Explain the origin and importance of blood groups

Over 400 red cell antigens have been described, the majority of which are
inherited in a simple mendelian fashion
Importance of blood group: individuals who lack a particular blood group
may produce antibody reacting to that antigen and may develop
transfusion reaction if transfused with blood containing that antigen.
However the biological role is not known.
Most important red cell antigens are ABO and Rh. Other antigens are less
important because the antigens are weak and their antibodies develop
only after multiple exposures. While others react only at low temperatures.

ABO system:

The ABO antigens are controlled by three allelic genes A, B, and O genes.
The A and B genes control the synthesis of enzymes required for the
addition of specific carbohydrate residues to the basic antigenic
glycoprotein, the H antigen.
The ABO antigens are complex carbohydrates

The core antigen is the H antigen, which is a glycoprotein precursor with Lfucose as the terminal sugar.
The A antigen is formed when N-acetylgalactosamine is added to the
terminal group of the H antigen.
The B antigen results when D-galactose is added to the H antigen.
Natural antibodies (IgM) to A and/or B antigens are found in the plasma of
subjects who have red cells that lack the corresponding antigen.
Two subgroups A1 and A2 are recognized on group A red cells. Both
subgroups agglutinate with anti-A antibody, but only group A1
agglutinates with lectin from Dolichos biflorus, a plant seed, and anti-A1
antibody.
This difference in agglutinability depends on the number of antigen sites
reduced in group A2 cells.
Individuals with group O red cells lack both A and B antigen, but have the
H antigen, and also anti-A and anti-B antibodies.
Individuals with the AB group possess both A and B antigen, but lack both
anti-A and anti-B antibodies.
The frequency of blood groups in the Caucasian population is:
o A (45 per cent),
o O (43 per cent);
o B (9 per cent),
o AB (3 per cent),
80 per cent of group A with strong antigens (A1) and 20 per cent with
weak antigens (A2).
Three allelic genes, A1, B1 and O1, and a pair of allelic genes, H and h,
determine the formation of the ABO antigens.
Gene H is responsible for the enzyme, -L-fucosyltransferase, which
attaches fucose to the glycoprotein precursor to form the H antigen.
Genes A and B are responsible for -N-acetyl-D-galactosaminyl transferase
and -D galactosyltransferase, which attach galactosamine and galactose
to substance H, respectively and thus determine the antigenic specificity
of the A and B antigen.
A1, B1 and H antigens are present in the red cells as well as most other
body cells, including white cells and platelets.
These antigens are also present in the body fluids of 80 per cent of the
population (who possess the secretor gene) in a water-soluble form. These
water-soluble antigens are present in plasma, saliva, semen, urine, gastric
juice, tears and bile, but not in the cerebrospinal fluid.
As the ABO antigens are stable, their detection in dried blood and other
body fluid stains are used in forensic serology.

THE RH SYSTEM

The Rhesus blood group system is next to the ABO in clinical importance.

The Rh system is named after the Rhesus monkey, the red cells of which
stimulated antibodies when they were injected into rabbits and guinea
pigs.
Although the Rhesus monkey antigen is similar to the Rh D antigen of
human red cells, it is not identical.
Human red cells have both the monkey antigen and a separate Rh
antigen.
The Rh D antigen is a protein of mol. wt. 30,000 Da.
The serological activity of the Rh antigen is governed by amino acid
sequence and the presence of specific phospholipids.
The red cell antigens of the Rhesus system are determined by three
closely linked pair of alleles.
The main alleles are Dd, Ce and Ee, and the three antigenic groups are
derived from one gene complex.
The term Rh +ve refers to the presence of the D antigen.
Each antigen is defined by a specific antibody (anti-C, anti-c, anti-E and
anti-e) except for d antigen. Anti-d does not exist.
Rh antigens, unlike the ABO groups, are only present on red blood cells.
Rh antibodies rarely occur naturally. Most Rh antibodies are immune,
'warm' and IgG in origin.
The D group is the most important, as 85 per cent of the population
possess the D antigen (Rh +ve).

OTHER BLOOD GROUP SYSTEMS

Other blood group systems are clinically less important.

The MN antigenicity depends on the terminal amino acid sequence of

glycophorin (a red cell membrane protein).

The P, Lewis and MN systems are not uncommon, but the naturally
occurring antibodies only react at low temperatures and have antigens
with low antigenicity.

The P antigen is determined by the conversion of trihexosyl ceramide to

globoside (P antigen) found on the red cell membrane.
The main feature of the antigens of the Lewis system is that they are
soluble substances present in saliva and plasma, which are adsorbed onto
the surface of red cells from plasma.
The Kell system is the third most important blood group system after the
ABO and Rh systems.
The K antigen is present on red cells, leukocytes and platelets. Although
the K antigen is immunogenic, it is of relatively low frequency and
therefore may only cause iso-immunization in patients who have had
multiple blood transfusions.

Outline the constituents and functions of plasma

Plasma is the fluid medium of the intravascular compartment

and is important for the transport of materials between tissues
and the internal environment
Plasma constitutes about 4% of total body weight (~ 4050ml/Kg)
Water and electrolytes
Approximately 93% of plasma is water.
The principal plasma cation is Na+ (140mmol/L), whilst
other important cations include K+ (4mmol/L), Ca++
(1mmol/L) and Mg++ (2mmol/L).
About one-third to one-half of the divalent cations are
complexed with proteins (e.g. albumin) or low-molecularweight anions, and carry negative charges as they are on
the alkaline side of their isoelectric points at a pH of 7.4.
Organic acids such as lactate and pyruvate make up the
remaining plasma cations.
Plasma carbohydrates
Blood glucose is the main carbohydrate in plasma (3.5-6
mmol/L), with variable small amounts of fructose,
galactose and mannose.
Complex carbohydrates are present in small amounts in
plasma; glycoproteins are formed by the covalent
attachment of carbohydrate to the amino acids,
asparagine, serine or threonine.
Plasma lipids
Lipids generally are complexed with plasma proteins in the
circulation.
A small fraction of the total fatty acids in plasma is
unesterified, usually associated with albumin.
The remaining fatty acids form the triglycerides and are
found in plasma as lipoproteins.
The lipoproteins complex with phospholipids and
cholesterol to form chylomicrons, very low-density
lipoproteins, low-density lipoproteins and high-density
lipoproteins.

Plasma proteins
Plasma proteins are a diverse group structurally and
functionally, with the total plasma protein concentration
ranging from 60 to 80 g/L.
All plasma proteins are globular molecules and range from
simple unconjugated proteins such as albumin to complex
proteins such as lipoproteins, glycoproteins and
metalloproteins.

Albumin
Albumin (mol. wt. 67000 Da) is the most abundant plasma
protein, with a concentration of 40 g/L.
It is synthesized in the liver, has a half-life of 20 days, and
is metabolized in the liver, kidneys and gut.
Approximately 13g of albumin is synthesized and
catabolized per day.
Its main functional role is the transport of a wide range of
substances and maintenance of plasma colloidal osmotic
pressure.
1 GLOBULINS
1 Antitrypsin
o 1 Antitrypsin is a serine protease inhibitor (serpin)
produced mainly by the liver.
o It is a potent inhibitor in plasma of trypsin,
chymotrypsin, activated plasmin and other
proteases.
1-Lipoproteins
o 1-Lipoproteins are associated with 1-globulins, and
contain 45-55 per cent lipid.
o The plasma lipoproteins may be divided into four
classes, namely the chylomicrons, very low-density
lipoproteins (VLDL), low-density lipoproteins (LDL)
and high-density lipoproteins (HDL).
o The chylomicrons are the largest of the lipoproteins,
consisting mainly of triglycerides (80-90 per cent)
with only 1-2 per cent protein. They are mainly

derived from dietary fat and serve to carry

exogenous triglycerides from the gut to the tissues
for utilization or storage.
o The VLDL transport endogenous triglycerides from
the liver to the peripheral tissues for storage or
utilization.
o The LDL transport cholesterol to the tissues
o The HDL return excess cholesterol from the
peripheral tissues to the liver
1-Acid glycoprotein
o 1-Acid glycoprotein is an acute phase protein which
is present in low concentrations, but its physiological
role is unknown.
o Pharmacologically, it is important as it binds to basic
drugs.
2-GLOBULlNS
Various proteins belong to this group of globulins:
2-Macroglobulin
o 2-Macroglobulin is a protease inhibitor in plasma,
and is the major protein in the 2-globulin fraction
(approximately 80 per cent).
o It has inhibitory functions on plasma trypsin,
chymotrypsin and plasmin.
o The primary function of 2-macroglobulin may be to
inhibit proteases produced by infectious organisms.
Prothrombin
o Prothrombin is a clotting factor synthesized by the
liver.
o Some 60 per cent of the extracellular pool of
prothrombin is in the plasma and 40 per cent in the
extravascular space.
o It has a rapid turnover.
Haptoglobulin
o Haptoglobulin is a heterogeneous group of globulins
that bind free haemoglobin and transport it to the
liver.
Caeruloplasmin
o Caeruloplasmin is a plasma protein that carries
copper, and is produced in the liver.

o It also functions as an oxidase enzyme and oxidizes

ferrous to ferric ions before the binding of iron to
transferrin.
o As an acute-phase protein, it may modulate
inflammation by its free radical scavenging
properties.
GLOBULINS
Transferrin
o Transferrin is the plasma protein that transports iron.
o Apotransferrin, its precursor, is produced in the liver.
o One molecule of transferrin will bind two ferric ions
and is normally approximately one-third saturated
with iron.
Haemopexin
o Haemopexin is a -globulin that binds to haem and
releases it to the reticuloendothelial system.
FIBRINOGEN
Fibrinogen is a large protein that is produced by the liver
and has an important role in blood coagulation.
-GLOBULINS
The -globulins consist of immunoglobulins. The
immunoglobulins are produced by plasma cells of the
bone marrow, spleen, lymph nodes and gut.
IgG accounts for 76 per cent of the total plasma
immunoglobulins. IgG can bind to complement, and its
main action is against soluble antigens.
IgA forms 16 per cent of the circulating antibodies, and is
present in seromucous secretions. It does not fix
complement and the main function is protection against
secretory mucosal surfaces.
IgM accounts for 7 per cent of plasma immunoglobulins, is
rapidly synthesized in response to particular antigens, and
can fix complement to break down foreign surfaces.
IgE is present in very low concentrations in normal
individuals, and is involved in hypersensitivity reactions by
binding to mast cells in capillaries and tissues.

Biological functions
Plasma has numerous functions. It is important for the carriage
of dissolved oxygen and carbon dioxide, glucose, amino acids
and excretory waste products such as urea and creatinine. The
bicarbonate in plasma, derived from the red cell, is an
important buffer system in blood.
The plasma proteins are a diverse group of proteins and have a
wide range of biological functions.
TRANSPORT FUNCTIONS
Many plasma proteins are carriers of hormones, metals,
vitamins, metabolites and excretory products in the body.
Albumin transports many substances and renders them
water-soluble. It transports bilirubin, free fatty acids, Ca+
+ and hormones such as thyroid hormone and cortisol,
and acidic drugs (e.g. barbiturates) .
The globulins transport a wide variety of substances.
and -lipoproteins transport triglycerides, cholesterol and
fat-soluble vitamins. Iron is transported by transferrin, and
copper by caeruloplasmin. Thyroxine is also transported
by thyroidbinding globulin, and cortisol by transcortin.
Transcobalamin is an important carrier for vitamin B12.
BLOOD COAGULATION
Various plasma proteins, including prothrombin and
fibrinogen, are involved in the coagulation cascade.
ENZYMES
Various enzymes are present in plasma, including plasma
cholinesterase and the acute-phase proteins such as 1acid glycoprotein and the anti -proteolytic enzymes, 1antitrypsin and 2-macroglobulin.
ONCOTIC PRESSURE
The plasma proteins exert an oncotic pressure (28 mmHg),
which contributes to the total osmotic pressure (5610
mmHg) of plasma. Plasma oncotic pressure is important
in the control of fluid balance between the vascular and
the interstitial compartments. Quantitatively albumin is
the most important plasma protein for oncotic pressure

because of its low molecular weight and high conc.

compared with other plasma proteins.
BUFFERING ACTION
Plasma proteins are amphoteric and dissociate in the pH
range of 7-7.8, with a net negative charge. Thus they can
accept H+ ions although the buffering function is minor
compared with other buffering systems in blood.
Describe the processes involved in haemostasis and
coagulation
4 main processes arrest bleeding post-vascular injury:
smooth muscle constriction / vascular spasm
platelet adhesion / aggregation (primary haemostasis)
coagulation (secondary haemostasis)
finbrinolysis & re-endothelialisation

PLATELET FUNCTION:
Non-nucleated cytoplasmic fragments
Derived from megakaryocytes ~ 2-4 m diameter
Average lifespan ~ 8-10 days
About 30% sequestered in the spleen
Important Platelet factors:
o PF3 - platelet phospholipid procoagulant activity
o PF4 - cationic alpha-granule protein which
neutralizes heparin

 Platelet Plug: platelet haemostatic function is divided into

3 phases,
o Adhesion
o Activation
o Secretion
o Aggregation
Platelet : Adhesion
o Subendothelial microfibrils contain large multimers of
vWF
o vWF react with adhesion receptor on the surface of
platelet GPIb-IX complex

o GPIIb / GPIIIa complex is exposed and binds vWF and

fibronectin leading to spreading of platelets on the
subendothelial matrix

Platelet : Activation
o Complex biochemical events activating
phospholipase A2 and C DAG & IP3 activation of
protein kinase C protein phosphorylation
cytoplasmic calcium Reorganization of
cytoskeleton transformation of shape from disc to
sphere and long pseudopods spreading onto
subendothelial matrix.
Platelet : Secretion
o Release of procoagulants and ligands from alpha and
dense granules
o Results in further activation and platelet adhesion
o Dense granules immediately release ADP, serotonin
and epinephrine
o granule contents:
PF4 (heparin inhibitor)
fibronectin, thrombospondin
platelet derived growth factor
fibrinogen, plasminogen, factors V, VIII, and vWF
o arachidonic acid products - PGG2, PGH2, TXA2

 Platelet : Aggregation
o ADP and TXA2 promote aggregation at the site of
vascular injury
o ADP not only promotes platelet to platelet adhesion
but also release of more ADP and TXA2 causing
secondary platelet aggregation and positive feedback
mechanism
o ADP also increases no. of fibrinogen receptors (IIbIIIa) on platelet surface
o Fibrinogen and vWF released from the granules
enhance platelet adhesion and aggregation
o Thrombospondin released from granules stabilizes
the platelet aggregate contact with collagen &
thrombin releases ADP, serotonin and TXA2

COAGULATION
Classical description, with division into intrinsic and extrinsic
systems:

 Common pathway from activation of factor X

"Classical" division into intrinsic & extrinsic systems is not
applicable to humans in vivo:
o no coagulopathy, nor disease state, is associated
with deficiencies of several of the proteins of the
intrinsic system
Cell based theory of coagulation has been proposed.
Cell based theory of coagulation:

Factor VIIa plays an important role at various stages.

In this model, the process that leads to the explosive
generation of thrombin that is required for the formation of a
stable clot and haemostasis may be divided into three stages:
(l) initiation, (2) amplification, and (3) propagation.

Thrombin Activity
Cleaves fibrinopeptides A & B from fibrinogen to yield
soluble fibrin
Both free and fibrin bound thrombin cleave fibrinogen
Propagation of thrombus at the site of injury
Thrombin activates Factor XIII cross-links fibrin,
increasing mechanical stability & reducing susceptibility to
lysis
Binds to thrombomodulin, on the endothelial surface
resulting in:

o activation of protein C
o protein C, in the presence of protein S, inactivates
Factors Va and VIIIa
Stimulates release from endothelial cells of products
involved in endogenous thrombolysis:
o Tissue plasminogen activator (tPA), and
o plasminogen activator inhibitor type 1
thrombin is also an effector molecule:
o Inducible receptors for thrombin are found on
endothelial & vascular smooth muscle cell surfaces
o direct effects on cell proliferation:
smooth muscle cell proliferation
endothelial cell proliferation
o influences cellular mechanisms for matrix protein and
collagen production
o direct effects on WBC's
IL-1 from macrophages
Promotes neutrophil degranulation

Describe the normal mechanisms of preventing thrombosis

including endothelial Factors and natural anticoagulants
In order to prevent uncontrolled disseminated coagulation,
there are a number of physiological inhibitors of coagulation.
Serine protease inhibitors include antithrombin III (inhibits IIa
and Xa), C1 inhibitors (inhibit contact factors), 2 macro
globulins (inhibit IIa and contact factors), 2 antiplasmin and
2 antitrypsin which inhibit circulating serine proteases.
n antithrombin III
u 2-globulin synthesized by the liver, tb ~ 70 hrs
u principal antagonist of the serine proteases
F XIIa, XIa, Xa, IXa, VIIa
F thrombin, plasmin & kallikrein
u ~ 85% of the plasma inhibition of thrombin
u heparin binds to lysine on ATIII
protease inhibition, especially Xa
u ATIII tb is reduced markedly by heparin, as the
complex is removed by the RES
F probably accounts for resistance to
anticoagulation with prolonged therapy

Factors Va and VIIIa are regulated by proteins C and S, which

are vitamin-K-dependent serine proteases. Protein C destroys
factor V and VIII, whilst protein S enhances protein C by
binding it to the platelet surface. Protein C is activated by
thrombomodulin formed by the binding of thrombin to the
endothelial cell surface.

Describe fibrinolysis and its regulation

Fibrinolysis is a process where fibrin and fibrinogen are
cleared by plasmin. Plasminogen (a -globulin) in blood
and tissue fluid is converted to plasmin (a serine

protease) by intrinsic activation (via vessel wall

activators or extrinsic activation via tissue activators).
Plasminogen activators are produced by endothelial
cells.
Plasmin cleaves fibrin and fibrinogen to fibrin
degradation products by hydrolysing arginine and lysine
bonds.
D-dimers are cleavage products of cross-linked fibrin.
Various split products such as fragments X, Y,D and E
may be produced

outline the methods for assessing coagulation, platelet

function and fibrinolysis
Bleeding time

 Simplate II
- modified Ivy technique
- torniquet @ 40 mmHg & standard template incision
- normal range < 9 minutes, operator dependent
Duke or Ivy - less reproducible than Simplate II
Platelet count ~ 150-400 x 109/L
Thrombin time
normal range 14-16s
tests final conversion of fibrinogen fibrin
bypasses intrinsic & extrinsic systems, and is abnormal in,
o afibrinogenaemia, hypofibrinogenaemia, dysfibrinogenaemia
o heparin therapy - corrects with protamine
o elevated FDP's - partially corrects with protamine
International normalised ratio / prothrombin time
tests the extrinsic pathway, normal range ~ 13-17s
platelet poor citrated plasma is recalcified & brain thromboplastin added
time taken to clot is measured as a ratio of control reagent
standardised control reduces inter-laboratory variation
recommended Australasian Reference Thromboplastin, ART
abnormal in
o VII deficiency
o liver disease,
o warfarin therapy,
o vitamin K deficiency
Activated partial thromboplastin time
Normal range ~ 25-35 s
Screens for coagulation factor deficiency, except VII & XIII
recalcified, platelet poor citrated plasma, plus an activator & platelet
substitute
varies with reagents used and laboratory
interpret with clinical findings and prothrombin time
factor deficiency corrected by the addition of normal plasma
factor inhibitor not corrected by normal plasma
heparin therapy therapeutic range ~ 1.5-2.5 x baseline

Fibrin/fibrinogen degradation products

Blood collected into a tube containing thrombin & a fibrinolytic inhibitor
latex agglutination test against fibrinogen-related Ag in serum
standard FDP's don't differentiate between 1 and 2 fibrinolysis
XDP's measure D-dimer which indicates fibrinolysis after fibrin
formation
FDP, XDP
- local lysis of fibrin, DIC
- malignancy, systemic infection, SIRS
FDP - primary fibrinolysis
normal XDP's helps exclude pulmonary thromboembolic disease
Fibrinogen
N: 1.5-4.0 g/l
Test either based upon thrombin clotting time, heat precipitation or
immunological methods
Discrepancies between functional and immunological methods found in
the presence of FDP's and dysfibrinogenaemia
decreased production
- hereditary a/hypo-fibrinogenaemia
- liver disease
- severe malnutrition syndromes
increased consumption
- DIC
- fibrinolysis
Euglobulin lysis time
normal range > 90 minutes
time reflects the presence of activators of the fibrinolytic system
Thromboelastography
functional assessment of the entire coagulation cascade & fibrinolytic
system
results may take up to several hours
requires multiple samples run sequentially throughout procedure
frequently require treatment prior to availability of results

Explain the physiological consequences of acute and chronic anaemia

Physiological responses to acute anaemia depend upon wether it is
hypovolumic or isovolumic. In case Hypovolumic state, the response is
similar to blood loss discussed in CVS section. Response to isovolumic
state has been discussed below:
The WHO definition of anaemia is a haemoglobin (Hb) concentration <130
g/L in men, and <120 g/L in women.

PHYSIOLOGIC RESPONSE TO ACUTE ANEMIA

The maintenance of tissue oxygen delivery during an acute reduction in
red blood cell concentration depends on both an increase in cardiac
output and an increase in blood oxygen extraction. These two
mechanisms require the preservation of an ample circulating blood
volume.
The cardiac output response
Cardiac output increases during isovolemic anemia and the extent of this
response appears to be closely related to the decrease in hematocrit. The
cardiac output response is usually due to an increase in stroke volume
and, to some extent, an increase in heart rate. The decrease in blood
viscosity plays a fundamental role in the rise in stroke volume by
increasing venous return and decreasing total peripheral vascular
resistance. These changes in cardiac loading conditions lead to improved
myocardial functioning and a direct enhancement of myocardial
contractility has also been described. The decrease in total peripheral

vascular resistance results, essentially, from reduced blood viscosity, but

may also be related to the decreased scavenging capacity of blood to
inactivate nitric oxide.
The oxygen extraction response
The aim of the second compensatory mechanism is to better match
oxygen delivery to oxygen demand at the tissue level. This mechanism,
which permits the extraction of blood oxygen to increase, involves
physiologic alterations at both the systemic and the microcirculatory level.
At the systemic level:
Matching oxygen delivery to tissue oxygen demand requires the
redistribution of blood flow to areas of high demand (e.g. the brain and
heart) in order to more effectively utilize oxygen held in venous blood.
Several experimental studies have demonstrated that cerebral and
coronary vasodilatation occurs during acute anemia; as a result, blood
flow in these areas increases out of proportion to the rise in CO. This
excepional increase in blood flow to the brain and heart occurs because
these organs are flow-dependent tissues, in contrast to other organs
(e.g. the splanchnic area, kidneys, and skin) that are flowindependent
tissues. Flow-dependent organs extract most of the oxygen available,
even under basal conditions, and are unable to increase oxygen extraction
further to meet their metabolic requirements. Coronary blood flow
increases even more than cerebral blood flow as myocardial oxygen
demand increases during anemia. When the hematocrit is reduced to 1012%, myocardial oxygen consumption more than doubles. Under these
conditions, coronary vasodilatation is nearly maximal. When the
hematocrit is below 10%, coronary blood flow can no longer match the
increased myocardial oxygen demand and ischemia develops, resulting in
cardiac failure. Excess perfusion to the brain and heart occurs at the
expense of flow-independent organs. Relative vasoconstriction occurs in
some tissues so that renal, mesenteric, and hepatic blood flows are
proportionately less than the total cardiac output response. This regional
blood flow redistribution among organs is partly due to alpha-adrenergic
stimulation, but is unaltered in the presence of -adrenergic blockade.
At the microcirculatory level:
Several physiological adjustments contribute markedly to providing a
more efficient utilization of the remaining oxygen in the blood. The main
effect of hemodilution on the microcirculation is an increase in red blood
cell velocity that allows red blood cell flux in the capillaries to be
maintained up to a systemic hematocrit of 20%. This increased flow

velocity stimulates arterial vasomotion and provides a more homogeneous

distribution of red cells in the capillary network. By shortening the transit
time, the increase in red blood cell velocity may also reduce the loss of
oxygen before it reaches the capillaries and, thereby, improve oxygen
transfer to the tissues. An increase in the ratio of microcirculatory to
systemic hematocrit has also been demonstrated. This phenomenon is
related to complex interactions between axially migrating red blood cells
(the Fahraeus effect) and the heterogeneous nature of the
microcirculatory network.

Finally, changes in the dynamics of the hemoglobin molecule can result in

more efficient tissue oxygen delivery in anemia. Indeed, a right shift of the
oxyhemoglobin dissociation curve - which enhances oxygen release at a
constant oxygen tension - begins at a hemoglobin level of 9 g/dL and
becomes more prominent when levels are <6.5 g/dL. This phenomenon
results from increased synthesis of 2,3 diphosphoglycerate and appears
with declining hemoglobin after 12 to 36 hours
Outline the production of blood constituents including red blood
cells, haemoglobin, plasma proteins, white blood cells and
platelets
Formation of RBC
Site of RBC production bone marrow
Most primitive cell is proerythroblast
Over a period of 5 days, the pro-erythroblast gives rise to series of progressively
smaller normoblasts by undergoing cell division and maturation
The erythroblasts progressively contain more haemoglobin while the nuclear
chromatin becomes more condensed
Eventually a pyknotic nucleus is extruded from the late erythroblast to form a
reticulocyte which is the first red cell to enter the circulation
The reticulocyte is released from the bone marrow into the peripheral circulation and
circulates for 1-2 days and matures when the RNA is completely lost
RBC life span ~ 120 days
Removed by the reticuloendothelial system
RBC biconcave disc with average dimensions : 7.5 wide, 2 thick
Peculiarity of RBC metabolism: red cell generate ATP by the anerobic glycolytic
pathway for energy, called embden Meyerhof pathway. 2 mol. Of ATP produced for
each mol of glucose being converted to lactate. This pathway also generates NADPH
which is required by methaemoglobin reductase to reduce methaemoglobin to
haemoglobin

Factors governing RBC production :

Erythropoetic activity is regulated by erythropoietin (t1/2~ 6-9 hrs) which is produced
90% in the kidney (peri-tubular complex) and 10% in the liver
It increases the rate of differentiation of stem cells
The final maturation of RBC requires vit B12 and folic acid for synthesis of
thymidine phosphate which is required for DNA synthesis

Heamoglobin production
Haem synthesis occurs in the mitochondria by a series of biochemical reaction
Protoporphyrin is synthesized from condensation of glycine and succinyl co A under
the influence of -aminolaevulinic acid (ALA) synthetase with pyridoxal phosphate as
a co-enzyme
Protoporphyrin + Fe++ Haem
Haem combines with a globin chain formed in the ribosomes
A Hb molecule is formed by tetramer of four globin chains each with its own haem
group in a hydrophobic pocket

Adult Hb = 22
chain synthesis starts after 6 months of age
Fetal Hb = 22 (1% of adult Hb)
HbA2 = 22 (1.5-3% of adult Hb)

WBC production:

Platelet production

PLATELET PRODUCTION
Pluripotent Stem Cell

CFU mega
Mitosis
Megakaryoblast
Mitosis

Basophilic Megakaryocyte
Mitosis
Granular Megakaryocyte

Mature Megakaryocyte
Fragmentation
PLATELETS

Describe abnormal haemoglobins and their clinical significance

Clinically important hemoglobin mutations

Sickle syndromes (abnormal polymerization)
Homozygous sickle cell disease (HbSS)
Sickle-Hemoglobin C (HbSC)
Sickle- thalassemia

Thalassemic disorders - reduced production of a normal globin chain

Structural mutants also resulting in a thalassemic phenotype (Hb Lepore, Constant Spring,
Hb E)
Decreased solubility (crystallization, Hb C disease)
Unstable hemoglobins (congenital Heinz body hemolytic anemia, Hb Koln)
Abnormal oxygen affinity mutations
High affinity hemoglobin (familial erythrocytosis, Hb Chesapeake)
Low affinity hemoglobin (familial cyanosis, Hb Kansas)

M hemoglobins (familial cyanosis/congenital methemoglobinemia)

SICKLE CELL SYNDROMES

The sickle cell syndromes are caused by a mutation in the -globin gene
that changes the sixth amino acid from glutamic acid to valine.
HbS (22 6 Glu:Val) polymerizes reversibly when deoxygenated to form a
gelatinous network of fibrous polymers that stiffen the erythrocyte
membrane, increase viscosity, and cause dehydration due to potassium
leakage and calcium influx. These changes also produce the
characteristic sickle shape. These abnormalities provoke unpredictable
episodes of microvascular vasoocclusion and premature RBC
destruction (hemolytic anemia).
UNSTABLE HEMOGLOBINS
Amino acid substitutions that reduce solubility or increase susceptibility
to oxidation produce unstable hemoglobins that precipitate, forming
inclusion bodies injurious to the red cell membrane. Representative
mutations are those that interfere with contact points between the and
subunits [e.g., Hb Philly], alter the helical segments [e.g., Hb Genova ],
or disrupt interactions of the hydrophobic pockets of the globin subunits
with heme [e.g., Hb Koln]. The inclusions, called Heinz bodies, are
clinically detectable by staining with supravital dyes such as crystal violet
(Heinz body test).
HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY:

High-affinity hemoglobins [e.g., Hb Yakima] bind oxygen more readily but

deliver less O2 to tissues at normal capillary PO levels. Mild tissue
hypoxia ensues, stimulating RBC production and erythrocytosis
Low-affinity hemoglobins [e.g., Hb Kansas] bind sufficient oxygen in the
lungs, despite their lower oxygen affinity, to achieve nearly full
saturation. At capillary oxygen tensions, they lose sufficient amounts of
oxygen to maintain homeostasis at a low hematocrit (pseudoanemia).
Capillary hemoglobin desaturation can also be sufficient to produce
clinically apparent cyanosis.
METHEMOGLOBINEMIAS Methemoglobin is generated by oxidation of
the heme iron moieties to the ferric state, causing a characteristic bluishbrown muddy color resembling cyanosis. Methemoglobin has such high
oxygen affinity that virtually no oxygen is delivered to tissues. Levels >
50 to 60% are often fatal. Congenital methemoglobinemia arises from
globin mutations that stabilize iron in the ferric state [e.g., HbM Iwata ] or
from mutations that impair the enzymes that reduce methemoglobin to
hemoglobin (e.g., methemoglobin reductase, NADP diaphorase).
Acquired methemoglobinemia is caused by toxins that oxidize heme
iron, notably nitrate and nitrite-containing compounds
thalessaemia
The four classic -thalassemias, most common in Asians, are:
-thalassemia-2 trait, in which one of the four -globin loci is deleted;
-thalassemia-1trait, with two deleted loci;
HbH disease, with three loci deleted;
hydrops fetalis with Hb Barts, with all four loci deleted
-Thalassemia-2 trait is an asymptomatic, silent carrier state. Thalassemia-1 trait resembles -thalassemia minor. Offspring doubly
heterozygous for -thalassemia-2 and -thalassemia-1exhibit a more
severe phenotype called HbH disease.
In HbH disease, HbA production is only 25 to 30% of normal.
Fetuses accumulate some unpaired chains. In adults, unpaired
chains accumulate and are soluble enough to form 4 tetramers called
HbH. HbH forms few inclusions in erythroblasts but does not precipitate
in circulating red cells. Patients with HbH disease have thalassemia
intermedia characterized by moderately severe hemolytic
anemia but milder ineffective erythropoiesis. Survival into mid-adult
life without transfusions is common.

The homozygous state for the -thalassemia-1 cis deletion (hydrops

fetalis) causes total absence of -globin synthesis. No physiologically
useful hemoglobin is produced beyond the embryonic stage.
Excess globin forms tetramers called Hb Barts (4), which has an
extraordinarily high oxygen affinity. It delivers almost no O2 to fetal
tissues, causing tissue asphyxia, edema (hydrops fetalis), congestive
heart failure, and death in utero
carboxyhaemoglobin
Hemoglobin binds to carbon monoxide preferentially compared
to oxygen (approx 240:1), so effectively, COHb will not release
the carbon monoxide, and therefore hemoglobin will not be
available to transport oxygen from the lungs to the rest of the
body. COHb has a half-life in the blood of 4 to 6 hours, but in
cases of poisoning, this can be reduced to 70 to 35 minutes
with administration of pure oxygen (the lower number applying
when oxygen is administered with 4 to 5% CO2 to cause
hyperventiliation).
Describe the pharmacology of heparin and low molecular weight
heparins

Heparin

Physicochemical
Structure

glycosaminoglycan found in the secretory granules of mast cells.

300040000daltons[1dalton

wtofhydrogen]

Heparan Sulfate.
Heparan sulfate is synthesized from the same repeating
disaccharide precursor (D-glucuronic acid linked to N-acetyl-Dglucosamine) as is heparin.
Source.
Heparin is commonly extracted from porcine intestinal mucosa or
bovine lung, and preparations may contain small amounts of other
glycosaminoglycans

Class
Presentation

The USP unit is the quantity of heparin that prevents 1 ml of

citrated sheep plasma from clotting for 1 hour after the addition of
0.2 ml of 1% CaCl2.

Prepared as unfractionated (5000 25000 Da)or low molecular

weight heparin (2000 8000 Da)
o LMWH is derived from depolymerised heparin (chemical or
enzymatic degradation)
- Unfractionated: Presented as a Na or Ca salt 1000 25000 IU/ml
o IU used 2 heterogeneity of compound variable potency
- LMWH: Presented in mg/ml
o More consistent in terms of potency

Pharmacodynamics
MOA

Heparin catalyzes the inhibition of several coagulation proteases

by antithrombin.
Antithrombin is synthesized in the liver and circulates in plasma. It
inhibits activated coagulation factors of the intrinsic and common
pathways, including thrombin, Xa, and IXa; however, it has
relatively little activity against factor VIIa.
Heparin increases the rate of the thrombin-antithrombin reaction
at least a thousandfold
Thrombin is more sensitive to binding unfractionated heparin binds to
both ATIII and thrombin
Once thrombin has become bound to antithrombin, the heparin
molecule is released from the complex.
Heparin molecules containing fewer than 18 monosaccharide units
(5400 daltons) also do not catalyze inhibition of thrombin by
antithrombin. Molecules of 18 monosaccharides or greater are
required to bind thrombin and antithrombin simultaneously. Lowmolecular-weight heparin preparations produce an anticoagulant
effect mainly through inhibition of Xa by antithrombin, because the
majority of molecules are of insufficient length to catalyze
inhibition of thrombin.
When the concentration of heparin in plasma is 0.1 to 1 units/ml,
thrombin, factor IXa, and factor Xa are inhibited rapidly (half-lives
less than 0.1 second) by antithrombin. This effect prolongs both
the aPTT and the thrombin time (i.e., the time required for plasma
to clot when exogenous thrombin is added); the PT is affected to a
lesser degree.

Use

initiate treatment of venous thrombosis and pulmonary embolism

initial management of patients with unstable angina or acute
myocardial infarction, during and after coronary angioplasty or
stent placement, and during surgery requiring cardiopulmonary
bypass. Heparin also is used to treat selected patients with
disseminated intravascular coagulation.
Low-dose heparin regimens are effective in preventing venous
thromboembolism in certain high-risk patients.
The principal advantage of low-molecular-weight heparin over
standard heparin is a more predictable pharmacokinetic profile,
which allows weight-adjusted subcutaneous administration without
laboratory monitoring. Thus, therapy of many patients with acute

venous thromboembolism can be provided in the outpatient

setting. Other advantages of low-molecular-weight heparin include
a lower incidence of heparin-induced thrombocytopenia and
possibly lower risks of bleeding and osteopenia.

Dose

Full-dose heparin therapy usually is administered by continuous

intravenous infusion.
Treatment of venous thromboembolism is initiated with a bolus
injection of 5000 units, followed by 1200 to 1600 units per hour
delivered by an infusion pump. Therapy routinely is monitored by
the aPTT.
Very high doses of heparin are required to prevent coagulation
during cardiopulmonary bypass. The aPTT is infinitely prolonged
over the dosage range used. Another coagulation test, such as the
activated clotting time, is employed to monitor therapy in this
situation.
Low-dose heparin therapy is used prophylactically to prevent deep
venous thrombosis and thromboembolism in susceptible patients.
(Until recently a suggested regimen for such treatment was 5000
units of heparin given every 8 to 12 hours.)

For LMWH The target therapeutic range for venous

thromboprophylaxis is 0.1-0.2 anti-Xa U/ml and for DVT
therapy is 0.5-0.8 anti-Xa U/ml
GIT
CVS

CNS
Respiratory
Other

Side
effects/
adverse
effects

High doses of heparin can interfere with platelet aggregation and

thereby prolong bleeding time.
Heparin "clears" lipemic plasma in vivo by causing the release of
lipoprotein lipase into the circulation. Lipoprotein lipase hydrolyzes
triglycerides to glycerol and free fatty acids.
Bleeding. Mild bleeding due to heparin usually can be controlled
without the administration of an antagonist. If life-threatening
hemorrhage occurs, the effect of heparin can be reversed quickly
by the slow intravenous infusion of protamine sulfate, a mixture of
basic polypeptides isolated from salmon sperm. Approximately 1
mg of protamine for every 100 units of heparin remaining in the
patient; it is given intravenously at a slow rate (up to 50 mg over
10 minutes).

Heparin-Induced Thrombocytopenia.
Heparin-induced thrombocytopenia (platelet count 150,000/ml or
a 50% decrease from the pretreatment value) occurs in about
0.5% of medical patients 5 to 10 days after initiation of therapy
with standard heparin.

The development of IgG antibodies against complexes of heparin

with platelet factor 4 (or, rarely, other chemokines) appears to
cause all of these reactions. These complexes activate platelets by
binding to FcIIa receptors, which results in platelet aggregation,
release of more platelet factor 4, and thrombin generation. The
antibodies also may trigger vascular injury by binding to platelet
factor 4 attached to heparan sulfate on the endothelium.
Other Toxicities. Abnormalities of hepatic function tests occur
frequently in patients who are receiving heparin intravenously or
subcutaneously. Mild elevations of the activities of hepatic
transaminases in plasma occur without an increase in bilirubin
levels or alkaline phosphatase activity. Osteoporosis resulting in
spontaneous vertebral fractures can occur, albeit infrequently, in
patients who have received full therapeutic doses of heparin
(greater than 20,000 units per day) for extended periods of time
(e.g., 3 to 6 months). Heparin can inhibit the synthesis of
aldosterone by the adrenal glands and occasionally causes
hyperkalemia, even when low doses are given. Allergic reactions to
heparin (other than thrombocytopenia) are rare.
Heparin Resistance.
The dose of heparin required to produce a therapeutic aPTT varies
due to differences in the concentrations of heparin-binding
proteins in plasma, such as histidine-rich glycoprotein, vitronectin,
and platelet factor 4; these proteins competitively inhibit binding
of heparin to antithrombin. Occasionally a patient's aPTT will not
be prolonged unless very high doses of heparin (50,000 units per
day) are administered. Such patients may have "therapeutic"
concentrations of heparin in plasma at the usual dose when values
are measured by other tests (e.g., anti-factor Xa activity or
protamine sulfate titration). These patients may have very short
aPTT values prior to treatment because of the presence of an
increased concentration of factor VIII and may not be truly
resistant to heparin. Other patients may require large doses of
heparin because of accelerated clearance of the drug, as may
occur with massive pulmonary embolism. Patients with inherited
antithrombin deficiency ordinarily have 40% to 60% of the normal
plasma concentration of this inhibitor and respond normally to
intravenous heparin. However, acquired antithrombin deficiency
(concentration less than 25% of normal) may occur in patients with
hepatic cirrhosis, nephrotic syndrome, or disseminated
intravascular coagulation; large doses of heparin may not prolong
the aPTT in these individuals.
Hypotension with rapid IV administration
o 2 vasodilation effect of heparin
Adrenal insufficiency (hypoaldosteronism)
- Allopecia
- Osteoporosis 2 heparin complexing with mineral substances ( risk LMWH)
- Allergy / Anaphylaxis ( risk bovine)
- Drug displacement (competes for protein binding)

Interaction
s
Pharmacokinetics
Absorption

Heparin is not absorbed through the gastrointestinal mucosa and

therefore is given by continuous intravenous infusion or
subcutaneous injection.
considerable variation in the bioavailability of heparin given
subcutaneously, and the onset of action is delayed 1 to 2 hours;
low-molecular-weight heparins are absorbed more uniformly.
Following subcutaneous administration, the bioavailability
(measured as anti-Xa activity) of LMWH is nearly 100% regardless
of dose, compared with about 30% for UFH

Distribution %plasmabound:95
Incontrast,UFHbindstonumerousplasmaproteinssuchasPF4,histidinerich
glycoprotein,fibrinonectin,vitronectin,vonWillebrandfactor,fibrinogen,andother
acutephaseproteinswhoselevelscanvarygreatly,aswellastomacrophagesand
endothelialcells.

Vd low (40-100ml/kg)

Metabolism

When doses of 100, 400, or 800 units/kg of heparin are injected

intravenously, the half-lives of the anticoagulant activities are
approximately 1, 2.5, and 5 hours, respectively
Heparin appears to be cleared and degraded primarily by the
reticuloendothelial system; a small amount of undegraded heparin
also appears in the urine.

Clearance of UFH is dose-dependent and occurs in two phases: a

dose-dependent rapidly saturable phase involving uptake and
degradation by vascular endothelium, and a slower nonsaturable
phase that reflects renal clearance
elimination of LMWH is primarily renal, and follows first-order kinetics.
Therefore, the plasma half-life of LMWH is two to four times longer
than that of UFH, ranging from 2-4 hours (vs.0.5-1hour) after
intravenous injection and from 3-6 hours after subcutaneous injection
Excretion

hypothermiaandrenalimpairmentdelaysheparinelimination

Kidney:
o Small amount unfractionated heparin unchanged in urine
o 1 route of elimination LMWH (not suitable for renal failure)

Evidence

Low-Molecular-Weight Heparin Preparations.

Enoxaparin
Dalteparin
tinzaparin
ardeparin
nadroparin
reviparin
Low-molecular-weight heparins (1000 to 10,000 daltons; mean, 4500 daltons, or
15 monosaccharide units) are isolated from standard heparin by gel filtration
chromatography, precipitation with ethanol, or partial depolymerization with
nitrous acid and other chemical or enzymatic reagents.
The biological activity of low-molecular-weight heparin is generally measured
with a factor Xa inhibition assay, which is mediated by antithrombin.
More predictable pharmacokinetic properties of low-molecular-weight heparins,
however, permit administration in a fixed or weight-adjusted dosage regimen
once or twice daily by subcutaneous injection.
Synthetic Heparin Derivatives.
Fondaparinux (ARIXTRA) is a synthetic pentasaccharide based on the structure of
the antithrombin binding region of heparin. It mediates inhibition of factor Xa by
antithrombin but does not cause thrombin inhibition due to its short polymer
length. Fondaparinux is administered by subcutaneous injection, reaches peak
plasma levels in 2 hours, and is excreted in the urine with a half-life of 17 to 21
hours. It should not be used in patients with renal failure. Because it does not
interact significantly with blood cells or plasma proteins other than antithrombin,
fondaparinux can be given once a day at a fixed dose without coagulation
monitoring. Fondaparinux appears to be much less likely than heparin or lowmolecular-weight heparin to trigger the syndrome of heparin-induced
thrombocytopenia

Comparison between Unfractionated Heparin and LMWH

Mean Molecular Weight

Saccharide units (mean)
Anti-Xa to anti-IIa activity
Affinity for plasma protein binding
Binds to endothelium
Dose-dependent clearance
Bioavailability at low doses
Inhibits platelet function
Increases vascular permeability

Standard Heparin

LMWH

12000-15000
40-50
1:1
High
Yes
Yes
Poor
Strong
Yes

4000-6500
13-22
2:1 to 4:1
Low
Weakly
No
Good
Moderate
No

Table 3. Some Commercially Available Low Molecular Weight Heparins

International

Method of

Anti-factor

Mean molecular

Plasma

Non Proprietary

Production

Xa/IIa ratio

weight (Daltons)

(min)

2.7:1

4500

129-180

acid

2.0:1

5000

119-139

3.2:1

4500

132-162

1.9:1

4500

111

2.0:1

6000

200

20:1

6500

1100

Name (Trade
Name)
Enoxaparin

Benzylation

(Clexane,

Alkaline

Covenox)

Hydrolysis

Dalteparin

Nitrous

(Fragmin)

depolynerization

Nadroparin

Fractionation /

(Fraxiparin)

Nitrous

acid

depolymerization
Tirizaparin

Heparinase

(Logiparin)

digestion

Ardeparin

Peroxidative

(Normiflo)

cleavage

Danaparoid
(a heparinoid)
(Comoparin,
orgaran)

Describe the pharmacology of protamine

half-life

Structure

Physicochemical
Protamines are simple protein principles obtained from
the sperm of salmon and certain other species of fish

Class
Presentation

Protamine Sulfate Injection, USP

10 mg/mL, 5 mL single-dose

MOA

Use
Dose

GIT

Pharmacodynamics
When administered alone, protamine has an anticoagulant
effect. However, when it is given in the presence of
heparin (which is strongly acidic), a stable salt is formed
which results in the loss of anticoagulant activity of both
drugs.
Protamine sulfate has a rapid onset of action.
Neutralization of heparin occurs within five minutes
after intravenous administration. Although the metabolic
fate of the heparin-protamine complex has not been
elucidated, it has been postulated that protamine
sulfate in the heparin-protamine complex may be
partially metabolized or may be attacked by fibrinolysin,
thus freeing heparin.
treatment of heparin overdosage
1 mg for 100 U (upto 50 mg over 10 min)
Because of the anticoagulant effect of protamine,
it is unwise to give more than 100 mg over a short
period unless there is certain knowledge of a larger
requirement.

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

Too-rapid administration of protamine sulfate can

cause severe hypotensive and anaphylactoid-like reactions

Interaction
s
Pharmacokinetics
Absorption
Distribution
Metabolism
Excretion
Evidence

describe the pharmacology of warfarin

warfarin

Structure

Physicochemical
Oral anticoagulant (coumarin).
2 stereoisemers - racemic mixture S-warfarin/
(4 x as potent)

R-warfarin

Class
Presentation

Tablets 0.5 / 1 / 3 / 5mg

Pharmacodynamics
MOA

Use

Dose

Prevents the hepatic synthesis of factors II, VII, IX, X

which are vitamin K dependent
Vitamin K is oxidised during the carboxylation of
the glutamic acid residues of the above factors.
Vitamin K reductase (and Vitamin K epoxide
reductase) returns oxidised Vitamin K to reduced
form in order to allow reactions to progress
Warfarin competitively inhibits these enzymes
prevents the reduction of Vitamin K
Results in depletion of the Vitamin K dependent
factors
Also inhibits Protein C & S production Effect faster
than inhibition of coagulation factors **initial
procoagulant effect
Half life - Vll = 6 hrs.; X = 40 hrs; lX = 24 hrs, ll =
60hrs
Prophylaxis of thrombosis / embolus formation in
patients with
o Articifial heart valves
o Arrhythmias (AF)
o h/o PE, peripheral arterial thrombosis, DV

2-5 mg/ day PO until INR therapeutic then stabilize

GIT
CVS

CNS
Respiratory
Other

Factors associated with speed of onset & offset of biological

activity of warfarin
ONSET
biological effect (INR) not seen for ~3-5 days
- Vitamin K Stores

o Rapid onset: Loading dose regimen; low vit K stores (seen

perioperatively); liver failure
o Slow onset: High vit K stores, high vit K diet
- Circulating clotting factors
o Rapid onset: perioperatively (dilution of circulating factors); liver failure
(synthesis)
- Warfarin
o Rapid onset /free fraction: alb (postop / liver failure / sepsis);
displacement reactions (amiodarone); cytP450 activity (Cimetidine)
o Slow onset: cytP450 activity (phenytoin, barbiturates)
- Other anticogulants (NSAIDs, heparin) risk haemorrhage

OFFSET
Speed of Offset:
- Rapid (min-hrs): FFP, Prothrombinex (cryoprecipitate) II, IX,
X concentrate
- Day: 1mg vitamin K (10mg impairs anticoagulation for days)
Side
effects/
adverse
effects

Special precaution:
Need responsible patients.
The following decrease INR - hereditary resistence/ VitK/ hypothyroid/ diuretics (spironolactone,
chlorthlidone) / barbiturates + rifampicin (induce hepatic enzymes) / cholestyramine (binds warfarin)
The following increase INR - decreased metabolism of S-warfarin by phenylbutazone/ metronidazole /
miconazole / trimethoprim/ sulphamethoxazole/ sulfinpyrazone
- decreased metabolism both isomers - amiodarone/ disulfiram / cimetidime.
Aspirin (decreases platelet function)
cephalosporins (decrease GI bacteria producing VitK)
Hepatic disease (decrease clotting factors)
Hypothyroid (decrease survival clotting factors).

ADVERSE EFFECTS
1. Haemorrhage.
2. Tissue Necrosis - venous thrombosis
leading to haemorrhagic infarction secondary to protein C
inhibition (cutaneous, breast)
Interaction
s
Pharmacokinetics
Absorption 100% bioavailability.
Warfarin detectable in plasma at 1hr post administration
o Peak 4-8 hrs post administration
Distribution >99% albumin bound therefore Vd = albumin = 0.12L/kg
Metabolism t 40hrs; metabolised in liver
R-warfarin (reduced) / S-warfarin (oxidized) to inactive metabolites by
glucuronide conjugation
Excretion
Enterohepatic circulation.
Excreted in urine and stool

Evidence

Classify and describe the pharmacology of anti-platelet drugs

Aspirin

Structure
Class

Physicochemical
Acetylsalicylic acid
Non-opiod analgesic. Synthesized 1853.
NSAID.
Used from 1899

Presentation

Pharmacodynamics
MOA
Irreversible blockade of platelet COX-1 prevents conversion of arachidonic acid
to TXA2
o platelet adhesion / activation / aggregation
o vasoconstriction
- Reversibly inhibits COX-1 / COX-2 throughout body
o Endothelium (prostacyclin)
o Renal arteries PG production
o GIT PG production

2. Inhibits kallikrein system (via inhibition kallikrein mediated

PG)
3. Analgesic i) decreased inflammatory mediators ii) ?
subcortical site
4. Antipyretic i) vasodilatation ii) decreased IL-1 by
macrophages: decreased
hypothalamic response

Use

Dose

Duration of Action
- Platelet activity is irreversible therefore action is present
for the lifespan of the platelet (7-10 days)
1. Analgesia
2. Anti-inflammatory
3.
Anti-pyretic
4. Platelet inhibition
5. Recurrent miscarriages
6.
HELP syndrome
Primary and secondary prophylaxis MI , TIA, CABG,
Analgesic/antipyretic dose 600-1200mg / d.
Anti-inflammatory dose > 4g / d

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

Related to non-specific blockade of COX-1 and COX-2 PG synthesis

- GIT: gastric acid production, mucosal barrier peptic ulceration / GIT upset
- Renal: PG-dependent renal artery dilatation renal impairment / failure in those
susceptible; papillary necrosis is chronic users . Decreased GFR via decreased PGI
2
- Resp: O2 consumption / CO2 production at therapeutic doses uncouples
oxidative phosphorylation

- OverDose: RR resp alkalosis 2 direct stimulation of respiratory centre,

metabolic acidosis 2 nature of drug
Salicylism - tinnitus, vertigo, decreased hearing ( usually chronic intoxication).
Low dose decreased secretion uric acid. High dose increases secretion - uricosuric.
Confusion, fever, dehydration with increased dose
TOXICOLOGY
Serious intoxication > 150-175mg /kg
1. Respiratory alkalosis - peripheral - increased CO2 produced by skeletal muscle
central
- stimulates medullary respiratory centre causes
increased RR and VT
2. Renal compensation - for resp alkalosis.
3. Further metabolic acidosis - i) salicylic acid dissociation giving increased H+
ii) deranged CHO metabolism leads to lactilactic,
pyruvic, acetoacetic acid accumulation
iii) decreased vasomotor function causes decreased
renal function and increased PO4/SO4 levels.

Interaction
s

. Increased risk salicylate intoxication with acetazolamide.

2. EtOH increased risk GI bleeding.
3. Displaces from protein binding - phenytoin, tolbutamide,
methotrexate.
4. Decreased activity spironolactone.
5. Decreased tubular secretion penicillin
Pharmacokinetics
Absorption 1. Absorbed rapidly in stomach (acid pH) and also
duodenum.
2. High mucosal cell concentration - leads to damage.
3. Buffering gastric pH > 3.5 decreases irritation.
Distribution Absorbed as ASA, hydrolyzed to acetic acid and salicylate
(blood and tissue esterase).
2. Bound to albumin - increased serum concentration increased free salicylate.
Metabolism
Hepatic:
o 50% metabolised via saturable enzyme pathway to salicyurate
o 20% glucoronidated (also saturable)
First order kinetics - half life 3 - 5 hours.
At high doses Zero order kinetics - half life > 15 hours due to
saturation of hepatic enzymes
Excretion
o Dose dependent elimination
Renal excretion - increased by alkalinizing urine.
Evidence

Clopidogrel
Physicochemical
Structure
Class

thienopyridine
antiplatelet

Presentation

75mg tablet

MOA

Pharmacodynamics
Irreversible blockade of the ADP receptor on the surface of
platelets
- ADP released from dense granules during platelet release
reactions (activation phase)
o Role of ADP platelet aggregation
o Prevents GP IIb/IIIa receptor transformation into active form
Duration of Action
- Due to irreversible blockade of ADP receptor action lasts
for lifespan of platelet (7-10 days)

Use
Dose

75 mg per day with or without an initial loading dose of 300

mg

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

Bone marrow suppression 2 ADP receptor blockade

o Neutropaenia
- Thrombotic thrombocytopaenic purpura
- Haemorrhage cerebral / GIT (risk with coadministration
with aspirin)

Interaction
s
Pharmacokinetics
Absorption rapidly absorbed after oral administration
peak plasma levels (appx. 3 mg/L) of the main circulating
metabolite occurring approximately one hour after dosing.
The pharmacokinetics of the main circulating metabolite are
linear (plasma concentrations increased in proportion to
dose) in the dose range of 50 to 150 mg of clopidogrel.
Distribution Clopidogrel and the main circulating metabolite bind
reversibly in vitro to human plasma proteins (98% and 94%,
respectively).
Metabolism
Clopidogrel is a pro-drug activated in the liver by cytochrome P450
enzymes, including CYP2C19. The active metabolite has an
elimination half-life of about eight hours and acts by forming a

Excretion

disulfide bridge with the platelet ADP receptor.

Extensive hepatic metabolism carboxylic acid derivative /
glucoronide (not active)
Elimination of metabolites in urine
Evidence

dipiridamol

Physicochemical
Structure
Class
Presentation

Pharmacodynamics
MOA

Inhibition of platelet adenosine uptake

o platelet adhesion to damaged vessels
- Potentiates effects of endothelial prostacyclin
o vasoconstriction
- Reversible inhibition of phosphodiesterase activity in platelet (Phosphodiesterase
metabolises cAMP AMP)
o cAMP Ca2+ phospholipase A2 activity arachidonic acid TXA2
formation
platelet aggregation
Smooth muscle relaxation
- Platelet adhesion > platelet aggregation

Use
Dose
CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects
Interaction
s

Vasodilation / Hypotension

Pharmacokinetics
Absorption
Distribution
Metabolism
Excretion

1 hepatic metabolism (glucoronidation)

- Excretion via bile
- Negligible renal excretion
Evidence

Tirofiban/ abciximab

Physicochemical
Structure
Class
Presentation
MOA

Pharmacodynamics
Block final common pathway of platelet aggregation
Prevents cross-linking of vWF/ fibronectin
- Abciximab monoclonal antibody high affinity for GP
IIb/IIIa R
- Tirofiban intermediate receptor affinity
Dont block platelet adhesion / activation / release reactions
Duration of Action
- Abciximab up to 15 days
- Tirofiban short (hours)

Use

Dose

Tirofiban: When given according to the recommended

regimen, >90% inhibition is attained by the end of the 30minute infusion. Platelet aggregation inhibition is reversible
following cessation of the infusion
AGGRASTAT (tirofiban), in combination with heparin, is
indicated for the treatment of acute coronary syndrome,
including patients who are to be managed medically and
those undergoing PTCA or atherectomy. In this setting,
AGGRASTAT has been shown to decrease the rate of a
combined endpoint of death, new myocardial infarction or
refractory ischemia/repeat cardiac procedure
In most patients, AGGRASTAT should be administered
intravenously, at an initial rate of 0.4 mcg/kg/min for 30
minutes and then continued at 0.1 mcg/kg/min.
Patients with severe renal insufficiency (creatinine clearance
<30 mL/min) should receive half the usual rate of infusion

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

Allergy
- Thrombocytopaenia
- risk bleeding with abciximab

Interaction
s
Pharmacokinetics
Absorption
Distribution Unbound fraction of tirofiban in human plasma is 35%. The

Metabolism
Excretion

steady state volume of distribution of tirofiban ranges from

22 to 42 liters
Abciximab unknown
- Tirofiban metabolism is limited.
Tirofiban has a half-life of approximately 2 hours. It is cleared
from the plasma largely by renal excretion, with about 65%
of an administered dose appearing in urine and about 25% in
feces, both largely as unchanged tirofiban.
Evidence

Describe the fibrinolytic pathway and outline the pharmacology of the

thrombolytic agents

Extrinsic activators of plasminogen-plasmin system:

Streptokinase
Urokinase
Rtpa
Intrinsic activators of plasminogen-plasmin system
Factor XII a
Kallikrien
Tpa
Extrinsic inhibitors:
EACA
Tranexamic acid
Aprotinin

Intrinsic inhibitors:
2 antiplasmin
2 macroglobulin
Streptokinase
Physicochemical
Structure
Class

Fibrinolytic drug.

Presentation
MOA

Pharmacodynamics
Produced by streptococci.
Urokinase produced by kidney
SK combines with plasminogen = activator complex.
Sk/plasminogen catalyzes plasminogen producing plasmin.
1. Fibrin --- fibrin split products.
2. Fibrinogen --- FDP.
Urokinase directly converts plasminogen to plasmin.
APSAC = anisoylated plasminogen: SK activator complex - after administration acyl group hydrolizes
therefore enzyme active site exposed

Use

Dose

. Acute myocardial infarction.

2. Pulmonary embolism - especially with significant CVS /
resp compromise.
3. Proximal DVT (decreased post phlebytic syndrome).
4. IV access de-clotting
MI - 1.5millionUnits over 30-60 mins.
PE - 250, 000Units over 30 mins --- 100,000Units/hour for 24
hrs
IV cannulae - 250,000Units in 2mls - leave in situ for 2 hrs.

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

CONTRAINDICATIONS
1. Recent haemorrhage or haemorrhagic diathesis.
2. Recent (within 2/12) CVA; intracranial or intraspinal
surgery.
3. Recent surgery up to 10th post-op day
4. High anti-strepAbs (e.g. Rheumatic fever, post-strep GN)
5. Uncontrolled hypertension > 200/110.
6. Other instrumentations or past bleeding conditions
SPECIAL PRECAUTIONS
1. Can be repeated within 5 days, but not between 5 days to
1 year (?longer).

2. Hypotension - slowing or cessation of infusion.

3. Reperfusion arrythmias in 1-10%
ADVERSE EFFECTS
1. Bleeding - treatment is stopping infusion / pressure/ FFP.
May use aminocaproic acid which inhibits plasminogen
--plasmin.
2. Anaphylaxis - secondary to antistreptococcal antibodies.
3. Hypersensitivity reactions.
Interaction
s

Increased bleeding with other anticoagulants

Pharmacokinetics

Absorption IV infusion
Distribution Intravascular space
Metabolism
Half life =- 23 mins
In part inactivated by antistreptococal antibodies
Excretion
No known metabolites - mechanism of elimination unclear
Evidence

tPA

Class

Physicochemical
Recombinant tissue-type plasminogen activator.
Fibrinolytic - recombinant via mammalian cell culture.

Presentation

50mg vial

Structure

MOA

Use

Dose

Pharmacodynamics
Converts plasminogen -- plasmin - fibrin -- FSP.
High affinity for fibrin therefore supposedly activates plasmin
in clot- bound fibrin.
Supposedly causes local fibrinolysis >> systemic fibrinolysis.
1. Acute myocardial infarction.
2. Pulmonary embolism.
especially indicated if
streptokinase
3. Proximal / central DVT.
contraindicated due
to previous exposure.
4. IV canually de-clotting.
100mg t-PA over 180 mins.
10mg in 1-2 mins
50mg in 1hr
40mg in 2hrs

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

CONTRAINDICATIONS
1. Haemorrhagic diathesis.
2. Recent internal bleeding.
3. Cerebral bleeding - within 2/12 of intracranial /
instraspinal surgery.
4. Major operation within 10 days.
5. Uncontrolled hypertension >200/110
6. Infective endocarditis.
7. Acute pancreatitis
Adverse effect:
Bleeding- may require coagulationfactors and possibly
aminocaproic acid

Interaction
s

With oral anticoagulants causing increased bleeding

Pharmacokinetics

Absorption IV infusion.
Distribution
Metabolism
Cleared via liver metabolism.
In 5 mins plasma level is decreased by 50%/ 10mins by 80%/
20mins by >90%.
Excretion
Evidence

Outline the pharmacology of antifibrinolytic agents such as epsilon

aminocaproic acid, tranexamic acid and aprotinin
Aminocaproic acid (EACA)
Similar to the amino acid lysine
Synthetic inhibitor of fibrinolysis.
Competitively inhibits plasminogen activation
Rapidly absorbed orally and is cleared from the body by the kidney.

Oral dose: 6 g four times a day.

IV dose: 5 g loading dose should be infused over 30 minutes to avoid
hypotension.
50% is excreted unchanged in the urine within 12 hours
Tranexamic acid
Analog of aminocaproic acid and has the same properties.
Oral dose: 15 mg/kg loading dose followed by 30 mg/kg every 6 hours,
Clinical uses:
adjunctive therapy in haemophilia
as therapy for bleeding from fibrinolytic therapy
as prophylaxis for rebleeding from intracranial aneurysms.
Postsurgical gastrointestinal bleeding and postprostatectomy bleeding
and bladder hemorrhage secondary to radiation- and drug-induced
cystitis.
Adverse effects:
intravascular thrombosis from inhibition of plasminogen activator,
hypotension, myopathy, abdominal discomfort, diarrhea, and nasal
stuffiness. The drug should not be used in patients with disseminated
intravascular coagulation or genitourinary bleeding of the upper tract, eg,
kidney and ureters, because of the potential for excessive clotting.
Aprotinin
Serine Protease Inhibitors
inhibits fibrinolysis by free plasmin and may have other antihemorrhagic
effects as well.
It also inhibits the plasmin-streptokinase complex in patients who have
received that thrombolytic agent.
Aprotinin will reduce bleedingby as much as 50%from many types of
surgery, especially that involving extracorporeal circulation for open
heart procedures and liver transplantation.
It is currently approved for use in patients undergoing coronary artery
bypass grafting who are at high risk of excessive blood loss.
adverse effects:
A more recent study indicated an increased risk of myocardial infarction,
stroke, and renal damage in aprotinin-treated patients. In larger studies,
a possible association with anaphylaxis has been reported in < 0.5% of
cases. Therefore, a small test dose is recommended before the full
therapeutic dose is given.

describe the pharmacology of heparin alternatives/analogues with

particular reference to direct thrombin inhibitors
ADVANTAGES OF DIRECT THROMBIN INHIBITORS OVER HEPARIN
Unfractionated Low molecular

Direct Thrombin

Heparin

inhibitor

weight
Heparin

Pharmacokinetic
Binding to plasma

Yes

Partial

No

Yes

Partial

No

Xa=IIa
Yes

Xa>IIa
Yes

IIa
No

effects
Inactivate clot bound

no

no

yes

thrombin
Platelet inhibition

yes

limited

Yes, thrombin

rare

induced
no

protein/ endothelium
Inactivated by heparinases
Biological Effects
Anticoagulant effects
Antithrombin dependent

Thrombocytopenia

yes

DIRECT THROMBIN INHIBITORS

These drugs can be divided into those that bind bivalently to thrombin at exosite 1(the
substrate recognition site) as well as the active site, and those that bind univalently to the
active site of thrombin only.
Bivalent DTIs that have been evaluated clinically are hirudin (desirudin) and bivalirudin
(previously hirulog).The low molecular weight univalent DTIs include argatroban
(Novastan), melagatran (the active form of ximelagatran), efegatran and inogatran.

HIRUDIN

Hirudin, a 65 amino acid polypeptide, with a molecular mass of 7000 Da. was originally
isolated from the saliva of the medicinal leech, (Hirudo medicinalis) in the late 1950s (now
available through recombinant DNA technology
- binds to thrombins active site by its globular amino-terminal domain and to thrombins
substrate recognition site ( exosite 1) via its carboxy-terminal domain.
- binding tightly to the enzyme.
- almost irreversible nature of this complex,
- no available antidote should bleeding occur.
not absorbed via the gastrointestinal tract and must be administered intravenously or by
subcutaneous injection
Clearance of hirudin is predominantly via the kidneys, so it should not be used in patients
with renal insufficiency.
- plasma half life of approximately 60 minutes after IV injection and approximately 120
minutes after subcutaneous injection.
Accidental over-dosage of hirudin in patients with renal insufficiency can be managed with
dialysis using polymethyl-methyl acrylate (PMMA) membranes which bind hirudin with high
avidity, thereby clearing it from the circulation. Dialysis with other membranes does not
effectively remove hirudin. .
MONITORING
monitored with the activated partial thromboplastin time (aPTT), recommended target aPTT
1.5-2.5 times the median of the laboratory normal range) This should be determined before
treatment, 4 hrs after the start of intravenous hirudin therapy, 4 hrs after each dosage change
and then at least once daily.

Unfortunately there are problems when aPTT is used to monitor hirudin therapy. There is a
variable response between patients and the lack of a linear correlation with plasma hirudin
levels.
at higher doses, use of the ecarin clotting time may be more appropriate, as its

correlation with plasma hirudin levels is more linear. aPTT monitoring may also
become an inaccurate measure if the patients baseline aPTT is raised due to a nonspecific lupus inhibitor or as a result of hepatic dysfunction.
--currently licensed for treatment of arterial or venous thrombosis complicated by
heparin-induced thrombocytopaenia and as an alternative to heparin for
cardiopulmonary bypass surgery in these patients. It has also been evaluated in acute
coronary syndromes and for venous thromboprophylaxis although further data is
required before is it approved for acute coronary syndromes because of its narrow
therapeutic window and concerns about the risk of bleeding.

BIVALIRUDIN
Bivalirudin is a synthetic 20-amino-acid polypeptide, with a molecular mass of 2000 Da. It is
comprised of an active site directed moiety, D-Phe-Pro-Arg-Pro which is linked via 4 Gly
residues to a dodecapeptide analogue of the carboxy terminal of hirudin, that interacts with
exosite 1 on thrombin.
It is a bivalent inhibitor of thrombin, and once bound, produces transient inhibition of the
active site of thrombin as thrombin cleaves the Pro-Arg bond within the amino terminal of
bivalirudin. With the removal of this bond within the amino segment, the carboxy terminal
portion of bivalirudin bound to thrombins substrate recognition site makes it a much weaker
thrombin inhibitor.
It has a plasma half life of 25 mins after intravenous infusion, with only a small amount of
the drug excreted via the kidneys,) suggesting that endogenous peptidases may contribute to
its clearance.
Consequently bivalirudin may be a safer alternative to hirudin in patients with renal failure
Bivalirudin has been approved as a heparin alternative in patients undergoing percutaneous
coronary angioplasty based on the results of a phase III study that compared bivalirudin with
heparin in 4098 patients with unstable angina undergoing percutaneous angioplasty.

ARGATROBAN
Argatroban, an arginine derivative, is a synthetic small molecule with a molecular mass of
508.7, initially described in 1978.
It is a univalent competitive inhibitor of thrombin, forming a non covalent bond with the
active site of thrombin to form a reversible complex.
Argatroban has a plasma half life of 39 to 51 mins
extensively metabolized by the liver into 4 mostly inactive metabolites, M1-M4, M1 having
3-5 times weaker activity. It is eliminated in the faeces after biliary secretion.
The recommended dosage of argatroban in the treatment of HITTS is 2ug/kg/min and dosing
should be adjusted to maintain the aPTT at 1.5 to 3 times the patients baseline, with a
maximum infusion rate at 10 ug/kg/min.
As argatroban relies on the liver as its primary mode of elimination , it can be administered
safely to patients with advanced renal insufficiency without the need for dosage adjustments.
In hepatic insufficiency the initial starting dose should be reduced to 0.5ug/kg/min and dose
adjusted according to aPTT . Argatroban may be preferable to the other agents in either
treatment of HITTS or during percutaneous intervention due to a combination of (1)
predictable dose-dependent therapeutic response that can be monitored with aPTT (2) the use
in renal failure patients and (3) its short half life

XIMELAGATRAN
Ximelagatran, a prodrug of melagatran, is an uncharged lipophilic univalent drug that has
negligible intrinsic activity against thrombin
Melagatran is a dipeptide mimetic of the portion of fibrinopeptide A that interacts with the
active site of thrombin.
melagatran is poorly absorbed from the gastrointestinal tract,

ximelagatran is well absorbed, has an oral bioavailability of about 20

It has a plasma half life of 3 hours and is administered twice daily.

The active agent, melagatran is eliminated via the kidneys,

One of the side effects of ximelagatran is elevation of liver transaminases, which occurs in
approximately 6% of patients. These levels decrease spontaneously during continued
treatment or following treatment discontinuation. With its predictable pharmacokinetics, wide
therapeutic window and no food and drug interactions, this orally active agent does not
require laboratory monitoring,) and has the potential to replace warfarin in patients with
nonvalvular atrial fibrillation. It may also prove suitable for extended use in both the
prophylaxis and treatment of venous thromboembolism.

Adverse effects.
One of the drawbacks of anticoagulants is that haemostasis is impaired and thus entails a risk
of bleeding.
no specific antidote has been recommended at present. Experimental studies reveal several
possibilities exist, using prothrombin complex concentrates, or fresh frozen plasma which
antagonize the anticoagulant effects and furnish thrombin, which then forms an inactive
complex with the thrombin inhibitor. However these antidotes still need careful preclinical
and clinical evaluation

PROPERTIES OF Direct Thrombin Inhibitors

PROPERTY
SITES OF

HIRUDIN
Active site

BIVALIRUDIN
Active site and

INTERACTION

and exosite exosite 1

WITH THROMBIN
PREDOMINANT

1
renal

Proteolysis at

ARGATROBAN
Active site

XIMELAGATRAN
Active site

hepatic

renal

MECHANISM OF

sites other than

CLEARANCE

kidneys and

ROUTE OF

IV

liver
IV

IV

Oral

ADMINISTRATION
HALF-LIFE,MIN
APPROVED

60
HITTS

25
Heparin

45
HITTS

240
None

INDICATIONS

alternate during
percutaneous
coronary
interventions

Describe the pharmacology of activated protein C, and activated factor VII.

FACTOR VII
Structure
Class

Physicochemical
Recombinant factor 7 of mol. Wt ~ 50000 da produced by genetic
engineering from baby hamster kidney
Coagulation factor/pro-coagulant

Presentation

Freeze dried white powder in vials

After reconstitution ~ 1mg / ml or 50000 IU/ml

MOA

Use

Dose

Pharmacodynamics
The role of FVIIa in the induction of haemostasis includes the
direct activation of FIX into FIXa and FX to FXa following
binding of FVIIa to exposed tissue factor, initiating conversion
of prothrombin into thrombin. Thrombin leads to the
activation of platelets and factor V and VIII at the site of
injury and formation of haemostatic plug by converting
fibrinogen into fibrin.
Pharmacological doses of FVIIa results in direct activation of
FX on the surface of activated platelets, at the site of injury
without the need for tissue factor large amount of prothombin is converted into thrombin. In summary FVIIa results
in rapid and large local production of FXa, thrombin and fibrin
Control of bleeding and surgical prophylaxis in pt:
With inhibitors to FVIII or IX
With congenital FVII deficiency
With glanzmanns thrombasthenia, who have antibody to GP
IIb-IIa and/or HLA and with past or present refractoriness to
platelet transfusions
Bolus injection of 80-120 g/Kg at dosing interval of <2.5
hours with a minimum of three doses to secure effective
haemostasis

CVS

CNS
Respiratory
Other
Side
effects/
adverse
effects

Body as a whole: fever , headache, pain

Platelets, bleeding, and clotting: haemorrhage, decreased
fibrinogen, DIC, increased fibrinolysis
Skin and musculoskeletal: haemarthrosis, pruritus, purpura,
rash

Interaction
s
Pharmacokinetics
Absorption Only given IV
Distribution Vd ~ 130-160 mL/Kg

Metabolism

Mean terminal half life ~ 3.9-6 hours

FVII coagulant half life~ 2.5 hrs

Excretion
Evidence

Activated protein C

Structure

Physicochemical
drotrecogin alfa (activated)) is a recombinant form of human activated
protein C
serine protease with the same amino acid sequence as human
plasma-derived activated protein C

Class
Presentation

MOA

Use

Dose

CVS

CNS
Respiratory
Other

supplied as a sterile, lyophilized, white to off-white powder for

intravenous infusion. The 5 and 20 mg vials of Xigris contain 5.3
mg and 20.8 mg of drotrecogin alfa (activated), respectively.

Pharmacodynamics
Activated protein C exerts an antithrombotic effect by
inhibiting Factors Va and VIIIa. In vitro data indicate that
activated protein C may have indirect profibrinolytic activity
through its ability to inhibit plasminogen activator inhibitor-1
(PAI-1) and may exert an anti-inflammatory effect by limiting
the chemotactic response of leukocytes to inflammatory
cytokines, an inhibitory process mediated by leukocyte cell
surface activated protein C receptor. In addition, in vivo data
suggest activated protein C may reduce interactions between
leukocytes and the microvascular endothelium. In vitro
bacterial phagocytosis by neutrophils and monocytes is not
affected.
indicated for the reduction of mortality in adult patients with
severe sepsis (sepsis associated with acute organ
dysfunction) who have a high risk of death (e.g., as
determined by APACHE II score 25)
should be administered intravenously at an infusion rate of
24 mcg/kg/hr (based on actual body weight) for a total
duration of infusion of 96 hours.

Side
effects/
adverse
effects

Bleeding is the most commonly reported adverse reaction

Anaphylaxis
Contraindication:

Active internal bleeding

Recent (within 3 months) hemorrhagic stroke

Recent (within 2 months) intracranial or intraspinal

surgery, or severe head trauma

Trauma with an increased risk of life-threatening

bleeding

Presence of an epidural catheter

Intracranial neoplasm or mass lesion or evidence of

cerebral herniation

Interaction
s
Pharmacokinetics
Absorption
Distribution
Metabolism inactivated by endogenous plasma protease inhibitor
the median clearance of drotrecogin alfa (activated) was 40 L/hr
(interquartile range of 27 to 52 L/hr) in adults with severe sepsis. The
median Css of 45 ng/mL (interquartile range of 35 to 62 ng/mL) was
attained within 2 hours after starting the infusion. In the majority of
patients, plasma concentrations of drotrecogin alfa (activated) fell
below the assay's quantitation limit of 10 ng/mL within 2 hours after
stopping the infusion. Plasma clearance of drotrecogin alfa (activated)
in patients with severe sepsis is approximately 50% higher than that in
healthy subjects.
Excretion
Evidence

Describe the pharmacology of blood products

Whole blood and red cell preparations
Normally 400-480 ml of blood is taken with 63 ml anticoagulant.
The anticoagulant dilutes the plasma by about 20%
Packed red cells are obtained after removing 200-250ml plasma after centrifugation or
sedimentation of 1 unit of whole blood. PRBC have hematocrit ~ 0.75 or higher

ANTICOAGULANTION:
Citrate-phosphate dextrose (CPD: sodium citrate-1.66g, anhydrous dextrose 1.46 g,
citric acid monohydrate 206mg, sodium acid phosphate 158mg, and water to 63ml)
CPD adenine (sodium 1.66g, anhydrous dextrose 1.82 g, citric acid monohydrate
206mg, sodium acid phosphate 158mg , adenine 17.3 mg, water to 63ml)
SAG M
ADSOL: shelf life extended to 42 days (saline, adenine, glucose and mannitol)
Components of standard anti-coagulants:
o Citrate
prevents clotting by binding Ca++
o Phosphate
pH ~ 5.5, acts as a buffer against the large fall in [H+] at 1-6C
? also may increase 2,3-DPG levels
o Dextrose
Allows continued glycolysis & maintenance of ATP
o Adenine
improves RBC survival by adding substrate for ATP synthesis
increases survival from 21 35 days
STORAGE OF BLOOD:
o Blood is stored at 4-6C
o Frozen storage: Frozen red cells are treated with glycerol (3.8M) as the
cryopreservative and stored in liquid nitrogen. The red cells must be thawed and
washed extensively with electrolyte solutions to remove glycerol before transfusion .
freezing & washing process decreases HLA antigens
Effect of storage on whole blood:
Red cells:
o As storage time increases, some red cells become spherical, due to metabolic
changes, with an associated increase in cell rigidity. If red cells are transfused
at the maximum recommended storage time, 10-20 % may be destroyed within
24 hrs
White cells:
o Granulocytes lose their phagocytic and bactericidal properties within 4-6 h
after collection but maintain their antigenic properties
Platelets:
o Become non-functional within 48 hrs in blood stored in 4C
Factor V and VIII:
o Levels decrease with the storage of whole blood. Factor V decreases 50% by
21 days, while factor VIII decreases exponentially to 75% by 24 hrs after
collection and 30 % after 21 days of storage
ATP and 2,3 DPG
o Concentrations fall with time but at different rates
o With CPD-A blood, 2,3 DPG decreases to 50% at 14 days and to 5% at 28
days. ATP decreases slowly to 75% at 28 days

Potassium levels
o After the first 48 hrs, there is slow potassium loss from the red cells into the
plasma, so that the plasma K+ conc. reaches approx 30mmol/L at 28 days
Metabolic effects
o glucose / dextrose / ATP / 2,3-DPG, and lactate
o PaCO2 , pH, HCO3
o Na+ / K+
o Oxidant damage to membranes with spherocyte formation
o 2,3-DPG O2 affinity
o changes occur earlier & to greater extent in whole blood cf. packed cells
Microaggregates
o conventional filters remove particles > 170 m
o aggregates of platelets/fibrin/leukocytes range from 20 to > 170 m
o clinical significance of microaggregates debated
o most would no longer use a micropore filter
o no change in the incidence of ARDS

INDICATIONS FOR TRANSFUSION

1. increase the O2 carrying capacity of blood DO2
2. increase circulating blood volume, when DO2 is low
NB: Hct at which transfusion indicated is age & disease dependent,
otherwise healthy patients rarely require transfusion at Hct > 30%,
whereas transfusion is usually required at Hct < 21% (RDM)
COMPLICATIONS OF BLOOD TRANSFUSION:
o Rapid or Massive Transfusion
impaired O2 transport
haemostatic failure
electrolyte & metabolic disturbance
vasoactive reactions
serological incompatibility
impaired reticuloendothelial function
o Oxygen Transport
o Transfusion Coagulopathy
Dilutional Thrombocytopenia
Low Factor V & VIII Activity
Disseminated Intrvascular Coagulation
o Metabolic Effects
citrate toxicity
hyperkalaemia
hypothermia
acid-base
o Transfusion Reactions
Immune Reactions
Non-Immune Reactions

Haemolytic Transfusion Reactions:

Acute Haemolytic Transfusion Reactions
Delayed Haemolytic Transfusion Reaction
Nonhaemolytic Transfusion Reactions
o Post-Transfusion Jaundice
o Infective Complications
Human Immunodeficiency Virus
Hepatitis Viruses
Cytomegalovirus
HTLV-1
Syphilis
Malaria

Rapid or Massive Transfusion

massive transfusion 1 times the patients blood volume
time-frame 1BV per 24 hours
BV per 2 hours
o

impaired O2 transport
defective rbc function
impaired Hb function
fluid overload / underload
DIC
ARDS
MOSF
microaggregates
haemostatic failure
dilution
depletion / consumption
decreased production
DIC
electrolyte & metabolic disturbance
hyperkalaemia / delayed hypokalaemia
sodium overload
acid-base disturbances
citrate toxicity
hypothermia
vasoactive reactions
kinin activation
damaged platelets & granulocytes
serological incompatibility
immediate generalised reaction
delayed transfusion reaction
Impaired reticuloendothelial function

Oxygen Transport

HbO2 dissociation pH, Temp., PaCO2 and 2,3-DPG

citrate is metabolised to HCO3 L-shift
hypothermia L-shift
stored blood deficient in 2,3-DPG L-shift
CO2 / H+ load R-shift
good correlation between decrease in rbc 2,3-DPG and P50 after 7 days storage,
2,3-DPG 4.8 mol/l 1.2 mol/l
P50 26.5 mmHg 18 mmHg

Transfusion Coagulopathy
Dilutional Thrombocytopenia
o total platelet activity in stored whole blood
~ 60-70% after 6 hrs,
~ 5-10% after 48 hrs
o effects of dilution depend upon,
initial platelet count
risk of haemorrhage depends upon acute versus chronic,
acute loss < 50,000-75,000
chronic disease < 10,000-15,000
volume transfused
~ 2 BV's in children
thrombocytopathy with massive transfusion
Low Factor V & VIII Activity
o Respectively, these decrease to ~ 15% and 50% of normal activity in whole
blood after 21 days
o packed cells minimal quantities
o however, only 5-20% FV and 30% FVIII activity are required for normal
haemostasis
o therefore these factors rarely decrease below those levels required for
coagulation
Disseminated Intrvascular Coagulation
o microvascular thrombosis occurs rarely
o bleeding is common, but usually originates from sites of local pathology
o DIC is associated with a high mortality, 2 underlying disease severity
o ? may be regarded as an incidental pre-terminal event in many patients
Metabolic Effects
o citrate toxicity
o citrate itself is nontoxic
o Produceshypocalaemia to citrate content of unit & rate of infusion,
hyperventilation
o 1.5-2.0 ml/kg/min rarely a problem
o FFP has higher % citrate than WB
o decreases in Ca++ are transient and are restored immediately following TX
o Factors 'g citrate toxicity
hypothermia (~ 50%, 3731C)

 hypovolaemia
liver disease, transplantation
o hyperkalaemia
o generally only with whole blood to the shelf-life of the unit
o 19-30 mmol/l after 21 days
o rate of infusion important 1.5-2.0 ml/kg/min
o hypothermia
o L-shift of HbO2 curve
o all banked products stored at ~ 2-6C and TX should be warmed 38-40C
o reduction of core temperature < 30C 's cardiac irritability and impairs
coagulation
o decreases of 0.5-1.0C may induce postoperative shivering & MRO2 ~
400%
o 42C results in RBC destruction
o warming with radiofrequency warmers is OK, microwaves result in rbc
damage
o Acid-base
o pH of CPD ~ 5.5
o freshly collected blood pH ~ 7.0-7.1, decreasing to pH ~ 6.9 after 21 days
o most acid in WB is CO2 ~ 150 mmHg lungs
o metabolic acidosis is still present when this is removed by adequate ventilation
o however, metabolism of citrate generates HCO 3 and acidosis is rarely a
problem, provided hypovolaemia is avoided and liver function is adequate
PLATELETS
1. random donor platelets - pooled from 6-8 donors
each bag contains ~ 40-50 ml 5-6 x 1010 platelets
stored at room temperature and are viable for ~ 3-5 days
filters with pore sizes < 170 m remove significant numbers
2. single donor platelets - collected by plateletpheresis
requires HLA matched donor to minimise antigenic differences
Special platelet bag made of polyolefin plastic enable better aeration of platelelts and
increases shelf life to 5 days if stored at 20-26with constant agitation.
Requirement for platelets depends upon cause and rate of development
Effects of transfusion variable, depending upon cause & preceding transfusion, t~ 10 days,
a. 1 unit of platelets ~ 7,000-11,000 / mm3 / m2 SA increase
b. 0.1-0.3 units/kg ~ 20,000-70,000 / mm3 (standard dose)
Although platelets contain only HLA class I antigens, contamination by leukocytes, and red
blood cells can cause alloimmunization. Thus ABO and Rh compatible platelets are usually
transfused.
Nearly 1/3rd of transfused platelets are sequestered in the spleen

Indications:
1. platelet count < 10,000 x 109/l * varies between institutions
2. platelet count < 50,000 x 109/l + spontaneous bleeding or surgery
3. platelet dysfunction, irrespective of count + spontaneous bleeding or surgery
Important points,
a. antibody production is to units transfused
limited effectiveness of future transfusions
b. not all hospitals have platelets readily available
c. they should be administered immediately preoperatively
d. they should not be run through a micropore filter

FRESH FROZEN PLASMA

200 ml standard volume contains all factors, including,
1. VIII:C ~ 200U - may be harvested prior to freezing
- noted on unit label
2. IX ~ 200U
3. fibrinogen ~ 400 mg
Prepared within 6 hrs, after which the labile factors (V/VIII) begin to diminish, stored -30C
For same reason should be used ASAP upon thawing
Shelf life of 1 year
Contains proportionally more citrate than whole blood
Administered as ABO compatible transfusion, volume ~ 200 ml/unit

Indications for use,

1. isolated factor deficiencies - laboratory proven
2. Massive blood transfusion 3. Reversal of warfarin effect
4. antithrombin III deficiency - thrombotic state
5. Immunodeficiency states - source of globulins
6. Thrombotic thrombocytopenic purpura
7. Haemophilia A
8. von Willebrand's disease
Dose: The usual starting dose is 10-15 ml/kg (equivalent to four packs of FFP for a 70kg
person), which raises the coagulation levels 12-15%.

Cryoprecipitate
fresh plasma frozen & thawed at 4-8C ~ 3% fails to redissolve, the cryoprecipitate
then warmed to room temperature with 20-50 ml of supernatant plasma
single donor preparation, stored for up to 6 months at -30C
contains,
i. VIII:C ~ 20-85% of the original levels
~ 80-120 units / 10-15 ml of plasma, or
~ VIII:C activity of FFP in 1/10th the volume
~ 120 ml for RX acute bleed in haemophilia A

ii. fibrinogen ~ 3-10x original plasma / ml

~ 250 mg / 10-15 ml of cryo, cf. 200 ml of FFP
- may result in hyperfibrinogenaemia in haemophiliacs
paradoxical bleeding
iii. VIII:vWF ~ 40-70% original plasma
iv. F-XIII ~ 3-10x original plasma / ml
v. fibronectin
Each unit contains a volume of 20-40 ml. It is stored at 30oC for up to 12 months and is
thawed to 37oC immediately before use.
Ten units of cryoprecipitate should increase fibrinogen level by 1 g/litre.
Indications,
1. haemophilia A
factor VIII:C deficiency principal use
not indicated for haemophilia B, as minimal content of factor IX
2. fibrinogen deficiency
preferrable to commercial fibrinogen preparations, which are pooled from
500-5000 donations and carry a high infection risk
massive transfusion plasma fibrinogen < 0.8 g/l
10 units increase plasma levels ~ 1 g/l in an adult (N:1.5-4.0 g/l)
Prothrombinex
contains factors II, IX and X ~ 250U / 10 ml for each factor
has low levels of VII
prepared from human donor plasma
presented as a freeze dried powder, requiring reconstitution with water
screened for HBV, HBC and heat treated for HIV
average dose ~ 1 ml/kg for acute haemorrhage, then 0.5 ml/kg each 24 hours