Secondary Hemostasis

• The fibrinolytic system is activated by plasminogen activators, which convert plasminogen to plasmin.
• Coagulation is a sequential process by which multiple plasma enzymes, cofactors, and other related substances interact to form an insoluble fibrin clot
• Fibrinolysis is the final stage of coagulation where the fibrin clot is digested by plasmin
• Procoagulants are proteins that promote clotting and may be subdivided into zymogens and cofactors
• Regulatory proteins maintain a balance between thrombosis and abnormal bleeding
• Physiologic inhibitors like Anti-thrombin III, Complement inhibitor, A2 macroglobulin, and A1 anti-trypsin are present in the plasma
• During clotting, procoagulants are sequentially activated, producing a localized thrombus
• Factors in the coagulation process are named using Roman Numerals in the order of their initial description or discovery
• Fibrinogen is the highest concentration factor and the heaviest
• Factor XIII is not included in the common pathway of coagulation
• Tissue Factor Pathway Inhibitor is the principal regulator of the tissue factor pathway
• Antithrombin neutralizes all serine proteases except Factor VIIa
• Protein C, when activated by thrombin-thrombomodulin, inactivates Factor Va and VIIIa
• Fibrinolysis is the systemic and accelerated hydrolysis of fibrin by bound plasminogen
• Plasmin digests clots by hydrolysis of arginine-related and lysine-related peptide bonds
• Tissue Plasminogen Activator (TPA) and Urokinase Plasminogen Activator (UPA) are activators of plasmin
• Plasminogen Activator Inhibitor 1 (PAI-1) is the principal inhibitor of plasminogen activation
• Alpha-2-antiplasmin is the primary inhibitor of free plasmin
• Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is activated by the thrombin-thrombomodulin complex
• Coagulation Tests
• Clotting Time:

  • Measures the period required for blood to clot after it has been removed from the body
  • Used to screen for problems in the blood clotting or coagulation mechanism
  • Methods:
  • Capillary Blood Method:
  • Slide drop method
  • Normal value: 2 - 4 mins
  • Capillary method (Dale and Laidlaw's)
  • Whole Blood (Lee and White Method):
  • Normal value: 7 - 15 mins
• Prothrombin time:

  • Monitors coagulation factors of the extrinsic and common pathways
  • Prolonged PT results indicate an abnormality of one or more extrinsic or common pathway coagulation factors
  • Used to monitor oral anticoagulant therapy (Coumadin/Warfarin - vitamin K antagonists)
  • Sample: PPP - Platelet-poor plasma (2000g x 10 mins)
  • Reagents:
  • Thromboplastin
  • Calcium chloride - initiates clotting
  • INR - International Normalized Ratio:
  • Standardized way of reporting PT results
  • INR of 2.0 - 3.0 must be achieved
• Activated Partial Thromboplastin Time (APTT):

  • Monitors coagulation factors of the intrinsic and common pathways
  • Prolonged APTT results indicate an abnormality of one or more intrinsic or common pathway coagulation factors
  • Used to monitor heparin therapy (heparin - antithrombin)
  • Sample: PPP (2000g x 10 mins)
  • Reagents:
  • Activators (MECK):
  • Micronized silica, Elagic acid, Celite, Kaolin
  • Phospholipids
  • Calcium Chloride
• Stypven Time:

  • Aka Russell's Viper Venom Time
  • Monitors coagulation factors of the common pathway
  • Prolonged results indicate an abnormality of one or more common pathway coagulation factors
  • Used to detect deficiencies in:
  • Fibrinogen
  • Prothrombin
  • Factor V
  • Factor X
  • Reagent: East Indian Viper venom (Vipera russelli)
  • Normal value: 6 - 10s
• Thrombin Time:

  • Detects fibrinogen deficiency
  • Sensitive test in detecting heparin therapy
  • Sample: PPP (2000g x 10 mins)
  • Reagents:
  • Thrombin
  • Calcium chloride
• Reptilase Time:

  • Reptilase - thrombin-like enzyme from the reptile Bothrops atrox
  • Detects fibrinogen deficiency
  • Unaffected by heparin therapy
  • Reagent: Reptilase/Atroxin (Bothrops atrox)
• Duckert's Test:

  • Aka 5M Urea Solubility Test
  • Detects Factor XIII deficiency
  • Sample: PPP (2000g x 10 mins)
  • Reagents:
  • 5M Urea
  • 1% monochloroacetic acid
  • Calcium chloride
• Mixing Studies:

  • Aka substitution studies
  • Procedure to identify specific factor deficiencies by mixing correction reagents with a patient's plasma and performing PT and APTT
  • Correction Reagents:
  • Fresh Plasma: All factors are present
  • Fresh Serum: All factors, except 1, 5, 8, 13, 2 (20% left)
  • Aged serum: All factors, except 1, 2, 5, 8, 13
  • Adsorbed Plasma: All factors, except 9, 10, 7, 2
  • Aged Plasma: All factors, except 5, 8
• Fibrinolysis Tests
• Whole Blood Clot Lysis Time:

  • Clot should remain intact for approximately 48hrs at 37C
  • Results:
  • Normal = clot lysis in >48hrs
  • Increased fibrinolysis = clot lysis in <48hrs
• Euglobulin Clot Lysis Time:

  • Screening procedure for the measurement of fibrinolytic activity
  • More sensitive than whole blood clot lysis time
  • Results:
  • Normal = clot lysis in >2hrs
  • Increased fibrinolysis = clot lysis in <2hrs
• Protamine Sulfate Test:

  • Detects the presence of fibrin monomers by causing the formation of fibrin strands or gel-like clots (paracoagulate)
  • Reagent: Protamine sulfate
  • Results:
  • Normal - no gel formation
  • Abnormal - gel formation
• Ethanol Gelation Test:

  • Less sensitive but more specific test than protamine sulfate test
  • Detects the presence of fibrin monomers
  • Reagent:
  • 50% ethanol
  • Sodium hydroxide
  • Results:
  • Normal - no gel formation
  • Abnormal - gel formation
• Latex D-dimer test:

  • Used to evaluate the presence of specific fragments arising from degradation of fibrin
  • D-dimer - evidence of intravascular fibrin formation
  • Results:
  • Single test to differentiate primary and secondary fibrinolysis