Diagnostic approach to hemostasis disorders Hugo ten Cate ... · TG in patients with (unexplained)...

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