Haemostasis (part 1: primary haemostasis)

Haemostasis (part 1: primary haemostasis)

1. Introduction

   • The pysiological response to blood vessel damage that acts to minimise blood loss
   • Balance between protecting from haemorrhage and thrombosis
   • Primary haemostasis = platelet plug formation
   • Secondary haemostasis = fibrin clot formation
   • General mechanism
     • Blood vessel injury
       • Platelet aggregation
         • Platelet activation
         • +
         • +
       • (Neural) vessel constriction
         • Reduced blood flow
           • Stable haemostatic plug
       • (Tissue factor) coagulation cascade
         • Fibrin formation

2. Primary haemostasis players

   • Endothelium
     • Normally the endothelium prevents thrombus formation by actively exhibiting antiplatlet, anticoagulant and profibrinolytic properties
       • Antiplatelet properties
         • The endothelium serves as a barrier between the highly pro-thrombotic ECM and the platelets in circulation
         • Secrete NO (nitric oxide) and prostaglandin I-2 (PGI2) which promotes vasodilation and inhibits the activation of platelets
         • Also expresses adenylate diphosphatase (ADPase) which degrades circulating ADP (an activator of platelets)
       • Anticoagulative
         • The endothelial cells feature membrane-bound thrombomodulin
           • Thrombomodulin binds to thrombin (converting it from a pro-thrombotic to anti-thrombotic protein)
             • This activates protein C (an inhibitor of the coagulation cascade)
               • Protein C (with protein S) inhibits coagulation factors Va and VIIIa
         • Endothelial membrane-bound heparin-like molecules act as cofactors
           • Bind as cofactors to antithrombin(III)
             • Activated antithrombin(III)
               • Inactivates thrombin
               • Inactivates factors IXa and Xa
       • Profibrinolytic
         • Endothelial cells produce t-PA (tissue plasminogen activator)
           • t-PA cleaves plasminogen into plasmin (which degrades fibrin - degrades thrombi)
     • Prothrombotic qualities (following injury)
       • Loss of endothelium reveals ECM collagen-bound vWF -> platelets biond vWF (via the platelet glycoprotein GPIb)
       • Procoagulative
         • In response to inflammatory cytokines (TNF, IL-1) endothelial cells prodcue tissue factor
           • Tissue factor activates the extrinsic coagulation cascade
       • Anti-fibrinolytic
         • Under the right circumstances (injury etc.) endothelial cells produce inhibitors of plasminogen activator (PAI's)
           • Prevents plasmin-mediated fibrinolysis
   • Platelets
     • Disc-shaped, anucleate cell fragments (from megakaryocytes - about 2-3,000 per megakaryocyte)
       • Function depends on their activation; shape change and degranulation
     • Feature granules (alpha and delta granules)
       • Alpha-granules feature P-selectins in the membrane (become externalised - adhesion); TGF-beta; PDGF; fibronectin and coagulation factors
         • Clotting factors in alpha-granules = fibrinogen (I); V and VIII
       • Delta-granules contain ADP, ATP, ionised Ca(2+), histamine, seratonin and adrenaline
     • Normally platelets are inactivated
       • Following vessel endothelial damage
         • ECM (with vWF is exposed)
           • Platelets bind to this via their surface GPIb glycoprotein
             • Also able to bind other ECM components (e.g. fibronectin)
             • Secretion of both alpha and delta-granules
               • Alpha-granule products promote coagulation and adhesion of platelets
               • Delta-granule products activate the coagulation cascade (Ca(+) and ADP also promotes platelet aggregation)
               • Platelet aggregation at the injury site (aided by ADP and thromboxane A2 (TxA2) release
                 • This aggregation leads to the formation of the primary haemostatic plug
                   • Held together by the interaction between platelets (GPIIb-IIIa - fibrinogen - GPIIb-IIIa) and platelets with the ECM (via ECM vWF - GPIb)
                   • This is followed by the activation of the coagulation cacade which ultimately produces fibrin (forms a meshwork around the platelet aggregation)
                     • Platelets release coagulation factors (fibrinogen (I), V and VIII)
                     • Platelets release pro-coagulative factors (Ca(+))
                   • Activated platelets contract their cytoskeletons to form a tight-knit mass of cells
                 • ADP activates other platelets
                   • Causes conformational change in a large platelet surface glycoprtein (GPIIb-IIIa)
                     • GPIIb-IIIa binds fibrinogen (from alpha-granules) and links activated platelets together
                     • Loss = Glanzmann's thrombasthenia (another bleeding disorder)
                 • TxA2 is a potent vasoconstrictor
           • Loss of this factor results in von willebrand disease (bleeding disorder)
             • Loss of the GPIb receptor = Bernard-soulier syndrome (bleeding disorder)

3. Testing platelets

   • Full blood count (with mean platelet volume (MPV), blood film)
   • PFA-100 (platelet function analyser)
     • Full blood sample is drawn up through a capillary tube towards a membrane with a hole in the end
       • The membrane is coated in collagen with either; ADP or adrenaline
         • If normal platelets -> should be activated by the membrane and form a clot
           • Blood should be inhibited from passing through the hole at the end
     • Measures high shear haemostasis
   • Platelet aggregation assay
     • In vitro assays
       • Measure platelets clumps in platelet-rich plasma (PRP) or whole blood when exposed to aggregation agonists
         • Collagen, ADP and thrombin all bind to their own specific receptor resulting in activation
           • All receptors are implicated in different diseases (e.g. GPIb, GPIIb-IIIa)
           • Activation = shape change, granule release and aggregation
     • Activation-induced change depends on the normal platelet function
       • Useful data; lag phase, aggregation rate and amplitude
   • Platelet secretion assay
     • Platelet ADP/ATP release (from delta-granules) can be measured
       • Valuable diagnostic tool
       • Methods: HPLC (high performance liquid chromatography) luminescence; Firefly lantern preps (luminescence due to the ATP concentration)
   • Flow cytometry
     • Uses whole blood (2ul minimum) - therefore its a physiological activity assay
       • Whole blood sample prevents artificially induced platelet aggregation
     • All blood cell properties can be analysed simultaneously
       • (Not just platelets)
     • New Ab's or dyes can be easily introduced into samples
     • High degree of sensitivity
       • Future - aggregation by flow?
         • I have no idea what this means either...

4. Examples of platelet disorders

   • Thrombocytopaenia
     • Signs/symptoms
       • Common
         • Spontaneous skin pupura (/ecchymoses)
         • Mucous membrane bleeding (/epistaxis)
         • Protracted bleeding after trauma/surgery
         • Menorrhagia / postpartum haemorrhage
       • Uncommon
         • Haemorrhages in the following places
           • Intracranial, gastrointestinal, retinal and genitourinary
     • Causes (aetiology)
       • Drugs
       • Infection
       • DIC
       • Immune
   • Glanzmann's thrombasthenia
     • Loss of platelet GPIIb-IIIa (binds platelets together via fibrinogen bridge)
       • >40 mutations ID'd (most type I involve the GPIIb gene; others = GPIIIa gene)
     • Bleeding disorder with a normal platelet count
     • Test results
       • Normal
         • Platelet count
         • Platelet size / morphology and lifespan
       • Very long bleeding time and PFA result
     • Autosomal recessive
       • Often severe bleeding, but variable phenotype
         • Subtypes (1 = complete IIb-IIIa loss; 2 = partial loss of IIb-IIIa)
   • Treatment options in platelet disorders
     • Specialist care
     • Avois aspirin, NSAIDs
     • Hormonal control on menses
     • Supportive care - tranexamic acid
     • Platelet transfusion
     • Recombinant rVIIIa (clotting factor)
   • Thrombocytosis (= high concentration of platelets in the blood)
     • Causes
       • Inflammatory response
       • Fe-deficiency / bleeding
       • Bone marrow disorder (e.g. cancer)
     • Antiplatelet / anticoagulant agents
       • Heparin (binds antithrombin(III) and inactivates thrombin)
       • Aspirin, NSAIDs (inhibit COX2)
       • Clopidogrel (ADP receptor inhibitor)

5. Von willebrand factor (vWF)

   • Large multimeric glycoprotein (exists as a series of multimers)
     • Extensively processed (multimer assembly)
       • Constituitively expressed
   • Functions
     • Carrier protein for factor VIII (protects VIII against premature proteolytic degradation)
     • Adhesive protein in primary haemostasis (platelet adhesion)
     • Binds a number of ligands
       • Collagen; platelet GPIb and GPIIb-IIIa; sulfatides; heparin
   • Clinical features
     • Highly heterogenous phenotypic expression
       • Mild-moderate bleeding tenedency
         • Bruising, epistaxis, gingival haemorrhage, post-surgical/dental bleeding
       • Mucosal bleeding
   • Laboratory diagnosis
     • Screening test
       • Increased bleeding time, increased partial thromboplastin time (doesnt invovle tissue factor)
     • Specific assay
       • Decreased: factor VIII, vWF (via Ab) and vWF activity
   • Criteria
     • Type 1 vWD (von willebrand disease)
       • Inherited bleeding disorder
         • Diagnosis based on three criterias - symptoms, lab tests and inheritence pattern
           • If all three criteria not met = possible type 1 VWD