Haemostasis Flashcards
(154 cards)
1
Q
What is haemostasis?
A
The cellular processes that enable both the SPECIFIC and REGULATED cessation of bleeding in response to vascular insult.
2
Q
What is the purpose of haemostasis? (x2)
A
To prevent blood loss from intact and injured vessels AND enable tissue repair.
3
Q
Why is haemostasis referred to as a balance?
A
Balance between pro-coagulant and anti-coagulant mechanisms, how these mechanisms respond to bleeding.
4
Q
What causes the haemostatic balance to tilt towards anti-coagulation? (x4)
A
□ Increase in fibrinolytic factors. □ Increase in anticoagulant proteins. □ Decrease in coagulant factors. □ Decrease in platelets.
5
Q
What the haemostatic balance to tilt towards pro-coagulation? (x4)
A
□ Decrease in fibrinolytic factors. □ Decrease in anticoagulant proteins. □ Increase in coagulant factors. □ Increase in platelets.
6
Q
OVERVIEW of haemostatic plug formation and coagulation in response to endothelial cell lining injury. !!! (x4 parts)
A
- VESSEL CONSTRICTION: vascular smooth muscle cells contract locally and limits blood flow to injured vessel. 2. PRIMARY HAEMOSTASIS: formation of an unstable platelet plug from platelet adhesion and aggregation. This LIMITS blood loss and provides a surface for coagulation. 3. SECONDARY HAEMOSTSIS: stabilisation of the plug with fibrin, caused by clotting cascade. This STOPS blood loss. 4. VESSEL REPAIR and DISSOLUTION OF CLOT: cell migration/proliferation and fibrinolysis.
7
Q
What are the structural components of an artery/venous wall? Coagulant nature?
A
□ LUMEN is found in the centre.
□ TUNICA INTIMA: single layer of endothelial cells – anticoagulant.
□ TUNICA MEDIA: Basement membrane, ECM (which gives the vascular wall its integrity), smooth muscle cells, and elastic laminae. These components are pro-coagulant – collagen, elastin and tissue factor on VSMCs.
□ TUNICA ADVENTITIA: connective tissue (mainly collagen) and fibroblasts, also with tissue factor. This layer is also pro-coagulant.
8
Q
What is the nature of the vessel wall under normal conditions, that promotes anti-coagulant activity?
A
Endothelium intact and contains thrombomodulin, EPCR and TFPI which are anti-coagulant.
9
Q
Where is vessel constriction significant in the bleeding cessation?
A
Small blood vessels.
10
Q
How large are platelets?
A
2-4 um.
11
Q
What are the characteristics of platelets?
A
Anuclear and life span of around 10 days.
12
Q
What is the body’s platelet count?
A
150-350 x 10^9/L.
13
Q
Where are platelets derived?
A
From megakaryocytes in the bone marrow.
14
Q
What are the two main cellular characteristics of megakaryocytes?
A
Lobular nucleus and granulated cytoplasm.
15
Q
What is the process of synthesis and maturation of platelets?
A
Differentiate from haemopoietic stem cells in the bone marrow into PROMEGAKARYOCYTES, then megakaryocytes. In the process of maturation, these cells fragment into platelets. When this fragmentation occurs, megakaryocyte migrates towards vessel wall and sends out proplatelet protrusions from which platelets leave and can enter the circulation.
16
Q
How many platelets are synthesised from a single megakaryocyte?
A
4000.
17
Q
What are the ultrastructural features of platelet membranes? How does this link to function? (x7)
A
□ GP VI is a glycoprotein receptor that can interact with collagen.
□ Alpha-IIb-Beta-3 integrin (Glycoprotein IIb/IIIa) interacts with fibrinogen.
□ Alpha-2-beta-1 integrin (glycoprotein Ia) interacts with collagen.
□ GP I b is essential for platelet capture via VWF.
□ TP receptor responds to thromboxane.
□ PAR receptor responds to thrombin.
□ PTY 1/12 responds to ADP.
Makes platelets very responsive to agonists that they may encounter in their environment.
18
Q
What is the ultrastructure of the platelet cell (intracellularly? (x4 parts) Link to function.
A
□ Alpha granules: contain growth factors, fibrinogen and VWF.
□ Dense granules: contain ADP, ATP, Serotonin and Ca2+.
□ Phospholipid membrane is extremely dynamic: negatively charged phospholipids get exposed in activation, making platelets pro-coagulant.
□ Has lots of microtubules and dynamic cytoskeleton which allows platelets to change shape during activation – with lots of projections.
19
Q
What are the roles of platelets? (x5)
A
□ Haemostasis and thrombosis. □ Cancer. □ Atherosclerosis: involved in development of this. □ Infection: platelets interact with leukocytes to promote infectious clearance. □ Inflammation.
20
Q
What are the two processes of platelet adhesion to site of endothelial injury?
A
□ FIRST MECHANISM: VWF (von Willebrand factor) is a protein found in blood in a globular form, such that platelet binding sites are concealed. □ When subendothelial collagen is exposed from endothelial damage, VWF binds to collagen, tethers and extends itself into a linear structure from the rheological shear forces of flowing blood. This exposes platelet binding sites. □ Platelets bind to these VWF binding sites using GPIb (glycoprotein found on platelet surface), where they roll and adhere. □ SECOND MECHANISM: Platelets can also bind directly to collagen via their GPVIA glycoproteins and alpha-2-beta-1 integrins (GPIa), but ONLY under low shear conditions i.e. not in arteries or capillaries.
21
Q
What are the processes associated with platelet activation in primary haemostasis? (x4)
A
□ Collagen is a potent platelet activator, activating platelets primarily through GPVI glycoproteins. This causes platelets to change shape and secrete platelet agonists: □ Thrombin is released from platelets resulting in coagulation and activation of further platelets. It also catalyses the formation of FIBRIN which is essential for clot stabilisation in secondary haemostasis. □ Platelets bound to collagen/VWF release ADP and thromboxane from their granules which activates platelets and allows further recruitment. □ Once platelets are activated, they change membrane composition and expose -ve phospholipids which promote coagulation in secondary haemostasis by recruiting clotting factors.
22
Q
What is the mechanism of platelet aggregation?
A
Platelets aggregate using alphaII-beta3 integrin (glycoprotein IIb and IIIa) via fibrinogen and Ca2+.
23
Q
What is the purpose of platelet aggregation? (x2)
A
Helps slow bleeding by providing a somewhat physical barrier (primary platelet plug), and provides a surface for coagulation in the clotting cascade (secondary haemostasis).
24
Q
What is the process of platelet shape change in activation and aggregation? (x4)
A
□ In normal state, platelets are disc-shaped.
□ During adhesion and interaction with VWF/collagen, they become rolling ball-shaped with projections.
□ Then, hemisphere shaped allowing firm but reversible adhesion.
□ Platelet then spreads and secures itself to site of injury irreversibly.
25
What are the two broad physiologies of platelet disorders?
Don’t have enough platelets OR platelets are not functional.
26
When does thrombocytopenia become problematic?
□ Thrombocytopenia is reduced platelet count. □ Normal range = 150-350 x10^9/L. □ Patients can have platelet count of 100 x 10^9/L with no spontaneous bleeding, because physiologically, humans actually have more than enough platelets. NB that there would be issues with bleeding in trauma though! □ Therefore, thrombocytopenia is problematic below 40x10^9/L = spontaneous bleeding.
27
What are the pathological causes of thrombocytopenia? (x2)
Treatment of leukaemias and auto-ITP.
28
Where are the proteins involved in secondary haemostasis (coagulation) synthesised?
□ THE LIVER – plasma haemostatic proteins. □ Endothelial cells – VWF, TM (thrombomodulin), TFPI. Covered in an earlier flashcard. □ Megakaryocytes – VMF and Factor V (FV).
29
What are three types of clotting factor?
□ SERINE PROTEASES that circulate as ZYMOGENS (inactive precursors). When these are activated, they are labelled with an ‘a’.
□ COFACTORS.
□ INHIBITORS: note that Protein C is a serine protease, and Protein S is a cofactor. READ PHOTO.
30
How are zymogens activated biochemically?
Activated by proteolysis, removing activation peptide.
31
What is characteristic of all serine protease clotting factors?
All have homologous serine protease domain which catalyses proteolysis of target substrate. They contain a catalytic triad of amino acids – His/Asp/Ser.
32
What triggers the secondary haemostatic cascade?
The trigger is the formation of factor-VIIa-tissue factor complex: FVII has a Gla domain which gives factor the ability to interact with platelet surface. FVII interacts with Tissue Factor where is it activated into serine protease FVIIa = cascade is TRIGGERED.
33
Where are levels of expression of Tissue Factor varied across the body?
Found more densely in certain organs e.g. lungs, brain, heart, testes – TF in these locations provide further haemostatic protection in these organs.
34
What is the structure of Factor VII, FIX, FX and Protein C? (x3 components)
Gla domain, 2x EGF-like domains and serine protease domain.
35
What is the relationship between FVII:FVIIa?
1% of FVII is found in its active form, FVIIa. This is VITAL for haemostatic system, though we do not know why.
36
What is the purpose of the EGF domain in serine proteases?
Involved in protein-protein interactions.
37
What is the purpose of the Gla domain?
Allows factors to interact with -ve phospholipid surfaces.
38
What coagulation proteins contain the Gla domain? (x6)
FVII, FX, Prothrombin, FIX, Protein C and Protein S.
39
How is the Gla domain formed? How does this give Gla domains its function?
□ Gla domain so-called because it contains Glutamic acid residues which get post-translationally modified by Vitamin K-dependent Carboxylase, and carboxylic acid is added to form gamma-carboxyglutamic acid or Gla. □ It gives these Glutamic acid residues an affinity for Ca2+ - it is this ability that causes Gla-domain clotting factors to fold up and bind -ve charged phospholipids.
40
What is the action of Warfarin as an anticoagulant?
Warfarin is an anticoagulant drug because it is a Vitamin K antagonist, so prevents body from gamma-carboxylating our clotting factors, so factors do not have Gla domain, and so cannot bind -ve phospholipid surfaces.
41
Once activated, how does the clotting cascade (secondary haemostasis) produce thrombin?
1. When the TF-FVIIa complex is formed, FIX and FX are converted into their active forms (FIXa and FXa) by proteolysis of activation peptide on each factor.
2. Simultaneously, there’s a dampening mechanism mediated by regulatory factors. TFPI turns stage 1 off almost straight away, meaning that activation of coagulation gets turned off.
3. However, if stimulus is big enough, FXa can produce some THROMBIN from prothrombin.
4. The small amount of thrombin produced feedbacks on the system and activates FVIII and FV into FVIIIa and FVa.
5. FVIIIa (a cofactor) and FIXa (a serine protease enzyme), forms a complex with phospholipids from the PLATELETS (provided from primary haemostasis) and Ca2+ and catalyses the conversion of FX --\> FXa.
6. FVa and FXa (a cofactor and protease respectively), forms a complex with phospholipids from the platelets and Ca2+ too and catalyses formation of Thrombin from Prothrombin.
7. Thrombin catalyses the conversion of FXI (a new coagulation factor) to FXIa which catalyses conversion of FIX to FIXa (and its subsequent complex).
8. NOTE which is the EXTRINSIC and INTRINSIC pathway. The COMMON pathway concerns Factor V, X, Thrombin and Fibrin as these factors are used by both extrinsic and intrinsic pathways.
42
What is the action of thrombin?
Enzyme that catalyses formation of insoluble fibrin from soluble fibrinogen.
43
What Factor deficiencies is Haemophilia A and B associated with?
HAEMOPHILIA A is FVIII deficiency; HAEMOPHILIA B is FIX deficiency.
44
What is the familial inheritance of haemophilia?
X-linked recessive so primarily affects boys.
45
How is the coagulation cascade regulated? (x3 mechanisms)
□ TFPI regulates initiation phase of coagulation as described.
□ Protein C (Activated Protein C (APC) pathway and Protein S regulates FVa and FVIIIa.
□ Antithrombin inhibits Thrombin and FXa.
46
What is FI?
Fibrinogen.
47
What is FIII?
Tissue factor.
48
What is FIV?
Calcium ions.
49
What is another name for prothrombin and thrombin?
These are serine proteases aka FII and FIIa respectively.
50
How does Tissue Factor Pathway Inhibitor (TFPI) work in regulating initiation of coagulation?
□ Tissue factor forms complex with FVIIa. This complex binds FX, which activates it to FXa.
□ The FXa that is generated can bind to TFPI, where it is inhibited.
□ TFPI has three Kunitz domains, and it is the SECOND domain that binds to FXa.
□ This FXa-TFPI complex docks back onto the FVIIa-TF complex, inactivating the complex by locking all the proteins in.
51
What are the levels of TFPI in the body and subsequent purpose?
There are only small amounts of TFPI in the body, so its purpose is only to dampen the coagulant response in small injuries.
52
How does the Protein C pathway work to regulate the coagulation cascade? Purpose of pathway?
□ Thrombin converts FII to FIIa, but where it encounters an endothelial cell, it binds tightly to thrombomodulin to produce thrombin-thrombomodulin complex.
□ Protein C is found localised to the endothelial surface by EPCR receptors. Thrombin in this state cleaves Protein C to release activation peptide --\> Activated Protein C (APC).
□ APC inhibits thrombin generation by proteolytically (cleaving) inactivating procoagulant cofactors FVa and FVIIIa.
□ Protein S acts as a cofactor for Protein C.
□ This prevents further coagulation outside of the site of injury, because where thrombin encounters an endothelial cell, it shows that it has reached a non-injured area. This keeps clotting inside the ‘plug’.
53
How does Antithrombin regulate the clotting cascade?
□ It is a serine protease inhibitor (SERPIN). □ Inactivates many activated coagulation serine proteases, but primarily FXa and thrombin. □ Any of these serine proteases that wash off the clot are at risk of triggering the clotting cascade elsewhere. □ So Antithrombin ‘mops up’ any free proteases that escape site of vessel damage.
54
How does Heparin work as an anti-coagulant?
Binds to antithrombin factor and enhances its efficiency at inactivating FXa and thrombin – i.e. it is an antithrombin cofactor.
55
What are anticoagulant agents?
Inhibit the coagulation cascade by targeting clotting factors in secondary haemostasis.
56
Relative importance of TFPI?
Doesn’t have much practical effect, but is essential for life.
57
What is the process of fibrinolysis?
□ Plasminogen is converted to PLASMIN by tPA (tissue plasminogen activator). □ Plasmin cuts up fibrin fibres in the clot, forming fibrin degradation products, FDP which circulate in the blood and are cleared in the liver.
58
When is tPA used therapeutically?
Therapeutic thrombolysis for STEMIs, ischaemic stroke… - called clot busters.
59
List three anticoagulation drugs?
Warfarin, Heparin and DOAC (oral) medications.
60
How do DOACs work?
They directly inhibit FXa and taken orally.
61
Name two anti-platelet agents? Basic mechanism of action?
Aspirin and P2Y12 blockers. Bind to sites on platelet surface to suppress its function.
62
What is the mechanism of aspirin?
Irreversibly inhibits the COX enzyme resulting in reduced platelet production of thromboxane (vasoconstrictor and initiates platelet release).
63
What tests can be used monitor haemostasis? (x3)
□ COAGULATION (PT, APTT): monitor haemostatic ability. □ PLATELET FUNCTION TESTS: used in bleeding patients to detect whether platelets are working. □ D-DIMER TESTS: d-dimer is a fibrinolytic product, so measuring d-dimer levels tells you how much coagulation has been happening in an individual.
64
How can cancer manifest a thrombosis?
Tumours can cause thromboembolisms, as tumours can break up from their site, and occlude downstream venous vessels.
65
What are antiplatelet agents?
Decrease PLATELET AGGREGATION and inhibit thrombus formation.
66
What are antiplatelet agents used for?
Used in prevention and treatment of arterial thrombosis e.g. thrombotic cerebrovascular or cardiovascular disease.
67
What are anticoagulant agents used for?
Used to treat atrial fibrillation, coronary heart disease, DVT, ischaemic stroke and STEMIs. In these cases, anticoagulant drugs can prevent formation of dangerous clots or prevent growth of clots.
68
How are anticoagulant agents significant in laboratory use?
Test tubes used for laboratory blood tests, and bags used to collect blood donations contain anticoagulants to prevent blood clotting.
69
What is abnormal bleeding? Just read this one: it is quite interesting.
□ Abnormal bleeding can be considered as easy bruising, gum bleeding, frequent nosebleeds (epistaxis), menorrhagia (heavy periods). □ When you compare these symptoms between individuals with and without bleeding disorders, there is higher symptomatic incidence in blood disorder patients. □ However, when abnormal bleeding is redefined criteria is epistaxis for more than 10 minutes, more than 1 bruise, large areas of bruising, the symptomatic incidence difference is much starker. □ Therefore, taking a detailed history is so important when studying haemostatic disorders. □ Otherwise, a healthy subject may seem like a patient with a haemostatic disorder.
70
What are the different things that can be affected in disorders affecting primary haemostasis? (x3)
□ Platelets disorder. □ Disorder of Von Willebrand Factor. □ Disorder of the vessel wall.
71
What platelet abnormalities cause disorders of primary haemostasis? (x2)
□ Already mentioned in a previous flashcard. □ Low numbers – THROMBOCYTOPENIA. □ Impaired function.
72
What are the causes of thrombocytopenia? (x3 categories, x2 examples for each apart from third category)
□ FAILURE OF PLATELET PRODUCTION in bone marrow failure e.g. leukaemia, B12 deficiency. □ SHORTENED HALF-LIFE of platelets from accelerated clearance e.g. immune (ITP), DIC. □ Increased pooling and destruction of platelets in an enlarged spleen e.g. hypersplenism.
73
How does leukaemia lead to bone marrow failure?
Proliferation of abnormal haemopoietic cells means that healthy haematopoietic cell stores are depleted, so normal haematopoiesis is reduced, and less healthy platelets are produced.
74
How does B12 deficiency lead to bone marrow failure?
B12 deficiency means that cells megakaryocytic cells continue to grow but don’t have the means to synthesise DNA to divide.
75
What is auto-ITP?
Auto-immune Thrombocytopenic Purpura – antiplatelet antibodies coat and ‘tag’ the platelet for removal by macrophages – mostly in the spleen.
76
What can cause impaired function in platelets? (x2 (x2, x1))
□ HEREDITARY: absence of glycoproteins important in platelet binding and interactions, OR problems with storage granules. □ ACQUIRED: due to drugs: aspirin, NSAIDs, clopidogrel. Usually, this is therapeutic and deliberate e.g. in stroke, though it can become pathological if it is an unwanted side-effect.
77
What is a cause of disorder of Von Willebrand Factor?
Von Willebrand disease.
78
What are the causes of Von Willebrand Disease? (x2)
□ HEREDITARY mutation results in decrease in quantity or function (common). □ ACQUIRED due to antibody (rare).
79
What is the pathophysiology of Von Willebrand Disease? !!!
□ VWF has two functions in haemostasis – binding to collagen and capturing platelet, and transport carrier for FVIII. □ Deficiency of VWF means that initial recruitment steps of platelets are dysfunctional, so bleeding is not initiated. □ It also means that FVIII levels fall, which causes haemophilia-like symptoms.
80
How is VWD staged? (x3)
□ TYPE 1: deficiency of VWF where you don’t make SOME. □ TYPE 2: VWF with abnormal function. □ TYPE 3: complete deficiency of VWF – autosomal recessive.
81
What are the two causes of disorders of vessel walls in primary haemostasis? Examples? (x2 and x4)
□ HEREDITARY (rare): connective tissue disorders e.g. hereditary haemorrhagic telangiectasia and Ehlers-Danlos syndrome. □ ACQUIERD: scurvy (Vitamin C deficiency), steroid therapy (steroids cause reduction of filling of connective tissue holding the vessel walls together), ageing (senile purpura – red/purple spots on skin from vessel wall weakness causing bleeding), and vasculitis (inflammation of blood vessels).
82
What is purpura?
Red/purple discoloration of skin from underlying bleeding, common in elderly.
83
What are the characteristics of bleeding in primary haemostatic disorders?
□ Immediate bleeding. □ Prolonged bleeding from cuts. □ Epistaxes. □ Gum bleeding. □ Menorrhagia. □ Easy bruising. □ Prolonged bleeding after trauma or surgery.
84
What symptom is typical of thrombocytopenia?
Petechiae. Caused by minor bleed from broken capillary vessels – usually spontaneous in primary haemostatic disorders.
85
What tests can be used to test for primary haemostatic disorders? (x4)
□ Platelet count, platelet morphology using electron microscopy. □ Bleeding time (PFA100 in lab): but very invasive and not useful; involves stabbing someone and testing how long bleeding stops. □ Assays of VWF: levels measured. □ Clinical observation – obvious.
86
How does thrombin generation compare in patients with haemophilia?
This occurs in patients with deficient FVIII/FIX – severity of this depends on which clotting factor is deficient. Not all clotting factor deficiencies lead to such severe effects.
87
Why do bleeding symptoms differ between primary and secondary haemostatic disorders?
Primary platelet plug is sufficient for small vessel injury, but insufficient in larger vessels where fibrin formation is required to stabilise the platelet plug. Hence, secondary haemostatic disorders don’t tend to affect small bleeds/vessels.
88
What are the two broad physiologies of abnormal secondary haemostasis?
□ LACK OF SPECIFIC FACTORS from failure of production or increased consumption/clearance. □ DEFECTIVE FUNCTION OF A SPECIFIC FACTOR from genetic or acquired defect.
89
What are the causes of failure of production of clotting factors? (x2) Examples (x1 and x3)
□ HEREDITARY: Factor VIII/IX – haemophilia A/B. □ ACQUIRED: liver disease, dilution and anticoagulant drugs.
90
Use examples to explain why coagulation factor deficiencies are not all the same. (Do not need to learn specifics!)
□ FACTOR VIII and IX (Haemophilia): severe but compatible with life. □ PROTHROMBIN (Factor II): lethal – mice die in utero. □ FACTOR XI: bleed after trauma but not spontaneously. □ Factor XII: no excess bleeding and almost normal haemostasis.
91
How does liver disease lead to disorder of secondary haemostasis?
Most coagulation factors are synthesised in the liver, so liver disease results in deficiency of these factors.
92
How does dilution lead to disorder of secondary haemostasis? Physiology?
Red cell transfusions no longer contain plasma, and therefore no longer contain platelets and clotting factors. Major transfusions without consideration of plasma transfusions means that clotting factors and platelets become diluted in the blood and clotting abilities in the patient are compromised.
93
Examples of disorders of increased consumption of clotting factors? (x2)
DIC (Disseminated Intravascular Coagulation) and Immune (autoantibodies).
94
What is DIC?
□ Coagulation is normally highly controlled and regulated. However, in DIC, there is unregulated activation. □ Generalised activation of coagulation from abnormal and excess Tissue Factor-FVII complex formation. □ Consumes and depletes coagulation factors, so patient susceptible to spontaneous bleeding. □ Platelets consumed. □ Simultaneous activation of fibrinolysis depletes fibrinogen. □ Deposition of fibrin in vessels causes organ failure.
95
How is DIC treated?
Treat the cause.
96
What are the symptoms of bleeding in secondary haemostatic disorders?
□ Superficial cuts do not bleed. □ Bruising is common, nosebleeds are rare. □ Spontaneous bleeding is deep, into muscles and joints (larger vessels). □ Bleeding after trauma may be delayed and is prolonged. □ Frequently restarts after stopping because the primary platelet plug initially stops bleeding, but easily broken down.
97
What symptom of bleeding is a hallmark sign of haemophilia?
Hemarthrosis – bleeding into a joint cavity.
98
What method of drug administration should be avoided in haemophiliacs?
Intra-muscular injections – results in extensive bruising.
99
What tests can be used to detect secondary haemostatic disorders? (x3)
□ Screening tests – CLOTTING SCREENING, which uses prothrombin time (PT) and activated partial thromboplastin time (APTT). □ Factor assays. □ Test for inhibitors.
100
How does the APTT and PT test work?
□ APTT looks at intrinsic factors – XII, Xi, VIII, IX and factors in the common pathway (V, X and II), and activates the intrinsic pathway of coagulation; measuring the length of time it takes for insoluble fibrin to form (fibrin can be seen as it forms).
□ PTT looks at extrinsic factors – VIIa, TF and those in the common pathway. Similarly, the extrinsic pathway of coagulation is activated and length of time for fibrin to form is measured.
□ Longer-than-normal times of coagulation indicates a deficiency of a clotting factor in that pathway.
101
What are the limitations of APTT and PT tests? (x6)
□ Mild factor deficiencies are not detected. □ Does not detect VWD because it just studies clotting factors. □ Does not detect FXIII deficiency (cross linking). □ Does not detect platelet disorders. □ Does not detect excessive fibrinolysis. □ Does no detect vessel wall disorders.
102
What are the two causes of fibrinolysis? (x2) Examples? (x1 and x2)
□ HEREDITARY: antiplasmin deficiency. □ ACQUIRED: drugs such as tPA (this is therapeutic and deliberate – clot-busting drug); DIC (unregulated coagulation is simultaneous with unregulated fibrinolysis).
103
What are the genetics of haemophilia?
X-linked recessive. However, it is possible for female carriers to become mild haemophiliacs due to inactivation of the X-chromosomes.
104
What are the genetics of VWD?
Autosomal – Type 1 and 2 are autosomal dominant; Type 3 is autosomal recessive.
105
What are the genetics of deficiencies of other clotting factors?
Autosomal recessive and therefore much less common.
106
How is abnormal haemostasis treated when it is caused by failure of production/function? (x2)
□ REPLACE MISSING FACTORS/PLATELETS: either prophylaxis or therapeutic. Prophylaxis is usually done so that if a patient bleeds, risk of death/complications are lower. □ STOP DRUGS e.g. anticoagulants.
107
How is abnormal haemostasis treated when it is caused by immune destruction? (x2)
□ Immunosuppression. □ Splenectomy for ITP as this is the site of most platelet destruction.
108
How is abnormal haemostasis treated when it is caused by increased consumption? (x2)
□ Treat cause. □ Replace as necessary.
109
How may factor replacement therapy be delivered? (x4)
□ PLASMA: contains all coagulation factors. □ CRYOPRECIPITATE: rich in fibrinogen, FVIII, VWF and Factor XIII (fibrin stabilising factor). □ FACTOR CONCENTRATES: clotting factors that have been removed from plasma and administered on their own. They are available for all factors except FV. Prothrombin complex concentrates (PCCs) contain Factors II, VII, IX and X. □ RECOMBINANT FORMS OF FVIII AND FIX.
110
How is warfarin overdose treated?
PCC – Prothrombin complex concentrate.
111
How may gene therapy be used to treat abnormal bleeding disorders?
Haemophilia A and B.
112
How is DDAVP used to treat abnormal bleeding disorders?
DDAVP (Desmopressin) is a synthetic analogue of ADH used to treat Von Willebrand Disease (VWD). It causes endothelial cells to release endogenous VWF stores and hence, can only be used to treat mild forms of the disease.
113
How is Tranexamic acid used to treat abnormal bleeding disorders?
Antifibrinolytic – works by competing with fibrin for binding to tPA. It therefore inhibits fibrinolysis and used to stabilise clots in haemophiliacs and not used to reduce bleeding in trauma settings generally.
114
When are blood transfusions needed? (x2)
• When there is massive bleeding and saline is not sufficient. • If patient is anaemic, but iron/B12/folate therapy is not appropriate/sufficient e.g. if a patient is severely anaemic from iron deficiency, they may be given a pint of blood to lift them off the floor, and then iron infusions after for recovery so their body can start making Hb again.
115
What are the biochemical differences that distinguish the ABO blood group?
• All red blood cells have a common glycoprotein and fucose stem. • A and B antigens on red blood cells are formed by adding a sugar residue to this common stem. • ‘A’ antigens: have an N-acetylgalactosamine residue added. • B antigens: have a galactose residue added. • O blood group: have nothing added to the common stem. • AB blood group: have a mix of A and B antigens.
116
What are the genetics of the ABO blood group?
• Genes code for TRANSFERASE enzymes. • A genes code for an ENZYME which adds N-acetylgalactosamine to common glycoprotein and fucose stem. • B genes code for an ENZYME which adds galactose. • A and B genes are CO-DOMINANT. • O gene codes for nothing; is RECESSIVE e.g. person is blood group A – genes could be AA or AO.
117
What ABO antibodies are present in the blood?
IgM antibodies of the antigen(s) that is absent. They are given the prefix, ‘anti-‘ e.g. antibody that binds to A antigen is called ‘anti-A’.
118
What is the mechanism that causes death when the wrong blood group is transfused?
IgM antibodies form a pentameter structure and are ‘complete’, meaning that they fully activate the complement cascade, causing haemolysis of RBCs. This releases Hb into the blood, which is toxic to the kidneys. Release of bilirubin from Hb breakdown in the liver gives patient jaundice, and activation of the cascade causes a cytokine storm. Cytokine storm results in fall in BP and shock in patients --\> death.
119
Why is the fact that ABO antibodies are IgM helpful in the laboratory?
IgM antibodies, when cross-linked with RBCs, agglutinate well and forms a clump which collects at the bottom of testing tubes. This clump formation makes it easy to identify a patient’s blood type in laboratory tests and is also QUICK which is good for emergencies.
120
What is the epidemiology of the ABO blood groups in the UK?
47% O, 42% A, 8% B, 3% AB.
121
What blood group is the universal acceptor?
AB is the universal acceptor because they have no antibodies in their plasma for A or B antigens. This is because they already have A and B antigens on their RBCs.
122
What blood group is the universal donor?
O is the universal donor because they have no antigens on their surface, so regardless of the antibodies present in the recipient’s body, it will not cross-link with O blood.
123
What is the RH blood grouping?
Describes patients who are RhD positive or RhD negative. RhD positive means you have the D antigen, while negative means you do not.
124
What are the genetics of the RhD positive or negative groups?
D codes for D antigen on RBC; d codes for no antigen and is recessive. Therefore, DD or Dd = RhD positive; dd = RhD negative.
125
What is the epidemiology of RhD blood group?
RhD positive = 85% of people; 15% are RhD negative.
126
How is the ABO and RH blood groupings shortened?
Patients’ ABO and Rh D groups usually shortened e.g. O positive means ABO group O and RhD positive.
127
Why is RH blood grouping clinical important to understand?
D antigen is the next immunogenic (sets off antibodies) after the AB antigens.
128
Presence of RhD antibodies (anti-D antibodies)? !!!
People who LACK the RhD antigen (RhD negative) CAN make anti-D antibodies only AFTER they have been exposed to the RhD antigen – either by transfusion of RhD positive blood, or in women if they are pregnant with an RhD positive foetus. Those who are RhD positive will have the D antigen and NO anti=D antibodies in their system, even after exposure to RhD-positive blood through transfusion or pregnancy.
129
What class of antibody is produced against the RhD antigen?
IgG.
130
What happens when a patient with anti-D antibodies is transfused with RhD positive blood?
• NOTE: this can only occur after the 2nd RhD positive transfusion. Remember, patient does not have anti-D antibodies from birth; anti-D antibodies are only found in the blood after an initial exposure to RhD positive blood IN RhD NEGATIVE PATIENTS. • IgG antibodies will cause haemolysis of RBCs, but this is a DELAYED HAEMOLYTIC TRANSFUSION REACTION (takes around 5-10 days). • IgG antibodies attach to the RBCs but do not activate the complement cascade straight away. Damage occurs as the RBCs pass through the spleen: resident macrophages recognise the RhD positive blood and IgG antibodies and destroy RBCs. • Results in fall in Hb (anaemia), high bilirubin, jaundice and in some cases renal failure. • This is all less dramatic and less immediately fatal than incorrect ABO transfusion.
131
What are the implications that should be considered in patients where anti-D antibodies are known? (x2)
• FUTURE TRANSFUSIONS: patient must, in future, have RhD negative blood, otherwise they will develop delayed haemolytic transfusion reaction. In any case, where blood group is known, RhD negative patients should always be given RhD negative blood to avoid EVER making anti-D. • HAEMOLYTIC DISEASE OF THE NEWBORN (HDN): if RhD negative mother has anti-D and in the next pregnancy, the fetus is RhD positive, mother’s IgG anti-D antibodies can cross the placenta and cause haemolysis of foetal red blood cells.
132
What causes death in haemolytic disease of the newborn? (x2)
• Bilirubin circulates in toxic levels from increased breakdown of Hb in the liver from IgG-mediated haemolysis. Causes brain damage from bilirubin staining of brain stem columns – clinical signs include hyperextension of legs, arms and neck of NEWBORN BABY. • Hydrops fetalis: FOETUS develops anaemia from IgG-mediated haemolysis of foetal RBCs. The heart therefore pumps a much greater volume of blood to deliver the same amount of oxygen. The increased demand on CO leads to heart failure and corresponding oedema.
133
What are anti-D injections?
Injection of anti-D antibodies, given to RhD neg mothers during pregnancy when exposed to RhD pos blood from their baby (remember, during pregnancy, there is some sharing of blood). Anti-D injections take RhD positive blood out of the mother’s system before the body can amount an immune response and produce anti-D antibodies of their own. Injections given when a small amount of blood has been shared e.g. torn vagina and some sharing of mother and baby blood there.
134
What other red cell antigens are there?
RhC/c/E/e etc.
135
What are the clinical implications of patients who develop antibodies against less common red cell antigens e.g. RhC/c/E/e? How is this screened?
• Once patient forms antibodies against an antigen, they must use antigen-negative blood of the corresponding antigen, or they risk delayed haemolytic transfusion reaction. • Plasma is screened for red cell antibodies BEFORE transfusion. If, after the ANTIBODY SCREEN, an antibody to one of these antigens is found, antigen-negative blood is given. However, only 8% will form an antibody to one of these antigens, so it is not really an issue.
136
When blood is donated and collected, what is it mixed with in the bag?
Anticoagulant to prevent clotting. Once clotted, becomes useless.
137
How is blood split INITIALLY once donated? Three groups?
• Split one unit of blood by centrifuging whole bag – red cells bottom, platelets middle and plasma on top. • Then, each layer is squeezed into satellite bags. • This is all processed in a closed system to prevent bacteria entering the system – of which blood is a great culture.
138
Why is it not practical to give whole blood to patients? Why is it beneficial to separate blood into its components and give separately? (x3)
• More efficient: less waste as patients don’t need all components. • Some components degenerate quickly if stored as whole blood. • If patient needs red cell transfusion because they are anaemic, giving them a red cell infusion is more practical than infusing them with red cells and all the associated plasma. This is because, infusing them with whole blood will overload them with fluid faster. Having red cells concentrated means that a smaller volume can be administered for the same oxygen-carrying capacity.
139
How can plasma be processed once separated from whole blood? (x3 option)
• FRESH FROZEN PLASMA (FFP): Frozen to preserve coagulation factors and stop degeneration. • Make CRYOPRECIPITATE: This is FFP which is THAWED in a FRIDGE, leaving a liquid component and a cryoprecipitate. The cryoprecipitate is rich in fibrinogen (FI) and F8, so it is useful for patients who need just FI or FVIII. • Plasma is FRACTIONATED (like in the oil industry): separating plasma into albumin, haemophilia factors (usually FVIII and FIX to treat Haemophilia A and B respectively, and Factor VIII for von Willebrand’s disease), and immunoglobulins.
140
List possible immunoglobulins that can be extracted from fractionation, and their clinical significance? (x2)
• Specific immunoglobulins such as tetanus, anti-D and rabies. • IVIg – a soup of antibodies given to immunosuppressed individual so they can fight infections. It is also particularly good at quelling with ITP (immune thrombocytopenic purpura – autoimmune antibodies against platelets) and AIHA (autoimmune haemolytic anaemia – autoantibodies against own RBCs).
141
What is the clinical significance of Albumin extracted from fractionation? (x3) Two types?
• 4.5% ALBUMIN: useful in people who are losing a lot of water and proteins because of severe burns. Also used in plasma exchanges. • 20% ALBUMIN: for certain severe liver and kidney conditions where you are losing proteins in the blood.
142
How are red cells stored?
1 unit from 1 donor is stored at 4 degrees in a fridge. It has a shelf-life of 5 weeks.
143
How are red cells administered to patients?
Given through a ‘blood giving set’ which has a filter to remove clumps and cell debris.
144
When are red cells stored? Shelf-life? Problems? Benefit?
Red cells are RARELY frozen because it can result in crystallisation and burst RBCs. There is also poor recovery on thawing, and you would typically lose a third of the cells. However, it is useful for rare blood groups/antibodies where a standard shelf-like of just 5-weeks would mean that will be wasted.
145
How is FFP stored? Shelf-life?
Stored at -30 degrees within 6 hours of donation to preserve coagulation factors. Shelf life of three years.
146
Why is it important to know blood group of patient before administration of FFP?
Because the plasma is what contains the antibodies of the donor blood. Therefore, must make sure that FFP does not contain antibodies corresponding to any of the recipient’s antigens.
147
When is FFP administered to a patient? (x2)
• If patient is bleeding and has ABNORMAL coagulation test results – both PT and APTT coagulation tests are prolonged --\> if both PT and APTT are prolonged, it suggests multiple factors are missing, so FFP should be administered as oppose to individual factors. • Reversal of anticoagulants e.g. warfarin. Used just before urgent surgery. However, not practical because it’s basically over-kill: Patient should only be given factors that are being suppressed by the anticoagulant. FFP is used when the factors that are deficient are not known, or when there is no preparation available that will replace just the factors lost, so all factors must be given instead.
148
How is cryoprecipitate stored? Shelf-life?
Same as FFP: Stored at -30 degrees within 6 hours of donation to preserve coagulation factors. Shelf life of three years.
149
When is cryoprecipitate used? (x2)
• Massive bleeding and fibrinogen is very low. • Hypofibrinogenemia.
150
How are platelets stored? Shelf-life?
22 degrees (room temperature) and constantly agitated. Without agitation (shake), they just clump and become useless. Shelf life of just 5 days ONLY – because of risk of bacterial infection.
151
Why is it important to know blood group of patient before administration of platelets? (x3)
Platelets have low levels of ABO antigens on them. But, wrongful administration is NOT deadly. The problems are:
* If a patient was blood group B, and given group A platelets, the patient’s anti-A antibodies will destroy the platelets, and the platelets will not last long in a patient.
* Platelets must be bathed in some plasma once donated, or they don’t really survive. This means that platelet donations will also contain some of the donor’s antibodies e.g. if Group O platelets are given to a Group A patient, the platelets would not be destroyed, but anti-A antibodies in the plasma of the donor will destroy some of the recipient’s blood.
* Need to know RhD group as well: This is because there is always some blood contamination in collected plasma – you can’t help it. If the donor is RhD pos, platelets should not be given to RhD neg recipients, as it would lead to RhD sensitisation and anti-D production.
152
When are platelets used clinically? (x4)
• Bone marrow failure – as bone marrow produces platelets. • Massive bleeding – because patients will lose some platelets. • Acute DIC. • If patient has low platelet count and about to go for surgery e.g. they were previously on anti-platelet drugs.
153
If a patient is bleeding post-surgery, their PT and APTT are prolonged, and fibrinogen is low, what components should be administered?
This is a TRICK QUESTION: Low fibrinogen means administer cryoprecipitate. However, notice that BOTH PT and APTT are low. If just fibrinogen was low, PT would be normal. Therefore, we instead administer FFP and only administer cryoprecipitate on top of this if fibrinogen is exceptionally low.
154
How are blood donors screened? (x2)
• Test for infections to protect recipient e.g. PCR to test for Hepatitis and HIV etc. However, there is a ‘window period’ for infections and not all infections will be picked up in tests if, for example, they have just caught the disease. Therefore, donors are questioned for high risk behaviour and excluded accordingly e.g. IVDU. • Also aim to prevent harm to donors, so donors are excluded if they are at risk of damaging their healthy by donating e.g. have heart problems.
