Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Lecture 2

This lecture includes:
• The blood clotting cascade
• Fibrinogen activation
• Thrombin activation

2
...
1 Overview
Haemostasis (blood clotting) occurs as a
result of several sequential processes
...
The association is mediated
by von willebrandt factor, A large
multimeric plasma protein with a molecular
mass of about 10^4 Kd
...

A Zymogen is an inactive form of a enzyme
which becomes activated following
proteolysis
...
A thrombosis is the throwing of a blood
clot in which can lead to heart attacks and
strokes
...
The beauty of the
system is that a very small amount of material
leads to a very ledge amount of material
being formed
...
This is commonly thought of as the surface tha is exposed when you
damage tissue
...
As the platelets start to aggregate they
bring about the release of other compounds such as seratonin and thobane A2
...
Its only after all this has happened that a
plaque will be able to form
...
These are inactive forms of enzymes that become active as soon as there proteolysed
...
This means they can be put into activity very
quickly
...

The zymogens will have serene residue present in there active sites that are important to there function
...
The activation of some of these proteins is represented by a subscript a to indicate
activation
...
g
...
Most of the factors are also given roman numerals which indicates
there discovery rather than there order
...
The serine proteases near the C terminus contains 250 residues contains the active site of
the enzyme
...
So the
active bit is similar to many others in the body
...
This means that the blod clotting proteases
are a bit more specific in there activation
...
The bit of the zymogen thats cleaved is 150-582 residues in length and also stays
associated with the C terminal
...
There is a theory that its to do with maintiang the specificity of the enzymes
...

2
...
Fibrin itself is made from a soluble protein fibrinogen
...
Fibrinogen itself is made
up molecular weight 340Kda comprising around 3-4% protein in the serum (quite high percentage)
...
The alphaalpha betabeta and gamma gamma chain
...
That has the effect of
releasing two peptides:
A - 18 residue in length from each of the alpha chains
...

A fibrinogen molecule has now lost the fibrino peptides A and B is called a
fibrin monomer
...

The activation of fibrinogen then is the cleavage of these four bonds to loose
these A and B particles to form the monomers
...
The banding
of the fibrin monomers has bands about 23nm apart this is half the length of a
fibrinogen molecule at 46nm
...

So why do fibrin monomers aggregate?
The consensus is that they come togather in the half staggered array and the
bands are present from the more dense globular regions lining up
...
The
main reason is the fibrin peptides normally act to cover the sites that bind the peptides in the array
...
The fibrin terminals have a more positive
center and negatively charged terminals
...
The distribution of charges allows the monomers to aggregate and come
togather to form an array
...
This can be seen that when you poke a
new clot it can be quite elastic in nature
...

The conversion of the soft clot to a hard clot is facilitated by factor 13 (also mown as fibrin stabilising factor)
...
The enzyme that dose this is called a transglutanimase (factor
13) Which causes a cross linking between the glutamine and lycine residues between fibers
...
This
stabilising reaction is quite an important one and people who have a deficiency in this enzyme have a tendency to
bleed
...
3 Thrombin activation
Thrombin is a fairly central enzyme to this system because it not only activates the factor 13 enzyme but also
fibrinogen
...
It has quite a lot of similarities o trypsin
...
Thrombin has two chains A and B that are linked by a
disulphide bond
...

Human thrombin is synthesised as a 579-residue zymogen celled prothrombin (factor 2)
...
The cleavage of prothrombin’s Arg 274Thr 275 and Arg323-Ile 324 bonds releases the A and B chains from one anouther
...


Prothrombin's pro-peptide consists of three domains; an N terminal, a 40-residue Gla domain
...
Gla and kringle domains occur in several of the proteins involved in clot
formation and lysis
...

The Kringle domains get there name from there shape and are curled by by disulphide bonds
...
There significance need a look
back to the actual synthesis of the molecule
...
If you don't have vitamin K in your diet then prothormbin can
only be produced into a form that cant be swell activate by stuart factor (factor 10)
...
This is difficult to understand because the analysis of these proteins shows that they are the
same in terms of primary sequence
...
Studying the
structure of the normal prothrombin using NMR showed the presence of these glutamate residues
...
Because the acid hydrolysis turns the carboxyl glutamate back
to just glutamate
...
Dicumerol is a
substance found in sweet clover thats gone off
...
Warfarin is used as a
vitamin K antagonist, it was also used as a rat poison as the rats at sit and it thinned there
blood causing internal bleeding
...
This is
because the blood doesn't circulate as efficiently they create blood clots
...
It prevents the pooling blood to clot called
thrombosis which can have adverse affects of blocking circulation
...


So why are these Carboxy glutamate residue important form the formation of blood clots? what do thy actually do?
The normal prothrombin with its gamma-carboxyglutamate residues was found to be able
to bind calcium ions (important)
...
The binding of calcium ions provides a means of anchoring the prothrombin to a
phospholipid membrane
...
This means that
the prothrombin will only bind to damaged cells that have exposed internal structures
...
So only an open wound will activate
the production of thrombin
...
It intact works further down the molecule sort of central to release thrombin
...
but the
binding to the phospholipids with calcium ions present it stimulates the conversion by a fold x20,000
...
That is for its own activation it acts on the Factor 5 molecule (proaccelerin)
...
However the
activated proacclerin is subject to degradation by thrombin
...
This shows a way of stopping the clot propagating away from the sight of injury by shutting the
clotting system down
...
Factor X activation
These two pathways come from the locations of the proteins involved in the blood clotting process
...
The extrinsic pathway gets
its name from the fact that one of its major proteins is only present in the tissues rather than in the blood
...
When
blood is collected today for donation they are collected in bags that have been treated so that the don't exchange
chargers
...

1
...

3
...


Hageman factor (factor XII)
Prekallikrein
Plasma Thromboplastin antecedent (PTA or Factor XI)
High Molecular Weight Kininogen (HMK)

Absorption to a suitable surface (one with a negative
charge) actives hageman factor which in the presence of
HMK proteolyses Prekallikrein to its active form (Kallikrein)
...
Now that we have activated hageman factor we can
proceed with the activation of PTA (factor XI)
...
Factor XIa itself catalyses the activation of
factor IX
...
This is
known as one of two forms of haemophilia
...
Christmas Facto of factor
IX is the protein which participates in the last stage of the
intrinsic pathway where Factor X is catalysed into its active
from Factor Xa
...
Factor VIII itself is proteolytically activated by
thrombin thus it is activated by injury as thrombin is
activated by the association of prothrombin with
phospholipids found on the insides of damaged cells
...

When injury occurs and it is released and the extrinsic pathway is initiated by its proteolysis
...
The activation of Factor VII, it
turn, mediates the activation of factor X with factor III ( the tissue factor or thromboplastin)
...
The gene for this protein is found on the X chromosome which is why only men
are susceptible to the disease
...
We can
also see that the extrinsic pathway is one that is mediated by calcium and phospholipids n fact the rate of this is
about x16,000 more in the presence of calcium and phospholipids
...
That is tissues with a good blood supply like the brain the lung and

muscles
...
However there two pathways with some
noticeable differences
...
If you add tissue factor then
this will initiate blood clotting in about 12 seconds
...
The
extrinsic one would show then that it is the most important one but for condition like haemophilia there are severe
consequences showing that it also very important to blood clotting
...
We
dont want it to kick off when its not needed but also we do want it a lot when it is needed
...
There are thus many things that control this system, things like thrombin which will activate
proaccelerin but also degrades it after a while to slow the process down
...
You tend to find that the
blood flow sweeps away these active forms and the liver takes out the active forms to stop them from circulating
...

Some of these inhibitors contain things like antithrombin which inhibits all the active proteases in the blood cascade
accept factor VII
...
Reactivity of the
antithrombin is also enhanced x700 by a protein heparin
...
It thus tends to shut the system down and also
enhances the mechanism to prevent excessive clotting because of injury
...
The activated Protein C then, in turn, protelyticlaly inactivates
proaccelerin and also inactivates factor VIII
...
This is thus an
important control factor
...
However in vevo that doesn't happen, there must be something in vivo that lacking in vitro
...
We now know there is another 74Kda glycoprotein called
thrombomodulin again present in vascular tissues
...
thrombomodulin modulates thrombin by binding and converting it to a form of
thrombin of decreased ability to form clots but an increased ability about 1000 times its ability to activate protein C
...

This is an example of biochemical amplification and this can be illustrated by the various components of the extrinsic
pathway
...
So you can see this biochemical amplification that a small amount of proconvertin can lead to
a large amount of fibrinogen
...
Clot lysis
We know that clots rant permanent structures, there role is to form temporary repair until such time as the tissue has
had chance to repair it self properly
...
The enzyme responsible for this is
called plasmin
...

Plasmin is a serine protease that specifically cleaves the structure of fibrin itself
...
Plasmin is formed by the proteolytic activation of plasminogen, an
86Kda zymogen
...
TPA consists of a domain structure with two kringle domains
...


The fibrinolysis system doesn’t just consist of its zymogen and its activators it also incorporates a
number of inhibitors, this process also has inhibits and one main one is alpha2-antiplasmin
...
Whats the importance of this then
...
This might be expected but plasminogen activators
see quite a lot of medical attensio
...
If this dose occurs we would like to get rid of this clot quickly before e it
causes harm
...
Its not actually an enzyme it just forms a tight one to one bind with a plasminogen and by
binding tends to activate the plasminogen and favour the production of plasmin
...
If you could give people TPA seems to be abetter
candiate for a plasminogen activator
...
can greatly reduce the damage it may cause
...
Protein Targeting
Think of proteins in your cells in your body there not randomly distributed
...
We should understand how this specificity occurs but also what happens when it goes wrong
...
A few example of this going wrong can lead to things like these storage diseases
...
Sometimes the substrates are mis-targeted,
the substrate continues to go there but there isn't an enzyme to deal with it so it just gets stored up
...
Protein synthesis occurs on the ribosomes, these ribosomes
sometimes tend to cluster on one mRNA sequence and this cluster is called a polysome
...
Some are present in the cytosol and some are associated with the
endoplasmic reticule forming the rough endoplasmic reticulum (RER)
...
What then disides where it goes then either the cytosol or the
membrane
...
In the 1970s two guys led to the signal
hypothesis where a number of amino acids on the end terminal of the polypeptide chain known as the signal
sequence
...

Evidence soon came along from cigar mils tine who defied a way to show that mouse myaloma cells that secret large
amount of immunglobulin had this signal
...
In
order to prove this theory he thought to take the ribosomes away from the RER and provided them with everything
they needed to sequence the proteins and they were now longer
...
The ribosomes still synthesis poplypeptides but now about 20 amino acids longer which is thought to
be the signal they were looking for
...
People new now there was an evidence for signalling excretory proteins and they found them in a lot of
other cells
...


1
...

2
...

3
...
Alanine (A), leucine
(L), valine (V), isoleucine (I) and phenylalanine (F) are
common in this region
...




---