Post on 21-Sep-2020
Diagnostic approach to hemostasis disorders
Hugo ten Cate, CARIM and MUMC+,
Maastricht, the Netherlands
Thrombin generation in unexplained bleeding
Disclosures
• Chairman of board, Federation of Dutch Anticoagulation Clinics (unpaid)
• Research support: Bayer, Boehringer, Pfizer/BMS; 2M
• Grants: Dutch Heart Foundation (CVON; CONTRAST), Marie Curie ITN TAPAS and TICARDIO (EU-ITN Marie Curie)
• Advisory board: Bayer, Daiichi, BMS/Pfizer
Bleeding and hemostasis
What is unexplained bleeding?
• Oftentimes referred to as vaginal bleeding that is longer or more excessive, or irregular (menorrhagia or metrorrhagia)
• That is not in proportion to the trauma, or to the medication used (more than expected)
• That occurs spontaneously
• Causes?
• Medical history essential; additional laboratory testing may be warranted (starting with essentials: platelet count, aPTT, PT)
“Reduce” complexity in capacity assay PFA MPV
Multiplate
ETP Thrombin generation
TEG/ROTEM Clot formation and lysis
Platelet Cone
Thrombin generation curve
• Calibrated Automated Thrombogram (CAT) assay
• Allows quantitative automated assessment of thrombin generation in platelet-rich and poor plasma
• Established pre-analytic conditions and attempted to standardize among labs (Loeffen et al, JTH 2012; Dargaud et al, Thromb Res 2012)
• Can distinguish between normal, hypo- and hypercoagulable states (better than with clotting assays)
Thrombosis
time
thro
mbin
Normal
Bleeding
Courtesy prof Hemker
Reference Value for Thrombin Generation
Table 1. Thrombin generation parameters in the reference sample and study population
Reference sample (n=1210) Study population (n=4843)
1pM TF Male (n=541) Female (n=669) p-value Male (n=2471) Female (n=2372) p-value
Lag Time [min] 5.07 (4.67/5.67) 4.67 (4.33/5.33) <0.0001 5.33 (4.74/6.07) 5.00 (4.33/5.67) <0.0001
ETP 1 [nM.min] 1047 (216) 1099 (203) <0.0001 1068 (267) 1139 (279) <0.0001
Peak Height [nM] 108 (51.0) 115 (48.7) 0.014 112.9 (51.92) 122 (56) <0.0001
5pM
Lag Time [min] 2.67 (2.33/3.00) 2.39 (2.33/2.67) <0.0001 2.67 (2.40/3.00) 2.67 (2.33/3.00) <0.0001
ETP [nM.min] 1322 (196) 1318 (212) 0.71 1352 (267) 1389 (281) <0.0001
Peak Height [nM] 236 (52.2) 259 (53.3) <0.0001 238 (59.27) 263 (67) <0.0001
The reference sample as presented was defined as apparently cardiovascular healthy subjects without history of CVDs, presence of
CVRFs or use of antithrombotic agents, oral contraceptives or hormonal replacement therapy. In addition, individuals with coagulation
abnormalities were excluded from the reference sample. Medians (interquartile range) of lag time and means (standard deviation) of
ETP and peak height are presented. P-values below 0.05 are printed bold.
Van Paridon P, et al, Wild, Spronk H. Gutenberg Health Study. 2018 Unpublished Data
TG in patients with (unexplained) bleeding (tendency)
• Hemophilia
• Unexplained bleeding: excess menstrual bleeding
• Peri-operative bleeding
• High risk for bleeding and thrombosis (ICU)
• Patients with thrombocytopenia and/or hematologic malignancy
• All patients on antithrombotic medication
Prototypic bleeding disease: hemophilia
Beltran-Miranda et al, Haemophilia 2005
Thrombin Generation and Factor VIII
Lewis SJ, et al. B J Haematol. 2007: 138; 775-782
Correlation between ETP and Bleeding
Dargaud Y, et al. Haemophilia 2018: 24; 619-627.
Personalized management of breakthrough bleeds in a patient on prophylaxis with emicizumab.
Yesim Dargaud et al. Haematologica 2018;103:e181-e183
Thrombin generation in hemophilia
• Potential to improve management of patients, in particular in complex situations (inhibitors, combined agents)
• HA: FVIII; TFPI and FV negative determinants
• HB: FIX; TFPI, AT , PS negative determinants (Chelle et al, Haemophilia 2019)
• Useful for monitoring effects of new therapeutic agents (eg emicizumab, anti-TFPI ab etc)
TG in patients with (unexplained) bleeding (tendency)
• Hemophilia
• Unexplained bleeding: excess menstrual bleeding
• Peri-operative bleeding
• High risk for bleeding and thrombosis (ICU)
• Patients with thrombocytopenia and/or hematologic malignancy
• All patients on antithrombotic medication
Szczepaniak et al, PlosOne 2015
Increased Plasma Clot Permeability and Susceptibility to Lysis Are Associated with Heavy Menstrual Bleeding of Unknown Cause: A Case-Control Study
Prevalence of hemostatic defects in subjects with unexplained heavy menstruation
Eising et al, Int Lab Hem 2018
In subjects with unexplained heavy menstruation
• Limited evidence of prolonged lagtime in relatively older women; no abnormal ETP in younger and older women. Uncertain how much TG analysis adds in diagnostic workup.
• Defining age and sex dependent normal ranges is essential
• Clot lysis may be accelerated in relatively young women
• Variable degree of platelet function defects and low vWF
TG in patients with (unexplained) bleeding (tendency)
• Hemophilia
• Unexplained bleeding: excess menstruation bleeding
• Peri-operative bleeding
• High risk for bleeding and thrombosis (ICU)
• Patients with thrombocytopenia and/or hematologic malignancy
• Patients on antithrombotic medication
1200
1600
2000
2400
2800
G2 (BL 930mL)G1 (BL 930mL)
ET
P (
nM
*min
)
(P=0.000)
1200
1600
2000
2400
2800
G2 (BL 930mL)G1 (BL 930mL)G1 (BL 930mL)
ET
P (
nM
*min
)
(P=0.000)
ET
P (
nM
*min
)
800
1200
1600
2000
2400
G2 (BL 930mL)G1 (BL 930mL)
(P=0.01)
ET
P (
nM
*min
)
800
1200
1600
2000
2400
G2 (BL 930mL)G1 (BL 930mL)G1 (BL 930mL)
(P=0.01)
T6 (post protamine adm) T1 (base-line)
Bosch, et al, J Cardiothorac Surg 2013; Bosch et al, Thromb Res 2013
TG prior to and 6 hrs following CT surgery related to bleeding
Low thrombin generation during major orthopaedic surgery fails to predict the bleeding risk in inhibitor patients treated with bypassing agents
Mancuso et al. Haemophilia, 2016,
TG in patients with (unexplained) bleeding (tendency)
• Hemophilia
• Unexplained bleeding: excess menstruation bleeding
• Peri-operative bleeding
• High risk for bleeding and thrombosis (ICU)
• Patients with thrombocytopenia and/or hematologic malignancy
• Patients on antithrombotic medication
Other high risk for bleeding populations
• ICU
• Hematologic malignancies (± thrombocytopenia)
• Use of antithrombotic agents
Clinical bleeding and thrombin generation in admissions to critical care with prolonged prothrombin time: an exploratory study • 306 patients; 101 bleeding events in 46 patients.
• Many patients with prolonged PT had endogenous thrombin potential (ETP) within the normal range (120/251 patients, 47.8%) or even elevated (8%).
• There was no suggestion by receiver operating characteristic analysis that variables of conventional TG were sensitive at predicting bleeding. No bleeding events were documented in patients defined as ETP high, despite elevated PTR.
Stanworth et al, Transfusion 2018
Thromboelastography and Thrombin Generation Assay for Bleeding Prediction in Patients With Thrombocytopenia and/or Hematologic Malignancies.
Kim et al, Ann Lab Med 2017
TG in patients with (unexplained) bleeding (tendency)
• Hemophilia
• Unexplained bleeding: excess menstruation bleeding
• Peri-operative bleeding
• High risk for bleeding and thrombosis (ICU)
• Patients with thrombocytopenia and/or hematologic malignancy
• Patients on antithrombotic medication
Cohort of vulnerable patients with CAD on DAPT
Overall bleeding complications during DAPT at T1, T2 and T3
T1, N (%) T2, N (%) T3, N (%) No bleeding 101 (80,2%) 99 (78,6%) 84 (80,0%) BARC 1 17 (13,5%) 24 (19,0%) 16 (15,2%) BARC 2 7 (5,6%) 3 (2,4%) 4 (3,8%) BARC 3 1 (0,8%) 0 (0%) 1 (1,0%) Total (N) 126 126 105 T1 = consultation 1-2 months after PCI, T2 = consultation 6 months after PCI, T3 = consultation 12 months after PCI, PCI = Percutaneous Coronary Intervention, N = number of patients, BARC = Bleeding Academic Research Consortium
No bleeding 59%
Minor bleeding
28%
Major bleeding
13%
Nobleeding
Minorbleeding
Breet, Olie et al, abstract NIV 2019
Platelet function testing in patients on DAPT; relation to outcome
Mean test results of LTA, Multiplate and VerifyNow in patients with clopidogrel and complete follow-up, patients with bleeding complications defined as BARC ≥ 1 and patients without bleeding complications with reference lines for cut-off values from prior studies.
Table 1
TG 1 and 6 months after PCI in PPP
1 month after PCI
Bleeding (N=8) Mean
(±SD)
No bleeding (N=85) Mean
(±SD)
p-value
ETP
nM*minute
s
837,3 (111,2) 1136,2 (229,6) <0.001
Peak nM 93,6 (17,0) 138,6 (42,0) 0.004
6 months after PCI
Bleeding (N=4) Median
[IQR]
No bleeding (N=64) Median
[IQR]
p-value
ETP
nM*minute
s
807,2 [608,1 – 1006,3] 1030,2 [911,2 – 1149,1] 0.072
Peak nM 88,9 [63,8 –114,0] 127,5 [104,2 –150,8] 0.039 Maj
or b
leed
ing
Non
-maj
or b
leed
ing
0
5
10
15
20
E
Tim
e t
o p
ea
k (
min
ute
s)
Maj
or b
leed
ing
Non
-maj
or b
leed
ing
0
5
10
15
D
La
g t
ime
(m
inu
tes)
Maj
or b
leed
ing
Non
-maj
or b
leed
ing
0
500
1000
1500
2000
A
ET
P (
nM
*min
)
Maj
or b
leed
ing
Non
-maj
or b
leed
ing
0
20
40
60
80
100
C
Ve
loc
ity
in
de
x (
nM
/min
ute
s)
Maj
or b
leed
ing
Non
-maj
or b
leed
ing
0
100
200
300
B
Pe
ak
(n
M)
Thrombin generation 1 month after PCI in PPP, patients with (N=8) and without (N=85) major bleeding between 1-12 months after PCI (N=93) A. ETP, B. Peak, C. Velocity index, D. Lag time, E. Time to peak. ETP = Endogenous Thrombin Potential, PPP = Platelet Poor Plasma, pM = Picomolar, N = number of patients
Breet, Olie et al, abstract NIV 2019
Platelet function testing in patients on DAPT; relation to outcome
• PRP: • Major bleeding during follow-up: decrease in ETP and peak over time
• Non-major bleeding: increase in ETP and peak over time
• ‘Delta PRP 1 and 2’: ETP (p=0,030) and peak (p=0,107)
Majo
r ble
eding
Majo
r ble
eding
Non-majo
r ble
eding
Non-majo
r ble
eding
0
500
1000
A
ET
P (
nM
*min
)
Change in thrombin generation parameters in PRP over time (measured 1 and 6 months after PCI) in patients with (N=8) and without major bleeding (N=47). A. ETP, B. Peak. Standard deviations are indicated by lines. ETP = Endogenous Thrombin Potential,
Maj
or ble
edin
g
Maj
or ble
edin
g
Non-m
ajor b
leed
ing
Non-m
ajor b
leed
ing
0
50
100
B 1 month after PCI6 months after PCI
Pe
ak
(n
M)
Breet, Olie et al, abstract NIV 2019
Conclusions 1: Potential use of thrombin generation analysis (TGA) in relation to bleeding in hemophilia
• for the clinician treating patients with hemophilia, TGA may be more sensitive to differences in hemostatic capability of severe hemophiliacs with identical levels of FVIII yet distinct bleeding phenotypes.
• ex vivo studies suggest TGAs may be a reliable tool for monitoring therapeutic effect in patients with hemophilia treated with bypassing agents, although this has yet to be confirmed in vivo.
• TGAs may have a role in the monitoring of emerging therapeutic agents although this has yet to be rigorously evaluated.
Teichman et al, Transfusion and Apheresis Sci 2018
Conclusions 2: Potential use of thrombin generation analysis (TGA) in relation to bleeding in other conditions
• To explore mechanisms
• In patients undergoing CT surgery, TGA is linked to post-operative bleeding; why?
• In patients with thrombocytopenia or double platelet inhibition, TGA in ppp suggests a “second hit” in etiology of bleeding
• Antithrombotic therapy: added value over assays that monitor drug effects?
Thank you
Renske Olie Yvonne Henskens Yvonne Bosch Henri Spronk Pauline van Paridon Philipp Wild, Mainz Synapse
Effects of Post-Trauma Idarucizumab in Animals Treated With Dabigatran
Grottke O, et al. J. Am. Coll. Cardiol. 2015; 66(13): 1518–1519.
With TG one can follow correction of DOAC associated anticoagulation with antidote; Effects of Post-Trauma Idarucizumab in Animals Treated With Dabigatran
Grottke O, et al. J. Am. Coll. Cardiol. 2015; 66(13): 1518–1519.
Potential indications for thrombin generation analysis
• Explore mechanisms of thrombosis and bleeding
• In vitro: mechanisms of cellular reactivity
• Individual:
Control treatment (eg factor replacement, anticoagulant dosing and reversal)
Estimating risk of recurrent thrombosis (in conjunction with d-dimer and clinical characteristics)
Thrombin Generation and Bleeding Risk
Bloemen S, et al. PLoS ONE. 2017; 12(5): e0176967.
Does TG in whole blood relate to bleeding in patients on oral anticoagulants (VKA)?
- 40 - Bloemen, et al, PlosOne 2017
Bridging patients from VKA to LMWH and back
-3 -2 -1 0 1 2 3 4 5
0
5 0 0
1 0 0 0
1 5 0 0
D a y
ET
P (
nM
x m
in)
E T P 1 p M
E T P 1 p M T M
E T P 5 p M
Similar patterns for PH Eijgenraam et al, Thromb Res 2016
The most important finding of our study was that ETP and D-dimer were independently associated with the risk of recurrence. Eichinger et al, Clin Chem 2008
Is the predictive quality of TG in consecutive patients after DVT increased by repetitive measurements?
Idiopathic events Recurrence Non-recurrence
B1 B2 B3 B1 B2 B3 P
LT 5.1
(3.7-6.1)
4.9
(4.1-8.3)
4.9
(4.1-8.3)
5.3
(4.7-6.3)
5.3
(4.3-6.3)
5.3
(4.3-6.3)
ns
PH 325.4*
(297.8-398)
310.3
(150.9-414.5)
257.8
(74.6-440.9)
268.9*
(222.4-326.8)
259.7
(215.5-342.8)
248.9
(176.1-325)
0.036
ETP 184.3
(171.1-
227.2)
175
(88.2-223.2)
223.2
(43.8-241.3)
183.1
(142.1-211.3)
167.5
(155.7-198.5)
164.8
(137.8-196.1)
ns
After adjustment both the idiopathic character of the event and
PH at B1 were independent predictors for recurrence,
with respective OR’s of 6.5 (95%CI 0.6-69.7) and 29.5 (95%CI 2.3-363.3).
Ten Cate-Hoek, et al, unpublished
44
80 µl PPP 80 µl PPP
20 µl PPP-Reagent (5 pM TF, 4 µM Phospholipids)
20 µl Substrate (+ CaCl2)
20 µl Substrate (+ CaCl2)
20 µl Calibrator
Record Calibrated Automated Thrombogram
CAT technique
Thrombin as key regulator of the coagulation cascade
Borissoff, Spronk, ten Cate, N Engl J Med. 2011; Ten Cate and Hemker, JAHA 2016
From traditional to innovative laboratory management? • Why?
• How?
• Any answers so far?
Why would one measure TG ?
To detect risk of thrombosis
To detect risk of bleeding
To monitor and adjust therapy
Mechanistic studies in vitro, human or animal experimental studies
From: Muller,... Spronk,... Renne T. Cell 2009.
Apixaban unbound and total concentrations.
Deborah Siegal et al. Blood Adv 2017;1:1827-1838
© 2017 by The American Society of Hematology
Study Group ETP (%) OR norm
Mega Q 1 82 0,57
Q 2 95 0,74
Q 3 105 1,26
Q 4 118 2,29
ABO O 93 0,7
Non O 106 1,2
Kyrle < median 90 0,5
> median 110 1,5
Azar INR 2.5 35 0,2
Conclusion : The more thrombin
The more thrombosis
Relation between ETP and odds ratio venous thrombosis
Courtesy, Prof Coen Hemker
Prediction of individual factor VIII or IX level for the correction of thrombin generation in haemophilic patients
Prediction of individual factor VIII or IX level for the correction of thrombin generation in haemophilic patients, First published: 29 June 2018, DOI: (10.1111/hae.13539)
A first‐in‐human study of the safety, tolerability, pharmacokinetics and pharmacodynamics of PF‐06741086, an anti‐tissue factor pathway inhibitor mAb, in healthy volunteers
Journal of Thrombosis and Haemostasis, Volume: 16, Issue: 9, Pages: 1722-1731, First published: 16 June 2018, DOI: (10.1111/jth.14207)
Prevalence of hemostatic defects in subjects with unexplained heavy menstruation
The final study group consisted of 58 females ranged in age from 40 to 60 years (median age: 48.4). They self-identified their ethnicity as Caucasian (100%).
A total of 60 Caucasian patients with unexplained HMB, who are treated between January 2010 and January 2013 with second- generation endometrial ablation (SGEA) at the gynaecology clinic of the teaching hospital of Apeldoorn (Gelre Hospitals) in the Netherlands, were invited to participate in this study. Patients did not take any drugs, estrogens, progestogens, aspirin or other non- steroidal anti-inflammatory drugs (NSAIDs) for at least 2 weeks before blood drawing.
Eising, Roest, et al, Int Lab Hem 2018
Eising, Roest, et al, Int Lab Hem 2018
Increased Plasma Clot Permeability and Susceptibility to Lysis Are Associated with Heavy Menstrual Bleeding of Unknown Cause: A Case-Control Study
Szczepaniak et al, PlosOne 2015