HEMOSTASIS Department of Surgery FEU-NRMF MEDICAL CENTER
HEMOSTASIS balance of the physiological processes prevent excessive bleeding after vessel injury maintain a viable circulation by keeping the blood in an uncoagulated state “Self-sealing” system to prevent excessive bleeding and potentially life threatening states
Four components Vessel function constriction of injured vessels Platelet function formation of platelet plugs Coagulation formation of fibrin plugs Fibrinolysis dissolution of blood clots
Normal Hemostasis Means stopping of blood – prevents blood loss Imbalance in one direction may lead to excessive bleeding, imbalance in the other may lead to thrombus formation
Overview of blood coagulation PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS
Hemostasis Primary Hemostasis Formation of platelet plug Secondary Hemostasis Formation of blood clot response is dependent on overlapping and interdependent responses
Vascular Endothelium Blood vessel wall integrity is essential for prevention of blood loss – provides potent anticoagulation surface.
Vascular Endothelium ECs form monolayer resting on continuous basement membrane; constitutes first barrier of defense from hemostasis and thrombosis
EC Products Secreted into the Subendothelium LUMEN EC surface products secreted into the blood stream
ECs provides numerous proteins to subendothelium essential for cell–to-cell interactions and formation of diffusion barrier to prevent blood loss. Vascular Endothelium
EC Products Secreted into the Subendothelium ● basement membrane ● elastin ●fibronectin ● collagen III and IV ● lamilin ● mucopolysaccharides ● microfibrils ● vibronectin ● vWf ● protease inhibitors These proteins essential for cell–to-cell interactions and formation of diffusion barrier to prevent blood loss into extravascular spaces.
Vascular Endothelium ECs also secrete numerous substances into the vascular lumen to prevent clotting (e.g. heparin) which promote fluidity of the blood
Procoagulants Anticoagulants ● PGI2 ●glycosaminoglycans ● tissue factor ● EDRF ● ATIII/heparin sulfate ● vWf ● t-PA ● protein kinase/thrombomodulin ● Factor V ● urokinase ● plasminogen activators ● inhibitors (PAI-1, PAI-2 ● nitric oxide ATIII) ● IL1, TNFα ● endothelin-1 ● PAF EC surface products secreted into the blood stream These substances promote fluidity of the blood.
Vascular Endothelium Normal endothelium acts as a potent anti-coagulation surface, upon stimulation or injury, endothelium transforms into a potent pro-coagulation surface as subendothelial collagen is exposed
Primary Hemostasis response to vascular injury that produces a platelet plug at the site of damage immediately limit bleeding through the formation of a loose platelet plug
Platelets Minute round or oval discs (1-4 mm diameter). Do not have a nucleus and can not divide. Formed from megakaryocytes in bone morrow Normally 150-450,000 per ml of blood
Platelets Adhering to the endothelial wall at the site of injury Releasing potent anticoagulant compounds Aggregating to form a plug Providing a phospholipid surface for activated coagulation enzyme complexes
Platelets Have many functional characteristics of whole cells Contains contractile proteins (actin, myosin and thrombosthenin) Residuals of ER and Golgi apparatus Mitochondria and enzymes to form ATP and ADP Can synthesis prostaglandins (thromboxane A2) Forms fibrin stabilizing factor Forms growth factors
Platelets Cell membrane contains glycoproteins that reduces adherence to normal endothelium yet promotes adherence to injured areas of vessel wall, especially injured ECs and collagen of subendothelium. Membrane also contains phospholipids that play activating role at multiple points in the clotting process. Platelets have a half life of 8-12 days Eliminated from the circulation mainly by tissue macrophages, especially in spleen.
Platelets Endothelial cell injury exposes subendothelial collagen which causes change in platelet shape and adherence. vWf- bridges platelet membrane glycoprotein to the exposed collagen at site of injury.
Vascular Injury INJURED
Vasoconstriction an immediate reflex Initial response to injury diminishing blood loss Local constriction of smooth muscle
Vasoconstriction Transient, typically lasts minutes (severed limbs) Result of release of local humoral actors, neural reflexes (caused by pain), local myogenic spasm Small blood vessels, release of vasoconstrictor thromboxane A2 from platelets is primarily responsible
Platelet adhesion Exposed collagen from the damaged site will promote the platelets to adhere undergo degranulation and release cytoplasmic granules Occurs within 15 secs after injury
Multimeric plasma glycoprotein required for normal hemostatic platelet plug formation
Forms a bridge between platelet glycoprotein IB and exposed collagen in the subendothelium
It is the “glue” that binds platelets to collagen.
vWF is also responsible for the binding and transport of factor VIII (antihemophilic factor), a procoagulant protein in plasma.
Platelet adhesion cytoplasmic granules serotonin ADP and Thromboxane A2
Platelet adhesion serotonin, a vasoconstrictor ADP attracts more platelets to the area thromboxane A2 promotes platelet aggregation, degranulation, and vasoconstriction
EM of normal and activated platelets Normal Aggregated Platelets
ADP and thromboxane A2 promote more platelet adhesion therefore more ADP and thromboxane “Second wave”
Platelet plug The positive feedback promotes the formation of a platelet plug
Primary platelet plug is only good for stopping bleeding in the first minute- it can not sustain hemostasis. Heparin does NOT interfere with this reaction
Arachidonic acid pathway ASPIRIN and other NSAID
Primary hemostasis Defects : associated with mucocutaneous bleeding, characterized by epistaxis, ecchymosis, genitourinary bleeding, or gingival bleeding “first wave” of aggregation “second wave” ( granule release )
Secondary Hemostasis Cascade of enzymatic reactions that ultimately results in the conversion of fibrinogen to fibrin monomers cross-linked into insoluble strands that serve to stabilize the loose platelet clot formed in primary hemostasis
Secondary Hemostasis triggered by the release of tissue factor from epithelial cells Platelets, vascular wall and multiple circulating or membrane-bound coagulation factors
Formation of Prothrombin Activator Prothrombin activator formed in two ways: Extrinsic pathway Intrinsic pathway In both pathways, inactive forms of blood clotting factors are converted to active forms. Designated by Roman numerals, small “a” for active
The common pathway Vessel Injury Prothrombin Activator Complex (rate limiting step) Ca++ Note that both the Extrinsic and Intrinsic Pathways converge on Factor X
Characteristics of clotting reactions in the cascade A proteolytic enzyme generates the next enzyme in the cascade by cleavage of a proenzyme The reactions occur on a phospolipid surface ( platelet membrane ) Each reaction requires a helper protien to bring the enzyme and substrate together
Clotting Factors in the Blood
Extrinsic Pathway Process begins with trauma to vascular wall initiated by tissue factor release by damaged tissue initiated by contact with Factor VII and platelets with collagen in vessel wall Clotting begins within seconds
Extrinsic Pathway Release of Tissue Factor (thromboplastin). Functions as a proteolytic enzyme complex Activation of Factor X. Tissue Factor further complexes with Factor VII which in the presence of Ca++ activates Factor X to Xa. Formation of Prothrombin Activator Xa combines with phospholipids and Factor V to form prothrombin activator complex In the presence of Ca++, this splits prothrombin into thrombin in the Common Pathway
Extrinsic Pathway Release of Tissue Factor (thromboplastin). Functions as a proteolytic enzyme complex
Extrinsic Pathway Activation of Factor X. Tissue Factor further complexes with Factor VII which in the presence of Ca++ activates Factor X to Xa.
Extrinsic Pathway Formation of Prothrombin Activator Xa combines with phospholipids and Factor V to form prothrombin activator complex
Extrinsic and common pathways Tissue Trauma Tissue Factor
Intrinsic Pathway Initiated by trauma to blood or exposure of blood to vascular wall collagen Takes 1-6 minutes to cause clotting All components are “intrinsic to the circulating plasma” and NO surface is required to initiate clotting
Intrinsic Pathway Causes activation of Factor XII (to XIIa) and release of platelet phospholipids Activation of Factor XI by Factor XII. This reaction also requires High Molecular Weight (HMW) kininogen and is accelerated by prekallikrein.
Intrinsic and common pathways Prothrombin Activator
Factor X activation Extrinsic Xase TF- VIIa Intrinsic Xase VIIIa-XIa 50x more effective at catalyzing factor X activation
Functions of thrombin Conversion of fibrinogen to fibrin Activation of factors V, VIII, XI and XIII
Prothrombin unstable plasma protein that splits in half to form thrombin. Made by liver and requires vitamin K
Common Pathway In the presence of Ca++, this splits prothrombin into thrombin in the Common Pathway
Common Pathway Fibrinogen large plasma protein formed in liver Acted on by thrombin (removes 4 peptides) to form fibrin which polymerizes into fibrin fibers
Common Pathway Fibrin-stabilizing factor from platelets trapped in clot is activated which causes formation of covalent bonds between fibrin monomer molecules
INTRINSIC PATHWAY (12-11-9-8-10) XII (Hageman Factor) XIIa XI XIa + Ca ++ IXa + VIII + Ca++ + phospholipid, F3,Plt Xa Prothrombin (Factor II) Thrombin Fibrinogen Fibrin Clot XIII a EXTRINSIC PATHWAY Tissue thromboplastin + VII + Ca++ V
All coagulation factors synthesized in the liver except for thromboplastin, Ca++
Factors 2,7,9,10 – vitamin K dependent
The coagulation system Regulated system In addition to Clot formation : Balanced the propagation of clot Feedback inhibition of cascade Mechanism of fibrinolysis
Feedback inhibition of cascade Tissue factor pathway inhibitor (TFPI ) Antithrombin III Protien C system