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Principles of Hemostasis




                            Marlies Ledford-Kraemer,
                             MBA, BS, MT(ASCP)SH

Revised: mmvi                                          1
Topics for Discussion

                            I nt
                                 ro d
                          Vi r        uct
                               cho        ion
                        P ri        w’s
                             ma          Tri
                                  ry
                       Sec
                           on        He ad
                     Ph         dar mos
                        ysi          yH        tas
                   Lab olog             em         is
                        o ra       ic C      ost
                  Fib        tor        oag asis
                      rin         yC
                          ol y
                               sis    oag ulatio
                                           ul a       n
                                                tio
                                                    n




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Virchow’s Triad




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Virchow’s Triad



                           Changes in blood coagulability
                   Platelets, Coagulation Factors & Inhibitors, Fibrinolysis




          Changes in vessel wall                  Changes in blood flow
          Endothelial changes due to                  Rheology in vessels
          inflammation or atherogenesis

www.CLOT-ED.com                                                                4
Vessel Wall
                                                   Tunica intima
                                                    Endothelium - inner most layer of cells
                                                    that separate the remainder of the
                                                    vessel from the lumen
                                                    Basement membrane – thin layer of
                                                    spongy connective tissue that secretes
                                                    elastic collagen

                                                   Tunica adventitia
                                                    Surrounds the tunica media
                                                    Connective tissues – produce elastic
http://www.alegent.com/122711.cfm                   and non-elastic collagen fibers
 Tunica intermedia                                  Prevents ballooning of vessel with high
                                                    systolic blood pressure
    Surrounds the tunica intima
                                                    Aneurysm – weaknesses in the tunica
    Smooth muscle – layer of smooth muscle cells    adventitia
    that are under involuntary control and can
    dilate or constrict
    Connective tissue – produces collagen fibers
    whose elasticity is reduced by hypertension
www.CLOT-ED.com                                                                               5
Rheology
Region 1                                                                Region 2
In this range of shear rates:                                           As shear rate increases:
   Cells     are    in     large                                          Cells are disaggregating
   aggregates         (rouleaux                                           Applied     forces    (yield
   formation)                                                             stress) are forcing cells to
   As shear rate increases,                                               orient and deform
   size of aggregates diminish                                            Blood viscosity decreases
   Viscoelasticity is strongly
   influenced by aggregation
   tendency of RBCs
                                                                        Region 3
                                                                        With increasing stress:
                                                                          Cells deform
                                                                          With normal deformability,
                                                                          cells will form layers that
Adapted from :                                                            slide on layers of plasma
http://www.vilastic.com                                                   and align in the direction
/tech10.html                                                              of flow




      Science of the deformation and flow of matter
      Blood is a fluidized suspension of elastic cells that demonstrates both a viscous
      and elastic effect
       – Elastic effect makes blood a non-Newtonian fluid (plasma is a Newtonian fluid)
       – Blood has a yield stress that depends on hematocrit and fibrinogen concentration
www.CLOT-ED.com                                                                                          6
Shear & Vessel Wall
                  Endothelial Cells




 At Wall Surface: Greatest                Platelet                 Flowing Blood
 Shear Rate & Least Velocity

                                                                          Shear
 At Vessel Center: Greatest                             Platelet
                                                                          Flow
 Velocity & Least Shear Rate

 Shear Stress
 Pressure- induced Force
 Between 2 Laminae
                                      Laminae (Arrow Length = Velocity)
             Endothelial Cells




www.CLOT-ED.com                                                                    7
Hemostasis
    Process by which blood is maintained in a fluid state and
    confined to the circulatory system
    Goal is to stop bleeding and to do so only at the site of
    injury
    Components
     – Platelets
           • Involved in Primary Hemostasis
     – Coagulation system
           • Involved in Secondary Hemostasis
     – Fibrinolytic system                      Platelet / fibrin mesh

     – Inflammatory processes
     – Wound healing processes
www.CLOT-ED.com                                                          8
Primary Hemostasis




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Primary Hemostasis
    First physiological response to vascular injury, which is
    mediated by platelets, in order to arrest bleeding
    Mechanism
     – Activation of platelets via stimulators such as thrombin
     – Adhesion of platelets to subendothelium via interaction
       between GPIb and von Willebrand Factor (VWF)
     – Release of platelet granule products in order to recruit
       more platelets to the injured site
     – Aggregation of platelets via interaction between
       GPIIb/IIIa (αIIbβ3) and fibrinogen to form the initial plug
    Triggers secondary hemostasis (coagulation proteins)
    Affected by medications, platelet function status, and
    vessel wall status
www.CLOT-ED.com                                                      10
Platelets
    Disk-shaped cell
    fragments produced in                       GPIIb/IIIa
    the bone marrow by
    megakaryocytes             Alpha                          Dense
    Life span ~ 10 days       granules                       granules

    Glycoprotein Ib (GPIb)
    is involved in adhesion
    Glycoprotein IIb/IIIa
    (GPIIb/IIIa) is the
    primary receptor for
    fibrinogen (aggregation
    phase)
    Provide procoagulant
    surface on which
    coagulation proteins          Coagulation
    can interact                   proteins                   GPIb

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Platelet Adhesion

              Platelet




                           GPIb

  Endothelium              VWF                                                 Injury site
                                                                           collagen exposed

                                                        COL
                                        COL
                                              COL     CO
                                                        L           COL

                                      VWF                            VWF
                                                    VWF
                                                              VWF
Subendothelial extracellular matrix
www.CLOT-ED.com                                                                               12
Platelet Aggregation




      Activated
       Platelet                  GPIb                                             Fibrinogen
   (Granule contents released)


                                 VWF                                         GPIIb/IIIa

                                                          COL
                                          COL
                                                COL     CO
                                                          L           COL

                                        VWF                            VWF
                                                      VWF
                                                                VWF
Subendothelial extracellular matrix
www.CLOT-ED.com                                                                            13
Tests for Primary Hemostasis
    Bleeding Time
     – Assesses all components of Virchow’s triad
     – in vivo test – performed directly on patient
     – Has fallen into disrepute and replaced by instruments that perform “in
       vitro” bleeding times
    Platelet Aggregation studies
     – Measure ability of platelets to aggregate, in vitro, when subjected to
       various stimulators (agonists)
     – Predominantly assesses function of platelet glycoprotein IIb/IIIa
       receptor
    Von Willebrand Factor (VWF) assays
     – Measure amount and function of VWF, a protein that works with
       platelets so that they adhere to site of injury
     – Assesses function of VWF ligand in its interaction with platelet
       glycoprotein Ib receptor
www.CLOT-ED.com                                                                 14
Platelet Appearance / Function
       Electron Micrographs            Aggregation Tracings




         Resting platelets




                                 Normal platelet   Aspirin-like defect
                                    function

         Activated platelets

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Secondary Hemostasis




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Secondary Hemostasis
    Process of blood coagulation
    Mechanism
     – Coagulation proteins work in
       concert to generate thrombin
     – Thrombin converts fibrinogen to
       fibrin
     – Fibrin consolidates the platelet plug
       made in primary hemostasis such         Credit: Weisel JW. University of Pennsylvannia

       that a thrombus (secondary
       hemostatic plug) is formed
    Prevents further blood loss from
    the injury site

www.CLOT-ED.com                                                                                 17
Coagulation Factors
    Factor        XII (FXII)             activated   FXII (FXIIa)
    Factor        XI (FXI)               activated   FXI (FXIa)
    Factor        X (FX)                 activated   FX (FXa)
    Factor        IX (FIX)               activated   FIX (FIXa)
    Factor        VIII (FVIII)           activated   FVIII (FVIIIa)
    Factor        VII (FVII)             activated   FVII (FVIIa)
    Factor        V (FV)                 activated   FV (FVa)

   Factor II (prothrombin) is converted to thrombin (FIIa)

                  Factor I (fibrinogen) is converted to fibrin

www.CLOT-ED.com                                                       18
“in vivo” v “in vitro” Coagulation
    Physiologic (“in vivo”) coagulation is dependent upon
    the tissue factor pathway
     – Goal is to form a thrombus
    Laboratory (“in vitro”) coagulation is dependent upon
    the contact system
     – Classical “waterfall” or cascade concept
           • Step-by-step biochemical reactions in which an inactive proenzyme
             is converted to a reactive enzyme which, in turn, converts another
             proenzyme to its active form
     – Amplification process (very minute amounts of Factor XII
       yield large amounts of thrombin)
     – Goal is to form a clot

www.CLOT-ED.com                                                              19
Waterfall Scheme of Coagulation

    FXII                   FXIIa


             FXI                      FXIa
                                  FVIIIa
                        FIX      Ca++ PL        FIXa             FVIIa            FVII
                                                                  TF
                                             Ca++ PL

                                   FX                     FXa            FX

                                                         FVa
                                               FII     Ca++ PL   Thrombin
                                                                                     TF = Tissue Factor
                                                                                     Ca+ += Calcium ion

After Macfarlane RG. Nature 1964;202:498-9
                                                     Fibrinogen          Fibrin      PL = Phospholipid

www.CLOT-ED.com                                                                                          20
Physiologic Coagulation


     Thrombus Formation

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Initiation Phase
                  Monroe DM, et al. ATVB 2006;26:41-8




                                                           “Extrinsic Pathway”




www.CLOT-ED.com                                                                  22
Amplification Phase
                  Monroe DM, et al.
                  ATVB 2006;26:41-8




www.CLOT-ED.com                                         23
Propagation Phase
                  Monroe DM, et al.
                  ATVB 2006;26:41-8




 “Intrinsic Pathway”
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Recapping Secondary Hemostasis
Tissue Factor complexed with FVIIa initiates coagulation at site
                          of injury

Small amounts of thrombin are generated that activate platelets

      Coagulation factors form complexes on platelet surfaces

        Very large amounts of thrombin are formed to convert
                        fibrinogen to fibrin

       Fibrin reinforces platelet plug (primary hemostasis) and
                        hemostasis is achieved

www.CLOT-ED.com                                                   25
Laboratory Coagulation


                  Clot Formation

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First the Specimen
    Blood is collected into     Plasma is used for testing
    a tube that contains         – PLASMA contains
    sodium citrate, an             FIBRINOGEN
    anticoagulant
                                 – Serum does not contain
    Blood fluidity is              Fibrinogen
    maintained because
    sodium citrate binds        Plasma is “platelet poor”
    calcium ions, which are     since platelets remain in
    critical to the             buffy coat
    coagulation process
    Tube is centrifuged in
    order to separate
    plasma from buffy coat                       Plasma

    (white blood cells &                        Buffy Coat
    platelets) and red
    blood cells                                 Red Cells



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Coagulation in the Laboratory

         Intrinsic Pathway                                 Extrinsic Pathway

                       XII
                             XI                      VII        Tissue Factor
                                  IX
       APTT                         VIII
                                                                   PT
     Intrinsic                             X                    Extrinsic
         +
                               Common      V    Pathway             +
     Common
                                           II                   Common



                  Fibrinogen                              Fibrin Clot

www.CLOT-ED.com                                                                 28
Routine Coagulation Assays
    Prothrombin Time (PT)
    Activated Partial Thromboplastin Time (APTT)
    Quantitative Fibrinogen (FIB)
    Thrombin Time (TT)
    Assays for specific coagulation factors
     – Factors assessed by a PT-based test system: FVII, FV, FX,
       and FII
     – Factors assessed by an APTT-based test system: FXII,
       FXI, FIX, and FVIII




www.CLOT-ED.com                                                29
Prothrombin Time (PT)
                       Time for clot formation
                           ~ 12 seconds

            Incubate at 37 oC for ~3 minutes

            0.1 ml Thromboplastin + Ca++

            0.1 ml Plasma



PT Reagent Composition
   Thromboplastin
                                                                                    Credit: PNAS; Collet JP and
     − Tissue Factor (recombinant/human or animal brain)                 Weisel JW. Un Pennsylvannia

     − Lipid (source of phospholipid since platelets were removed from plasma)
     − CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate
   Historically referred to as “complete” since both phospholipid and apoprotein
   make up the reagent

www.CLOT-ED.com                                                                                                   30
Causes for Prolonged PT
    Deficiencies or abnormalities in:
     – FVII (Extrinsic Pathway)
     – FV, FX, FII (prothrombin), and FI (fibrinogen)
           • Both PT and APTT will be prolonged
    Vitamin K antagonists
     – PT sensitive to reductions in three of four vitamin K-dependent
       procoagulant proteins: FVII, FX, and FII
           • FIX measured by APTT
     – Pharmacologic anticoagulants that modify vitamin K-dependent
       proteins such that they do not bind calcium thereby reducing blood
       coagulability
    Liver disease
     – Site for synthesis of vitamin K-dependent proteins
     – Site for clearance of coumarins (warfarin) and coagulation proteins

www.CLOT-ED.com                                                              31
Biochemistry: Vit K-Dep Proteins
    Addition of an extra carboxyl group to glutamate (Glu)
    residues at their amine termini gives rise to a novel
    amino acid called gamma-carboxyglutamate (Gla)
     – Presence of Gla enables proteins to undergo a calcium-
       dependent conformational change that allows for their
       binding to
       phospholipid
       surfaces and
       generation of
       membrane
       bound
       macromolecular
       complexes

www.CLOT-ED.com                                                 32
Vitamin K Antagonists (AVK)
    DICUMAROL: 3,3’-methylenebis (4-hydroxycoumarin)
     – Isolated by Karl Link (University of Wisconsin-1939) as the anti-
       vitamin K agent responsible for hemorrhagic disorder in cattle
     – Coumarin derivatives
           • Bishydroxycoumarin (Dicumarol)
           • Warfarin (Coumadin®)
                  – Analog #42 of many coumarins synthesized by Dr Link and named by him as
                    WARFarin for the Wisconsin Alumni Research Foundation and coumARIN
    Require monitoring because:
     – Vitamin K-dependent proteins have different half-lives
     – Differences in drug absorption and clearance
     – Levels affected by concomitant medications, comorbid conditions,
       changes in diet, patient compliance
     – PT (thromboplastin) reagents vary in their reaction to clotting defects
       produced by AVK (warfarin)
www.CLOT-ED.com                                                                           33
Warfarin Sodium
    Oral anticoagulant of choice in North America
    Pharmacologic properties more favorable than Dicumarol
     – Warfarin is 5-10x more potent
    Racemic mixture (~1:1) of R & S isomers
     – S isomer is 5x more potent than R isomer
           • Hepatic microsomal enzyme cytochrome P450 2C9 is responsible for the
             oxidative metabolism of S isomer
    Bioavailability
     –   Absorbed from gastrointestinal tract
     –   Maximal blood concentrations reached in 90 minutes
     –   Half-life (T½) is 36-42 hours
     –   At therapeutic concentrations, 99% of warfarin is bound to albumin
         and 1% is free and can bind to its receptor on hepatic cells
    Anticoagulant effects are reversed by administration of vitamin K
    or biologic products that contain vitamin K-dependent proteins
www.CLOT-ED.com                                                                     34
Warfarin Mode of Action
                           CO2          H2O
                            O2
   Des-Carboxy                                    Prothrombin
 Prothrombin (“Glu”)       γ-Carboxylase             (“Gla”)



    Vitamin K                                          Vitamin K
  hydroquinone                Vitamin K               2, 3 epoxide
(active form K1H2)                                (inactive form K1O)
                           epoxide reductase
                                (VKOR)

  Regeneration
  of active form    NAD+                       NADH
  is sensitive to
     warfarin                WARFARIN
www.CLOT-ED.com                                                    35
International Normalized Ratio
                                             ISI
                            Patient PT
                  INR =
                          Mean Normal PT

    “Normalizes” the PT by mathematically considering
    differences in PT reagents (thromboplastins)
    Only to be used to monitor long term anticoagulant
    effects for patients stabilized on oral anticoagulant
    therapy
     – Prevent recurrence of thrombosis caused by under
       anticoagulation
     – Prevent hemorrhagic complications caused by over
       anticoagulation
www.CLOT-ED.com                                             36
Activated Partial Thromboplastin Time

                                                                      Time for clot formation
                                                                          ~ 30 seconds

                                                            0.1 ml CaCl2
                                                            Incubate at 37 oC for ~5 minutes
                                                            0.1 ml Activator
                                                            0.1 ml Plasma



APTT Reagent Composition
    Activator to convert FXII to FXIIa
    Phospholipid (replaces “in vivo” platelet surface on which coagulation reactions
    occur)
    CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate
    Referred to as “partial thromboplastin” since no Tissue Factor is used
      − Two-stage assay (activation and re-calcification)
www.CLOT-ED.com                                                                                 37
Intended Use for APTT
    Screening test
     – Intrinsic (and severe common pathway) factor deficiencies
    Laboratory monitoring
     – Unfractionated heparin
     – Other antithrombotic agents (Direct Thrombin Inhibitors)
    Laboratory detection of the Lupus Anticoagulant




www.CLOT-ED.com                                                   38
Causes for Prolonged APTT
    Most common causes
     – Heparin (contamination from lines or therapeutic)
     – Lupus Anticoagulant
    Other causes
     – Deficiencies of coagulation factors
           • FVIII (Hemophilia A or Von Willebrand Disease), FIX, FXI, FXII
     – Liver disease (site of production for most coagulation
       factors)
     – Consumption of coagulation factors as seen in
       Disseminated Intravascular Coagulation (DIC)



www.CLOT-ED.com                                                               39
Heparin
    Heparin is a heterogeneous group of straight-chain anionic
    mucopolysaccharides (glycosaminoglycans)
     – Molecular weights range from 5 – 40 kiloDaltons
     – Composed of alternating D-glucosamine residues linked 1 → 4
       to either L-iduronic acid or D-glucuronic acid
    Heparin is highly acidic therefore binds to positively charged
    amino acids such as arginine & lysine
     – Pentasaccharide sequence
           • Comprisies ~30% of heparin
           • Binds to antithrombin (AT)
           • Accelerates AT inhibition of
             activated factors XII, XI, IX, X, and II (thrombin)
             thus serving as an anticoagulant
    Pharmaceutical heparins are extracted from pig intestinal
    mucosa (source of mast cells)
www.CLOT-ED.com                                                          40
Heparin and Antithrombin




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APTT Monitoring of Heparin
    Assumes antithrombotic (anti-IIa) effect parallels
    anticoagulant effect
    Limitations
     – Pre-treatment APTT of patient
           • Baseline APTT of patient prolonged due to Lupus Anticoagulant
           • Baseline APTT of patient sample below or at low end of reference
             interval due to high levels of FVIII (apparent “heparin resistance”)
     – APTT reagents vary in sensitivity to heparin
           • Laboratories must determine responsiveness of their APTT reagent
             to unfractionated heparin
           • Determine APTT therapeutic interval (seconds) for reagent used to
             monitor heparin therapy



www.CLOT-ED.com                                                                 42
Direct Thrombin Inhibitors
    Hirudin [Lepirudin (rDNA)–trade name: Refludan®]
     – Approved in USA, Canada, and EU for Heparin Induced
       Thrombocytopenia (HIT) complicated by thrombosis
     – Target APTT is 1.5-2.5 x patient baseline APTT
     – In absence of severe thrombosis, some experts recommend a target
       APTT of 1.5-2.0 x patient baseline APTT and monitor every 4 hours
    Argatroban [non-US trade name: Novastan]
     – Approved for HIT with or without thrombosis and also for
       anticoagulation during percutaneous coronary intervention (PCI) in
       patients with, or at risk for, HIT
     – Target APTT 1.5-3.0 x patient baseline APTT (maximum 100 seconds)
    Hirulog [Bivalirudin-trade name: Angiomax™]
     – Undergoing evaluation for use as an anticoagulant for “on-pump” and
       “off-pump” cardiac surgery in patients with HIT
     – Target APTT is 1.5-2.5 x patient baseline APTT
www.CLOT-ED.com                                                            43
“Recap”ping the PT and APTT
    PT and APTT are screening assays to determine if a
    patient, when challenged, has a potential to bleed
     – If warfarin or heparin are not present in sample, then:
           • Prolonged PT and normal APTT = deficiency of FVII
           • Normal PT and prolonged APTT = deficiencies in any of the intrinsic
             pathway factors (FVIII, FIX, FXI, or FXII)
           • Prolongation of both PT and APTT = deficiencies of factors common
             to both pathways (FX, FV, FII, or fibrinogen)
    PT, via the INR, is used to monitor oral anticoagulant
    therapy (warfarin)
    APTT is used to monitor heparin anticoagulant therapy
    APTT is affected by inhibitors such as Lupus Anticoagulant


www.CLOT-ED.com                                                                    44
Tests for Fibrinogen
      Quantitative Fibrinogen                Thrombin Time (TT)
    Measures the amount of              Assesses the functionality of
    fibrinogen present in plasma        fibrinogen in plasma
    Low levels, termed                  TT clotting time prolonged
    hypofibrinogenemia, can be           –   HEPARIN
    inherited but generally are due      –   Direct thrombin inhibitors
    to acquired causes such as           –   Hypofibrinogenemia
    DIC, liver disease, or               –   Dysfibrinogenemia
    fibrinolytic therapy
                                         –   Elevated fibrin split products
    High levels are seen in
    inflammatory states since                                     Time for clot formation
                                                                      ~ 15 seconds
    fibrinogen is an acute phase
    reactant                                            Incubate at 37 oC for ~2 minutes
                                                        0.2 ml Diluted Thrombin
                                                        0.2 ml Plasma


www.CLOT-ED.com                                                                             45
Quantitative Fibrinogen
                                                         Calibrator plasma is serially diluted
                      Time for clot formation            (1:5, 1:10, 1:20, and 1:40) to establish
                         ~ 5-14 seconds                  a reference curve (see graph below)
                                                         Patient plasma is diluted 10 fold (1:10)
                                                         in buffer
            Incubate at 37 oC for ~3 minutes
            0.1 ml Thrombin
            0.2 ml Diluted Plasma
                                                                        100



     Patient clotting time in seconds is
                                                Clotting Time-Seconds
                                                                        35.0
     read against the reference curve
                                                                        18.3
       − Patient clotting time of 8.0                                    9.5
         seconds equates to 281 mg/dl                                    8.0
                                                                         5.1     Patient
         fibrinogen in example at right
     Fibrinogen concentration is inversely
                                                                          1
     proportional to clotting time                                        1
                                                                            10
                                                                            10      58       115
                                                                                           100       230 281   460   1000



                                                                                  mg/dl Fibrinogen
www.CLOT-ED.com                                                                                                         46
Coagulation: A Balancing Act




                  Generate   Inhibit Thrombin
                  Thrombin      Formation

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Inhibitors
    Naturally occurring inhibitors
     – Protein C (activated) and Protein S
           • Inhibit coagulation cofactors FVIIIa and FVa
     – Antithrombin
           • Inhibits FXIa, FIXa, FXa, FVIIa/TF, and thrombin (IIa)
    Pathologic inhibitors
     – Acquired or autoimmune antibodies to specific
       coagulation factors
    Pharmacologic inhibitors
     – Heparin and Low Molecular Weight Heparin
     – Warfarin
     – Direct Thrombin Inhibitors
www.CLOT-ED.com                                                       48
Fibrinolysis




www.CLOT-ED.com   49
Hemostasis: A Delicate Balance




          Generates                            Generates
          Thrombin                              Plasmin


                        Form a    Dissolve a
                       Thrombus   Thrombus

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Fibrinolytic System



                                             TAFI
                           Thrombin     TM
                                                             Thrombin
                                     TAFIa                      TM
                                                          PC          APC        ‡



                  Fibrinogen FIBRIN
                                                 tPA      Plasminogen
        FXIII
                                  Plasmin                            PAI-1   ‡

       Fibrin(ogen) Degradation
               Products                     Antiplasmin
www.CLOT-ED.com                                                                      51
Breakdown of Fibrin(ogen)

                                FPB
                                FPB       FPA & B
                                          FPA & B
          Fibrinogen                                        Fibrinogen

                                                                       Fibrin
                                Thrombin                Fibrin Monomer Clot
                                                        Fibrin Monomer
         Fragment X
         Fragment X

                                                        Fibrin Polymer
                                                        Fibrin Polymer
         D
         D            Y
                      Y
                                               FXIII          FXIIIa

                               PLASMIN              D
                                                    D   E
                                                        E    D
                                                             D    D
                                                                  D      E
                                                                         E   D
                                                                             D
         D
         D        E
                  E       D
                          D


                                D-dimer    D
                                           D     D
                                                 D      E
                                                        E    D
                                                             D    D
                                                                  D      E
                                                                         E   D
                                                                             D



www.CLOT-ED.com                                                                  52
Fibrinolytic Agents
    All currently available thrombolytic agents are
    plasminogen activators (PA)
     – Convert patient plasminogen to plasmin which then acts
       on fibrin within a thrombus
     – Additionally can breakdown fibrinogen (fibrinogenolysis)
           • Commonly referred to as the lytic state (systemic lysis)
           • Therapeutic doses of PA overwhelm PAI-1 and α2-antiplasmin
    Beneficial effect is reduction of thrombus size
    (thrombolysis)
    Negative effect is that hemostatic plugs are also lysed
    Most commonly used agents are: Streptokinase (SK),
    Alteplase (tPA), Reteplase, and Tenecteplase (TNK-tPA)
www.CLOT-ED.com                                                           53
Summaries




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Time Frame for Hemostasis

           Platelets         Coagulation Factors     Fibrinolytic Proteins

         Primary
          Primary               Secondary
                                 Secondary             Fibrinolysis
                                                       Fibrinolysis
        Hemostasis
        Hemostasis              Hemostasis
                                Hemostasis
 •• Vessel constriction
    Vessel constriction     •• Activation of
                                Activation of       •• Activation of
                                                        Activation of
    occurs immediately
    occurs immediately         coagulation
                                coagulation            fibrinolytic
                                                        fibrinolytic
                               factors occurs in
                                factors occurs in      proteins happens
                                                        proteins happens
 •• Platelet adhesion
    Platelet adhesion          seconds
                                seconds                immediately
                                                        immediately
    occurs in seconds
    occurs in seconds
                            •• Fibrin forms in
                               Fibrin forms in      •• Dissolving the
                                                        Dissolving the
 •• Platelet aggregation
     Platelet aggregation      minutes
                               minutes                 thrombus
                                                        thrombus
    takes minutes
     takes minutes                                     requires hours
                                                        requires hours


www.CLOT-ED.com                                                              55
Bleeding: Balance is Disrupted




    Presence of Inhibitors*
•Pharmacologic (warfarin, heparin)
•Allo or Auto-antibodies to Factors
www.CLOT-ED.com                                                    56
Thrombosis: Balance is Disrupted




                                               Inhibitors*
                                          • Activated Protein C
                                          • Protein S
                                          • Antithrombin
www.CLOT-ED.com                                              57
Conclusion
    Primary hemostasis, a platelet-dependent process, forms
    hemostatic plugs when a vessel is injured
    Secondary hemostasis, a coagulation factor-dependent
    process, begins with Tissue Factor exposure
     – Small amounts of thrombin are generated via FXa
       formation by the TF:FVIIa complex (“Extrinsic Pathway”)
     – Sustained thrombin generation depends on FXa
       formation via FIXa and FVIIIa-mediated complexes on an
       activated platelet surface
     – Amount of thrombin generated dictates bleeding or
       thrombotic risk

              The clinical history is the best “test” for hemostasis
www.CLOT-ED.com                                                        58
References
    Ansell J, et al. The pharmacology and management of the vitamin K antagonists: The Seventh ACCP Conference
    on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:204S-233S.
    Clinical and Laboratory Standards Institute (CLSI). One-stage Prothrombin Time (PT) test and Activated Partial
    Thromboplastin Time (APTT) test; Approved Guideline H47-A, 1996.
    Crowther MA, et al. Practical aspects of anticoagulant therapy (Chapter 89). In: Colman RW, ed. Hemostasis and
    Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001.
    Fairweather RB, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of
    Anticoagulant Therapy: Laboratory monitoring of oral anticoagulant therapy. Arch Pathol Lab Med
    1998;122(9):768-81.
    Greaves M, Preston FE. Approach to the bleeding patient (Chapter 48). In: Colman RW, ed. Hemostasis and
    Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001.
    Hirsh J, Raschke R. Heparin and low-molecular-weight heparin: The Seventh ACCP Conference on Antithrombotic
    and Thrombolytic Therapy. Chest 2004;126:188S-203S.
    Konkle BA. Clinical approach to the bleeding patient (Chapter 77). In: Colman RW, ed. Hemostasis and
    Thrombosis, 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006.
    Link KP. The discovery of Dicumarol and its sequels. Circulation 1959;19(1):97-107.
    Olson JD, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant
    Therapy: Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med 1998;122(9):782-98.
    Physicians’ Desk Reference, 60th ed. Montvale: Thomson PDR, 2006.
    Poller L. Prothrombin Time (Chapter 6) and Activated Partial Thromboplastin Time (Chapter 5). In: Jespersen J,
    ed. Laboratory Techniques in Thrombosis-A Manual, 2nd ed. Dordrecht: Kluwer Academic Publishers, 2000.
    Tran HAM, Ginsberg JS. Anticoagulant therapy for major arterial and venous thromboembolism (Chapter 116). In:
    Colman RW, ed. Hemostasis and Thrombosis, 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006.
    van den Besselaar, AMHP, Gralnick HR, Lewis SM, Editors. Thromboplastin Calibration and Oral Anticoagulant
    Control. Dordrecht: Martinus Nijhoff Publishers, 1984.
www.CLOT-ED.com                                                                                                 59

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Hemostasis principles

  • 1. Principles of Hemostasis Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH Revised: mmvi 1
  • 2. Topics for Discussion I nt ro d Vi r uct cho ion P ri w’s ma Tri ry Sec on He ad Ph dar mos ysi yH tas Lab olog em is o ra ic C ost Fib tor oag asis rin yC ol y sis oag ulatio ul a n tio n www.CLOT-ED.com 2
  • 4. Virchow’s Triad Changes in blood coagulability Platelets, Coagulation Factors & Inhibitors, Fibrinolysis Changes in vessel wall Changes in blood flow Endothelial changes due to Rheology in vessels inflammation or atherogenesis www.CLOT-ED.com 4
  • 5. Vessel Wall Tunica intima Endothelium - inner most layer of cells that separate the remainder of the vessel from the lumen Basement membrane – thin layer of spongy connective tissue that secretes elastic collagen Tunica adventitia Surrounds the tunica media Connective tissues – produce elastic http://www.alegent.com/122711.cfm and non-elastic collagen fibers Tunica intermedia Prevents ballooning of vessel with high systolic blood pressure Surrounds the tunica intima Aneurysm – weaknesses in the tunica Smooth muscle – layer of smooth muscle cells adventitia that are under involuntary control and can dilate or constrict Connective tissue – produces collagen fibers whose elasticity is reduced by hypertension www.CLOT-ED.com 5
  • 6. Rheology Region 1 Region 2 In this range of shear rates: As shear rate increases: Cells are in large Cells are disaggregating aggregates (rouleaux Applied forces (yield formation) stress) are forcing cells to As shear rate increases, orient and deform size of aggregates diminish Blood viscosity decreases Viscoelasticity is strongly influenced by aggregation tendency of RBCs Region 3 With increasing stress: Cells deform With normal deformability, cells will form layers that Adapted from : slide on layers of plasma http://www.vilastic.com and align in the direction /tech10.html of flow Science of the deformation and flow of matter Blood is a fluidized suspension of elastic cells that demonstrates both a viscous and elastic effect – Elastic effect makes blood a non-Newtonian fluid (plasma is a Newtonian fluid) – Blood has a yield stress that depends on hematocrit and fibrinogen concentration www.CLOT-ED.com 6
  • 7. Shear & Vessel Wall Endothelial Cells At Wall Surface: Greatest Platelet Flowing Blood Shear Rate & Least Velocity Shear At Vessel Center: Greatest Platelet Flow Velocity & Least Shear Rate Shear Stress Pressure- induced Force Between 2 Laminae Laminae (Arrow Length = Velocity) Endothelial Cells www.CLOT-ED.com 7
  • 8. Hemostasis Process by which blood is maintained in a fluid state and confined to the circulatory system Goal is to stop bleeding and to do so only at the site of injury Components – Platelets • Involved in Primary Hemostasis – Coagulation system • Involved in Secondary Hemostasis – Fibrinolytic system Platelet / fibrin mesh – Inflammatory processes – Wound healing processes www.CLOT-ED.com 8
  • 10. Primary Hemostasis First physiological response to vascular injury, which is mediated by platelets, in order to arrest bleeding Mechanism – Activation of platelets via stimulators such as thrombin – Adhesion of platelets to subendothelium via interaction between GPIb and von Willebrand Factor (VWF) – Release of platelet granule products in order to recruit more platelets to the injured site – Aggregation of platelets via interaction between GPIIb/IIIa (αIIbβ3) and fibrinogen to form the initial plug Triggers secondary hemostasis (coagulation proteins) Affected by medications, platelet function status, and vessel wall status www.CLOT-ED.com 10
  • 11. Platelets Disk-shaped cell fragments produced in GPIIb/IIIa the bone marrow by megakaryocytes Alpha Dense Life span ~ 10 days granules granules Glycoprotein Ib (GPIb) is involved in adhesion Glycoprotein IIb/IIIa (GPIIb/IIIa) is the primary receptor for fibrinogen (aggregation phase) Provide procoagulant surface on which coagulation proteins Coagulation can interact proteins GPIb www.CLOT-ED.com 11
  • 12. Platelet Adhesion Platelet GPIb Endothelium VWF Injury site collagen exposed COL COL COL CO L COL VWF VWF VWF VWF Subendothelial extracellular matrix www.CLOT-ED.com 12
  • 13. Platelet Aggregation Activated Platelet GPIb Fibrinogen (Granule contents released) VWF GPIIb/IIIa COL COL COL CO L COL VWF VWF VWF VWF Subendothelial extracellular matrix www.CLOT-ED.com 13
  • 14. Tests for Primary Hemostasis Bleeding Time – Assesses all components of Virchow’s triad – in vivo test – performed directly on patient – Has fallen into disrepute and replaced by instruments that perform “in vitro” bleeding times Platelet Aggregation studies – Measure ability of platelets to aggregate, in vitro, when subjected to various stimulators (agonists) – Predominantly assesses function of platelet glycoprotein IIb/IIIa receptor Von Willebrand Factor (VWF) assays – Measure amount and function of VWF, a protein that works with platelets so that they adhere to site of injury – Assesses function of VWF ligand in its interaction with platelet glycoprotein Ib receptor www.CLOT-ED.com 14
  • 15. Platelet Appearance / Function Electron Micrographs Aggregation Tracings Resting platelets Normal platelet Aspirin-like defect function Activated platelets www.CLOT-ED.com 15
  • 17. Secondary Hemostasis Process of blood coagulation Mechanism – Coagulation proteins work in concert to generate thrombin – Thrombin converts fibrinogen to fibrin – Fibrin consolidates the platelet plug made in primary hemostasis such Credit: Weisel JW. University of Pennsylvannia that a thrombus (secondary hemostatic plug) is formed Prevents further blood loss from the injury site www.CLOT-ED.com 17
  • 18. Coagulation Factors Factor XII (FXII) activated FXII (FXIIa) Factor XI (FXI) activated FXI (FXIa) Factor X (FX) activated FX (FXa) Factor IX (FIX) activated FIX (FIXa) Factor VIII (FVIII) activated FVIII (FVIIIa) Factor VII (FVII) activated FVII (FVIIa) Factor V (FV) activated FV (FVa) Factor II (prothrombin) is converted to thrombin (FIIa) Factor I (fibrinogen) is converted to fibrin www.CLOT-ED.com 18
  • 19. “in vivo” v “in vitro” Coagulation Physiologic (“in vivo”) coagulation is dependent upon the tissue factor pathway – Goal is to form a thrombus Laboratory (“in vitro”) coagulation is dependent upon the contact system – Classical “waterfall” or cascade concept • Step-by-step biochemical reactions in which an inactive proenzyme is converted to a reactive enzyme which, in turn, converts another proenzyme to its active form – Amplification process (very minute amounts of Factor XII yield large amounts of thrombin) – Goal is to form a clot www.CLOT-ED.com 19
  • 20. Waterfall Scheme of Coagulation FXII FXIIa FXI FXIa FVIIIa FIX Ca++ PL FIXa FVIIa FVII TF Ca++ PL FX FXa FX FVa FII Ca++ PL Thrombin TF = Tissue Factor Ca+ += Calcium ion After Macfarlane RG. Nature 1964;202:498-9 Fibrinogen Fibrin PL = Phospholipid www.CLOT-ED.com 20
  • 21. Physiologic Coagulation Thrombus Formation www.CLOT-ED.com 21
  • 22. Initiation Phase Monroe DM, et al. ATVB 2006;26:41-8 “Extrinsic Pathway” www.CLOT-ED.com 22
  • 23. Amplification Phase Monroe DM, et al. ATVB 2006;26:41-8 www.CLOT-ED.com 23
  • 24. Propagation Phase Monroe DM, et al. ATVB 2006;26:41-8 “Intrinsic Pathway” www.CLOT-ED.com 24
  • 25. Recapping Secondary Hemostasis Tissue Factor complexed with FVIIa initiates coagulation at site of injury Small amounts of thrombin are generated that activate platelets Coagulation factors form complexes on platelet surfaces Very large amounts of thrombin are formed to convert fibrinogen to fibrin Fibrin reinforces platelet plug (primary hemostasis) and hemostasis is achieved www.CLOT-ED.com 25
  • 26. Laboratory Coagulation Clot Formation www.CLOT-ED.com 26
  • 27. First the Specimen Blood is collected into Plasma is used for testing a tube that contains – PLASMA contains sodium citrate, an FIBRINOGEN anticoagulant – Serum does not contain Blood fluidity is Fibrinogen maintained because sodium citrate binds Plasma is “platelet poor” calcium ions, which are since platelets remain in critical to the buffy coat coagulation process Tube is centrifuged in order to separate plasma from buffy coat Plasma (white blood cells & Buffy Coat platelets) and red blood cells Red Cells www.CLOT-ED.com 27
  • 28. Coagulation in the Laboratory Intrinsic Pathway Extrinsic Pathway XII XI VII Tissue Factor IX APTT VIII PT Intrinsic X Extrinsic + Common V Pathway + Common II Common Fibrinogen Fibrin Clot www.CLOT-ED.com 28
  • 29. Routine Coagulation Assays Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT) Quantitative Fibrinogen (FIB) Thrombin Time (TT) Assays for specific coagulation factors – Factors assessed by a PT-based test system: FVII, FV, FX, and FII – Factors assessed by an APTT-based test system: FXII, FXI, FIX, and FVIII www.CLOT-ED.com 29
  • 30. Prothrombin Time (PT) Time for clot formation ~ 12 seconds Incubate at 37 oC for ~3 minutes 0.1 ml Thromboplastin + Ca++ 0.1 ml Plasma PT Reagent Composition Thromboplastin Credit: PNAS; Collet JP and − Tissue Factor (recombinant/human or animal brain) Weisel JW. Un Pennsylvannia − Lipid (source of phospholipid since platelets were removed from plasma) − CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate Historically referred to as “complete” since both phospholipid and apoprotein make up the reagent www.CLOT-ED.com 30
  • 31. Causes for Prolonged PT Deficiencies or abnormalities in: – FVII (Extrinsic Pathway) – FV, FX, FII (prothrombin), and FI (fibrinogen) • Both PT and APTT will be prolonged Vitamin K antagonists – PT sensitive to reductions in three of four vitamin K-dependent procoagulant proteins: FVII, FX, and FII • FIX measured by APTT – Pharmacologic anticoagulants that modify vitamin K-dependent proteins such that they do not bind calcium thereby reducing blood coagulability Liver disease – Site for synthesis of vitamin K-dependent proteins – Site for clearance of coumarins (warfarin) and coagulation proteins www.CLOT-ED.com 31
  • 32. Biochemistry: Vit K-Dep Proteins Addition of an extra carboxyl group to glutamate (Glu) residues at their amine termini gives rise to a novel amino acid called gamma-carboxyglutamate (Gla) – Presence of Gla enables proteins to undergo a calcium- dependent conformational change that allows for their binding to phospholipid surfaces and generation of membrane bound macromolecular complexes www.CLOT-ED.com 32
  • 33. Vitamin K Antagonists (AVK) DICUMAROL: 3,3’-methylenebis (4-hydroxycoumarin) – Isolated by Karl Link (University of Wisconsin-1939) as the anti- vitamin K agent responsible for hemorrhagic disorder in cattle – Coumarin derivatives • Bishydroxycoumarin (Dicumarol) • Warfarin (Coumadin®) – Analog #42 of many coumarins synthesized by Dr Link and named by him as WARFarin for the Wisconsin Alumni Research Foundation and coumARIN Require monitoring because: – Vitamin K-dependent proteins have different half-lives – Differences in drug absorption and clearance – Levels affected by concomitant medications, comorbid conditions, changes in diet, patient compliance – PT (thromboplastin) reagents vary in their reaction to clotting defects produced by AVK (warfarin) www.CLOT-ED.com 33
  • 34. Warfarin Sodium Oral anticoagulant of choice in North America Pharmacologic properties more favorable than Dicumarol – Warfarin is 5-10x more potent Racemic mixture (~1:1) of R & S isomers – S isomer is 5x more potent than R isomer • Hepatic microsomal enzyme cytochrome P450 2C9 is responsible for the oxidative metabolism of S isomer Bioavailability – Absorbed from gastrointestinal tract – Maximal blood concentrations reached in 90 minutes – Half-life (T½) is 36-42 hours – At therapeutic concentrations, 99% of warfarin is bound to albumin and 1% is free and can bind to its receptor on hepatic cells Anticoagulant effects are reversed by administration of vitamin K or biologic products that contain vitamin K-dependent proteins www.CLOT-ED.com 34
  • 35. Warfarin Mode of Action CO2 H2O O2 Des-Carboxy Prothrombin Prothrombin (“Glu”) γ-Carboxylase (“Gla”) Vitamin K Vitamin K hydroquinone Vitamin K 2, 3 epoxide (active form K1H2) (inactive form K1O) epoxide reductase (VKOR) Regeneration of active form NAD+ NADH is sensitive to warfarin WARFARIN www.CLOT-ED.com 35
  • 36. International Normalized Ratio ISI Patient PT INR = Mean Normal PT “Normalizes” the PT by mathematically considering differences in PT reagents (thromboplastins) Only to be used to monitor long term anticoagulant effects for patients stabilized on oral anticoagulant therapy – Prevent recurrence of thrombosis caused by under anticoagulation – Prevent hemorrhagic complications caused by over anticoagulation www.CLOT-ED.com 36
  • 37. Activated Partial Thromboplastin Time Time for clot formation ~ 30 seconds 0.1 ml CaCl2 Incubate at 37 oC for ~5 minutes 0.1 ml Activator 0.1 ml Plasma APTT Reagent Composition Activator to convert FXII to FXIIa Phospholipid (replaces “in vivo” platelet surface on which coagulation reactions occur) CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate Referred to as “partial thromboplastin” since no Tissue Factor is used − Two-stage assay (activation and re-calcification) www.CLOT-ED.com 37
  • 38. Intended Use for APTT Screening test – Intrinsic (and severe common pathway) factor deficiencies Laboratory monitoring – Unfractionated heparin – Other antithrombotic agents (Direct Thrombin Inhibitors) Laboratory detection of the Lupus Anticoagulant www.CLOT-ED.com 38
  • 39. Causes for Prolonged APTT Most common causes – Heparin (contamination from lines or therapeutic) – Lupus Anticoagulant Other causes – Deficiencies of coagulation factors • FVIII (Hemophilia A or Von Willebrand Disease), FIX, FXI, FXII – Liver disease (site of production for most coagulation factors) – Consumption of coagulation factors as seen in Disseminated Intravascular Coagulation (DIC) www.CLOT-ED.com 39
  • 40. Heparin Heparin is a heterogeneous group of straight-chain anionic mucopolysaccharides (glycosaminoglycans) – Molecular weights range from 5 – 40 kiloDaltons – Composed of alternating D-glucosamine residues linked 1 → 4 to either L-iduronic acid or D-glucuronic acid Heparin is highly acidic therefore binds to positively charged amino acids such as arginine & lysine – Pentasaccharide sequence • Comprisies ~30% of heparin • Binds to antithrombin (AT) • Accelerates AT inhibition of activated factors XII, XI, IX, X, and II (thrombin) thus serving as an anticoagulant Pharmaceutical heparins are extracted from pig intestinal mucosa (source of mast cells) www.CLOT-ED.com 40
  • 42. APTT Monitoring of Heparin Assumes antithrombotic (anti-IIa) effect parallels anticoagulant effect Limitations – Pre-treatment APTT of patient • Baseline APTT of patient prolonged due to Lupus Anticoagulant • Baseline APTT of patient sample below or at low end of reference interval due to high levels of FVIII (apparent “heparin resistance”) – APTT reagents vary in sensitivity to heparin • Laboratories must determine responsiveness of their APTT reagent to unfractionated heparin • Determine APTT therapeutic interval (seconds) for reagent used to monitor heparin therapy www.CLOT-ED.com 42
  • 43. Direct Thrombin Inhibitors Hirudin [Lepirudin (rDNA)–trade name: Refludan®] – Approved in USA, Canada, and EU for Heparin Induced Thrombocytopenia (HIT) complicated by thrombosis – Target APTT is 1.5-2.5 x patient baseline APTT – In absence of severe thrombosis, some experts recommend a target APTT of 1.5-2.0 x patient baseline APTT and monitor every 4 hours Argatroban [non-US trade name: Novastan] – Approved for HIT with or without thrombosis and also for anticoagulation during percutaneous coronary intervention (PCI) in patients with, or at risk for, HIT – Target APTT 1.5-3.0 x patient baseline APTT (maximum 100 seconds) Hirulog [Bivalirudin-trade name: Angiomax™] – Undergoing evaluation for use as an anticoagulant for “on-pump” and “off-pump” cardiac surgery in patients with HIT – Target APTT is 1.5-2.5 x patient baseline APTT www.CLOT-ED.com 43
  • 44. “Recap”ping the PT and APTT PT and APTT are screening assays to determine if a patient, when challenged, has a potential to bleed – If warfarin or heparin are not present in sample, then: • Prolonged PT and normal APTT = deficiency of FVII • Normal PT and prolonged APTT = deficiencies in any of the intrinsic pathway factors (FVIII, FIX, FXI, or FXII) • Prolongation of both PT and APTT = deficiencies of factors common to both pathways (FX, FV, FII, or fibrinogen) PT, via the INR, is used to monitor oral anticoagulant therapy (warfarin) APTT is used to monitor heparin anticoagulant therapy APTT is affected by inhibitors such as Lupus Anticoagulant www.CLOT-ED.com 44
  • 45. Tests for Fibrinogen Quantitative Fibrinogen Thrombin Time (TT) Measures the amount of Assesses the functionality of fibrinogen present in plasma fibrinogen in plasma Low levels, termed TT clotting time prolonged hypofibrinogenemia, can be – HEPARIN inherited but generally are due – Direct thrombin inhibitors to acquired causes such as – Hypofibrinogenemia DIC, liver disease, or – Dysfibrinogenemia fibrinolytic therapy – Elevated fibrin split products High levels are seen in inflammatory states since Time for clot formation ~ 15 seconds fibrinogen is an acute phase reactant Incubate at 37 oC for ~2 minutes 0.2 ml Diluted Thrombin 0.2 ml Plasma www.CLOT-ED.com 45
  • 46. Quantitative Fibrinogen Calibrator plasma is serially diluted Time for clot formation (1:5, 1:10, 1:20, and 1:40) to establish ~ 5-14 seconds a reference curve (see graph below) Patient plasma is diluted 10 fold (1:10) in buffer Incubate at 37 oC for ~3 minutes 0.1 ml Thrombin 0.2 ml Diluted Plasma 100 Patient clotting time in seconds is Clotting Time-Seconds 35.0 read against the reference curve 18.3 − Patient clotting time of 8.0 9.5 seconds equates to 281 mg/dl 8.0 5.1 Patient fibrinogen in example at right Fibrinogen concentration is inversely 1 proportional to clotting time 1 10 10 58 115 100 230 281 460 1000 mg/dl Fibrinogen www.CLOT-ED.com 46
  • 47. Coagulation: A Balancing Act Generate Inhibit Thrombin Thrombin Formation www.CLOT-ED.com 47
  • 48. Inhibitors Naturally occurring inhibitors – Protein C (activated) and Protein S • Inhibit coagulation cofactors FVIIIa and FVa – Antithrombin • Inhibits FXIa, FIXa, FXa, FVIIa/TF, and thrombin (IIa) Pathologic inhibitors – Acquired or autoimmune antibodies to specific coagulation factors Pharmacologic inhibitors – Heparin and Low Molecular Weight Heparin – Warfarin – Direct Thrombin Inhibitors www.CLOT-ED.com 48
  • 50. Hemostasis: A Delicate Balance Generates Generates Thrombin Plasmin Form a Dissolve a Thrombus Thrombus www.CLOT-ED.com 50
  • 51. Fibrinolytic System TAFI Thrombin TM Thrombin TAFIa TM PC APC ‡ Fibrinogen FIBRIN tPA Plasminogen FXIII Plasmin PAI-1 ‡ Fibrin(ogen) Degradation Products Antiplasmin www.CLOT-ED.com 51
  • 52. Breakdown of Fibrin(ogen) FPB FPB FPA & B FPA & B Fibrinogen Fibrinogen Fibrin Thrombin Fibrin Monomer Clot Fibrin Monomer Fragment X Fragment X Fibrin Polymer Fibrin Polymer D D Y Y FXIII FXIIIa PLASMIN D D E E D D D D E E D D D D E E D D D-dimer D D D D E E D D D D E E D D www.CLOT-ED.com 52
  • 53. Fibrinolytic Agents All currently available thrombolytic agents are plasminogen activators (PA) – Convert patient plasminogen to plasmin which then acts on fibrin within a thrombus – Additionally can breakdown fibrinogen (fibrinogenolysis) • Commonly referred to as the lytic state (systemic lysis) • Therapeutic doses of PA overwhelm PAI-1 and α2-antiplasmin Beneficial effect is reduction of thrombus size (thrombolysis) Negative effect is that hemostatic plugs are also lysed Most commonly used agents are: Streptokinase (SK), Alteplase (tPA), Reteplase, and Tenecteplase (TNK-tPA) www.CLOT-ED.com 53
  • 55. Time Frame for Hemostasis Platelets Coagulation Factors Fibrinolytic Proteins Primary Primary Secondary Secondary Fibrinolysis Fibrinolysis Hemostasis Hemostasis Hemostasis Hemostasis •• Vessel constriction Vessel constriction •• Activation of Activation of •• Activation of Activation of occurs immediately occurs immediately coagulation coagulation fibrinolytic fibrinolytic factors occurs in factors occurs in proteins happens proteins happens •• Platelet adhesion Platelet adhesion seconds seconds immediately immediately occurs in seconds occurs in seconds •• Fibrin forms in Fibrin forms in •• Dissolving the Dissolving the •• Platelet aggregation Platelet aggregation minutes minutes thrombus thrombus takes minutes takes minutes requires hours requires hours www.CLOT-ED.com 55
  • 56. Bleeding: Balance is Disrupted Presence of Inhibitors* •Pharmacologic (warfarin, heparin) •Allo or Auto-antibodies to Factors www.CLOT-ED.com 56
  • 57. Thrombosis: Balance is Disrupted Inhibitors* • Activated Protein C • Protein S • Antithrombin www.CLOT-ED.com 57
  • 58. Conclusion Primary hemostasis, a platelet-dependent process, forms hemostatic plugs when a vessel is injured Secondary hemostasis, a coagulation factor-dependent process, begins with Tissue Factor exposure – Small amounts of thrombin are generated via FXa formation by the TF:FVIIa complex (“Extrinsic Pathway”) – Sustained thrombin generation depends on FXa formation via FIXa and FVIIIa-mediated complexes on an activated platelet surface – Amount of thrombin generated dictates bleeding or thrombotic risk The clinical history is the best “test” for hemostasis www.CLOT-ED.com 58
  • 59. References Ansell J, et al. The pharmacology and management of the vitamin K antagonists: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:204S-233S. Clinical and Laboratory Standards Institute (CLSI). One-stage Prothrombin Time (PT) test and Activated Partial Thromboplastin Time (APTT) test; Approved Guideline H47-A, 1996. Crowther MA, et al. Practical aspects of anticoagulant therapy (Chapter 89). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001. Fairweather RB, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy: Laboratory monitoring of oral anticoagulant therapy. Arch Pathol Lab Med 1998;122(9):768-81. Greaves M, Preston FE. Approach to the bleeding patient (Chapter 48). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001. Hirsh J, Raschke R. Heparin and low-molecular-weight heparin: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:188S-203S. Konkle BA. Clinical approach to the bleeding patient (Chapter 77). In: Colman RW, ed. Hemostasis and Thrombosis, 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006. Link KP. The discovery of Dicumarol and its sequels. Circulation 1959;19(1):97-107. Olson JD, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy: Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med 1998;122(9):782-98. Physicians’ Desk Reference, 60th ed. Montvale: Thomson PDR, 2006. Poller L. Prothrombin Time (Chapter 6) and Activated Partial Thromboplastin Time (Chapter 5). In: Jespersen J, ed. Laboratory Techniques in Thrombosis-A Manual, 2nd ed. Dordrecht: Kluwer Academic Publishers, 2000. Tran HAM, Ginsberg JS. Anticoagulant therapy for major arterial and venous thromboembolism (Chapter 116). In: Colman RW, ed. Hemostasis and Thrombosis, 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006. van den Besselaar, AMHP, Gralnick HR, Lewis SM, Editors. Thromboplastin Calibration and Oral Anticoagulant Control. Dordrecht: Martinus Nijhoff Publishers, 1984. www.CLOT-ED.com 59