Fibrinolysis

• Regulatory systems have to be in place to keep blood clot growth/expansion in check or remain in place especially as the tissue regeneration has taken place.
• Fibrin clots are degraded by enzymatic proteolysis.
• Similar to coagulation, fibrinolysis involves the coordinated activities of zymogens (e.g. Plasminogen), activators (tPA), cofactors (e.g. Fibrin), and inhibitors (e.g. PAI-1) - it is a localised, surface-bound process.
• The main roles of Fibrinolysis are:

  1. To function as a preventative measure against vascular occlusion by preventing fibrin formation above that required to prevent blood loss or rapidly removing excess fibrin
  2. Removal of fibrin clot as part of the tissue remodelling.
• Plasminogen is a 92-kDa protein that is present in blood as the inactive precursor of the serine protease plasmin
• 488,489 Plasminogen is converted to plasmin by cleavage at the Arg561-Val562 peptide bond by tissue-type or urokinase-type plasminogen activator (tPA and uPA, respectively).
• Activation of plasminogen by tPA is the major pathway that leads to lysis of fibrin clots.
• t-PA is secreted into the circulation by ECs as a single chain form that is rapidly cleared by the liver or inactivated by its fast-acting inhibitor PAI-1 (in a 1:1 ratio)
• The rate of t-PA and PAI-1 secretion is markedly increased by various physical and biochemical triggers, including:

  • strenuous exercise,
  • hypercoagulability
  • thrombin.
• Free t-PA is an insufficient activator of plasminogen in the absence of Fibrin
• The assembly of t-PA and plasminogen onto the fibrin surface forms a ternary complex - plasminogen and t-PA bind to the surface of fibrin via lysine residues, which induces a conformational change in t-PA and facilitates plasminogen activation.
• the native form of plasminogen contains a glutamic acid residue near its amino terminus - it is referred to as Glu-plasminogen
• kringles are 5 loop structures that are important to the localisation of the fibrinolytic response found on the Glu-plasminogen
• The kringles contain lysine binding sites promoting binding to lysine residues on the surface of the fibrin clot
• the Glu plasminogen is activated mainly by tPA to form two chain Glu-plasmin (which is fibrinolytically inactive) as its lysine binding sites are masked/obscured
• Glu-plasmin converts Glu-plasminogen to single chain Lys-plasminogen, which has a higher affinity for fibrin
• lys-plasminogen is converted by tPA to two chain Lys-plasmin which is the active serine protease as it can bind to fibrin and Lys-plasmin can also covert Glu-plasminogen to Lys-plasminogen and Glu-plasminogen can also autocatalyse to Lys-plasmin.
• the conversion of Glu-plasminogen to heavy chain and light chain of the lys-plasmin and it is this cleavage of the arginine 561 and Valine 562 which forms this peptide bond and exposes lysine binding sites etc.
• Urokinase – produced by the kidney and present in the urine.
• Two routes by which Lys-plasmin is generated – this is via autocatalysis from Glu-plasmin or conversion of Lys-plasminogen to Lys-plasmin via t-PA and U-PA.
• Glu-Plasminogen mainly activated by t-PA into two chain Glu-plasmin (Fibrinolytically inactive).
• Glu-plasmin undergoes autocatalysis to generate Lys-plasmin.
• Glu-plasmin coverts Glu-plasminogen to single chain lys-plasminogen which has a higher bindings affinity for fibrin.
• Kringles are structural protein domains that are characterised by a triple loop, disulphide bridge structure and conformation, shape is dictated by the number of H bonds and small sections of anti-parallel Beta-sheet secondary protein structures.
• The kringles are bound to the activation peptide.
• Lys-Plasmin can cleave, hydrolyse various substrates via cleavage of lysine-arginine bonds.
• When there is the formation of an in-situ clot and also enhancement of fibrinolysis the generation of fibrin degradation products (FDPs) in the form of D-dimers and these are the products of cross-linked fibrin.
• D Dimers are the fibrinolytic degradation products of cross-linked fibrin.
• Small protein fragments present in the blood after a blood clot is degraded by fibrinolysis and essentially small protein fragments are present in the blood and we can measure these using various immunological-based assays (e.g. ELISA) to assess the increase in fibrinolysis in patients
• D fragments of the fibrin protein joined by covalent cross-link (Isopeptide bond) via the action of factor XIIIa
• Fibrin is initially degraded to large aggregates (during fibrinolysis) composed of fragments denoted X and Y oligomers, and then to DDE fragments (containing the Central E domain and two D domains), these can then be further broken down to free E domains and D-Dimers.
• The detection of D-Dimers in the plasma indicates the presence of an in situ (taking place with its original location/site i.e. the damaged blood vessel) clot.
• The increase of D-dimers are due to an increase in fibrinolysis and are assessed to determine clot formation, and are also increased by the coagulation system
• Similar to thrombin generation the plasmin-generating potential of plasma is considerable and requires regulatory mechanisms to prevent the accumulation of a potentially lethal and destructive enzyme - serine protease can cause severe damage if not kept in check.
• The inhibitors target either plasmin itself or its activators/inducers - the main/primary inhibitor of plasmin is the serpin a2-antiplasmin (coded by the serpin 2 gene)
• serpin a2-antiplasmin exerts its effect in 2 ways:

  1. Formation of a stable 1:1 complex with Plasmin completely inactivating the plasmin
  2. slows fibrinolysis by masking/covering lysine–binding sites on glu-plasminogen and consequently interfering with plasmin binding to fibrin
• Lys-fibrinogen has a higher binding affinity for fibrin surface and is less susceptible to the effects of a2-antiplasmin.
• a2 antiplasmin deficiency (<20 cases) has been described as plasmin degrading blood clots as impaired plasmin activity leads to bleeding tendency, which has been severe in reported cases of a2-anti-plasmin deficiency and in liver cirrhosis there is a reduced production of a2-antiplasmin leading to decreased activity of plasmin and thus an increase in fibrinolysis
• a backup inhibitor (a2 macroglobulin) is an anti-protease large 720 kDa plasma protein - it is found in the blood and mainly produced by the liver and locally synthesised by macrophages and fibroblasts
• In humans encoded by the A2M gene, able to inactivate a numerous repertoire of proteases and functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein and inhibitor of coagulation via inhibition of thrombin