Thrombin Generation Assays

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Thrombin Generation Assays

• Technothrombin TGA Kit
• By Jennifer J. Kiblinger
• Technical Regulatory Affairs Manager
• DiaPharma Group, Inc.

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Technothrombin TGA Kit Thrombin Generation
AssayIntroduction

• DiaPharma introduces the Technothrombin TGA kit,
  a global research test for investigation of
  bleeding and thrombotic tendency. This thrombin
  generation assay from Technoclone is based on
  monitoring the formation of thrombin with a
  fluorogenic substrate upon activation of the
  coagulation cascade by tissue factor (TF).
• By providing varying amounts of TF and
  phospholipid, the Technothrombin TGA is a
  universal tool with multiple applications
• Hemophilia during inhibitor by-passing therapy
• Pro-coagulant microparticles
• Anticoagulant drugs
• Thrombophilia
• Features of the assay
• Reagent-complete kit includes thrombin calibrator
  and controls
• Easy to perform User-friendly software and
  instructions are provided
• Calibration required only once per lot number
• Step-by-step applications available for the
  Bio-TEK FLx800 TBI, as well as many other
  fluorometers
• Can be performed with plasma or whole blood, and
  in platelet rich plasma or platelet poor plasma

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Thrombin Generation AssaysOutline

• Measurement Principle / Methodology
• Applications (General)
• Introduction to Technoclone
• Technothrombin TGA
• Cevron Coagulation Analyzer
• Publications References

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Why Thrombin Generation Assays?

• TGA is to clotting assay as ECG is to pulse
• TGAs provide more info
• The amount of thrombin that generates, as well as
  the length of time that it is active, both count
• With routine clotting tests, only the initial
  phase of thrombin generation is measured

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Why Thrombin Generation Assays?

• More to come
• Clotting times only represent lag phase before
  thrombin generation ignores amount of thrombin
  that forms and time it remains active
• After clot formation, much of the thrombin action
  is still to come

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Why Thrombin Generation Assays?

• Describes the concentration of thrombin in
  clotting plasma
• Good physiological function test of thrombotic
  hemostatic system
• Measures thrombotic and bleeding tendency

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History of the Thrombin Generation Assay

• Choice of substrate
• Chromogenic vs. Fluorogenic
• Traditionally TG was measured with chromogenic
  substrate
• Substrate converted slowly, did not bind too
  tightly to thrombin Clotting mechanism not
  disturbed, velocity of substrate conversion is
  proportional to concentration of thrombin during
  course of thrombin generation

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History of the Thrombin Generation Assay

• BUTChromogenic thrombin substrates are also
  converted by the thrombin-a2-Macroglobulin
  complex
• Physiologically inactive must correct for this
  in order to calculate the actual free thrombin
• Fibrinogen must be removed (defibrinated)

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History of the Thrombin Generation Assay

• Fluorogenic substrate fluorescence not heavily
  influenced by turbidity appearance of a clot
  will not impair signal fibrinogen does not need
  to be removed
• Presence of platelets does not disturb
  measurement
• Can study role of platelets in coagulation in
  large series of samples

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History of the Thrombin Generation Assay

• Fluorogenic assays Tappenden et al. found that
  of the substrates tested, Z-Gly-Gly-Arg-AMC,
  appears the most appropriate fluorogenic
  substrate for monitoring thrombin generation.
• Ref Tappenden KA et al. Evaluation of
  Fluorogenic Substrates for the Monitoring of
  Thrombin Generation. ISTH Sydney 2005, P1897.

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History of the Thrombin Generation Assay

• Thrombin Calibrator allows calculation of molar
  concentration of thrombin

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Measurement Principle

• Remember coagulation starts when a vessel is
  wounded
• A vessel is wrapped in a layer that is full of
  cells containing TF
• After injury, this TF is exposed to blood, binds
  with FVIIa to form TFFVIIa complex (extrinsic
  pathway)
• Activates FX to FXa, which activates prothrombin
  to thrombin

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Measurement Principle

• Universal tool for analysis and monitoring of
  hemostatic system on an individual basis
  (universal monitor of clotting function)

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MEASUREMENT PRINCIPLE
The coagulation cascade is activated upon
addition of different concentrations of - tissue
factor - phospholipids TECHNOTHROMBIN TGA
is based on monitoring the fluorescence generated
by the cleavage of a fluorogenic substrate by
thrombin over time
ZGGR-AMC
AMC
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Measurement Principle

• Thrombin Generation Parameters
• Lag phase ( clotting time)
• From addition of reagents until first burst of
  thrombin
• Slope steepest rate of thrombin formation per
  min.
• Velocity Index (nM/s)
• peak thrombin
• Peak time lag time
• Peak Max FIIa formed
• Time to Peak
• AUC (area under curve) ETP (Endogenous thrombin
  potential) amount of work that can potentially
  be done by thrombin how much, and how long is it
  active?
• Inactivation Phase

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Measurement Principle

• General assay procedure
• Add reagents consisting of coagulation trigger
  (PL, TF) and plasma
• PPP need PL PRP dont necessarily need PL
• Add mixture of fluorogenic substrate and Ca2 to
  begin coagulation
• Instrument takes absorbance readings
• Software program calculates all parameters (Peak
  height/Max FIIa, lag time, etc.) expresses
  results in nM Thrombin

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Potential Advantages

• Taken together with routine clotting parameters,
  allows analysis of clotting defects
  thrombophilia in more detail
• Technically simple
• Few reagents required
• Adaptable to semi-automated instrumentation or to
  manual technology
• Step-by-step instructions provided
• Flexible
• Can be adapted for different purposes assay can
  be tweaked to suit many needs
• Applicable for both hemophilia A B
• Sensitive

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Potential Disadvantages

• Time-consuming
• 60 90 minutes per assay, unlike typical
  chromogenic clotting assays
• Full clinical utility still being investigated
• Does not necessarily replace specific tests for
  i.e. thrombophilia, etc.

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Applications

• Identify clotting defects / thrombophilic
  patients in more detail
• Obtain full picture of thrombotic tendency of
  patients at risk for thrombosis or bleeding
• Measure acquired thrombotic disorders like with
  use of oral contraceptives
• Monitor anticoagulant therapies
• i.e. warfarin, heparins, anti-platelet drugs
• Development of anti-thrombotic drugs
• Hemophilia A B
• Monitoring treatment such as with FVIII, FVIIa,
  FEIBA
• Microparticles

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Applications - Thrombophilia

• Thrombophilia
• Determine states of thrombophilia
• Clotting assays dont indicate thrombotic
  tendency of unknown origin
• Measurement of overall thrombin generation
  enables the assessment of simultaneous effects of
  numerous small changes in the hemostatic system
• May either reinforce or compensate each other

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Applications - Thrombophilia

• In some forms of thrombophilia, such as AT,
  Protein C, and Protein S deficiencies,
  TF-triggered clotting produces larger amount of
  thrombin compared to normal plasma
• Assaying thrombin generation in PPP can detect
  such deficiencies

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Applications - Thrombophilia

• Measurement of overall thrombin generation
  enables the assessment of simultaneous effects of
  numerous small changes, which may either
  reinforce each other or compensate each other
• Hemker HC et al. Calibrated Automated Thrombin
  Generation Measurement in Clotting Plasma.
  Pathophysiol Haemost Thromb 2003 334-15

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Applications - Thrombophilia

• Risk of recurrent thrombosis (DVT or PE)
• Relative risk of recurrent VTE correlates with
  peak thrombin concentration
• (Hron et al. Identification of patients with low
  risk of recurrent venous thromboembolism by
  measuring thrombin generation. www.technoclone.com
  )
• Patients can be grouped according to risk of
  recurrence using the TGA

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Applications - Thrombophilia

• Risk of recurrence 60 lower when peak thrombin
  is less than 400 nM

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Applications Anticoagulation Therapy Monitoring

• No single clotting test that reacts similarly to
  different types of anticoagulants
• Clotting tests measure length of initial stage of
  thrombin formation
• Clotting occurs at end of initiation pahse and is
  complete when gt95 of all thrombin is still to
  appear
• Length of initiation phase doesnt usually
  indicate the amount of thrombin activity still to
  come

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Applications Anticoagulation Therapy Monitoring

• Antithrombotic drugs diminish amount of thrombin
  that appears in clotting plasma, but not
  necessarily the lag time (i.e. clotting time)
• Lag time (clotting time) could be normal, but
  with less thrombin formed TGA will reflect this

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Applications Anticoagulation Therapy Monitoring

• Anticoagulation Monitoring
• Calculate INR values for patients
• Significant correlation between INR values
  determined with Technothrombin TGA and PTs
• Sensitive to presence of anti-platelet
  anti-thrombotic agents

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Applications Anticoagulation Therapy Monitoring

• Lag time, velocity of propagation phase relevant
  when indirect FXa inhibitor or rFVIIa are present
  in therapeutic concentrations
• Method can be useful for study of different
  anti-thrombotic agents
• Regardless of mechanism of action (LMWHs, direct
  indirect FXa inhibitors, direct FIIa inhibitors)

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Applications Anticoagulation Therapy Monitoring

• Using a calibration set (AK-Calibrant
  anti-vitamin K), Technothrombin TGA can be used
  for direct INR determination

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Applications Hemophilia A B

• Determine states of bleeding disorders
• Hemophilia A B
• Monitor therapy in hemophilia patients
• Monitor during Factor VIII inhibitor by-passing
  therapy (FVIIa, FEIBA)
• Post-infusion studies/characterization of
  hemostasis
• Monitoring gene therapy

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Applications Hemophilia FVIII Bypassing Therapy

• Hemophilia A patients with inhibitors generally
  treated with preparations containing activated
  coagulation factors to achieve hemostasis by
  bypassing FVIII

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Applications Hemophilia FVIII Bypassing Therapy

• TGA no thrombin generation observed in FVIII
  inhibitor plasma
• When spiked with FEIBA or rFVIIa (NovoSeven),
  rate and peak of thrombin generation increases
  dose-dependently
• Changes in TG properties coincide with
  administration of FEIBA
• FEIBA less sensitive to TF levels than rFVIIa

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Applications Hemophilia FVIII Bypassing
Therapy

• TGA enables monitoring of pharmacodynamic
  pharmacokinetic properties of by-passing
  therapies
• Can help optimize FEIBA or rFVIIa treatment
• TGA is suitable to assess hemostatic effects of
  both product in vitro in vivo

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Applications Microparticles

• Microparticles
• Circulating microparticles contribute to thrombus
  formation possible major risk factor for
  thrombosis in atherosclerotic patients
• Level of circulating microparticles currently
  determined by ELISA or FACS, based on
  flip-flopping of phosphatidylserine to surface
  of microparticles and membrane protein markers
  (using Annexin V as marker)

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Applications Microparticles

• Microparticles
• Determination of clotting activity of
  microparticles would allow to directly relate
  their circulating levels to the
  microparticle-induced thrombotic tendency.

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Applications Microparticles

• Microparticles generated during endotoxinemia are
  thrombogenic and can be measured by the TGA.

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Applications Microparticles

• Determine thrombogenic activity of apoptotic MPs
  using TGA
• Can see difference in activity of apoptotic MPs
  derived from ECs, SMCs, and U937Cs
• Analyze mechanisms leading to thrombin generation
  induced by these apoptotic MP
• May be difference in mechanisms leading to
  thrombin generation among apoptotic MPs

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Technothrombin TGA

• Rapid reliable measurement of coagulation
  parameters in patients undergoing anticoagulation
  therapy, in patients with hemophilic or
  thrombophilic disorders, and for the measurement
  of circulating microparticles.

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TECHNOTHROMBIN TGA
FOR RESEARCH USE ONLY
Kit components
In addition, TGA Reagent A (RA) without Tissue
Factor and a microparticle free control (MFC) are
available. Kit for whole blood samples also
available.
? All reagents are available on a modular basis.
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Technothrombin TGAReaders Software

• Readers with software available from Technoclone
• Bio-Tek FLx800 TBI Fluorometer
• BIO-TEK Technothrombin TGA package with KC4TM
  software
• Flexible and affordable solution for fluorescence
  measurements
• Ideal for use with Technothrombin TGA
• Black plates for the TGA determination on the
  fluorescence reader are available on request

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Technothrombin TGAReaders Software

• Readers with software available from Technoclone
• Applications for many fluorescent readers
  available from DiaPharma
• TECAN, Flurostar BMG, Fluoroscan Thermo Electron,
  VICTOR PerkinElmer, Molecular Devices SpectraMax
  (in development)
• More applications being developed see your
  DiaPharma representative for specific requests.

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Technothrombin TGA

• Whole blood or plasma
• Negatively charged phospholipids containing
  different amounts of TF and CaCl2
• Provides different TF concentrations, monitors
  whole kinetics of thrombin generation during
  initiation, amplification, and down-regulation of
  thrombin formation
• Can detect changes in thrombin formation

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Technothrombin TGA

• Based on monitoring fluorescence generated by
  cleavage of a thrombin substrate over time, upon
  activation of the coagulation cascade by
  different concentrations of PL and TF
• Concentration of thrombin is automatically
  calculated using thrombin calibrator and
  appropriate software
• Allows for visualization of the different phases
  of clot formation

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Technothrombin TGA Test Procedure

• Allow reagents to reach RT before use
• Fluorometer wavelength 360 nm/460 nm
  (excitation/emission)
• Temperature during measurment 37?C

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TECHNOTHROMBIN TGA
Test Procedure
Sample Preparation Citrated plasma (PPP or
PRP) PRP samples generally more
stable Color of the plasma does not
influence the TGA measurement
Mechanical agitation leads to significant changes
thrombin generation Thrombin Standard
Curve Calibration range 5-500 nM
thrombin Add thrombin calibrator
substrate (thrombin standard calibrated
against WHO preparation) Read for 10
minutes at 30 sec intervals Samples Add
sample reagent substrate Read for 60
minutes at 60 sec intervals 90 minutes
for FVIII inhibitor bypassing therapy Analysis
Software calculates thrombin generation,
results are given in nM Thrombin
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Technothrombin TGA Test Procedure

• Lag phase depends in general on amount of TF
  added, and therefore type of TGA reagent (TGA RB,
  RCLow, or RCHigh) used
• Slope and peak thrombin can depend on amount of
  PL in sample
• For RB, RCLow (low PL), value determined in PPP
  by number and composition of MPs present in sample

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Technothrombin TGA Test Procedure

• Anticoagulation Therapy RCHigh
• Thrombophilia PPP, RCLow
• Hemophilia FEIBA RB
• Hemophilia rFVIIa RCLow

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Technothrombin TGA Test Procedure

• Excess of TF will cause mechanism of
  prothrombinase formation to take EXTRINSIC
  pathway (FVII, V, X, TFPI)
• Decreased TF changes pathway of thrmobin
  formation to included FVIII, FIX, FXI
• Absence of TF (but high PL) thrombin generation
  occurs via INTRINSIC pathway
• In PPP, without PL, thrombin generation depends
  on PL present in plasma, i.e. presence of
  circulating pro-coagulant microparticles

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Technothrombin TGA Test Procedure

• Expected Ranges
• See pkg insert for expected values in PPP and PRP
• In general, PRP yields higher peak FIIa
• Lot-specific batch table will include reference
  ranges
• Plasma preparation very important for accurate
  results

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Ceveron Alpha Coagulation Analyzer

• Fully automated coagulation analyzer
• Allows routine measurements of all relevant
  coagulation parameters on one analyzer.
• Can perform clotting, turbidometric (LIA),
  fluorometric, and chromogenic assays
• Ideal for small to medium labs or as a back-up
  instrument

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Ceveron Alpha Coagulation Analyzer
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Ceveron Alpha Coagulation Analyzer

• Ideal for use with the Technothrombin TGA kit
• Thrombin generation is measured with a special
  adapted TGA fluorometric module which is placed
  over the cuvette motor
• UV emitter (360 nm) is placed in the module
• Optimal wavelength for specific substrate is
  determined by spectrometry
• TGA graphs can be shown for single evaluations
  and for multiple comparisons

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Summary

• Regular global clotting assays like the PT and
  aPTT do not discriminate patients with thrombotic
  tendency they dont tell the whole story
• The TGA is a physiological function test of the
  thrombotic-hemostatic system that can measure
  clotting and bleeding tendencies, as well as
  measure effects of treatment with a combination
  of drugs or a combination of pathological
  conditions

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Summary

• The Technothrombin TGA is a reliable global
  coagulation test suitable for discriminating
  patients with bleeding as well as thrombotic
  tendencies
• Use of a fluorogenic substrate along with a
  thrombin calibrator eliminates major assay
  interferences

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Publications References

• Kaufmann V et al. Validation of the thrombin
  generation assay Technothrombin TGA.
  www.technoclone.com.
• Varadi K et al. Applications of thrombin
  generation assay in characterizing FVIII
  Bypassing Agents. XXth Congress of the ISTH,
  Sydney, Australia, Aug. 8 12, 2005.
• Varadi K et al. Monitoring the bioavailability of
  FEIBA with a thrombin generation assay. J Thromb
  Haemost. 2003 Nov. 1 (11) 2374-80.
• Gerotziafas GT et al. Toward a standardization of
  thrombin generation assessment The influence of
  tissue factor, platelets, and phospholipid
  concentration on the normal values of the
  Thrombogram-Thrombinoscope assay. Thrombosis
  Journal 2005 Oct. 26 (3)16.
• Hemker HC et al. Continuous registration of
  thrombin generation in plasma, its use for the
  determination of the thrombin potential. Thromb
  Haemost 1993 70 617-624.
• Hemker HC et al. Thrombin generation, an
  essential step in haemostasis and thrombosis
  Haemostasis and Thrombosis. Edinburgh, Churchill
  Livingstone, 1994 477-490.
• Technothrombin TGA Package Insert.
  www.technoclone.com.
• Kaufmann V. et al.  TECHNOTHROMBIN TGA a novel
  Thrombin Generation Assay suitable for the new
  routine coagulation analyzer Ceveron alpha. ISTH
  Sydney 2005.
• Tappenden KA et al. Evaluation of Fluorogenic
  Substrates for the Monitoring of Thrombin
  Generation. ISTH Sydney 2005. P1897.