Coagulation Lecture 1 - Primary and Secondary Haemostasis Flashcards
What is Haemostasis?
The process/es that prevent excess blood loss after tissue injury
Maintains an equilibrium between bleeding and clotting (thrombosis)
Thrombosis - the formation of a blood clot
Roles of Haemostatic System
Keep blood fluid
Confine circulating blood to vasculature
Prevent excessive blood loss if damage occurs
Confine blood coagulation to the site of injury
Lyse and/or remove fibrin clots
Elements involved in Haemostasis
Vascular - endothelial cells - smooth muscle cells and fibroblasts Circulatory - platelets - leukocytes - plasma proteins - lipoproteins
Primary and Secondary Haemostasis - Introduction
Two stages of haemostasis:
- primary and secondary
Primary
- involves platelets
- platelets become activated
- formation of the primary haemostatic plug
Secondary
- involves the plasma coagulation factors
- complex series of reactions that results in the formation of fibrin
- stabilises primary haemostatic plug
- prevents ongoing loss of blood from the vasculature
- clot must (eventually) be removed/dissolved to allow vessel to resume/normal function
Overview of Primary Haemostasis
Endothelial damage => platelet activation
- adhesion
- shape change
- aggregation
- secretion
The platelet plasma membrane is the focus of interactions between extra and intracellular environments
Platelet Agonists
Substances that activates platelets
- ADP (nucleotide)
- thromboxane A2, platelet activating factor (lipids)
- collagen (protein)
- thrombin (enzyme)
Primary Haemostasis - Platelet Adhesion
Vascular injury exposes subendothelial collagen to blood
Platelets adhere to this collagen
Occurs quickly
Platelets bind to collagen-bound vWF in subendothelium via Gp 1b-V-IX
Collagen binding induces platelet activation which causes changes in platelet biochemistry and shape
Exposure of GP IIb-IIIa
Flip-flopping of membrane PL
von Willebrand Factor (vWF)
Protein Variably sized units 'multimers' Produced by ECs and megakaryocytes Storage - platelet alpha granules - Weibel-Palade bodies in ECs Released after - platelet activation - damage to endothelial cells - therapeutic treatments Also circulates as complex with factor VIII
Platelet Aggregation
Fibrinogen binds to GP IIb-IIIa on adjacent platelets
Measurement of platelet aggregation is the gold standard test to determine platelet activation
Laboratory Assays for Primary Haemostasis
Platelet concentration
Bleeding time
Platelet function analyser (PFA-100)
vWF
Laboratory Assays for Primary Haemostasis - Platelet Function Analysers
Mimics platelet adhesion and aggregation at a site of vessel injury - high shear rates - collagen exposure Membrane coating - collagen - agonist Time to membrane occlusion recorder Sensitive to platelet adhesion, aggregation deficiencies
Basic Concepts of Blood Coagulation
Its a sequential process of chemical reactions involving
- plasma proteins
- phospholipids
- calcium ions
Inactive forms of enzymatic factors are called ‘zymogens’
Active forms of enzymatic factors are called ‘serine proteases’
Clotting Factors I, II, III and IV
I - fibrinogen - function: thrombin substrate II - prothrombin - function: serine protease III - tissue factor - function: co-factor IV - calcium - function: mineral
Common Characteristics of Coagulation Factors - Proteins
Proteins that are clotting factors have four characteristics in common:
- a deficiency in the factor generally produces a bleeding tendency disorder
- the physical and chemical characteristics of the factor are known
- the synthesis of the factor is independent of other proteins
- the factor can be assayed in the lab
Common Characteristics of Coagulation Factors - Fibrinogen Group
Factors I, V, VIII and XIII
Factors V and VIII decrease during blood storage in vitro
Increase during pregnancy, inflammation, OC use
Common Characteristics of Coagulation Factors - Prothrombin Group
Factors II, VII, IX, and X
Relatively stable during blood storage in vitro
Vitamin K dependent factors
Vitamin K is available via dietary sources and intestinal bacteria
Inhibited by warfarin
Common Characteristics of Coagulation Factors - Contact Group
Factors XI, XII, prekallikrein, high molecular-weight kininogen (HMWK)
They are moderately stable during storage in vitro
Interaction of Coagulation Factors
Models have been proposed for the interactions of factors within and between pathways
Waterfall or Cascade
- characterised by sequential activation of coagulation factors in a series of pathways
Modern
- characterised by formation of complexes of coagulation factors
Coagulation Pathways
Extrinsic
- initiated by the entry of tissue thromboplastin into the circulating blood
Intrinsic
- involves the contact factors prekallikrein, high molecular-weight kininogen, factor XII and factor XI
Both the extrinsic and intrinsic pathways effectively activate the common pathway
Ultimately results in the formation of fibrin
Fibrin Formation
Clotting is the visible result of the conversion of plasma fibrinogen into a stable fibrin clot
Thrombin plays a major role in converting fibrinogen to fibrin
Fibrin formation occurs in three phases:
- proteolysis
- polymerisation
- stabilisation
Regulatory Mechanisms of Coagulation
Tissue factor pathway inhibitor (TFPI) - circulates with lipoprotein - binds to FXa - complex binds to FVIIa-TF Protein C pathway - regulates FVa and FVIIIa - protein C, protein S, thrombomodulin SERPINS (serine proteases inhibitors) - antithrombin: inhibits thrombin - heparin co-factor II: inhibits thrombin
Fibrinolysis
Digestion of a fibrin clot
Plasminogen is activated to plasmin
Plasmin => digestion of fibrinogen and fibrin
- formation of fibrinogen/fibrin degradation products (FDPs)
Inhibited by plasminogen activator inhibitors (PAIs)
Laboratory Assays For Secondary Haemostasis
Prothrombin time (PT) Activated partial thromboplastin time (aPTT) Thrombin time Fibrinogen assay Factor assays
Blood Samples for Coagulation Assays
Samples need to be anticoagulated to prevent clotting
Reverse anticoagulant effect to allow clotting under controlled conditions
Avoid:
- clotted specimens
- under-filled tubes
- EDTA: irreversible effect
PT and aPTT
Commonly used as ‘screening tests’
I.e non-specific tests that are used to provide evidence of the presence of a disorder of the coagulation system
May also be used to monitor:
- anticoagulant therapy
- progress of a disease and it’s treatment
Cascade Theory for the Intrinsic Pathway
Starts with a negatively charged surface Then FXII => FXIIa => FXI => FXIa => FIX => FIXa:FVII => FX of the common pathway
Cascade Theory for the Extrinsic Pathway
Starts with vessel injury
Then tissue factor (FIII) release =>
TF:FVIIa =>
FX of the common pathway
Cascade Theory for the Common Pathway
Starts with FX Then FXa:FV => Prothrombin => Thrombin => Fibrinogen => Fibrin
Laboratory Assessment of Fibrinolysis
Fibrin/fibrinogen degradation products - special tube with antifibrinolytic agent and thrombin - latex agglutination method - contain antibodies to fragments D and E - normal values < 10 ug/mL D-dimers - latex agglutination method - detects cross-linked fibrin D-dimers - normal values: < 200 mg/L
