Haemostasis (part 2: secondary haemostasis)

Haemostasis (part 2: secondary haemostasis)

1. Describe primary and secondary homeostasis

   • Primary homeostasis
     • Characterised by injured blood vessel constriction/spasm (different response (vasodilation) in uninjured vessels) - followed by thrombocytic plug
     • Depends on the platelet and blood vessel wall
       • Platelets aggregate at the site of injury to reduce and arrest the bleeding
   • Secondary homeostasis
     • When the clotting cascade is activated (by tissue factor from the surface of endothelial cells and platelet secretions)
       • Coagulation and coagulation factors
         • Platelets, vascular and clotting factors
         • Extrinsic pathway
         • Intrinsic pathway
         • Reversed by fibrinolysis

2. Clotting cascade

   • Intrinsic pathway (stimulated by exposed collagen)
     • XII (Hageman factor)
       • XIIa
         • +
           • Kallikrien
             • Prekallikrien
         • +
           • High molecular weight kininogen (HMWK)
         • +
           • XI
           • XIa
             • +
               • IX
               • IXa
                 • IXa + VIIIa (+ (Ca(2+) + phospholipids) = tenase complex
                   • +
                     • Xa
                       • Xa + Va (+ Ca(2+) + phospholipids) = prothrombinase complex
                         • Va
                           • +
                             • Thrombin (IIa)
                             • V
                         • +
                           • Prothrombin (II)
                           • Thrombin (IIa)
                             • +
                               • Fibrinogen (I)
                               • Fibrin monomer (Ia)
                                 • +
                                   • Fibrin clot
                                     • +
                                       • Digested fibrin products
                                       • Fibrinolysis
                                       • Plasmin
                                         • +
                                           • Plasminogen
                                           • Urokinase
                                           • tPA
                             • +
                               • XIII
                               • XIIIa
                         • COMMON PATHWAY
                     • X
                     • VIIa (+ tissue factor)
                       • +
                         • VII
                         • Tissue factor
                           • Tissue injury
                             • Extrinsic Pathway (stimulated by endothelial damage -> tissue factor release)
                   • VIIIa
                     • +
                       • VIII
                       • Thrombin (IIa)
                       • vWF
           • Thrombin (IIa)
     • +
     • +
     • Recruits a complex of; prekallikrien, XII and HMWK

3. Coagulation factors

   • Generated in the liver (except VIII - at least the vWF-interacting portion)
     • VII produced in multiple organs
   • Three types
     • The fibrinogen family
       • Fibrinogen
       • V
       • VIII
       • XIII
     • The prothrombin family
       • II
       • VII
       • IX
       • X
       • Protein C
         • Binds with thrombomodulin on endothelial cells
           • This complex becomes activated by portein S
             • Activated complex inhibits Va and VIIa
       • Protein S
     • The contact family
       • VII (Hageman factor)

4. Monitoring haemostasis

   • Bleeding time (Duke's method)
     • Patient skin is cleaned with alcohol wipe
       • Prick made with a lancet
         • Filter paper applied to the area every 30 seconds
           • BT = no. of blood spots (until blleding stopped) divided by 2
             • This gives the BT in mins (normal is 3-6 mins)
               • Measures time for primary haemostasis
                 • Not very reliable
                   • Varies greatly between patients
                   • In cold it is shortened (due to capillary constriction)
                     • In warmth = opposite
                 • Prolonged by
                   • Lack of platelets (thrombocytopaenia)
                   • Platelet disorders (heparin, aspirin)
                   • Severe anaemia
                   • vWD
                   • Collagen vascular disease
   • Clotting time - capillary tube method
     • Prep and prick the patient
       • Fill a capillary tube up with blood
         • Every 30 secs blot the capillary tube on filter paper and drag back slightly
           • Fibrin formation is identified when a fibrinous clot is dragged from the capillary tube
             • Seen as a fibrin thread
               • Normal clotting time = 5-8 mins
                 • It is increased in haemophilia
                 • Not relying on primary haemostasis alone (outside of the body)
   • Coagulation tests
     • Measuring the intrinsic system
       • Activated partial thromboplastin time (aPTT)
         • Performed at 37degressC
           • Plasma collected (9:1 ratio of trisodium citrate anticoagulate and blood)
             • Add kaolin/elgaic acid (source of phospholipids) and Ca(2+) - activate the intrinsic pathway
               • Measure the time for a clot to form
                 • Normal = 22-35secs
                   • Prolonged aPTT
                     • Heparin/direct thrombin inhibitors
                       • Factor deficiencies (intrinsic factors; XII, XI, IX, VIII)
                         • Common pathway factor deficiencies (X, V, II, I)
                           • Luopus anticoagulant
                             • Specific inhibitors (VIII or IX)
                               • Possible warfarin, liver dysfunction DIC?
     • Measuring the extrinsic system
       • Prothrombin time (PT)
         • Performed by adding thromboplastin and Ca(2+) to plasma
           • Thromboplastin mimicks tissue factor (activates VII - i.e. the extrinsic system)
             • Can be human or rabbit
           • Measure the time for clot formation
             • Can be performed automatically or manually
               • Normal PT time = 11-15 secs
                 • Prolonged PT time
                   • Factor deficiency
                     • VII (extrinsic pathway)
                     • or, II, V and X (common pathway)
                   • Warfarin
                   • High dose heparin
                   • Liver dysfunction
                   • Vitamin K deficiency
                     • VII requires vitamin K for activity
                     • (So does XI - but not part of the extrinsic pathway)
                   • Disseminated intravascular coagulation (DIC)
                   • Lupus anticoagulant
               • Considerations
                 • Underfilling with blood can alter the anticoagulant:blood ratio of 9:1
                 • Polycythaemia alters the anticoagulant:blood ratio of 9:1
                 • Errors in the optical end point analysis by...
                   • Lipaemia
                   • Haemolysis
                   • Hyperbilirubinaemia
                   • Hyperproteinaemia
               • Internationational normalised ratio (INR) - accounts for differences between different nationalalities
                 • Can be tested using a portable electronic system
                   • Calculation of the INR = (patients PT in secs / mean normal PT i secs)^international sensitivity index
                     • Thromboplastin reagents vary in sensitivity between labs - the INR allows for standardisation by introducing an international sensitivity index (IS)
                 • For the purpose of standardising the monitoring of warfarin therapy
     • Measuring the common pathway alone
       • Thrombin time (TT)
         • Performed by adding thrombin (IIa) to patients plasma
           • Measures the conversion of fibrinogen to fibrin
             • Normal = <20 secs
               • Shortened TT in dysfibrinogenemia
               • Prolonged TT in
                 • DIC, liver disease, hypofibrinogenemia, heparin, lupus
   • Measuring fibrinogen levels
     • Detects deficiencies of fibrinogen or the ability to convert to fibrin
     • Normal = 200-400mg/dL
       • May do titers to assist in evaluation
       • Levels decreased in
         • DIC (consumption of clotting factors)
         • Liver disease (decreased synthesis)
         • Massive transfusion (dilutional coagulopathy)
         • Hypo-, dys- and afibrinogenemia
       • Increased in
         • Age
         • Females; pregnancy, oral contraceptive and menopause
         • Acute phase reaction
         • Disseminated malignancy
   • Activated clotting time (ACT)
     • Measures clot formation in intrinsic and common pathways
       • Freshly obtained blood added to a tube containing negatively charged particles
         • Negative charge initiates intrinsic pathway
         • Type of charged particle affects the normal length of ACT
           • e.g. Kaolin (90-150secs); glass (110-190secs)
           • Prolonged in
             • Heparin, hypothermia, platelet dysfunction, haemodilution, cardioplegic solutions, hypofibrinogenemia, factor 9intrinsic and common) deficiencies
         • Used in cardiopulmonary bypass; haemodialysis; vascular and general surgery

5. Bleeding disorders

   • Haemophilia A
     • X-linked (affects mostly males)
     • Severity associated with the level that factor VIII is affected
     • Bleeding - joints, gut, trauma, intracerebral haemorrhage at birth
     • Treated with recombinant factor VIII
     • Lab results
       • Long aPTT
       • Reduced fVIII activity
   • Anticoaglants
     • Why?
       • Prevent thrombus formation -> embolus
         • e.g. DVT and pulmonary embolism
     • Over-anticoagulation
       • Reversal urgent!
         • Heparin overdose
           • Give: protamine sulphate
           • Inhibits thrombin formation
         • Warfarin overdose
           • Give: vitamin K
           • Inhibits vitamin K activating enzymes (vitamin K-epoxide reductase)
             • Certain clotting factors arent activated (II, VII, IX, X)
       • Unstoppable bleeds (prolonged clotting times etc.)
     • Duration of anticoagulant therapy is important
       • First DVT/PE (previous risk factors) = 3 month therapy
       • First DVT/PE (idiopathic = spontaneous) = 6-12 months
       • >2 episodes = indefinite therapy
     • Targetting Xa
       • Why?
         • Pivotal upstream location - at the start of the common pathway
         • Primary site of cascade amplification
         • Rate-limiting component in the production of thrombin
       • Inhibtion of Xa
         • One molecule of Xa inhibited prevents ~138 molecules of fibrin being generated
       • Function of Xa - only to promote coagulation (compared to thrombin)
       • Indirect Xa inhibitors
         • Mediated through the binding to and activation of antithrombin(III) - e.g. heparin-like molecules
       • Direct Xa inhibitors
         • Bind directly to Xa (without the need for antithrombin(III)
         • Currently undergoing clinical trials
         • Inhibit Xa bound to activated platelets
           • i.e. trapped within a forming thrombus at the prothrombinase (Xa+Va+Ca(2+) + phospholipids)
           • Possible advantage over heparin and indirect Xa inhibitors
           • Appears to inhibit thrombus formation, but allow sufficient thrombin formation - mainting the ability to form a haemostatic plug elsewhere
             • Lower bleeding risk
     • Inhibitors of thrombin
       • Heparin (indirectly through antithrombin(III)
       • Direct = Bivalirudin
         • Binds directly to exosite 1 or active site
         • Does not bind platelet factor 4
         • Few other interactions
         • Wide therapeutic window
         • Expensive