Haemostasis Overview

Question 1

Definition of haemostasis

Answer 1

Mechanism which the body maintains blood in a fluid state within blood vessels
Upon injury the body can arrest bleeding with a blood clot
How the clot is removed after healing

Question 2

In vivo it’s a balance between…

Answer 2

Not bleeding und not clotting

Question 3

What is required for normal haemostasis?

Answer 3

Normal blood vessels (vasculature)
Number of functional platelets
Produce fibrin via the ‘coagulation cascade’
Normal fibrinolysis system
Levels of natural anticoagulants

Question 4

Primary haemostasis

Answer 4

Occurs initially the formation of a platelet aggregate, Within 5 minutes of bleeding

Question 5

Secondary haemostasis

Answer 5

Formation of stable fibrin clot within 10 minutes of bleeding

Question 6

Primary haemostasis

Answer 6

Platelet adhesion
Platelet activation
Platelet aggregation

Question 7

Definition of a platelet

Answer 7

Anucleated cell fragment derived from bone marrow MK’s
150-400 x 109/litre
7.7 -11.2 fl
Lifespan of 10 days 30% sequestered in spleen
They circulate as quiescent cells surveying integrity of vascularture
Undergo explosive activation upon vessel wall damage

Question 8

Normal platelets are circles

Answer 8

A nuclear platelets are small red dots

Question 9

What happens when a blood vessel is disrupted?

Answer 9

Collaged fibres and smooth muscle is exposed to blood. Platelets are attracted to this area, then attach to collages fibres by Von Willebrand factor (vWF). Platelets are activated = change shape, release granular contence = attracts further platelets

Question 10

Blood can still flow through blood vessel

Answer 10

Surface for secondary hemostasis

Question 11

Platelet adhesion

Answer 11

Exposior of collagen initiates binding of VWF which also binds to glycol protein 1b complex /IX/V
Locks platets to sight of injury = no blood clot in head

Question 12

von Willebrand factor

Answer 12

Proteins synthesized by mega karyocytes (mks) and endothelial cells
Found in plasma, platelets, endothelial cells, subendothelium
Largest multimers = most effective in Haemostasis
260 kD
Undergoes conformational charge = can bind to Gp 1b

Question 13

Primary haemostasis

Answer 13

Exposure of collagen fibres attract VWF and they bind. Then attracts platelets which bind to GPIb-IX receptor-complex
Release granular contents.
glycoproteins
vWF
fibrinogen
coagulation factors
ADP
serotonin
calcium

Question 14

Secondary homeostasis

Answer 14

Blood forms a stable clot

Question 15

What is a clot made from?

Answer 15

Mesh of fibrin and platelets which form a haemostatic plug at sight of injury in vessel wall

Question 16

Coagulation

Answer 16

Process by which fibrin is produced

Question 17

Fibrinolysis

Answer 17

Process by which fibrin is broken down

Question 18

Coagulation factors - labile plasma proteins which are activated to become these proteins…

Answer 18

Serine proteases- hydrolyse peptide bonds - II, VII, IX, X
Cofactors- tissue factor, V, VIII
Transglutaminase- XIII

Question 19

Coagulation Cascade

Answer 19

When 1 factor is activated, it acts on a factor further down the system = activation of that factor till thrombin is produced = converts fibrinogen to fibrin

Question 20

Intrinsic pathway

Answer 20

Activated by, contact factors
Things which are exposed when blood vessels are disrupted

Question 21

Extrinsic pathway

Answer 21

Damaged tissue release tissue factors = directly activates factor VII
= small amounts of thrombin produced

Question 22

Cell based model of haemostasis

Answer 22

Initiation
Amplification
Propagation
Generates thrombin burst = haemostatic fibrin clot

Question 23

Initiation phase

Answer 23

Tissue factor bearing cells
Binds to factor VII a = promotes activation of factor X to Xa in conjunction with cofactor V which converts prothrombin to thrombin
Factor IX is activated by tissue factor VII complex to produce small amounts of activated factor IX

Question 24

Amplification

Answer 24

Thrombin produced activates further platelets, + releases factor VIII from its vWF complex. It’s bound to surface of platelets which combines with factor IX which was released in initial stage.

Question 25

Propagation

Answer 25

Thrombin and activated platelets utilise factor IX to activate factor XI which acts of facto IIX = produces burst of thrombin

Question 26

Clot formation

Answer 26

Once Fribrinogen has been converted to the fibrin monomer by the action of thrombin, these monomers polymerise with hydrogen bonding, but this fibrin polymer is still an unstable polymer, and it needs the action factor 13 to cross-link these fibrin polymers to cross linked fibrin to give an insoluble fibrin that will combine with a platelets to form a stable fibrin platelet clot

Question 27

Fibrinogen - fibrin monomer - fibrin polymer - cross-linked fibrin

Answer 27

Fibrinopeptide release 2. Hydrogen bonding 3.Transamidase bonding

Question 28

What is fibrinolysis?

Answer 28

Mechanism the fibrin clot is broken down into smaller fragments for disposal. most important of these fibrin degradation products is the D-dimer Diagnosis of DVT deep vein thrombosis DIC (Disseminated intravascular coagulation)

Question 29

Process of fibrinolysis

Answer 29

Plasminogen - plasminogen - fibrin - fibrin degradation products

Question 30

Summary

Answer 30

Define coagulation and the process involved in clot formation, The importance of coagulation factors What are the stages/pathways involved in coagulation? How do we best describe what happens in vivo? Examine the role thrombin plays in secondary haemostasis. Define fibrinolysis.

Question 31

Laboratory tests

Answer 31

Samples must be suitable for parameter being measured Test system be regulated Quality control Results compared to locally established normal ranges

Question 32

Things required for abnormal haemostasis

Answer 32

History of abnormal haemostasis Family history Preventative screening Monitoring of anticoagulant therapy

Question 33

Abnormalities =

Answer 33

Vasculature Platelets Functional fibrin via coagulation cascade Fibrinolysis Functional natural anticoagulants

Question 34

Vasculature

Answer 34

No specific tests Imagining techniques Markers of inflammation e.g. CRP, ESR + vWF

Question 35

Platelets

Answer 35

Count number + size using automated blood cell analysers Check morphology using blood film Bleeding time Platelet function analysers PFA Platelet aggregation tests Platelet nucleotides

Question 36

Fibrin production

Answer 36

Prothrombin time PT Activated partial thrombophlebitis time APTT Thrombin time TT Fibrinogen level Fib

Question 37

Fibrinolysis

Answer 37

Clot lysis times D-diner levels Plasminogen levels Alpha -2- antiplasmin levels

Question 38

Natural anticoagulants

Answer 38

Function + absolute levels Protein C, S Antithrombin Activated protein C resistance/factor V Leiden

Question 39

PFA-100 platelet function analyser

Answer 39

Mimics primary haemostasis

Question 40

Platelet aggregation

Answer 40

Centrifuge blood gently to obtain platelet rich plasma Stirred in a cuvette at 37 oC between a light source and photocell Addition of agonist = platelet aggregation = platelets clumps = transmission increases + absorbacne decreases Addition of different agonists at a range of concentration allows detection of certain defects Agonists bind to their own specific receptors resulting in activation. Shape change, granule release, aggregation

Question 41

Fibrin formation

Answer 41

Measures secondary haemostasis Routine test = coagulation screen (CS) 300-500 daily

Question 42

Screening tests

Answer 42

Prothrombin time (PT) Clotting pathway initiated with damaged tissue How to make a reagent Emulsify in a blender ! Wash with chloroform Sieve ! Dilute in buffer Standardise

Question 43

Screening tests

Answer 43

100ul plasma (in sodium citrate, which binds calcium) Warm to 37oC 200ul Tissue factor + Phospholipid (to replace platelets removed by centrifugation) + Calcium (removed above) Time for clot formation Establish local normal range

Question 44

Screening tests PT abnormal in

Answer 44

inherited deficiencies of fibrinogen, prothrombin, FV, FVII, FX Acquired deficiencies of above Consumptive - bleeding- over activation- liver disease Therapeutic - warfarin Acquired inhibitors to factors

Question 45

Screening tests international normalised ration INR

Answer 45

Used to standardise warfarin therapy around world INR {patients Prothrombin time (seconds)/ control Prothrombin time (seconds)} isi

Question 46

Isi= international sensitivity index

Question 47

Screening tests Activated Partial Thromboplastin Time (aPTT)

Answer 47

Demonstration of intrinsic pathway + common pathway Activated using contact activation Need source of phospholipid Need calcium

Question 48

Screening tests aPTT abnormal in

Answer 48

Inherited deficiencies X Acquired deficiencies of above Consumptive Bleeding Over activation (Disseminated Intravascular Coagulation, DIC) Liver disease Therapeutic – Heparin Acquired Inhibitors to factors

Question 49

Thrombin Time (TT) Screening tests

Answer 49

Addition of diluted Bovine Thrombin to neat sample Direct conversion of Fibrinogen  Fibrin Abnormal in Patients with low fibrinogen Patient with non-functioning fibrinogen Patients with Inhibitors of Thrombin Anticoagulant - Heparin

Question 50

Fibrinogen estimation Screening tests

Answer 50

Similar addition of Thrombin (concentrated) to diluted plasma (1:5, 1:10, 1:20) Time taken to clot = Fibrinogen concentration in the sample Reported from comparison of standard curve Oxford range 1.5-4.0 g/l Abnormal in Inherited fibrinogen deficiency Acquired deficiency Bleeding DIC Liver disease

Question 51

D-dimer (DD) Screening tests

Answer 51

Breakdown product of clot formation Only found in cross-linked Fibrin breakdown Marker of rate of clot formation/breakdown Reported as ug/l FEU Oxford <500 ug/l FEU

Question 52

Summmary

Answer 52

What body does to prevent blood loss Understand processes overall Understand the difference between Primary and Secondary haemostasis Understand which routine laboratory tests can be used to investigate Haemostasis Understand test procedures