Monitoring Thrombin Generation: The Holy Grail of Laboratory Hemostatic Assessment.
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1 Monitoring Thrombin Generation: The Holy Grail of Laboratory Hemostatic Assessment. Marcus E. Carr, Jr., MD, PhD Professor of Medicine, Pathology, and BioMedical Engineering Director of the Central Virginia Center for Coagulation Disorders Virginia Commonwealth University Richmond, Virginia
2 New Insights into the Clotting Mechanism Emphasize: The central role of thrombin production The interplay between cellular and plasma protein components
3 Blood flow Platelet Endothelial cell Endothelial cell TF TF TF TF TF TF DM Monroe, WFH2002, Seville,Spain.
4 Blood flow Platelet Endothelial cell TF TF TF TF DM Monroe, WFH2002, Seville,Spain.
5 Blood flow Platelet Endothelial cell TF TF TF TF DM Monroe, WFH2002, Seville,Spain.
6 X Xa TF VIIa TF VIIa Xa Tissue factor-bearing cell TF IX VIIa IXa DM Monroe, WFH2002, Seville,Spain.
7 Xa TF VIIa TF X VIIa Xa Va II Tissue factor-bearing cell VIII/vWF VIIIa + free vwf IIa V Platelet V Va XI XIa TF VIIa XIa VIIIa Va Activated platelet DM Monroe, WFH2002, Seville,Spain.
8 Xa TFPI VIIa TF TF VIIa Xa Va Tissue factor-bearing cell VIII/vWF VIIIa + free vwf IIa V Platelet V Va XI XIa TF IX VIIa IX X II IIa IX XIa VIIIa Xa Va Activated platelet DM Monroe, WFH2002, Seville,Spain.
9 Blood flow Platelet Endothelial cell TF VIIa X Va Xa II IIa TF TF TF DM Monroe, WFH2002, Seville,Spain.
10 Blood flow Platelet X II IXa Xa XIa VIIIa Va Endothelial cell IIa IIa TF VIIa X Va Xa II IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa TF TF TF DM Monroe, WFH2002, Seville,Spain.
11 What Does This Mean for Our Understanding of Hemophilia? The primary problem in hemophilia is deficient platelet-based thrombin generation. This leads to deficient platelet function and altered clot structure.
12 Xa TFPI VIIa TF TF VIIa Xa Va Tissue factor-bearing cell VIII/vWF VIIIa + free vwf IIa V Platelet V Va XI XIa TF VIIa XIa VIIIa Va Activated platelet DM Monroe, WFH2002, Seville,Spain.
13 Xa TFPI VIIa TF TF VIIa Xa Va Tissue factor-bearing cell IIa Platelet V Va XI XIa TF IX VIIa X II IXa XIa Va Activated platelet DM Monroe, WFH2002, Seville,Spain.
14 Blood flow Platelet X II Endothelial cell TF X Va Xa II IIa TF TF TF DM Monroe, WFH2002, Seville,Spain.
15 Thrombin Generation Curve 80 [ Thrombin ] (nm) Time (minutes) DM Monroe, WFH2002, Seville,Spain.
16 Most Clotting Assays Have Been Plasma Based Plasma-based assays completely miss the impact of platelets (for which phospholipid has been added as a substitute) and platelet activation on thrombin generation. Such assays typically use clot formation as the end point and thus also miss the impact of altered thrombin generation on platelet function and clot structure.
17 Can These Plasma Assays Be Used to Monitor Factor-Replacement Therapy in Bleeding Patients? PPP-based assays can be used to measure clotting factor levels. In many cases, raising the factor level to normal will correlate with stoppage of bleeding and clinical benefit. However, with some clinically effective agents this is clearly not the case. A prime example is recombinant activated Factor VII (rfviia). PPP = platelet-poor plasma.
18 Can the PT or ACT Be Used for Monitoring rfviia? (NO) Lisman T et al. Blood. 2002;99:175. PT = prothrombin time; ACT = activated clotting time. Telgt DSC et al. Thromb Res. 1989;56:603.
19 Thrombin Generation in Congenital Hemophilia A Blood Contemporaneous control Contemporaneous control + 10 nm FVIIa Hemophilia A patient Patient + 10 nm FVIIa TAT (nm) Butenas S et. al. Blood. 2002;99: Time (min)
20 Laboratory Assays that Are Under Investigation Thrombogram Synapse BV Thromboelastogram (TEG) TEG Haemoscope Corporation ROTEG Pentapharm Ltd ReoRox MediRox AB Sonoclot Sienco, Inc. Platelet contractile force Hemodyne, Inc.
21 Laboratory Assays Under Investigation Thrombogram Uses a 96-well microtiter plate and a fluorometer to measure change in fluorescence as thrombin cleaves a fluorogenic substrate. Sample is PPP or PRP. Software produces kinetic curve of thrombin concentration (nm) versus time (mins). PRP = platelet-rich plasma.
22 Synapse BV Thrombogram (Dr. H.C. Hemker) The program is written for the Thermo Labsystems Ascent Reader The Thrombinoscope Program is available from Synapse BV The program comes with a comprehensive manual and lyophilized thrombin standard
23 Thrombogram : Measurement of Time Course of Thrombin Production Screenshot of a Thrombogram TM Measurement Normal Platelet- Poor Plasma A Normal Platelet- Poor Plasma B Blue: Signals from 4 identical experiments superimposed Green: Signals from 4 standards with fixed thrombin activity
24 Screenshot of a Thrombogram Experiment Each frame shows 4 identical experiments plus the median (bold) for details Donor A PPP Donor B PPP Donor A PRP Donor B PRP
25 Thrombogram Measurement in Platelet-Rich Plasma: Effect of FVIII Infusion 150 Haem. t=3-45% Normal control Thrombin (nm) Haem. t=½ - 32% Haem. t=½ - 7% Time (min) Haemophiliac before
26 Laboratory Assays Under Investigation ReoRox This method measures viscoelastic changes during clotting of whole blood. G', G", phase angle, damping, and frequency are measured. Clotting time and clot completion time are calculated based on preset changes in damping and frequency. (Outer sample surface moves. Sample in the cup effects movement.)
27 MediRox: ReoRox 4 Ramström S et al. Thromb Res. 2002;105:
28 ReoRox 4: Measurement of Sample Viscoelastic Properties Ramström S et al. Thromb Res. 2002;105:
29 ReoRox 4: Measurement of Sample Viscoelastic Properties Examples of tpa Treatment (Full Rheological Study) No Treatment Blood clotted with thromboplastin. Study over 180 min. Treatment Blood with 24 IU/ml of tpa clotted with thromboplastin.
30 Laboratory Assays Under Investigation Thromboelastogram This assay measures the coupling of a moving outer cup with a centrally placed piston during clotting of blood samples. As clot forms, it connects the inner piston and outer wall and the piston begins to move. The degree of coupling is dependent on adhesion and clot strength. Measured parameters are defined by changes in the kinetic curve.
31 Haemoscope TEG
32 Thromboelastogram: TEG Output
33 Thromboelastogram: Qualitative Analysis The TEG system documents the interaction of platelets with protein coagulation cascade from the time of placing the blood in the TEG analyzer until initial fibrin formation, clot rate strengthening, and fibrinplatelet bonding via GPIIb/IIIa, to eventual clot lysis.
34 Yoshioka and colleagues concluded that the TEG was more suitable than the APTT, PT, or FVII as a monitor of hemostatic effects during rfviia treatment. APTT = activated partial thromboplastin time; PT = prothrombin time. Yoshioka A et al. Haemostasis. 1996;26(Suppl 1):
35 Laboratory Assays Under Investigation ROTEG Like the TEG, the ROTEG assesses coupling of an inner piston to an outer wall during clotting of a blood sample. The ROTEG differs in that the inner piston is caused to rotate. The freedom of rotation of the piston is measured by an optical follower. As clot forms, the degree of rotation is reduced. Measured parameters are virtually identical to those defined by the TEG curve.
36 ROTEG : Multi-Channel TEG Analysis Inner Piston Rotates Light Source Mirror Inner Piston Heating Block Photo Detector Sample
37 ROTEG Output
38 Correlation Between ROTEG and TEG
39 In Hemophilic Blood, rfviia Shortens CFT and Increases MCF in ROTEG Ingerslev J et al. Blood Coag Fibrinol 2000;11(suppl 1):S25-S30. Addition of Buffer CFT = 1190 sec Addition of 10 U/mL rfviia CFT = 367 sec Addition of 20 U/mL rfviia CFT = 299 sec Addition of 60 U/mL rfviia CFT = 195 sec Addition of 120 U/mL rfviia CFT = 129 sec CFT = complement fixation test. MCF = median corpuscular fragility.
40 Laboratory Assays Under Investigation Sonoclot The Sonoclot measures viscoelastic properties during clotting of blood samples. A hollow tube is caused to vibrate up and down within a cup containing the clotting sample. The resistance to oscillation can be used to measure sample viscosity. Once clotting begins, the signal can no longer be used to measure viscosity, but changes in the signal may correlate with structural events within the clot. The signal is termed the clot signature.
41 Sonoclot
42 Sonoclot Output Liquid Phase Clot Rate Time to Peak Clot Retraction Clot Signature Hyperfibrinolysis
43 Laboratory Assays Under Investigation Hemodyne This instrument measures platelet contractile force (PCF), clot elastic modulus (CEM), and thrombin generation time (TGT) in a sample of clotting whole blood. PCF is the force generated by platelets during clot retraction.
44 Hemodyne Instrument Measures PCF, CEM, and TGT
45 Hemodyne Output is Continuously Displayed and a Final Report is Printed
46 TGT is Measured as the Initial Upswing in the PCF Kinetic Curve PCF (Kdynes) TGT TGT TIME (min)
47 TGT is Measured as the Initial Upswing in the PCF Kinetic Curve
48 TGT Occurs at the Moment of F1+2 Appearance 8 20 PCF (Kdynes) Volunteer 1 TGT=250 sec Prothrombin fragment F1+2 (nmol/l) (a) PCF (Kdynes) Volunteer 2 TGT=240 sec. PCF (Atroxin + CaCl2) Prothrombin F Prothrombin fragment F1+2 (nmol/l) (b) TIME (min) 0
49 Variation of TGT and PCF in Twenty-Five Normals 10 8 PCF (Kdynes) TIME (min)
50 TGT Is Prolonged in Hemophilia 10 PCF (Kdynes) Factor FIX Def. Factor VIII Def. Normal Healthy Volunteers TIME (min)
51 Response of Hemophilia to Increasing FVIII Concentration 10 PCF (Kdynes) U/ml U/ml U/ml U/ml U/ml TIME (min)
52 Prolonged TGT in Patient with Spontaneous FVIII Inhibitor 8 6 Patient Control TGT PCF (Kdynes) TIME (minutes)
53 Response to rviia Infusions in a Patient with Spontaneous High Level FVIII inhibitor - PAS PCF (Kdynes) Pre rfviia (Baseline) Post 180 ug/kg rfviia Post 280 ug/kg rfviia Normal Control TIME (min)
54 Abnormally Short TGT and High PCF in CAD and DM PCF (Kdynes) Diabetics with CAD Non-Diabetics with CAD Normal Healthy Volunteers TIME (min)
55 Response of PCF and TGT to a Direct Thrombin Inhibitor (Hirudin) 10 PCF (Kdynes) U/ml U/ml U/ml U/ml U/ml TIME (min)
56 Response to rviia in Patient with FVII Deficiency PCF (Kdynes) Patient pre rfviia (Baseline) Patient post 18 ug/kg rfviia Control TIME (min)
57 rfviia Has Minimal Effect on TGT, PCF, and CEM in Normal Blood (a) PCF (Kdynes) ug/ml (Baseline) ug/ml (80% rec. dose) ug/ml (160%) ug/ml (320%) ug/ml (380%) Time (sec) CEM (Kdynes/cm 2 ) PCF (Kdynes) Lag Time (min) CEM PCF TGT N7NC001 N7NC002 N7NC003 (b) rfviia (µ g/ml)
58 In Hemophilic Blood, rfviia Shortens TGT and Increases PCF and CEM PCF (Kdynes) (a) µg/ml (Baseline) µg/ml (80% rec. dose) µg/ml (160%) µg/ml (320%) µg/ml (380%) Time (sec) CEM (Kdynes/cm 2 ) PCF (Kdynes) Lag Time (min) CEM PCF TGT N7P006 N7P007 N7P001 (b) rfviia (µg/ml)
59 Prolongation of TGT by Bivalirudin and Partial Correction with rviia PCF (Kdynes) Baseline Bivaliarudin (25% rec. dose) Bivalirudin (25% rec. dose) + NovoSeven (200% rec. dose) TIME (min)
60 Thrombotic Risk short ( ) TGT and/or ( ) PCF Hemorrhagic Risk Long ( ) TGT and/or ( ) PCF 18 PCF (Kdynes) Hyperthrombotic Normal Hemophilia TIME (min)
61 Case Study The patient is a nine year old male, diagnosed with Severe FVIII deficiency when he bled from his Vitamin K injection site. Developed a high level inhibitor at age 3. Treated with Autoplex, FEIBA, and Porcine VIII for bleeds. Immune tolerance therapy failed to reduce the inhibitor. Continuing joint bleeds led to use of rviia.
62 Case Study (cont.) Therapy was instituted at the standard dose of 90 µg/kg. Mother felt dose was inadequate and increased it to > 200 µg/kg with what she felt was a better response. Physician was concerned this dose might be thrombogenic. At the physician and mother s request the following study was performed.
63 Response of TEG to rviia infusion Control Post 230µg/Kg Pre rviia R K MA G (sec) (sec) (mm) (dynes/cm 2 ) Control Patient Pre rviia (Baseline) >3600 >3600 * * Patient Post 230 µg/kg rfviia **Unable to measure
64 Response of ROTEG to rviia infusion Control CT:489 sec MCF: 62 mm Patient, Pre rviia CT:49 min MCF: 6 mm Patient, Post 230µg/kg rviia CT: 15 min MCF: 82.5 mm
65 Response of PAS to rviia infusion PCF (Kdynes) Pre NovoSeven Post 230 mcg/kg NovoSeven Normal Controls TIME (min)
66 CONCLUSIONS Whole blood assays that measure the rate of thrombin generation may allow detection of deficient coagulation. These same assays may also be useful in the monitoring of hemostatic agent therapy. The interplay of cellular elements and fluid phase components may be assessable by such methods.
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