Haematology Physiology

Dr Swapnil Pawar July 1, 2020 757

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    Haematology Physiology
    Dr Swapnil Pawar



 Briefly outline the components involved in haemostasis

Haemostasis is the natural process which allows circulation to respond to vessel injury in a way which minimises blood loss. There are three main components of haemostasis:

  1. Vasoconstriction:
    1. Trauma in vessel wall causes: local myogenic spasm and local factor release from traumatised tissue and platelets, as well as nervous reflexes initiated by pain impulses  smooth muscle contraction  vasoconstriction
    2. Thromboxane A2 released from platelets is responsible for much of the vasoconstriction
    3. The more severe the trauma the greater the amount of spasm. Vessel size is also important; vasoconstriction may be able to stop the bleeding in small vessels and it reduces the blood flow in medium to large vessels.
  2. Formation of platelet plug: we have outlined these steps in a previous podcast on haematology pharmacology. To briefly recap, formation of a platelet plug requires platelet adhesion, activation and aggregation. Endothelial disruption exposes subendothelial connective tissue to which platelets adhere via glycoproteins and von Willebrand factor. Platelet adhesion results in degranulation and activation of substances including ADP, vWF, phospholipase 2 and thromboxane A2. These substances promote platelet aggregation, vasoconstriction and provide positive feedback for further platelet activation and adhesion. Platelet aggregation is mediated primarily by fibrinogen which binds to glycoprotein 2b/3a receptors on platelets forming a platelet plug.
  3. Formation of blood clot via the coagulation pathway:
    1. Occurs in 15-20 seconds in severe trauma and in 1-2 minutes in minor
    2. Platelet plug is converted into definitive clot by fibrin. Formation of fibrin involves a number of reactions and clotting factors. The fundamental reaction is conversion of soluble fibrinogen to insoluble fibrin


Describe the coagulation pathways


Consists of 2 pathways which join to form a final common pathway leading to formation of fibrin clot

  • Intrinsic:
    • Activated by contact with negatively charged surfaces such as collagen and subendothelial connective tissue.
    • Plasma factor XII interacts with negatively charged surfaces and forms a complex with exposed collagen, prekallikrein and high molecular weight kininogen  this complex then activates factor XII to XIIa  activation of XI to XIa  activates factor IX in presence of calcium  IXa in presence of VIII, calcium and platelet phospholipid activates X  Xa
  • Extrinsic
    • Alternative mechanism for activation of factor X
    • Tissue factor found on surface of perivascular tissue cells binds factor VII  this activated factor VIIa and tissue factor complex then activates factor X.
    • This complex also activates factor IX which can also activate factor X
  • Common pathway:
    • Activated factor X (Xa) in the presence of factor Va, calcium and platelet phospholipid converts prothrombin (factor 2) to thrombin (2a).
    • Thrombin hydrolyses arginine-glycine bonds of fibrinogen to fibrinopeptide A and B to form fibrin monomers. Hydrogen bonds then link these monomers to form loose insoluble fibrin polymer
    • Factor XIII activated by thrombin stabilises the fibrin polymers via covalent bond cross links
    • Thrombin has direct effects on other clotting factors in addition to fibrinogen and factor XIII. It also amplifies the activation of factor V, VIII and XI
    • Factor Xa is very important: it is the only physiological activator of prothrombin


Outline the fibrinolytic pathway


The fibrinolytic pathway is a highly regulated enzymatic process that degrades and removes fibrin clot, and thus counterbalances the coagulation pathway. Fibrinolysis is also activated upon vessel injury but is inhibited by the thrombin generated by the coagulation cascade. Once the initial stimulus for clot formation is diminished or removed, thrombin generation is reduced and the balance favours fibrinolysis.


Fibrinolysis involves the release of plasminogen activators, plasmin formation from plasminogen and clot lysis.

  • Release of plasminogen activators
    • Injured endothelial cells express tissue plasminogen activator on their surface and secrete this into plasma. tPA is the primary plasminogen activator. The endothelial cells also activate factor XII, as outlined above, and releases bradykinin during the breakdown of high molecular weight kininogen.
    • Bradykinin stimulates tPA, nitric oxide and prostacyclin release from endothelial cells, all of which inhibit platelets
    • Factor XIIa and kallikrein are also direct plasminogen activators
  • Plasmin formation from plasminogen
    • Plasminogen binds to fibrin and the plasminogen activators cleave bound plasminogen to form plasmin. Plasminogen activators are only able to activate bound plasminogen.
  • Clot lysis
    • Plasmin cleaves crosslinked fibrin and fibrinogen resulting in fibrin degradation products such as D-dimer and degradation of the clot
    • Thicker strands or denser networks of fibrin are more resistant to fibrinolysis


Describe the body’s mechanisms of preventing thrombosis


Virchow’s triad describes three broad factors that contribute to the development of thrombosis. These are:

  • Stasis
  • Endothelial injury
  • Hypercoaguability


The body’s mechanisms to prevent thrombosis therefore targets these three factors:


Blood flow targets stasis

  • Blood flow washes away and dilutes active mediators

Endothelial factors targets endothelial injry

  • Vessels are smooth  prevent activation of the intrinsic system
  • Thrombomodulin: activates protein C system
    • All endothelial cells except those in cerebral microcirculation produce THROMBOMODULIN – a thrombin binding protein.
    • Thrombin is procoagulant, however when it binds to thrombomodulin it becomes an anticoagulant as thrombomodulin-thrombin complex activates protein C
    • Activated protein C and cofactor protein S inactivates factors V and VIII and inactivates an inhibitor of tissue plasminogen activator  increases formation of plasmin
    • Plasmin, as described earlier, is an active component of the fibrinolytic system which lyses fibrin and fibrinogen
  • Plasminogen receptors are located on surfaces of many types of cells including endothelium: when plasminogen binds to the receptors it becomes activated  discourages clot formation in the intact vessel
  • Absence of damage prevents blood being exposed to collagen  vWF cannot bind. Also without injury there is no exposure to tissue factor  extrinsic pathway not activated.
  • Glycocalyx: mucopolysaccharide coating vessels  repels platelets and coagulation factors
  • Prostacyclin: inhibits platelet aggregation by increasing platelet cAMP + relaxation of vascular smooth muscle
  • Nitric oxide: inhibits platelets by increasing platelet cGMP and causes vasodilation

Natural anticoagulants

  • Heparan sulphate is a linear polysaccharide expressed on cell surfaces which enhances the activity of circulating antithrombin 3. Antithombin 3 is a small glycoprotein which inactivates several enzymes in the coagulation cascade, most importantly thrombin and factor Xa.
  • Protein C and protein S as outlined earlier
  • Coagulation factors circulate in an inactive form and plasma contains inhibitor systems
  • Tissue factor pathway inhibitor: inhibits activity of VIIa-tissue factor complex and thus the activation of factor X


Outline how the following tests assess coagulation: prothrombin time, activated partial thromboplastin time, activated clotting time and ROTEM


Prothrombin time: extrinsic pathway

  • The PT specifically evaluates the extrinsic and common pathway
  • Blood is collected in a tube containing citrate to bind the calcium and prevent the clotting process from beginning before the test commences
  • Blood cells are separated from the plasma by centrifuge
  • Tissue factor and calcium is then added to the plasma sample and the time taken for the clot to form is measured
  • Rate of clotting via the extrinsic pathway is mainly influenced by the amount of factor VII available: has a short half-life and depends on vitamin K.
  • Testing PT: mainly function of vitamin K dependent enzymes: II, VII, X (IX is also vitamin K dependent but not tested) as well as factor V and fibrinogen.
  • Causes of an isolated prolonged PT include factor VII deficiency, warfarin, liver disease, paracetamol toxicity and mild vitamin K deficiency.


  • This is a ratio of a patent’s prothrombin time to a control prothrombin time e.g. INR of 2 means blood is clotting twice as slowly as normal

APTT: intrinsic pathway

  • This measures the activity of the intrinsic and common pathways of coagulation
  • Phospholipid calcium and an activator such as kaolin (negatively charged particles) are added to platelet poor plasma and time taken for formation of a fibrin clot is measured
  • This evaluates factors XII, XI, IX, VIII of the intrinsic pathway as well as X, V, II and fibrinogen from the common pathway
  • Causes of an isolated elevated APTT included factor 8, 9, 11 or 12 deficiency, von Willebrand disease, antiphospholipid antibodies, heparin and factor inhibitors to factors in the intrinsic pathway

Activated Clotting Time

  • Point of care test done when very high doses of anticoagulation, mainly heparin, are being used e.g. bypass or ECMO
  • Fresh whole blood is added to a tube containing a surface activator such as glass balls or kaolin. This activates the intrinsic and then common pathway.
  • Causes of a prolonged ACT include heparin, hypothermia, low levels of fibrinogen, clotting factor deficiencies (except factor VII or XIII), thrombocytopenia, platelet dysfunction


  • Rotational thromboelastometry is a whole blood viscoelastic haemostasis analyser
  • Whole blood is added to a heated cup at 37 degrees. A pin is suspended within the cup and oscillates.
  • As the blood clots, fibrin strands form between the cup and the pin which impedes rotation of the pin. This impediment is detected and a trace is generated.
  • ROTEM provides information about clot formation, propagation, strength and lysis, and can be used to guide transfusion strategy
  • ROTEM measures certain variables:
    • Clotting time: time from the start to when the waveform reaches 2mm above baseline. Prolongation may indicate a deficiency of coagulation factors or presence of anticoagulants.
    • Clot formation time: time from 2mm above baseline to 20mm above baseline. Increased CFT generally reflects either fibrinogen or platelet deficiency
    • Alpha angle: angle of tangent at 2mm amplitude. If this is low generally there is a fibrinogen or platelet deficiency. If this is high generally the patient is in a hypercoaguable state.
    • Maximum clot firmness: maximum strength of the clot. If this is low then there may be a deficiency of platelets or fibrinogen.
    • A5 or A10: amplitude at 5 or 10 minutes.
    • LI30: lysis index at 30 minutes – this is the percent reduction in maximum clot firmness that exists 30 minutes after clotting time
    • Maximum lysis: maximum fibrinolysis detected during measurement. If this is high it suggest hyperfibrinolysis.
  • ROTEM has six separate assays which help isolate different parts of coagulation.
    • INTEM: uses phospholipid and ellagic acid and is similar to APTT, tests the intrinsic pathway
    • EXTEM: contains tissue factor as an activator and is similar to PT, tests the extrinsic pathway
    • HEPTEM: contains heparinise which neutralises the effect of heparin. Therefore if the HEPTEM CT is much shorter than the INTEM CT it would suggest ongoing heparin effect.
    • APTEM: is similar to EXTEM except it contains aprotinin which inhibits fibrinolysis. If the CT is shorter and the MCF is higher with APTEM compared to EXTEM it would suggest hyperfibrinolysis.
    • FIBTEM: is similar to INTEM but contains cytochalasin D which inhibits platelet contribution to clot formation and therefore just assesses the fibrinogen component
    • ECATEM: contains ecarin which activates prothrombin. It will be prolonged in the presence of direct thrombin inhibitors but normal in the presence of heparin





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