin

Platelets and Thrombin Generation, Volume: 22, Issue: 9, Pages: 1381-

1389, DOI: (10.1161/01.ATV.0000031340.68494.34)
 Cell-based Coagulation

Tissue factor

thrombin
prothrombin

Platelets and Thrombin Generation, Volume: 22, Issue: 9, Pages:

1381-1389, DOI: (10.1161/01.ATV.0000031340.68494.34)
 Cell-based Coagulation in words
Initiation (on the surface of the tissue factor-bearing cell)
• In plasma leaking from broken blood vessel(s), a small amount of activated factor VII (FVIIa) binds to
tissue factor (TF) on the surfaces of cells outside of the vascular endothelium.
• The TF/FVIIa complex on the cell cuts factor X to form FXa and cuts factor IX to form FIXa.
• FXa cuts prothrombin (FII) to form thrombin (FIIa).
• The (as yet small amount of) thrombin then amplifies the signal = the “MATCH”.

Priming/amplification (on the surface of the platelet)

• Thrombin cuts and activates FV, FVIII and FXI at the platelet surface, which provides the
phospholipid “cofactor”.
• Thrombin also helps activate the platelets (see platelets slides).
• The activated platelet and its attached clotting factors are now ready to make lots of fibrin.

Propagation (on the surface of the platelet)

• FIXa (from “initiation”) binds to the platelet via FVIIIa and the FVIIIa-bound FIXa complex cuts many
FX to form many more FXa.
• FXIa also cuts FIX to make more FIXa, each of which can make many more FXa.
• FXa combines with FVa on the platelet.
• Each platelet-attached FVa/FXa complex cuts lots of prothrombin to produce lots more thrombin (FIIa).
• The large amount of thrombin can convert lots of soluble fibrinogen to insoluble fibrin to form a large
(but relatively unstable) clot in between the platelets of the platelet plug.
• Thrombin also cuts factor XIII and FXIIIa covalently joins the fibrin molecules to form a stable mesh
that can “catch” blood cells and further grow the clot.
• Thrombin continues to increase amounts of FVa, FVIIIa and FXIa, leading to more FXa, more thrombin
and more fibrin clot.
• Thus a very small signal can induce a very big and fast response.
 Fibrin Formation and Crosslinking

Covalent bond
between D peptides of
different fibrin
molecules

https://ahdc.vet.cornell.edu/sects/coag/test/Ddimer.cfm
https://www.youtube.com/watch?v=-
ulGunQMGpQ&list=PLkv9qVBSWseFg4CZy7jtl5
pBoqReWmoXA&index=67

https://www.youtube.com/watch?v=hr1Pgb3r
_cU&list=PLkv9qVBSWseFg4CZy7jtl5pBoqReW
moXA&index=68
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]
 Clot Retraction and Fibroblast Invasion
Once the clot is formed:
Platelets in the clot contract (actin & myosin)
 pulls the edges of the ruptured vessel closer together.
 fluid is squeezed out of the clot = serum (plasma minus
fibrinogen and clotting factors trapped in the clot)

Platelets secrete growth factors

 Fibroblast cells grow around and into the clot
 Scar tissue forms (1-2 weeks)
 Fibroblasts become endothelial cells
 Clot is removed (fibrinolysis)
Fibrinolysis

Tissue plasminogen activator (tPA)

plasminogen

plasmin

https://ahdc.vet.cornell.edu/sects/coag/test/Ddimer.cfm
 Fibrinolysis and Dissolution of the Clot
• Plasminogen is a plasma zymogen (inactive proteolytic enzyme),
produced by the liver.
• Plasminogen binds to fibrin during clot formation.
• Damaged endothelial cells slowly secrete another protease, tissue
plasminogen activator (tPA).
• When tPA binds to fibrin it becomes activated.
• Activated tPA cuts plasminogen to give the active enzyme plasmin.
• Plasmin cuts fibrin into many pieces that are released into blood.
• This removes the clot and opens the vessel so that blood can flow
again.
• D-dimers are fibrin fragments containing the cross-links between 2
different fibrin monomers.
• Increased concentrations of D-dimers in the blood show that a clot is
being broken down.
• As the fibrin dissolves, macrophages (and other WBCs) phagocytose
the clot debris, including RBCs  bilirubin ……yellow bruises…
 Summary of haemostasis – clot formation
Vessel injury  blood oozes out of the
vessel. To stop this: NEEDS
• Primary haemostasis= 1- collagen
2- Von Willebrand factor
• Vessel contraction
3- Platelets
• Formation of platelet plug 4- Platelet activators

• Secondary Haemostasis
• Cell-based coagulation
• starts with leaking blood contacting tissue TF-cell, clotting factors VII,
factor-bearing cells X, V, II, platelets, IX, VIII,
XI and I

Blue: Vit K dependent factors, Underlined: Haemophilia factors

 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Biochemical (laboratory)
measurement of clotting
Primary haemostasis:  Bleeding time

Secondary haemostasis:
Extrinsic pathway  Prothrombin Time (PT)
Intrinsic pathway  Partial Thromboplastin Time (PTT)
also  Whole blood clotting time
Bleeding Time Test Measures Platelet Function

• cut the underside of the subject's forearm with

blood pressure cuff above the wound
• measure the time it takes for bleeding to stop
(Normal < 9.5 minutes).
• = platelet function only

Disorders of clotting factors DO NOT AFFECT the

bleeding time test.
Prothrombin Time (PT) test – Extrinsic Pathway

Blood taken Add tissue factor,

in citrate or calcium,
EDTA tube phospholipids

Serum

Clot

Plasma Fibrin
Clot
Red
blood
cells
Prothrombin Time (PT) test – Extrinsic Pathway
Massive excess of tissue factor
binds and activates FVII
in liquid phase

FVIIa activates
lots of factor X

FXa combined with FVa

& added Ca2+ &
phospholipid activates
No platelets required
lots of thrombin
because of added
phospholipid.
lots of thrombin produces
No priming or propagation
lots of fibrin clot
steps required because of
excess tissue factor added = Extrinsic Pathway
 Extrinsic coagulation pathway in words
• Tissue factor (TF) is not present in blood from uninjured vessels.
• Factor VII in the plasma binds to tissue factor and becomes activated
FVIIa.
• The TF/FVIIa complex cuts many factor X molecules to form many
FXa.
• Each FXa combines with co-factors Ca2+, FVa and phospholipid to cut
many prothrombin (FII) molecules to form many thrombin.
• Each thrombin cuts many soluble fibrinogen molecules to form a mass
of insoluble fibrin.
Prothrombin Time (PT) test – Extrinsic Pathway
• Blood is collected into citrate or EDTA tube
(to chelate Ca2+). Removal of Ca2+ prevents Biochemical
coagulation in blood collection tube.
Coagulation Pathways
• Blood is centrifuged to separate the plasma.
• Plasma is mixed with Ca2+, tissue factor Tissue factor
added
and phospholipid.
• Time to make a clot is measured 
usual range = 12-15s.
• Tests function of factors VII, X, V, II &
fibrinogen = extrinsic pathway.
• PT is often expressed as INR (international
normalized ratio) or prothrombin ratio.
• INR = (Patient’s PT/Normal PT)
Normal INR = 0.8-1.2
 Partial Thromboplastin Time (PTT) test
– Intrinsic Pathway
Factor XII is a plasma
protein that activates (cuts)
Blood taken Add factor XII activator, itself after binding to
in citrate or calcium,
negatively-charged surfaces
EDTA tube phospholipids
and molecules, including
• in vitro: glass, silica, kaolin
etc.
• in vivo: collagen, platelets,
denatured proteins, nucleic
acids, microbes

FXIIa activates FXI and also

Plasma Fibrin stimulates inflammation.
Clot
 Partial Thromboplastin Time (PTT) test
– Intrinsic Pathway
Excess activation of factor XII
activates lots of factor XI
FXIa activates
lots of factor IX

FIXa combined with

FVIIIa & added Ca2+ &
phospholipid activates
lots of factor X
No platelets required
because of added FXa combined with FVa
phospholipid. & added Ca2+ &
phospholipid activates
lots of thrombin

lots of thrombin produces

lots of fibrin clot

= Intrinsic Pathway
 Intrinsic coagulation pathway in words
• Contact with negatively-charged surfaces (collagen, platelets, glass tube) converts
factor XII to activated factor XII (FXIIa) (by change of conformation and auto-
cutting)
• Each FXIIa cuts many factor XI molecules to form many FXIa molecules.
• Each FXIa combines with co-factor Ca2+ to cut many factor IX molecules to form
many FIXa.
• Each FIXa combines with co-factors Ca2+, FVIIIa and phospholipid to cut many
factor X molecules to form many FXa.
• Each FXa combines with co-factors Ca2+, FVa and phospholipid to cut many
prothrombin (FII) molecules to form many thrombin.
• Each thrombin cuts many soluble fibrinogen molecules to form a mass of insoluble
fibrin.
• Thrombin also cuts FV and FVIII to activate them.
 Partial Thromboplastin Time (PTT) test
– Intrinsic Pathway
• Blood is collected into citrate or
EDTA tube (to chelate Ca2+) and Biochemical
centrifuged. Coagulation Pathways
Activator added
• Plasma is mixed with phospholipid,
Ca2+ and a factor XII activator
(kaolin, silica…)
• Time to form a clot is measured -
usual range is less than 39 seconds.
• Tests function of factors XII, XI, IX,
VIII, X, V, II = intrinsic pathway
• These factors can also be activated
by inflammation
 Whole blood clotting time test
• Useful when no lab immediately
available.
• Blood collected without anticoagulant.
• Time taken by whole blood to form a
clot – tests ability to form fibrin, mostly
intrinsic coagulation pathway (tube
activates FXII).
• Laboratory tests of haemostasis do not
always reflect in vivo clotting or
bleeding risk, but can help identify
where problems are.
 Summary of haemostasis – clot formation
Vessel injury  blood leaks out of the
vessel. To stop this: NEEDS
• Primary haemostasis= 1- collagen
• Vessel contraction 2- Von Willebrand factor
• Formation of platelet plug 3- Platelets
4- Platelet activators
• Secondary Haemostasis
• Cell-based coagulation TF-cell, factors VII, X, V, II,
• starts with leaking blood contacting tissue platelets, IX, VIII, XI and I
factor-bearing cells

• Laboratory Coagulation cascades

• Extrinsic pathway TF, factors VII, X, V, II and I
• starts with Tissue Factor and FVII
• Intrinsic pathway Factors XII, XI
• starts with Factor XII activation VIII, IX, X, V, II and I

Blue: Vit K dependent factors, Underlined: Haemophilia factors

Anti-Coagulation Pathways and Therapeutics
 Anti-Coagulation Pathways and Therapeutics
Tissue factor
Cell-based
Coagulation

thrombin
prothrombin

This is a chain reaction!

How do we stop it?
 Mechanisms of Anticoagulation
Clots should not form in healthy/unbroken
blood vessels.
• 90% of thrombin is immobilised in the fibrin clot.

Endothelial cells inhibit coagulation by

• Their smooth endothelial surface and glycocalix layer,
which repel platelets and clotting factors
• Stimulating vasodilation (prostacyclin and NO)
• Stimulating fibrinolysis (secretes tPA)
• Producing molecules that inhibit clotting factors,
especially thrombin
• Producing molecules that break down clotting factors
 Anticoagulation - Thrombomodulin
Thrombomodulin
Inactive thrombin Active protein C
Thrombin Protein S

In the laboratory Protein C In the body

Active protein C/protein S complex

Tissue factor

prothrombin thrombin
 Anticoagulation – Thrombomodulin
Thrombomodulin – Thrombin modulator
• Produced by endothelial cell surface.
• Binds thrombin and inhibits activation of pro-coagulation factors (V,
VII, VIII and XI)  stops the chain reaction.
• Thrombomodulin-thrombin complex cuts Protein C to produce
activated protein C (APC).
• APC combines with a co-factor Protein S.
• Synthesis of both protein C and protein S (in the liver) requires
vitamin K (deficiency?).
• The protein C/protein S complex inactivates factors Va and VIIIa
(components of cell-based coagulation and the intrinsic and common
pathways in vitro).
 Anticoagulation - Antithrombin & heparin
Heparin
Antithrombin

In the laboratory In the body

Tissue factor

prothrombin thrombin
 Anticoagulation – Antithrombin & heparin
Antithrombin III is produced by the liver.
Heparin is produced by basophilic mast cells and attaches to the
surface of endothelial cells.
Heparin is the co-activator for antithrombin.
The antithrombin/heparin complex inactivates (all proteolytic
enzymes)
• Thrombin (FIIa, common pathway). This stops the chain reaction and clot
formation.
• Factor Xa (common pathway)
• Factor VIIa (extrinsic pathway)
• Factor IXa (intrinsic pathway)
• Factor XIa (intrinsic pathway)
• Factor XIIa (intrinsic pathway)
 Anticoagulation – Tissue Factor Pathway
Inhibitor (TFPI)
Tissue factor
pathway inhibitor

Factor Xa

In the laboratory In the body

Tissue factor

prothrombin thrombin
 Anticoagulation – Tissue Factor Pathway
Inhibitor (TFPI)

• Tissue factor pathway inhibitor (TFPI) is produced by

endothelial cells and is dissolved in the plasma.
• TFPI binds to factor Xa.
• The TFPI/Xa complex inactivates the tissue factor/FVIIa
complex
• This stops the initiation step of coagulation in vivo almost
immediately (extinguishes the match) and inhibits the extrinsic
pathway in vitro.
 Therapeutic Anticoagulants
• Vitamin K blockers, e.g. WARFARIN 
↘Factors II, VII, IX, X  decreases both
extrinsic (mainly) and intrinsic pathways
• HEPARIN  activates antithrombin  effects on
inhibits factors II, VII, IX, X, XI, XII  - PT?
mostly intrinsic pathway - PTT?
• ASPIRIN  inhibits formation of - bleeding time?
thromboxane A2  decreases platelet - cell-based?
activation and aggregation
• Also plasminogen activators  activate
dissolution of clots
• ADP receptor blocker  decreased
platelet activation.
Coagulation disorders
Hypercoagulability
Hypocoagulability
 Blood
Ursula Mariani, Blood Forum
Email: [email protected]

Coagulation disorders
Hypercoagulability
Hypocoagulability
Hypercoagulability (risk of thrombosis)

• 3 main causes:

1. Haemodynamics
2. Vessel injury
3. Excess pro-coagulants vs anticoagulants
 Hypercoagulability - Haemodynamics
Sluggish blood flow  markedly increases coagulation
risk Risk of
Deep Vein
• Venous blood stagnation (E.g. plaster cast, long Thrombosis
hospitalization, long flight) (DVT)

• Atrial fibrillation
• left atrium of heart does not fully expel the blood
• blood stagnates in left atrium, can form a clot there
• Clot expelled by the ventricle
• Can cause a stroke or a lower limb ischemia.
• Polycythaemia (too many red blood cells)  makes
blood more sluggish  higher coagulation risk
 Hypercoagulability - Vessel injury
Chronic vessel injury Activates
• Endothelial wall damage -Factor XII & intrinsic pathway
-Platelet aggregation
• no longer smooth/glycocalyx damaged
• collagen protrudes into the vessel
• Damage due to: cholesterol plaques, toxins (smoking,
diabetes), increased vessel stress (high blood pressure)
•  inappropriate coagulation at site of damage
•  increased risk of myocardial infarction, stroke, lower limb
ischemia.

 Prevention – anti-platelet or anti-coagulant treatment

• Aspirin = inhibits production of Thromboxane A2
• Clopidogrel = blocks ADP receptor that activates platelet
aggregation.
• Warfarin = blocks vitamin K (factors II, VII, IX and X)
Hypercoagulability - Excess Pro-coagulants vs
Anticoagulants
• Factor V Leiden mutation (resistance to activated protein
C): 5% of Caucasians are heterozygous for this mutation
• Protein C or protein S deficiencies (genetic)
• Antithrombin III deficiency (genetic mutation OR
premature babies)
• The oral contraceptive pill increases liver production
of pro-coagulant proteins  increases the risk of
imbalance  increased risk of DVT.
 Hypocoagulability (bleeding risk) - Platelets
Disruption of platelet function increases bleeding time, but PTT
and PT are normal.

• Genetic defect of von Willebrand factor  decreased

attachment of platelets to vascular wall  von Willebrand’s
disease

• Platelet dysfunction, particularly in people who have taken

aspirin or Clopidogrel.
• Low platelet count = thrombocytopenia caused by
• central (not enough production – e.g. leukaemia, toxins)
• peripheral (too much destruction)
 Hypocoagulability - Extrinsic Pathway
Disruption of factors in the extrinsic pathway prolongs
prothrombin time (PT) and increases INR
• Vitamin K deficiency: Vitamin K is essential for synthesis of
factors II, VII, IX and X.
• Vitamin K comes from intestinal bacteria and diet.
• Abdominal surgery or inflammatory bowel disease  decreased
absorption of vitamin K from GI tract.
• Neonates may have bacterial deficiency  haemorrhagic disease of
the newborn.
• Anti vitamin K treatments (AVKs, e.g. Warfarin) inhibit Vitamin K 
↓ factors II, VII, IX and X
• Ingestion of poisons based on AVKs (Rattex ®)
• Liver disease  decreased production of all clotting factors,
except tissue factor  affects both extrinsic and intrinsic
pathways  prolongs both PT and PTT.
 Hypocoagulability – Vitamin K-dependent
Factors

Vitamin K deficiency has a major disruptive effect on the extrinsic pathway because of its
effect on factor VII, but also disrupts the intrinsic pathway to a lesser extent because it
decreases production of factor IX, in addition to decreasing FX and thrombin.
 Hypocoagulability - Intrinsic pathway
Disruption of factors in the intrinsic pathway prolongs Partial
Thromboplastin Time (PTT)
1- coagulation factor deficits
• Haemophilia = X-linked genetic deficiency of Factor VIII or Factor IX.
• Genes are located on the X chromosome.
• Females have 2 X chromosomes, males have 1 X and 1 Y
chromosome, which has no genes for FVIII and FIX
• The mother is usually a healthy heterozygous carrier.
• Son has haemophilia if he inherits the mother’s X Chromosome
with the mutant gene.
• Clinical presentation: male, large haematomas, easy bruising,
severe joint bleeds that cause joint deformations.
2- Heparin Treatment  activates Antithrombin III  AT/heparin
complex blocks intrinsic pathway by inactivating factors Xa and IIa
first and then factors XIIa, IXa and XIa  increased PTT. PT is also
prolonged due to inactivation of factors IIa, VIIa and Xa.