Endocrinology: 10:00 - 11:00Scribe: Brittney Wise

Friday, October23, 2009Proof: Laura Adams

Dr. SteciukCoagulation 2Page1 of 5

**This transcript has the picture of the coagulation phase and it has all notes from the power point in italics**

[S1] Coagulation II: The Coagulation Cascade and Secondary Hemostasis
[S2]The Appropriate Level of Hemostasis - Hemostasis is a balance between bleeding and thrombosis
[S3]Too little or too much coagulation leads to problems
[S4 and S5]Clot Formation
Primary hemostasis is the injury, vessel wall contraction/response to injury, platelet adhesion, platelet aggregation, and platelet plug. After this you get the formation of fibrin.
Remember the fibrinogen forms the cross-links between the platelets within the platelet plug.
In secondary hemostasis what happens is that you have all this fibrinogen there and some of this fibrinogen gets converted to fibrin.
Fibrin is a very strong, long polymer that forms a mesh/net that puts the platelet plug into place and holds it down. It is a filamentous structure.
The final step right at the end of the coagulation cascade is getting fibrin (factor 1) and you cross-link it.
You really need both primary and secondary hemostasis and if you will have problems if you lack either.
Primary Hemostasis
Vasculature and Platelets
Acute bleeding, oozing/ Mucosal surfaces
Secondary Hemostasis
Coagulation Cascade
Delayed bleeding/Large wounds, deep sites, joints, surgery, dental procedures
[S6 and S7]Simple Hemostasis
This is a cartoon schematic of what he has been saying.
Primary hemostasis is the one that forms the platelet plug which will last for 6 hours. The coagulation phase gets you to fibrin which is like a brick wall and is really strong and will last. The fibrin will stay in place for 1-2 weeks or however long for the blood vessel wall that is injured to re-endothelialize (the endothelial cells will regenerate themselves.
This is showing that you need platelets and the coagulation phase to make fibrin and make a stable clot.
If you lack primary hemostasis you will lack the architecture on which to build clots and you will continue bleeding.
If you lack secondary coagulation you will have the platelet but this will eventually break down and you will see delayed bleeding.
Primary hemostasis is vessels + platelets. Vessels, platelets, and the coagulation cascade contribute to fibrin formation which leads to secondary hemostasis.
Primary hemostasis and Secondary hemostasis together lead to the formation of a “stable clot”.
[S8]Secondary Hemostasis
The first thing that happens in the coagulation phase is tissue factor. The tissue factor is present on the fibroblasts which underneath the endothelial cell layer. So, when you lose the endothelial cell layer you are exposed to vWF. Another thing that is sitting there in addition to vWF and collagen is tissue factor. It gets the whole cascade started.
Secondary Hemostasis: (1)Tissue Factor (2)Phospholipid complex expression (3)Thrombin activation (4)Fibrin polymerization
[S9 – S19]Simplified Coagulation Cascade (the cascade builds on itself so S19 is all inclusive)
There are 2 phases of coagulation:
Initiation Phase – begins with vessel injury exposing tissue factor (TF) on fibroblasts; this is secondary hemostasis; primary hemostasis has already occurred; you have to have primary hemostasis first because secondary hemostasis (which is defined by the coagulation cascade) happens on the surface of the platelet plug; just by itself,the initiation phase is not enough to get you a stable plot
tissue factor activates factor 7
factor 7 activates factor 10
factor 10 in the presence of factor 5 activates factor 2 (aka Thrombin)
So prothrombin becomes thrombin at this point
Thrombin is the central figure in the regulation of this phase
factor 2 (Thrombin) activates factor 1 (fibrin)
Besides its regulatory part it produces fibrin by converting fibrinogen to fibrin
factor 13 is responsible for cross-linking factor 1 (fibrin)

Propagation phase – helps to make the clot more stable; it increases the number of factors and it works as a positive feedback loop that works on itself
factor 2 (thrombin) activates factor 11 in the presence of Kallikreins which are present in vivo
IN VIVO = factor 2 + Kallikreins activate factor 11
IN VITRO = (Kallikreins are not present) so you have factor 12 + factor 2 to activate factor 11
factor 11 activates factor 9
factor 9 in the presence of factor 8 activates factor 10
Also, factor 7 activates factor 9 (this is not very efficient unless this propagation phase is going)
So, once this process gets started (once the initiation phase gets started) thrombin starts the propagation phase and you are basically just amplifying coagulation

Nomenclature: traditionally this cascade has been divided into several components.
Extrinsic – this name comes from the fact that tissue factor is extrinsic because it was on fibroblasts; we now know this is the initiation phase
Intrinsic – it used to be thought that this was the more important step; we now know that this is the propagation phase
Common – factors 10, 5, 2, and 1 are common to both

[S20]Important Negative Regulations
Read from slide. Anti-thrombin becomes important when we start to talk about treatment for thrombosis.
Antithrombin deactivates factors 2, 9, 10, 11, and 12.
Protein C and Protein S together act to deactivate factors 5 and 8 of the common pathway.
[S21]In vitro coagulation assays
Blood collected in 3.2% sodium citrate(in a sky blue tube)
Sodium citrate acts by binding and sequestering calcium which is an important cofactor in many of the coagulation cascade reactions(so if you don’t have calcium you don’t get coagulation so sodium citrate is just an anticoagulant; this serves to keep the blood static so that you can see what would have happened if you had tested the blood right away)
Ratio of blood to anticoagulant is very critical (9:1)
Prompt processing of sample is required

[S22]Simplified Coagulation Cascade
PT (pro-thrombin time)
Testing the extrinsic factors like factor 7
Deficiencies in factor 7 or any of the common pathway (10, 5, 2, or fibrin) factors will prolong your PT
So you need to have functioning factor 7, 10, 5, 2 and 1
Prolonged PT and normal PTT will result from an isolated deficiency in factor 7
PTT (partial thromboplastin time; sometimes you will see APTT which is activated partial thromboplastin time)
Testing more of the propagation phase factors (12, 11, 9, and 8)
This is going to test to see if you have factor 12,11, 9, and 8 and the common pathway, 10, 5, 2, and 1
Prolonged PTT and normal PT will result from an isolated deficiency in the propagation phase
BOTH prolonged PT and PTT will result from a deficiency in any of the common pathway factors
[S23]Physical manifestations of disorders of secondary hemostasis – factor deficiencies
Bleeding into joints
Bleeding into deep tissues: peritoneum, body cavities, muscle, CNS(also, retroperitoneum, intramuscular hematomas, joint arthrosis)
Bleeding with surgery and dental procedures(also trauma)
[S24] Clinical pictures
Top left is a hemophiliac that is bleeding into the joint space.
Bottom picture is a person with a hemarthrosis where they have to withdraw the blood from the joint.
[S25] Classification of Disorders of Secondary Hemostasis
Congenital Deficiencies (you are born without particular factors that you need)
Hemophilia A and B are common; all the rest are exceedingly rare
Acquired Deficiencies (more common than ANY of the congenital deficiencies; these can result from liver disease, Vitamin K deficiency, or Disseminated Intravascular Coagulation; the most common cause is iatrogenic because of drugs that we use
[S26 – S29] Disorders of Secondary Hemostasis
Congenital Deficiencies
Hemophilia A
A congenital deficiency of factor 8 which is part of the propagation phase (intrinsic pathway).
X-linked
Prolonged PTT, normal PT
Hemophilia B (Christmas disease)
Acongenital deficiency of factor 9 which is part of the propagation phase (intrinsic pathway)
X-linked(genes for factor 8 and 9 are on chromosome X)
Prolonged PTT, normal PT
Picture of Prince Charles is to note that the royal families of Europe had a big problem with these autosomal recessive disorders because they wanted to marry people from other royal families, so you have cousins marrying cousins, and they all interbred
Hemophilia C (Rosenthal Syndrome)
Very uncommon. It’s not x-linked so it’s seen in men and woman and it’s seen as a congenital deficiency of Factor 11. You will have a … and an normal PT.
Autosomal dominant
Prolonged PTT, normal PT
Factor 7 Deficiency
Only in initiation phase (not in propagation phase) so you will see a prolonged PT and a normal PTT
Factor 5, Factor 10, hypofibrinogenemia, dysfibrinogenemia – all rare
These are deficiencies in the common pathway so you will see prolonged PT and PTT
Factor 12 Deficiency –
Prolonged PTT (remember if you have a lot of factor 12 in vitro that you will do well on the PTT)
Prolonged PTT, normal PT; asymptomatic (this is picked up on routine PTT screenings and people who are getting ready for surgery)

Acquired Deficiencies
Liver Disease and Vitamin K deficiency
With either of the above you will get a deficiency of factors 2, 7, 9, and 10
The liver produces factors 2, 7, 9, and 10 in a vitamin K dependent manner
This results in a preferential prolongation of the PT. Why does it not affect PTT if it affects factor 2 and 10 which are part of the common pathway and factor 9 which is in the propagation phase? Only factor 7 is in the extrinsic phase which is what’s measures by the PT. The things at the top of the cascade, in vitro in particular, are the things that the test is going to be most sensitive too. So the first thing that happens in the tube is that factor 7 gets activated. If you don’t get factor 7 going then nothings gets going that’s in the PT.
The same thing occurs for PTT with factor 12. If you have a little deficiency in factor 12 everything is going to get thrown off because it’s a positive feedback loop.
The PT is most sensitive because 7 is at the beginning of the in vitro assay
Disseminated Intravascular Coagulation (DIC)
This is very deadly and is caused by sepsis. Sepsis is defined as bacteria in the blood. Bacteria have things on their surface that your coagulation system recognizes and they get the coagulation system going fast and hard. So, when you have bacteria in your blood the coagulation system is going to get activated whereverbacteria are present.
You can see coagulation in a lot of places with this. The first thing that is going to happen is that you are going to get coagulation in healthy blood vessels: examples include your liver, spleen, brain, or lungs. This is called thrombosis which is where blood clots up a vessel and prevents blood from getting to an organ causing it to quit from a lack of oxygen (hypoxia to the organ).
What makes sepsis worse and even more deadly is that it is very difficult to treat because you get all this coagulation everywhere and you suck up all the coagulation factors in your blood stream. So the places in your bloodstream where thrombosis is not occurring have coagulation factor levels that are really low. So everywhere else where thrombosis is not occurring you are going to bleed.
Sepsis is deadly for 2 reasons: you thrombose and you bleed. This keeps the doctors confused and then they don’t know what drug to give you. They can’t give you an anti-coagulant for the thrombosis because that will make you bleed more. You don’t want to treat them with a pro-coagulant because that’s going to make the thrombosis worse.
In sepsis DIC is caused by intravascular coagulation triggered by foreign organisms in the bloodstream
Intravascular coagulation (in undamaged vasculature) causes thrombosis of end-organs
Uncontrolled clot formation depletes coagulation factors from the circulation resulting in bleeding
Very difficult to treat because of the combination of bleeding and thrombosis

[S30]Thrombosis
Risk factors for thrombosis are age, immobilization, diet, hyperlipedemia, oral contraceptives, pregnancy, hormone replacement therapy, long bone fractures, hip, knee, gynecologic, and prostate surgery, smoking, inflammation, and placement of a central venous catheter.
Another example is when you have a thrombosis and you get deep vein thrombosis that can go to your lungs and form an infarct.
Other disorders that predispose you to thrombosis include anti-phospholipid syndrome and any cancer. Cancer is a pro-thrombotic state. If you have cancer somewhere, lots of times people are discovered to have cancer because they end up with a thrombotic event.
Protein C and Protein S inhibit the coagulation pathway so they are anti-coagulants. This will cause bleeding. You can get deficiencies in Protein C or S. If you have a lack of either of these you are going to be more likely to thrombose because you don’t have anything to anti-coagulate.
Recall that proteins C and S deactivate factors 5 and 8. So without proteins C and S you are going to have hyper-activated factors 5 and 8 and that is going to rev up coagulation and thrombosis.
One of the most common examples of this is activated protein C resistance via a mutation called Factor 5 Leiden. This is actually very common particularly in the Caucasian population of Northern European decent. This is a mutation in factor 5 that keeps it from being deactivated by protein C which prevents anti-coagulation and allows thrombosis.
Another thing you can get are activating mutations –most common of these is an activating mutation that occurs in prothrombin that makes it much more efficient at turning fibrinogen into fibrin. With this you just get too much coagulation so you are predisposed to thrombosis.
Protein C deficiency, protein S deficiency, activated protein C resistance ( factor V Leiden)
Recall proteins C and S deactivate factors V and VIII
Activating prothrombin mutation (G20210A)
[S31 and S32]Anticoagulation
When people have risk factors for thrombosis you treat them with drugs like Warfarin (Coumadin). Warfarin is a Vitamin K antagonist so it’s going to mimic liver disease or Vitamin K deficiency. It’s going to decrease production of factors 2, 7, 9, and 10.
Because you have thrombosis you want to block the coagulation phase that’s acting up by decreasing factors 2, 7, 9, and 10.
We monitor Warfarin with PT. Remember, this is like in liver disease, the factors 2, 7, 9, and 10. The factor 7 ends up being the most important because it’s at the top of the cascade, overpowering the effect of also inhibiting factor 7, 9, and 10.
The trick with Warfarin is the only oral anti-coagulant so it’s very commonly used. One problem with it is that is doesn’t just decrease 2, 7, 9, and 10 but it also decreases protein C and S because they are also produced by the liver. Remember protein S and C are anti-coagulants. So in the initial phases of treating someone with Warfarin for thrombosis the initial inhibition is more important for protein C and S, so in the initial phase of treating for thrombosis with Warfarin you are going to induce a pro-thrombotic state (this is bad). You have got someone who is clotting and you are actually going to give them a drug that causes more thrombosis. This is a major drawback of Warfarin therapy.
Heparin is an anti-coagulant and works via a different pathway. Anti-thrombin is a negative regulator of the coagulation cascade.
Anti-thrombin inhibits the activation of factors 2, 9, 10, and 11. So if you give heparin it activates anti-thrombin or potentiates the activity of anti-thrombin so if anti-thrombin is working at a level of say 1, if you give heparin, then that thrombin is going to be working at the level of say the thousands. So it really knocks down the activity in the bloodstream of these coagulation factors.
Remember, in addition to 11, 2, 9 and 10 anti-thrombin is also an inhibitor of factor 12. This is not important for coagulation in vivo, but it turns out because it is such an efficient inhibitor of factor12 it really works on the PTT. Remember PTT really starts with factor 12 in vitro, so when you give heparin it’s going to inhibit that 12, so the best way to measure heparin therapy is to measure the PTT.
Warfarin = measure with PT because of factor 7
Heparin = measure with PTT because of factor 12
When we start someone on Warfarin therapy because of the initial pro-thrombotic effect, we go ahead and bridge them with heparin. So, we will give them Heparin at the same time or even beforehand so we can get them anti-coagulated first and then start the Warfarin therapy. So this way, even though you are decreasing protein C and S and causing a pro-thrombotic state with the Warfarin it’s totally counteracted by the presence of Heparin being on board and that anti-thrombin really down regulating the activation of the coagulation cascade.
Warfarin
Warfarin is a vitamin K antagonist
Mimics liver disease and vitamin K deficiency by decreasing productions of II,VII,IX, X
Monitored by PT (very sensitive to factor VII)
Protein C and protein S are also produced by the liver in a vitamin K dependent manner and also decrease when warfarin is started
Unfortunately when Warfarin is started (presumably to prevent thrombosis) the protein C/S inhibition dominates and there is an initial prothrombotic effect
Heparin
Heparin is often used in hospitalized patients when initiating warfarin
Heparin acts by potentiating the effect of antithrombin
Recall antithrombin inhibits II, IX, X, and XI
Monitor heparin with the PTT (sensitive to factor changes in factor XI)
[S33]The End

[End 40:45minutes]