Haemostasis (part 1: primary haemostasis)

Introduction
1. The pysiological response to blood vessel damage that acts to minimise blood loss
2. Balance between protecting from haemorrhage and thrombosis
3. Primary haemostasis = platelet plug formation
4. Secondary haemostasis = fibrin clot formation
5. General mechanism
  1. Blood vessel injury
    1. Platelet aggregation
      1. Platelet activation
      2. +
      3. +
    2. (Neural) vessel constriction
      1. Reduced blood flow
        Stable haemostatic plug
    3. (Tissue factor) coagulation cascade
      1. Fibrin formation
Primary haemostasis players
1. Endothelium
  1. Normally the endothelium prevents thrombus formation by actively exhibiting antiplatlet, anticoagulant and profibrinolytic properties
    1. Antiplatelet properties
      1. The endothelium serves as a barrier between the highly pro-thrombotic ECM and the platelets in circulation
      2. Secrete NO (nitric oxide) and prostaglandin I-2 (PGI2) which promotes vasodilation and inhibits the activation of platelets
      3. Also expresses adenylate diphosphatase (ADPase) which degrades circulating ADP (an activator of platelets)
    2. Anticoagulative
      1. 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
      2. 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
    3. Profibrinolytic
      1. Endothelial cells produce t-PA (tissue plasminogen activator)
        t-PA cleaves plasminogen into plasmin (which degrades fibrin - degrades thrombi)
  2. Prothrombotic qualities (following injury)
    1. Loss of endothelium reveals ECM collagen-bound vWF -> platelets biond vWF (via the platelet glycoprotein GPIb)
    2. Procoagulative
      1. In response to inflammatory cytokines (TNF, IL-1) endothelial cells prodcue tissue factor
        Tissue factor activates the extrinsic coagulation cascade
    3. Anti-fibrinolytic
      1. Under the right circumstances (injury etc.) endothelial cells produce inhibitors of plasminogen activator (PAI's)
        Prevents plasmin-mediated fibrinolysis
2. Platelets
  1. Disc-shaped, anucleate cell fragments (from megakaryocytes - about 2-3,000 per megakaryocyte)
    1. Function depends on their activation; shape change and degranulation
  2. Feature granules (alpha and delta granules)
    1. Alpha-granules feature P-selectins in the membrane (become externalised - adhesion); TGF-beta; PDGF; fibronectin and coagulation factors
      1. Clotting factors in alpha-granules = fibrinogen (I); V and VIII
    2. Delta-granules contain ADP, ATP, ionised Ca(2+), histamine, seratonin and adrenaline
  3. Normally platelets are inactivated
    1. Following vessel endothelial damage
      1. 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)
Testing platelets
1. Full blood count (with mean platelet volume (MPV), blood film)
2. PFA-100 (platelet function analyser)
  1. Full blood sample is drawn up through a capillary tube towards a membrane with a hole in the end
    1. The membrane is coated in collagen with either; ADP or adrenaline
      1. 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
  2. Measures high shear haemostasis
3. Platelet aggregation assay
  1. In vitro assays
    1. Measure platelets clumps in platelet-rich plasma (PRP) or whole blood when exposed to aggregation agonists
      1. 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
  2. Activation-induced change depends on the normal platelet function
    1. Useful data; lag phase, aggregation rate and amplitude
4. Platelet secretion assay
  1. Platelet ADP/ATP release (from delta-granules) can be measured
    1. Valuable diagnostic tool
    2. Methods: HPLC (high performance liquid chromatography) luminescence; Firefly lantern preps (luminescence due to the ATP concentration)
5. Flow cytometry
  1. Uses whole blood (2ul minimum) - therefore its a physiological activity assay
    1. Whole blood sample prevents artificially induced platelet aggregation
  2. All blood cell properties can be analysed simultaneously
    1. (Not just platelets)
  3. New Ab's or dyes can be easily introduced into samples
  4. High degree of sensitivity
    1. Future - aggregation by flow?
      1. I have no idea what this means either...
Examples of platelet disorders
1. Thrombocytopaenia
  1. Signs/symptoms
    1. Common
      1. Spontaneous skin pupura (/ecchymoses)
      2. Mucous membrane bleeding (/epistaxis)
      3. Protracted bleeding after trauma/surgery
      4. Menorrhagia / postpartum haemorrhage
    2. Uncommon
      1. Haemorrhages in the following places
        Intracranial, gastrointestinal, retinal and genitourinary
  2. Causes (aetiology)
    1. Drugs
    2. Infection
    3. DIC
    4. Immune
2. Glanzmann's thrombasthenia
  1. Loss of platelet GPIIb-IIIa (binds platelets together via fibrinogen bridge)
    1. >40 mutations ID'd (most type I involve the GPIIb gene; others = GPIIIa gene)
  2. Bleeding disorder with a normal platelet count
  3. Test results
    1. Normal
      1. Platelet count
      2. Platelet size / morphology and lifespan
    2. Very long bleeding time and PFA result
  4. Autosomal recessive
    1. Often severe bleeding, but variable phenotype
      1. Subtypes (1 = complete IIb-IIIa loss; 2 = partial loss of IIb-IIIa)
3. Treatment options in platelet disorders
  1. Specialist care
  2. Avois aspirin, NSAIDs
  3. Hormonal control on menses
  4. Supportive care - tranexamic acid
  5. Platelet transfusion
  6. Recombinant rVIIIa (clotting factor)
4. Thrombocytosis (= high concentration of platelets in the blood)
  1. Causes
    1. Inflammatory response
    2. Fe-deficiency / bleeding
    3. Bone marrow disorder (e.g. cancer)
  2. Antiplatelet / anticoagulant agents
    1. Heparin (binds antithrombin(III) and inactivates thrombin)
    2. Aspirin, NSAIDs (inhibit COX2)
    3. Clopidogrel (ADP receptor inhibitor)
Von willebrand factor (vWF)
1. Large multimeric glycoprotein (exists as a series of multimers)
  1. Extensively processed (multimer assembly)
    1. Constituitively expressed
2. Functions
  1. Carrier protein for factor VIII (protects VIII against premature proteolytic degradation)
  2. Adhesive protein in primary haemostasis (platelet adhesion)
  3. Binds a number of ligands
    1. Collagen; platelet GPIb and GPIIb-IIIa; sulfatides; heparin
3. Clinical features
  1. Highly heterogenous phenotypic expression
    1. Mild-moderate bleeding tenedency
      1. Bruising, epistaxis, gingival haemorrhage, post-surgical/dental bleeding
    2. Mucosal bleeding
4. Laboratory diagnosis
  1. Screening test
    1. Increased bleeding time, increased partial thromboplastin time (doesnt invovle tissue factor)
  2. Specific assay
    1. Decreased: factor VIII, vWF (via Ab) and vWF activity
5. Criteria
  1. Type 1 vWD (von willebrand disease)
    1. Inherited bleeding disorder
      1. Diagnosis based on three criterias - symptoms, lab tests and inheritence pattern
        If all three criteria not met = possible type 1 VWD

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