MLAB 1227 Coagulation Keri BrophyMartinez

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MLAB 1227- CoagulationKeri Brophy-Martinez

• Unit 1 Primary Hemostasis

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Hemostasis

• Heme blood
• stasis to halt
• Process of retaining blood within the vascular
  system
• Repairs injury to blood vessels
• Stops or prevents blood loss

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Balance of Hemostasis
Fibrinogen
Procoagulant Factors
Regulatory Factors
Fibrin

• Balance of bleeding (hemorrhaging) and clotting
  (thrombosis)
• Imbalance in one direction can lead to
• bleeding hypocoagulable state OR
• thrombosis hypercoagulable state

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Hemostasis

• Components
• Vascular System
• Controls rate of blood flow
• Platelet System
• Interaction of vasculature and platelets form a
  temporary plug
• Coagulation System
• (i.e) fibrin forming
• Fibrinolytic System
• Fibrin lysing
• Coagulation Inhibition System
• Natural inhibitors
• Control fibrin formation and fibrin lysis

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• Failure or deficiencies in any of these five
  systems can leads to varying degrees of
  uncontrolled hemorrhaging or clotting

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Hemostasis

• The hemostatic components remain inert in the
  presence of intact vascular tissue or endothelium
• Following injury, each component must function
  optimally.

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Hemostasis OverviewConsists of three stages

• Primary Hemostasis
• Process of blood clotting in response to injury
  where blood vessels (vasculature) and platelets
  are the main players.
• Primary Hemostatic plug is formed
• Platelet plug temporarily arrests bleeding.
  Insoluble fibrin strands deposit on the initial
  plug to reinforce and stabilize. The fibrin
  originates from soluble plasma proteins.
• Secondary Hemostasis
• Actions of the protein coagulation factors form
  fibrin in response to injury
• At this time, blood has changed into a solid
  state
• Fibrinolysis
• Clot is removed following healing of wound

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• http//health.howstuffworks.com/adam-200077.htm

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Vascular System

• Blood Vessels
• Arteries
• Carry blood from the heart to capillaries
• Thickest walls of the vasculature
• Veins
• Return blood from capillaries to the heart
• Thinnest walls of vasculature
• Capillaries
• No vessel wall
• Do not contribute to hemostasis

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Vascular System Blood Vessels

• Construction
• Endothelium
• Single layer of endothelial cells, lining vessels
• Coated by glycocalyx
• Protects basement membrane
• Produces Von Willebrand's factor (vWF), a part of
  Factor VIII
• Secretes prostaglandins, plasminogen activators
• Negatively charged, repels circulating proteins
  and platelets
• Subendothelium
• Smooth muscle and connective tissue with collagen
  fibers

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Vascular System Blood Vessels

• Basement membrane
• Collagen material stimulates platelets
• Connective tissue
• Elastic fibers- provide support around vessels

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Vascular System Blood Vessels

• Function
• Endothelium
• Controls vessel permeability
• Controls blood flow rate
• Produces and releases substances that inhibit OR
  stimulate platelets, coagulation and fibrinolysis
• Subendothelium
• Collagen within is whats exposed upon injury

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Vascular Endothelium ProductsStimulators

• Produces vonWillebrand factor (vWF)
• Helps in platelet adhesion to collagen
• Carries factor VIII
• Tissue factor (TF) activates secondary hemostasis
  via extrinsic pathway
• Tissue plasminogen activator (tPA) is released
  activating fibrinolysis

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Vascular Endothelium ProductsInhibitors

• Release of tPA activates release of plasminogen
  activator inhibitor (PAI-1) to inhibit
  fibrinolysis
• Thromomodulin forms a complex with thrombin
• Platelet aggregation via prostacyclin production

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Vascular System Function Following Injury

• Initiate hemostasis
• Vasoconstriction of the arterioles
• Minimizes blood flow to injured area
• Prevents blood loss
• Immediate
• Short-lived

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Vasoconstriction

• Mechanism
• Neurogenic factors
• Regulatory substances
• Prolong vasoconstriction
• Serotonin ( made by platelet activation
  endothelium)
• Thromboxane A2 ( made by platelet activation
  endothelium)
• Endothelin-1 (made by damaged endothelial cells)

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Vasoconstriction

• Vasodilation Counteracts Vasoconstriction
• Endothelial cells
• Prostaglandin (PGI2)/ Prostacyclin
• Vasodilates to increase blood flow to bring fresh
  supplies of clotting substances
• Inhibits platelet aggregation
• Contraction of venules
• Causes gaps between them which pushes fluids
  causing edema or swelling

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Thought question

• Think about the last time you cut your finger
  with a piece of paper. Did your finger bleed
  immediately?
• If not, what might have prevented the bleeding?

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Answer..

• No, the finger probably did not bleed
  immediately, due to vasoconstriction of the blood
  vessels

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Discussion

• What actions of the endothelial cells prevent
  clotting from occurring within the blood vessels?

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Answers..

• Since the endothelial lining has a negative
  charge, it normally repels coagulation proteins
  and platelets in the circulation. It synthesizes
  products that help to inhibit fibrin formation.

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All About Platelets

• Second major component of the hemostatic system

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Platelets

• What is a platelet?
• Small 2-3 µm
• Anuclear
• Reddish-purple granules
• Fragments of megakaryocyte cytoplasm

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Platelets

• Life span
• 9-10 days
• Normal Range
• 150-440 x 109 /L

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Platelet Side noteSeen in conditions with
increased need and/or destruction

• Giant platelets

• Micromegakaryocytes Dwarf Megs

• May Hegglin anomaly, Bernard-Soulier syndrome,
  pregnancy, malignancy

• Seen in malignant disorders such as CML and MDS

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Anatomy of a Platelet

• Peripheral zone Responsible for platelet
  adhesion and aggregation
• Glycocalyx
• Contains glycoprotein receptors
• GPIb binds von Willebrands factor needed for
  platelet adhesion to collagen
• GPIIb/IIIa bind fibrinogen needed for
  aggregation
• Bind ADP and thrombin, promoting aggregation
• Factors I, V, VIII on surface, involved in 2o
  hemostasis
• Plasma membrane
• Exposed on platelet activation
• Layer called PF3 (platelet factor) surface for
  interaction of plasma coagulation factors
• Initiation of formation of thromboxane A2. This
  stimulates aggregation and vasoconstriction

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Anatomy of a Platelet

• Structural or Sol-Gel zone Responsible for
  platelet retraction/contraction functions and
  platelet shape
• Microtubules
• Cytoskeleton
• Binding protein
• Organelle zone Responsible for storage and
  platelet release functions
• Granules
• Dense bodies, alpha granules, lysosomal granules
  and microperoxisomes
• Mitochondria
• Glycogen

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Anatomy of a Platelet

• http//www.platelet-research.org/1/function_morpho
  .htm

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Production of Platelets

• Made in Bone marrow
• Need dictates the amount of platelets produced.
• Stimulus for production is the platelet mass in
  circulating blood 80 and megakaryocyte mass
  in bone marrow
• Originate from CFU-GEMM to form CFU-Meg
• Cytokines and growth factors such as IL-3 and
  GM-CSF influences progenitor stages

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Platelet Development

• Megakaryoblast
• 10-15 µm
• Increased nuclear cytoplasmic ratio
• Promegakaryocyte
• 80 µm
• Dense alpha and lysosomal granules
• Basophilic megakaryocyte
• Megakaryocyte

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Production of Platelets

• Precursor Cell Megakaryocyte
• Produces about 2000 platelets
• Platelets are released via sinuses of bone marrow

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Production of Platelets

• Thrombopoietin (TPO)
• Regulates platelet development
• Influences all stages of megakaryocyte production
• Produced in the liver, kidney and spleen

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Production of Platelets

• How does TPO work?
• Maintains a constant number of platelets in
  peripheral blood by binding Mp1 (platelet
  receptor). Bound TPO can not stimulate
  proliferation of bone marrow progenitor cells
• The higher the platelet count, the more TPO is
  bound and stimulation of bone marrow is
  decreased.

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Thought question

• If a patient had a low platelet count what will
  happen?

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Answer

• TPO increases the number of megakaryocytes in the
  bone marrow, increases size and DNA count of
  megakaryocytes and increases maturation rate

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Function of Platelets

• Surveillance of blood vessel continuity
• Checks endothelial lining for gaps and breaks
• Fill-in small gaps caused by separation of
  endothelial cells
• Formation of primary hemostatic plug
• Surface for coagulation factors to make secondary
  hemostatic plug
• Aid in healing injured tissue

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Formation of Primary Hemostatic Plug

• Once the platelets normal environment is
  changed, they become activated or adhesive
• Three stages of plug formation

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Stage 1 Platelet Adhesion

• Platelets attach to non-platelet surfaces, such
  as collagen fibers in the subendothelium
• Platelets move from the blood vessels and into
  the tissues.
• Exposure to surfaces in the tissues causes them
  to bind to collagen with the presence of von
  Willebrand factor ( vWF) and Glycoprotein IbIX,
  making a bridge formation, which triggers a shape
  change
• Reversible
• No ADP released

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Stage 1 Platelet Activation

• Platelets undergo a shape change from disc to
  spiny sphere with projections
• Activation required for 1O hemostatic plug
  formation
• Activation continues until Ca threshold met
• Outcome
• Activation of GPIIb/IIIa receptors for
  fibrinogen
• Secretion of granules within platelets into
  tissues

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Platelet Shape Change

• http//www.platelet-research.org/1/function_morpho
  .htm

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Stage 2 Platelet aggregation

• Chemical changes cause platelets to aggregate and
  stick to one another
• Newly arriving platelets become activated by
  agonists
• Exposure of GPIIb/IIIa sites bind fibrinogen
• Fibrinogen activated platelets serves as a
  bridge between two platelets
• Calcium must be present

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• Activated platelet membrane generates TXA2
• TXA2 stimulates release

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Adhesion Aggregation

• http//www.platelet-research.org

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Stage 3 Platelet Secretion Release

• Requires ATP
• Platelets release contents of their granules,
  causing vasoconstriction
• Granules trigger a secondary aggregation which is
  irreversible
• Granules consist of
• Alpha granules Factor V, Factor VIIIvWF,
  Fibrinogen, a2-antiplasmin, platelet factor 4
• Dense bodies ATP, ADP, serotonin, Ca

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Granules cont

• Factor V receptor on platelet surface for factor
  Xa prothrombin
• PF4 heparin neutralizing factor
• ADP agonist, continues to recruit and stimulate
  platelets by increasing cytoplasmic calcium

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Side note

• Heparin is used on patients who clot excessively.
  Endothelial cells make heparin-like molecules
  and expose them on their surface. PF4 binds
  these substances. Heparin can complex with bound
  PF4 and heparin will be neutralized.

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Final Stage Stabilization of Clot

• AKA primary hemostatic plug formation
• Thrombus formation
• Platelets release Factor V
• Expose factor III, accelerating coagulation
  cascade
• Promote activation of clotting factors

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Platelet System Additional Functions

• Provides the reaction surface for some
  coagulation system reactions, as well as platelet
  factor 3 (PF3) which is platelet phospholipid
• Supports and maintains endothelial lining
• Defective hemostasis can occur due to
• decreased number of platelets (quantitative)
• abnormally functioning platelets (qualitative)

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Blood clot
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Coagulation System

• Composed of 14 coagulation factors (serine
  proteases) which are interdependent (Factors I
  through XIII there is no Factor VI and PK and
  HMWK)
• Inactive form of each is an enzyme precursor
  which is usually designated by a Roman numeral
  but also given a name Ex. Factor I fibrinogen.
  Numbers correspond to order of discovery NOT
  order in cascade.
• Active forms are usually designated by the letter
  a after the Roman numeral and may also have a
  different name Ex. Ia Fibrin
• Cofactors are needed for many reactions in the
  cascade Ex. Calcium, platelet factor 3 (PF3)
• Each molecule must be present in sufficient
  quantity as well as functioning normally
• Final product is fibrin mesh or clot which
  completely stops bleeding
• Secondary hemostasis
• Slow contraction and lysis of the clot occurs

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Fibrinolytic System

• Plasminogen is converted to plasmin
• Plasmin enzymatically attacks the fibrin molecule
  producing fibrin degradation products (FDPs,
  sometimes called FSPs) that are cleared from the
  circulation by macrophages
• Fibrin is a product formed during hemostasis,
  tissue repair or inflammation
• Fibrin plays a temporary role
• Once injury heals, the fibrin clot is lysed

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Coagulation Inhibition System

• Provides balance and control of clotting
  mechanisms
• Natural inhibitors and anticoagulants circulate
  in the plasma to
• Prevent clotting when its not needed
• Limit or localize the clotting that is needed
• Examples Protein C and S, antithrombin III