WK 9

• Function of Haemostasis?

  1. Maintain blood in fluid state while circulating in vasculature
  2. Stop bleeding at site of injury by haemostatic plug formation
  3. Ensure plug removal post-healing
• Haemostasis components?

  1. Blood vessels
  2. Platelets
  3. Coagulation factors and their inhibitors
  4. Fibrinolytic system and its inhibitors
• Primary Haemostasis?

  1. Platelet adhesion
  2. Platelet activation + granule release
  3. Platelet aggregation
• Secondary Haemostasis?

  Coagulation + fibrin clot formation
• Blood Vessel: Intima, Media, Adventitia. Endothelial cells: luminal surface covered by glycocalyx (proteoglycan coat) (can activate ATIII, coag. inhibitor). Express coagulation proteins (thrombomodulin, endothelial protein c receptor), plasminogen activator, protein S, vWF, ADPase. Metabolises vasoactive peptides. Produces angiotensin II, prostacyclin, endothelin, thromboxane. Produces NO.
• Subendothelial blood vessel cells compose collagen, elastic tissues, proteoglycans, glycoproteins inc. fibronectin and vWF. These are exposed when endothelial wall in compromised to activate embolism.
• Vasoconstriction involves the contraction of the smooth muscle (tunica media) following injury to arrest blood loss. Also occurs in microcirculation where smooth muscle is absent
• Platelets are derived from megakaryocytes. No nucleus. 1.5-3.5um. Contain microtubules which maintain shape. Depolymerise during aggregation (malleable). Cytoplasm has delta, alpha and lysosomal granules. Contractile system: dense tubular system, circumferential microfilaments, actin, myosin and calmodulin to maintain the disc shape and then to change shape. Has a canalicular membrane system that communicates with the exterior.
• In primary haemostasis, platelets are involved in 1. adhesion. 2. activation with shape change and granule release. 3. aggregation
• ADHESION: Loss of endothelial cells --> exposure of collagen + vWF --> vWF etc deposit in damaged vessel, binding to collagen --> GPIb binds to vWF --> GPIa-IIA and GPVI bind to collagen and other platelet receptors --> GPIIb/IIIa binds fibrinogen and vWF
• vWF = large multimeric glycoprotein --> secreted + stored by endothelial cells, megakaryocytes and platelets. Bridging molecule at sites of vascular injury for normal platelet adhesion and promotes platelet aggregation under high shear conditions. (primary haemostasis). Carrier of factor VIII in circulation.
• ACTIVATION: Activated GIb receptors --> platelet shape change (disc>sphere with projecting pseudopods), spreading, secretion and clot retraction. + granule release (ADP, ATP, serotonin, calcium, vWF, FV, fibrinogen). Agonist react with specific receptors --> further activation inc. GpIIb/IIIa.
• Arachidonic acid released by PLA2 from membrane phospholipids due to disrupted endothelium. Cyclo-oxygenase 1 (PTGS1) metabolise AA to form cyclic-endoperoxides, mostly converted into TXA2 = local vasoconstrictor, mobilise Ca2+ and stimulates secondary granule release
• AGGREGATION: fibrinogen binds simultaneously to GpIIb-IIIA receptors on two different platelets = receptor cross-linking
• Platelet procoagulant properties:

  Membrane changes configuration-> negatively charged phosphatidyl-serine and inositol to surface --> facilitates complexation of coagulation factors. Membrane blebbing --> microparticle formation (-ve charge, rich in factor Va)

  Microparticles bind FVIII, Va, Xa --> form tenase and prothrombinase complexes on surface. Surface expression of P-selectin = recruitment of leukocytes
• Secondary Haemostasis = series of proteolytic reaction. All enzymes except FXIII are serine proteases. Local conc. of clotting factors at injury site. Surface mediated reaction on exposed collagen, platelet phospholipids and tissue factor
• Three stages of caogulation:

  Initiation

  Amplification

  Propagation
• Coagulation Initiation:

  Occurs on TF-bearing cells (eg fibroblast). FVIIa/TF complex and Ca2+ activate FX and FIX. FXa/Va and FII produce small amounts of Thrombin (IIa)
• Coagulation Amplification:

  Thrombin activates V, VIII and XI on the activated platelet surface. Consolidates production of FXa. Sets the stages for the procoagulant complex assembly.
• Coagulation Propagation:

  Intrinsic tenase (FVIIIa/FIXa FXa) is assembled on activated platelet surface. Generates a thrombin burst --> haemostatic fibrin clot at site of injury. Thrombin leaves fibrinogen to form fibrin. Fibrin meshwork is stabilised by FXIIIa.
• Inhibitors of Coagulation:

  Tissue factor pathway inhibitor (Quenches factor VIIa-TF complex)

  Antithrombin III (ATIII) (binds thrombin to form inactive complex. Incapacitates Xa, IXa, XIa, XIIa. Potentiated by heparin).

  Protein C (cleaves cofactor VIIIa and Va). Needs activation.

  Protein S (enhances protein C activity. does not require activation)

  Thrombomodulin (activated by thrombin. binds to alter confirmation. activates protein C)
• fibrinolysis is activated at time of coagulation. tissue plasminogen activator (tPA) and plasminogen bind fibrin. Activated to form plasmin. plasmin cleaves fibrin. thrombin-activated fibrinolysis inhibitor removes the binding site of plasminogen and tPA. Plasminogen activator inhibitor. Plasmin inhibitor (a2-antiplasmin)
• Disseminated Intravascular Coagulation

  Excessive + widespread coagulation activation = consumption of factors, loss of normal regulatory mechanisms. Prolonged PT, aPTT, prolonged PTT, reduced fibrinogen. Elevated D-dimer (fibrin degradation product)
• Haemophilia = Factor VIII deficiency (A). Factor IX deficiency (B/Christmas disease). X linked recessive. Soft tissue bleeding and haemarthrosis. Prolonged aPTT
• vWB disease:


  quantitative and qualitative vWF abnormality. Autosomal dominant. Mucosal bleeding. Usually increases VIII half life 5-fold. most common bleeding disorder (1% population). vWF synthesised by endothelial cells and megakaryocytes
• Lupus Anticoagulant

  Acquired inhibitor of phospholipid coagulation activation. inc. venous + arterial thrombotic risk. Prolonged aPTT
• Anticoagulation is indicated where it prevents: development of pathological thrombus (eg AF, peri-operative DVT) + progression of established thrombus (DVT + PE). Both to stop embolisation.
• Warfarin. Haemorrhagic component of sweet clover. inhibits vitamin K epoxide reductase complex in liver. reduced form of vitamin k isa co-factor of gamma carboxylation of FII, VII, IX, X, proteins C and S. Warfarin results in a decrease in functional 2,7,9,10.
• Warfarin is orally available, maximal effects at 2-4 days. Highly protein bound. Hepatic metabolism. mean plasma half-life ~40hours. 2-5 day (duration of effect).
• Warfarin is metabolised by hepatic CYP450 2C9 isoform. Antibiotics potentiate warfarin. Anticonvulsants inhibit warfarin (phenytoin, carbamazepine and phenobarbital are potent inducers of the CYP450 system). Toxic effects = bleeding, teratogenic.
• Warfarin reversal

  Oral/IV vitamin K. Prothrombin complex (2,9,10) or fresh frozen plasma.
• Warfarin monitoring: thromboplastin assigned a value ISI for calibration to reference thromboplastin. PT patient/PT normal ISI = INR (International normalised ratio). Venous thromboembolism 2-3, AF 2-3, mechanical heart valves 3-4.5
• Heparin is a heterogenous polymer (repeating disaccharide units). MW = 3000-30000. Unfractionated heparin = parental only
• Low molecular weight heparin (LMWH). mean MW ~5000. Eg Clexane, fondaparinux (synthetic pentasaccharide) Chemical or enzymatic derivative of unfractioned heparin.
• LMWH indirectly inhibits Xa and thrombin by potentiating antithrombin. LMWHs have higher anti-Xa/anti-IIa ratios, typically 2-4
• Blocks B2 receptors = decrease NA binding to B2 = decreased cAMP and promotion of Ca channel and Ca release from SR. Decrease Ca = decrease cross bridging = decrease contractility
• Block beta 1 adrenergic receptors

  -      Inhibit the binding of catecholamines

  -      Inhibit activation of G stimulatory protein

  -      Inhibit activation of adenylyl cyclase

  -      Huge reduction of conversion of ATP to cAMP

  -      Reduced activation of PKA

  -      Reduced phosphorylation of voltage gated Ca2+ channels

  -      Reduced Ca2+ entry -> reduced depolarisation of SA and AV node -> increased refractory period
•  B1 blockers Effect on contractility

  -      Inhibit the binding of catecholamines

  -      Inhibit activation of G stimulatory protein

  -      Inhibit activation of adenylyl cyclase

  -      Huge reduction of conversion of ATP to cAMP

  -      Reduced activation of PKA

  -      Reduced phosphorylation of voltage gated Ca2+ channels

  -  
• B1 blockers efect on contractility:

  Reduced Ca2+ entry -> reduced binding to Troponin C and decreased release of Ca2+ from SR due to decrease Ca2+ induced Ca2+ release and decreased activation/phosphorylation of SR ad myosin

  -      Reduced exposure of myosin binding sites on actin -> reduced formation of cross bridge and power stroke -> decreased contractility
• b1 blockers:

  Angina

  · Because they decrease heart rate, they will allow the heart to have more time to fill, and thus will increase blood supply to the heart, allowing more blood supply to the epicardium, myocardium, and endocardium -> decrease ischemia and present further infarction

  · Decreased inflammation -> less bradykinin and adenosine release -> decrease pain