Haematology 3 Flashcards
What’s VTE?
Venous Thromboembolism
Made up of:
Deep Vein Thrombosis (DVT)
Pulmonary Embolism (PE)
Virchow’s triad and thrombosis they predispose to
venous (stasis and hypercoaguability)
arterial (vessel wall)
Risk factors for VTE
- age
- previous VTE
- cancer
- immobility/paresis
- surgery/trauma
- other illness (e.g. Inflammatory bowel
disease; Behcet’s; Paroxysmal noctural
haemoglobinuria; myeloproliferative diseases) - COCP/HRT
- pregnancy/puerperium
- inherited thrombophilia
- antiphospholipid Abs
- obesity
- smoking
puerperium
the period of about six weeks after childbirth during which the mother’s reproductive organs return to their original non-pregnant condition
Definition of Hereditary thrombophilia
A genetically determined disorder of haemostasis which increases the probability of venous thromboembolism (VTE)
What are the 5 inherited thrombophilias
1965 Antithrombin 1 in 3,000 1981 Protein C 1 in 300 1984 Protein S 1 in 300 1993/4 Factor V Leiden 1 in 20 1996 PTG20210A 1 in 50
Function of factor Va
Factor Va orientates X and PT so the cleavage is better.
What’s AT and what does it do?
Anti-thrombin is a serine protease inhibitor (serpin) and inhibits Xa and IIa (thrombin)
What’s PC and what does it do when activated?
Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogen, the activated form of which plays an important role in regulating anticoagulation, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically inactivating proteins Factor Va and Factor VIIIa. APC is classified as a serine protease as it contains a residue of serine in its active site. The zymogenic form of protein C is a vitamin K-dependent glycoprotein that circulates in blood plasma.
What’s protein S?
A cofactor for protein C
Thrombin negative feed back
Thrombin bound to thrombomodulin activates protein C, an inhibitor of the coagulation cascade. The activation of protein C is greatly enhanced following the binding of thrombin to thrombomodulin, an integral membrane protein expressed by endothelial cells.
Inheritance of anticoagulation defects
Autosomal dominant
e.g AT, PC, PS
Only one allele leads to an increased risk of VTEs.
What’s factor V Leiden?
R506Q (arginine to glutamine)
Cleavage by PC takes place 10x more slowly
1 in 20 so common!
What’s PTG20210A?
The “G20210A” refers to the fact that the mutation is a guanine (G) to adenine (A) substitution at position 20210 of the DNA of the prothrombin gene. This mutation (or more accurately, single-nucleotide polymorphism or variant), is commonly associated with increased risk of occurrence and recurrence of the disease venous thromboembolism (VTE), including both deep vein thrombosis (DVT) and pulmonary embolism (PE).
The polymorphism is located in a noncoding region of the prothrombin gene (3’ untranslated region nucleotide 20210), replacing guanine with adenine. The position is at or near where the pre-mRNA will have the poly-A tail attached.
Genetic code causes the body to make too much of the prothrombin protein. By having too much prothrombin, it increases the chances the blood clotting.
Antiphospholipid Syndrome Clinical Criteria
Arterial or Venous Thrombosis (young people with strokes)
or
Pregnancy Morbidity (recurrent miscarriage)
• ≥ 1 fetal death(s) after 10 weeks
• ≥ 1 premature birth(s) (≤ 34 weeks) due to preeclampsia, eclampsia or placental insufficiency
• ≥ 3 consecutive spontaneous abortions < 10 weeks
Antiphospholipid antibodies
Antiphospholipid antibodies are not directed against phospholipids but against proteins that bind to negatively charged phospholipids
Antibodies to ß2glycoprotein I (ß2GPI) are probably pathogenic, antibodies to prothrombin probably not
Detected as:
Lupus Anticoagulant
Anticardiolipin Antibodies
Antiß2GPI Antibodies (binds cardiolipin)
Affect intracellular signalling.
Paradoxically slow clotting in test tube.
Signs and symptoms of DVTs
Pain and tenderness Swelling Pitting oedema Increased warmth Collateral superficial veins Change in skin colour
Wells score
Pretest probability assessment for DVT
active cancer 1 paralysis, plaster 1 bed > 3d, surgery within 4w 1 tenderness along veins 1 entire leg swollen 1 calf swollen > 3cm 1 pitting oedema 1 collateral veins 1 previous DVT 1 alternative diagnosis -2
Low <1 Moderate 1-2 High >2 Unlikely ≤1 Likely ≥2
What’s the D-dimer test?
A monoclonal antibody against D-dimers.
During clotting fibrinogen (D-domain, E-domain, D-domain) crosslinks to form fibrin (D-domain, E-domain, D-domain D-domain, E-domain, D-domain, etc).
D-domains are cleaved together.
Suggests there has been a clot.
Use of pre-test probability when
considering D-dimer result
Sens 0.96 spec 0.44 LR neg = 0.091
Useful for negatives.
If unlikely DVT based on wells score and D dimer negative then 0.5% chance. Don’t need to treat.
If likely DVT based on wells score and D dimer negative then 3.4% chance. Must treat
What type of DVTs is US good at detecting
Proximal DVTs (popliteal and above)
When to treat a DVT? Draw the flow chart
First do Wells score:
Unlikely -> D-dimer:
- if negative discharge
- if positive -> proximal US:
- if negative discharge
- if positive treat
Likely -> Proximal US
- if positive treat for DVT
- if negative -> D-dimer:
- if negative discharge
- if positive -> repeat US 6-8 days later: if neg discharge, if positive treat for DVT
Signs and symptoms of PE
Shortness of breath Chest pain - pleuritic Tachycardia Haemoptysis Circulatory collapse
Features of the ideal anticoagulant
- oral
- a wide therapeutic index
- predictable pharmacokinetics and dynamics negating the need for monitoring
- a rapid onset of action
- an antidote
- minimal non-anticoagulant side-effects
- minimal interactions with other drugs and food
Heparins
- Heparin is a mixture of glycosaminoglycan chains extracted from porcine mucosa.
- Unfractionated heparin (UFH) chains average 15,000 Da.
- Low molecular weight heparin (LMWH) is a mixture of smaller chains – average 5,000 Da.
- All heparins depend on a specific pentasaccharide sequence for binding to antithrombin. They bind AT and potentiates its action.
- 5 saccharides in a row where sulphides are in the right orientation are able to bind to AT
What is fondaparinux?
A chemically synthesised pentasaccharide that has the sulphates in the right position to bind antithrombin III and inhibit factor Xa.
Unlike heparin, it is selective for factor Xa.
LMWH preferentially inhibits…
IIa (thrombin) more than Xa
UFH only inhibits
IIa
Unfractionated heparin
Given by continuous intravenous infusion.
Requires monitoring by APTT with adjustment of infusion rate to keep in the therapeutic range (often quoted a 1.5 – 2.5 normal – with our laboratory reagent 45 – 70 seconds)
LMWH
Low molecular weight heparin
The key property of LMWHs is that they
produce a much more predictable anticoagulant response than UFH.
They also have very high bioavailability after s/c injection.
The dose can therefore calculated by
body weight and be given s/c od without
any monitoring or dose adjustment.
What’s protamine sulphate and when is it used?
It is a highly cationic peptide that binds to either heparin or low molecular weight heparin (LMWH) to form a stable ion pair, which does not have anticoagulant activity. The ionic complex is then removed and broken down by the reticuloendothelial system. In large doses, protamine sulfate may also have an independent—however weak—anticoagulant effect.
Works better with longer chains.
UFH – yes
LMWH – partial
Fondaparinux - no
Vitamin K
Vitamin K is needed to perform an essential posttranslational modification of key clotting factors (II, VII, IX, X)
Vitamin K antagonists (VKA) can therefore be used as anticoagulants
Warfarin mechanism
Warfarin inhibits the vitamin K-dependent synthesis of biologically active forms of the calcium-dependent clotting factors II, VII, IX and X, as well as the regulatory factors protein C, protein S, and protein Z. Other proteins not involved in blood clotting, such as osteocalcin, or matrix Gla protein, may also be affected. The precursors of these factors require gamma carboxylation of their glutamic acid residues to allow the coagulation factors to bind to phospholipid surfaces inside blood vessels, on the vascular endothelium. The enzyme that carries out the carboxylation of glutamic acid is gamma-glutamyl carboxylase. The carboxylation reaction will proceed only if the carboxylase enzyme is able to convert a reduced form of vitamin K (vitamin K hydroquinone) to vitamin K epoxide at the same time. The vitamin K epoxide is in turn recycled back to vitamin K and vitamin K hydroquinone by another enzyme, the vitamin K epoxide reductase (VKOR). Warfarin inhibits epoxide reductase
PT/INR
The PT expressed as an INR measures the degree of anticoagulation
INR
1.0 normal, not anticoagulated
1.0 – 2.0 under-anticoagulated
2.0 – 3.0 correct degree of anticoagulation for most
3.0 – 4.0 extra anticoagulation for some eg MHV
>4.0 over-anticoagulated
Reversal of warfarin
Major bleeding:
give PCC (30 u/kg)
and vitamin K 5 mg iv
Non-major bleeding:
give vitamin K 1-3 mg iv
INR > 8.0 without bleeding:
give vitamin K 1-5 mg orally
What’s PCC?
Prothrombin complex concentrate
Made up of factor II, IX and X (sometimes VII)
Acts within minutes
Warfarin is not the ideal anticoagulant because…
- very narrow therapeutic range
- unpredictable
- slow onset so cover with heparin
- interacts with other drugs and food
Direct oral anti coagulants that inhibit Xa
Rivaroxaban
Apixaban
Edoxaban
Direct oral anti coagulant that inhibits IIa
Dabigatran
Dabigatran
Target Half life Renal clearance Bioavailability Peak
Target IIa Half life 12-17 h Renal clearance 80% Bioavailability 6% Peak 2 h
Rivaroxaban
Target Half life Renal clearance Bioavailability Peak
Target Xa Half life 7-13 h Renal clearance 33% Bioavailability 80% Peak 2-4 h
Apixaban
Target Half life Renal clearance Bioavailability Peak
Target Xa Half life 10-14 h Renal clearance 25% Bioavailability 50% Peak 2-4 h
Edoxaban
Target Half life Renal clearance Bioavailability Peak
Target Xa Half life 8-10 h Renal clearance 50% Bioavailability 62% Peak 2-4 h
Antidote to dabigatran
Idarucizumab
- Monoclonal mouse antibody developed with high dabigatran binding affinity
- Monoclonal antibody was then humanized and directly expressed as a Fab fragment in mammalian cells (hamster)
Antidotes to Xa inhibitors that are in clinical trials
Andexanet
A truncated inactive recombinant FXa, expressed in CHO cells, that avidly binds Xa inhibitors.
• Lacks the GLA domain and replaces the activation peptide with a linker connecting light and heavy chains.
• Catalytically inactive because of a mutation (S419A) in the catalytic triad.
Stages of haemostatic plug formation
Vessel constriction
Formation of an unstable platelet plug
- platelet adhesion
- platelet aggregation
Stabilisation of the plug with fibrin
-blood coagulation
Dissolution of clot and vessel repair
-fibrinolysis
Primary haemostasis
Formation of an unstable plug
Secondary haemostasis
Stabilisation of the plug with fibrin
Why do people bleed?
• Almost always a defect of procoagulant effect – Acquired or congenital – Includes: • adhesive functions of platelet-VWF • stabilising effect of FXIII
• Acquired disorders are most common in hospital
• Very rarely excess anticoagulant effect
– except for anticoagulant drugs (warfarin, NOACs)
• Very rarely excess fibrinolytic effect
– except therapeutic
Types of defects of procoagulant function and how to test for them
• Too little protein (quantitative) – Usually inherited (specific assays) – Excess consumption (global pattern) – Liver failure (global pattern) – Drugs, antibody, hormonal (specific assays)
• Defective function (qualitative)
– Usually inherited (specific assays)
• Inhibited function
– Antibody present (specific assays)
– Drugs
Tests of haemostasis
• Screening tests
– Clotting screen (PT/APTT)
– Fibrinogen
– Full blood count (platelets)
- Specific factor assays (eg Factor VIII)
- Platelet function (PFA-100, platelet aggregation)
- Global tests: ROTEM, TEG, thrombin generation
Which clotting screen is used for the extrinsic pathway?
Prothrombin time (PT)
VIIa, V, X, II
Extrinsic pathway activated by exposed tissue
Which clotting screen is used for the intrinsic pathway?
Activated partial thromboplastin time (APTT)
XII, XI, VIII, IX, V, X, II
contact activated
Why are coagulation screens done? What’s the problem?
- Detect a bleeding tendency
- Detect a cause for bleeding
- Detect a systemic disease
- Detect a thrombotic disorder
- ‘Reassurance’
In fact they are reliable for none of these
things:
• Unphysiological
• Limited sensitivity & specificity
• Test a very limited portion of haemostasis
• i.e. practical but unreliable
Bleeding disorders not detected by routine clotting tests
- Mild factor deficiencies
- Platelet disorders
- von Willebrand disease
- Factor XIII deficiency (cross linking)
- Excessive fibrinolysis
- Vessel wall disorders
- Metabolic disorders (e.g. uraemia)
- (Thrombotic disorders)
Should you do pre-operative coagulation tests?
No!
Patients should have a bleeding history taken and do a coagulation test if cause for concern.
– previous surgery and trauma
– family history
– anti-thrombotic medication
Categories of coagulation disorders
Reduced production of coagulation factors
– Hereditary failure of production:
• Factor VIII/IX, haemophilia A/B
– Acquired:
• Liver disease
• Dilution
• Anticoagulant drugs – warfarin, direct oral anticoagulants
Increased consumption
– Acquired:
• Disseminated intravascular coagulation (DIC)
• Immune - autoantibodies
What proportion of each factor is sufficient for haemostasis?
Approx 40%
Features of haemophilia A and B
• Haemophilia
– A – deficiency factor VIII
– B – deficiency factor IX
• Congenital, X-linked recessive
– Inversion intron 22 (50% haemophilia A), point mutations, deletions
• Incidence
– A - 1 in 10,000
– B – 1 in 50,000
• Factor VIII and Factor IX deficiency are clinically indistinguishable using
– coagulation screen results
– pattern of bleeding
– severity
Platelet function testing
- Platelet number (FBC)
- Platelet aggregation (measure aggregation in response to standard agonists)
- Global tests (bleeding time, PFA)
- Platelet granule contents (ATP:ADP ratio)
What’s TEG and what’s it used for?
- Thromboelastogram
- whole blood in cuvette, contact activation next to pin, slows rotation of the blood
- developed for use with celite activator
- measures changes in clot strength
- includes all phases of haemostatic mechanism including fibrinolysis
Inheritance of haemophilia
• For a female heterozygote (carrier) – Half of sons are affected – Half of daughters are carriers • For a male haemophiliac – All daughters are heterozygotes – All sons are unaffected • Approx one third are new mutations
Abnormal test in haemophilia
APTT Increased
Pattern of bleeding in haemophilia
• Not from superficial cuts or small vessels (eg
nosebleeds)
• Delayed
• Deep: muscle and joint
Problems with primary haematostasis
Categories of bleeding in haemophilia
Severe (≤1%)
Joints and muscle
Bleeding often delayed, but prolonged
Risk of intracranial haemorrhage and chronic arthropathy (disability)
Moderate (2-5%)
Prolonged or excessive bleeding after minor trauma
Rarely bleeds into joints
Mild (6-40%)
‘Spontaneous’ bleeds
Excessive bleeding only after major trauma or surgery
History of haemophilia treatment
1950’s whole blood, plasma 1960’s cryoprecipitate 1970’s factor concentrates 1984 factor VIII cloned 1990’s recombinant FVIII and FIX 2000 protein free production
Half lives of factor replacements
Factor VIII 8-12 hours
Factor IX 18-24 hours
What’s ‘on demand’ factor replacement therapy?
Treat bleeds as they occur
What’s ‘prophylactic’ factor replacement therapy?
Treat before bleeds occur
Which haemophiliac patients are treated prophylactically?
Severe or moderates that behave as severe.
What are the non-concentrate treatments (not factor) for haemophiliacs?
- Local measures
- Tranexamic acid:
- Binds to plasminogen
- Blocks binding to fibrin
- Clot lysis is reduced
•Desmopressin (DDAVP)
- Vasopressin derivative
- Acts via V2 receptors
- 2-5 fold rise in VWF-VIII (VIII>VWF); DDAVP induces vWF secretion by endothelial cells by binding to V2R and activating cAMP-mediated signaling in endothelial cells
Complications of haemophilia
- Acute bleeds
- Long term arthropathy
- Development of inhibitors (antibodies) to FVIII or IX
- Transmission of hep B, hep C, HIV
- vCJD
What’s Von Willebrand’s disease?
• Quantitative or qualitative deficiency of von Willebrand factor (VWF) • Mostly autosomal dominant • Chromosome 22 • The four hereditary types of vWD described are type 1, type 2, type 3, and pseudo- or platelet-type.
– Quantitative • Type 1 – partial deficiency – most common (75%), prevalence 10^-3-10^-4 • Type 3 – complete deficiency – 5%, prevalence 10^-6
– Qualitative
• Type 2
What are the VWF functions and the related features of VWD?
– Bridge between collagen on damaged vessel wall and
platelets under shear (1° haemostasis)
• → platelet-type mucosal bleeding
– Stabilise and protect factor VIII (2° haemostasis)
• → coagulation defect bleeding
Type 2 VWD
Type 2 vWD (20-30% of cases) is a qualitative defect and the bleeding tendency can vary between individuals. Four subtypes exist: 2A, 2B, 2M, and 2N.
Type 2A
The vWF is quantitatively normal but qualitatively defective. The ability of the defective von Willebrand factors to coalesce and form large vWF multimers is impaired, resulting in decreased quantity of large vWF multimers and low RCoF activity. Only small multimer units are detected in the circulation. Von Willebrand factor antigen (vWF:Ag) assay is low or normal.
Type 2B
This is a “gain of function” defect. The ability of the qualitatively defective vWF to bind to glycoprotein Ib (GPIb) receptor on the platelet membrane is abnormally enhanced, leading to its spontaneous binding to platelets and subsequent rapid clearance of the bound platelets and of the large vWF multimers. Thrombocytopenia may occur. Large vWF multimers are reduced or absent from the circulation.
Type 2M
Type 2M vWD is a qualitative defect of vWF characterized by its decreased ability to bind to GPIb receptor on the platelet membrane and normal capability at multimerization. The vWF antigen levels are normal. The ristocetin cofactor activity is decreased and high molecular weight large vWF multimers are present in the circulation.
Type 2N (Normandy) This is a deficiency of the binding of vWF to coagulation factor VIII. The vWF antigen test is normal, indicating normal quantity of vWF. The ristocetin cofactor assay is normal. Assay for coagulation factor VIII revealed marked quantitative decrease equivalent to levels seen in hemophilia A. This has led to some vWD type 2N patients being misdiagnosed as having hemophilia A.
VWD treatment
• Generally “on demand” • Local – OCP (oral contraceptive pill), nasal cauterisation • Tranexamic acid • DDAVP – nasal, s/c, IV • Concentrates – plasma-derived – recombinant VWF now in clinical trial
What causes HDN?
- First described in 1894 by Townsend
- Identified as due to vitamin K deficiency in 1930s
- Vitamin K dependent coagulation factors: II, VII, IX, X
- Treatment and prophylaxis of neonates first described in 1939
Causes of low vitamin K in the neonate
- Poor placental transfer of vitamin K
- Low fetal vitamin K stores
- Low vitamin K content of breast milk (1-2 mg/L) compared to formula
- Absent bacterial vitamin K synthesis in neonatal gut
- Functional immaturity of fetal liver
