Lecture 12: Coagulation System Flashcards
Draw out the flow diagram of the blood coagulation in vivo
What are the 3 roles of thrombin?
1) The initial thrombin formation (from Xa activity) is to produce more thrombin
2) But it’s main job is to convert fibrin from fibrinogen which is the net that cross-links platelets to form a clot.
3) convert XIII to XIIIa which stabilises the fibin net by cross linking it.
What is the role of XIIIa?
stabilises the fibrin net by cross linking it.
Extrinsic pathway is ______ important
Extremely important (has the tissue factor in it)
What does the APTT test measure?
It measures the factors of the intrinisic pathway (so all factors except VII)
- Activated partial thromboplastin time (aPTT or APTT) is a medical test that characterizes blood coagulation, also known as clotting.*
- Apart from detecting abnormalities in blood clotting,[2] partial thromboplastin time is also used to monitor the treatment effects with heparin, a widely prescribed drug that reduces blood’s tendency to clot.*
The key clotting factors that is affected by APTT (so if APTT is prolongued you wonde about it), are f_actor 8, 9. 11 and 12._
(Deficient factor 12 does not cause you to bleed)
The intrinsic pathway does not include ______
Tissue factor/VIIa
What factors does the APTT measure?
All factors except VII
XII
XI
IX
VIII
Describe the steps to measure the activated Partial Thromboplastin time
APTT is performed by adding an activator (e.g. kaolin, micronised silica, ellagic acid) to platelet poor plasma in the presence of phospholipid and calcium.
- Venous blood sample collected into citrate (blue lid)
- Citrate stops blood from clotting in the tube because it removes calcium (chelation)
- Spin the sample down to collect the plasma
- Add phospholipid and an activator to plasma
- Add calcium to overcome the citrate
- Measure length of time to clot formation.
The normal clotting time is ~24-37 seconds, although this will vary depending on reagents and assay kits.
APTT is performed by adding an ______ (e.g. kaolin, micronised silica, ellagic acid) to _____________ in the presence of _______________________.
APTT is performed by adding an activator (e.g. kaolin, micronised silica, ellagic acid) to platelet poor plasma in the presence of phospholipid and calcium.
If you put blood in a glass or plastic tube, it coagulates because_______
Because of the Silica in the glass/plastic. (contact factor)
What is needed to test the APTT? (3)
1) Contact factor (e.g. kaolin, silica, ellagic acid)
2) Source of phospholipid
3) Calcium
What test can test the factors of the intrinsic pathway?
APTT
APTT measures all factors with the exception of ________\_
It is most sensitive to reductions in _________________
It is commonly prolonged in the haemophilias due to reduced levels of _______\_
This test measures all factors with the exception of factor VII.
- It is most sensitive to reductions in contact factors (XII and XI)
It is commonly prolonged in the haemophilias due to reduced levels of VIII and IX.
Why is the Prothrombin time NOT physiological?
Why?
In vivo, the tissue factor pathway is switched off quickly, and we need the IXa and XIIIa to act as an amplifer.
In PR/vitro, we add so much tissue factor that the amplification is by-passed/ this is not necessary.
Prothrombin time is prolonged in people with VII deficiency, X, V, Prothrombin or lacking in fibrinogen.- but is not prolonged in people who lack VIII or IX
Therefore this test is not physiological, as if you lack VIII or IX, you will have a severe bleeding disorder, but will have a normal Prothrombin ratio.
What drug can the Prothrombin time monitor?
Drug Monitoring
It is used to monitor therapy with oral anticoagulant drug warfarin. When prothrombin ratio is used to measure the effect of warfarin a correction factor is introduced, to account for different thromboplastin sensitivity to warfarin.
- Corrected PR used for warfarin monitoring is called international normalised ratio (INR).
- INR is therefore an adjusted PR facilitating interpretation of warfarin control by removing an important laboratory variable.
Prothrombin time is prolonged in people with_________________
but is not prolonged in people who lack______
Prothrombin time is prolonged in people with
1) VII deficiency,
2) X,
3) V,
4) Prothrombin
5) lacking in fibrinogen.
But is not prolonged in people who lack
1) VIII
2) IX
the Prothrombin time/ratio is sensitive to defects in …..
The test is sensitive to defects in three of four vitamin K dependent factors in extrinsic pathway, includes II (prothrombin), VII, X.
It is also sensitive to V, fibrinogen.
How do you calculate the prothrombin ratio from time?
The Prothrombin time (in sec) is often converted to the prothrombin ratio
This standardises the test to account for the diff lab methods and different normal ranges for the prothrombin time
= Prothrombin time (patient) / Prothrombin time (normal plasma)
The normal ratio is usually around 1.0 (0.9-1.2)
The Thrombin Clotting Time is used to measure _______________
The test is sensitive to abnormalities of fibrinogen.
It is also sensitive to inhibitory effects of heparin and FDP (fibrin degradation products).
What drugs can monitored with the Thrombin clotting time (TCT)?
Drug Monitoring
Thrombin clotting time very prolonged for both heparin and dabigatran.
With protamine addition, it corrects for heparin.
What do heparin and dabigatran do?
Inhibit thrombin and very significantly prolongues and diluted thrombin clotting time (TCT).
How do you measure the Prothrombin Time?
Method
The test is performed by adding tissue thromboplastin (tissue factor) to platelet poor plasma in presence of calcium.
The clotting time (PT) varies depending on the source of the thromboplastin (~12 to 15 seconds).
- Most routine laboratories use thromboplastin from rabbit brain extracts or recombinant tissue factor.
- Thromboplastin is a lipoprotein. This is a protein and phospholipid mixture.
How do you measure the TCT?
Thrombin Clotting Time (Fibrinogen, Heparin Inhibitor)
Method
TCT is performed by adding thrombin to p_latelet poor plasma_ and measuring the time taken for a clot to form. The clotting time will depend on the concentration of thrombin which is used in the test.
- When a more concentrated thrombin is used, the clotting time is 4-10 seconds, and the test is essentially only affected by the concentration or function of fibrinogen in the plasma.
- If a more diluted form of thrombin is used, the dilute thrombin time (dTCT) gives a normal clotting time of ~18 seconds (range 15-20 seconds).
Describe the ‘mixing studies’
The above tests (APTT, PR, TCT) are the basic screening tests used to evaluate a potential haemostatic abnormality.
If the APTT is prolonged, mixing studies is performed.
Introduction
Mixing studies is _1:1 mixing of patient plasma with normal plasma, t_hen incubation
-
Sample corrects to normal and remains normal, then factor deficiency
- __Now have at least 50% of all factors
- Sample does not fully correct (anything above the normal range), then an inhibitor is present
Specific assays of all the individual coagulation factors can be performed. The methods are based on APTT or prothrombin time depending on the factor measured.
Do the “inhibitors” in coagulation affect APTT?
NO
“Inhibitors” In Coagulation
Antithrombin, protein C and protein S are called inhbitors as they shut down coagulation (natural inhibitors).
They do NOT affect APTT. Inhibitors which affect APTT are different.
Describe 4 inhibitors that affect APTT 1+1
1) Lupus Anticoagulant
Inhibitors (prolonged 1+1) may commonly be due to presence of a lupus anticoagulant. This is paradoxically named as while it prolongs APTT, it does not cause bleeding. This is because a_ntibodies in plasma that interfere with APTT assay (phospholipid)._
- In the setting of thrombosis, a lupus anticoagulant may be associated with procoagulant disorder (excess clotting) called antiphospholipid syndrome. This can also be associated with recurrent miscarriage.
- However, more often transient l_upus-like inhibitors_ are seen in patients who are unwell with inflammation and these are not significant.
2) Factor Inhibitors
Factor inhibitors are autoimmune antibodies against clotting factor (usually VIII) (acquired haemophilia).
- Associated with significant bleeding including bruising. Can be life threatening.
- Rare but should be considered in the bleeding patient
3) Drugs: Heparin
Heparin causes prolonged APTT with an inhibitory pattern. Confirm by addition of protamine (corrects for heparin).
Heparin is an anticoagulant that works as an i_nhibitor by upregulating antithrombin._
- Biological glycosaminoglycan chains
- Natural GAGs activate antithrombin in vivo
- This does not affect the APTT!!
This may be administered therapeutically in hospital or be a contaminant in a centrally collected sample (heparin used to lock central lines).
- If you draw a blood sample, it will have heparin contamination in it, so it may affec their in-vitro clotting time
4) Drugs: Dabigatran
Dabigatran is a direct thrombin inhibitor which also causes a prolonged APTT with abnormal 1+1. Prolonged 1+1 does not correct with protamine.
** Thrombin Clotting Time is very prolonged for both heaprina nd dabigatran; with prothamine additiona, it corrects for heaprin
_______________ corrects for heparin (Increased TCT)
Protamine
How can you differentiate increased APTT 1+1 caused by heparin vs dabigatran?
Prolonged 1+1 can be corrected in heparin with prothamine
It does not correct - if caused by dabigatran.
A child comes in with relatively severe bleeding
What does this mean
1) APTT 85 sec
2) 1+1 is 34 sec (normal 25-37)
3) PR is normal (0.9)
The common pathway is normal. Therefore not due to deficiencies in fibrinogen, thrombin, V or X
Factor(s) deficiency
1) VIII
2) IX
3) XI (mild)
4) XII (asymptomatic)
(intrinsic)
because the bleeding is relatively severe, it’ll probably be a deficiency in factor VIII or IX
Interpret
APTT Prolonged, PT Normal
Deficiencies of factor(s) VIII, IX, XI, XII (intrinsic)
Interpret
APTT Normal, PT Prolonged
- Deficiency of factor VII (extrinsic)
- Occasionally mild deficiencies of II, V, X, I
- This is because PR is more sensitive to these factor deficiencies
Interpret
APTT Prolonged, PT Prolonged
- Deficiency of factor(s) II, V, X and I (common)
- Multiple factor deficiencies
Interpret
TCT Prolonged
- Deficiency of fibrinogen
- Thrombin inhibitor (eg dabigatran or heparin)
Describe the various causes of Acquired Multiple factor Deficiency
Acquired Multiple Factor Deficiencies (Most Common Scenario)
Acquired deficiencies are usually multifactorial and are more common than hereditary defects. It can be caused by:
-
Warfarin or vitamin K deficiency (II, VII, IX, X)
- Vit K deficiency- antibiotics, liver failure patients
- Massive blood loss due to loss of coagulation factors and dilution with fluids
-
Disseminated intravascular coagulation (DIC)
- __Where the clotting system has been triggered in the microvasculature eg. toxins from meningiacoccal sepsis
- Widespread activation of coagulation causing thrombosis followed by bleeding as clotting factors and platelets used up. Low fibrinogen often seen.
- Liver disease causes lack of production of coagulation factors and inhibitors (except VIII)
Disseminated Intravascular Coagulation (DIC)
“Disseminated intravascular coagulation is an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction.”
Disseminated intravascular coagulation (DIC) is widespread activation of coagulation (by another disease such as sepsis or malignancy activates). This causes thrombosis, followed by bleeding as clotting factors and platelets are used up.
Low fibrinogen are often seen.
Where are most of the coagulation factors made?
The liver
Therefore Liver disease causes lack of production of coagulation factors and inhibitors (except VIII)
What is the possible cause of this?
High APTT
High PT (PR = 2.5)
Normal Fibrinogen
Corrects with normal plasma (normal 1+1)
1) Wafarin (Low II, VII, IX. X) *
2) Vit K deficiency *
3) Liver disease *
What are the 5 causes of prolonged PT?
1) Extrinsic pathway
2) Wafarin (Low II, VII, IX. X)
3) Vit K deficiency
4) Liver disease
5) Low factor VII (if APTT is normal)
What is Wafarin used for?
How does it affect our haemostasis?
Warfarin is general name for several hydroxy-coumarin derivatives. It is used in atrial fibrillation, v_enous thromboembolism,_ other thrombotic disorders.
- Inhibits recycling of vitamin K
- Lack of carboxylated factors
PT is prolonged. APTT is also prolonged (although not routinely measured: warfarin reduces II, VII, IX and X).
Warfarin dose adjustments via INR.
Reversed by vitamin K (or more rapidly by replacing clotting factors in plasma products)
Describe INR
Prothrombin time; converted to a ratio- adjusted so internationally standardised
International Normalised Ratio
Used for Wafarin monitoring
How does the Wafarin affect the different coagulation tests?
Inhibits recycling of vitamin K
Lack of carboxylated factors
- PT is prolonged.
- APTT is also prolonged (although not routinely measured: warfarin reduces II, VII, IX and X).
How does Wafarin work?
Inhibit the recycling of vit K
Vit K - (Factors 2, 7, 9, 10)- lack of vit K- won’t bind to lipid bi-layer
Diagnose
A 23 year old man is knocked off his bike. Coagulation tests after 4 hours in hospital:
APTT 46s (25-37);
PT 38 secs (12-15s);
Fibrinogen 0.9g/L (1.5-4.0).
Multi-trauma patient (internal bleeding)
Trauma associated blood loss.
Transfusion without replacement of plasma.
Describe Hereditary Single Factor Deficiencies
Hereditary factor deficiencies are less common. Usually single factor is deficient.
- First take a _bleeding histor_y, then basic coagulation tests.
- Depending on these, single factor assays.
Describe Haemophillia
Haemophilia is an inherited condition with lack of one clotting factor.
Haemophilia A (factor VIII) and B (factor IX) are X-linked recessive inherited conditions and therefore patients are predominantly male.
- Haemophilia A is common hereditary disease with serious bleeding. Incidence is 1/5,000 live male births
- Haemophilia B is less common. Incidence is 1/30,000 live male births. Clinical features identical to Hemophilia A
Symptoms vary markedly with severity. In severe disease (factor levels of <1%), it can cause spontaneous joint bleeds (chronic arthropathy, joint destruction, deformity, arthritis), soft tissue bleeds (tissue damage (muscle, renal, gastroinstestinal, intracranial), nerve damage, deformity). Intracranial bleed was the commonest cause of death.
Haemophilia A (factor ____ deficiency ) and B (factor ____deficiency ) are________recessive inherited conditions and therefore patients are predominantly ___.
Haemophilia A (factor VIII) and B (factor IX) are X-linked recessive inherited conditions and therefore patients are predominantly male.
Describe the Treatment of Haemophillia
Treatment
Haemophilia is treated by r_eplacing the missing factor_.
In NZ, this is almost always using recombinant factors, which are made in the laboratory (due to HIV and HCV in 1980s and 1990s).
Prophylactic treatment is given especially in childhood and teenagers when severe to prevent joint damage.
Normal lives and joint outcomes. Except patients who develop inhibitors.
Describe Von Willebrand’s Disease
Von Willebrand’s Disease
Von Willebrand Factor
Von Willebrand factor is plasma glycoprotein. It is synthesized in megakaryocytes and endothelial cells
- Promotes platelet adhesion at vessel wall
- Serves as a carrier for factor VIII. Complexes with Factor VIII to stabilize and prevent degradation
Von Willebrand’s Disease
Von Willebrand disease is the most common inherited bleeding disorder. This is a defect of primary haemostasis (platelet tethering) and results from reduced levels, or abnormal function of von Willebrand factor.
- It is autosomal dominant, worldwide incidence of 1-3%.
- Qualitative or quantitative abnormality of VWF, can be mild through to very severe
Laboratory Tests
- Abnormal platelet function screen (platelet function assay)
- Marginally prolonged APTT (not all)
- Low factor VIII
- Low level of von Willebrand factor with specific assays
- Function defects of von Willebrand factor (rare), i.e. amount of protein (von Willebrand antigen) is greater than corresponding functional activity (collagen binding or ristocetin cofactor activity).
Symptoms
- Mucosal bleeding (lack of platelet tethering) (e.g. epistaxis, gum bleeds, GI blood loss)
- Bruising
- Menorrhagia
- Postpartum or perioperative bleeding
Treatment
- Mild von Willebrand’s disease is commonly treated with DDAVP (desmopressin), which trigger release of von Willebrand factor and factor VIII.
- In severe disease, plasma purified VIII and von Willebrand factor are given (biostate).
Diagnose
A 58 year old woman has presented for a hysterectomy for fibroids. Her GP has undertaken some coagulation testing: APTT 76 seconds (24-37); PT 13 seconds (normal); TCT 16 seconds (normal). No history of bleeding. APTT 1+1 34 secs (normal).
Factor assays show factor XII level 10% (normal >40%)
Diagnose
An 18 year old male is rushed to hospital from his GP with fever and a rash
On arrival he has a distinctive progressive rash and his blood tests show:
- Platelets 90 (150-400);
- APTT 42 seconds (24-37);
- PT 24 seconds (12-15);
- Fibrinogen 1.1 g/L (>1.5)
Diagnosis is ?meningococcal septicaemia.
Associated with disseminated intravascular coagulation. Many causes such as sepsis (lots of different bacteria), malignancy, organ damage (e.g. pancreatitis), trauma
