causation versus correlation? - virology...
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Causation versus Correlation? The pathway of altered coagulation as a
pathophysiological mechanism in HIV disease
Jason Baker, MD, MS
HIV Medicine and Infectious Diseases
Hennepin Healthcare Research Institute
Associate Professor of Medicine
University of Minnesota
I have no financial relationships to disclose
Outline:
Studies linking coagulation to clinical risk among persons with HIV
Potential causal pathway for HIV-association coagulopathy and disease
Experimental interventions targeting coagulation in persons with HIV
Central questions remaining, and future directions
Associations with All-Cause Mortality
Baseline LevelOR (4th/1st QRT)
UnivariateP-value
D-dimer 12.4 <0.0001
IL-6 8.3 <0.0001
hsCRP 2.0 0.05
D-dimer Levels Rise 75%
from Pre-HIV to Post-ART
with Viral Suppression2
N=249 U.S. Military HIV Natural History
1-Kuller et al PLoS Medicine 2008;5(10):e203
2-Freiberg et al PLoS One 2016;11(4):e0152588
3-Neuhaus et. al. JID 2010; 201(12):1788
D-dimer and Risk for Non-AIDS Co-morbidities
n=3766SMART/ESPRIT/SILCAAT with HIV VL <500
Event No.
Serous Non-AIDS
(SNA) or Death260
Death (all causes) 144
Cancer 99
Liver/Renal 31
CVD 84
MI 46
Stroke 19
Cardiac Death 19
PE* 11
DVT* 3
*not included in definition of SNA
HIV Studies Linking D-dimer to Risk forEnd-Organ Diseases:
• Mortality (1-6, 8)• CVD (6-9)• Renal, Hepatic (9)• Grade 4 events (10)• Frailty Phenotype (11)
1. Kuller et. al. PLoS Med 2008;5:e203
2. Ledwaba et. al. PLoS One 2012;7:e24243
3. Hunt et. al. JID 2014;210;1228
4. Byakwaga et. al. JID 2014;210:383
5. Lee et. al. JID 2017;215:1270
6. Tenorio et. al. JID 2014;210(8):1248
7. Duprez et. al. PLoS One 2012;7:e444548. Ford et. al. AIDS 2010;24:15099. Grund et. al. PLoS One 2016;11(5):e0155100
10.Hart et. al. JAIDS 2018;77:1
11.Onen et. al. J Frailty Aging 2014;3:158
Associations for Serious Non-AIDS or
Death by D-dimer and IL-6 Levels1
Control arms of SMART/ESPRIT/SILCAAT:
n=3766 on continuous ART with HIV RNA <500
Coagulation and Inflammation Represent Overlapping yet Distinct Pathways for Disease
✓ When both IL-6 and D-dimer are
considered together, associations
with risk for non-AIDS end organ
diseases are attenuated but persist.
✓ Both IL-6 and D-dimer contribute
independent information on risk.
1-Grund et. al. PLoS One 2016;11(5):e0155100
Altered Coagulation as a Pathophysiologic Mechanism for Disease Risk among Persons with HIV Infection?
HIV Infection Coagulopathy End-Organ
Diseases
CONFOUNDERS
MEDIATORS
? ?
Coagulation with Thrombin Generation and Inhibition.
Tripodi A, Mannucci PM. N Engl J Med 2011;365:147-156
Coagulation Cascade, Thrombin Generation and Inhibition
Figure 1 from Tripodi & Mannucci, NEJM 2011;365:147
(Fibrinolysis)✓ D-Dimers
Coagulation FactorsProcoagulation:
• FII (prothrombin)• FV• FVII• FVIII• FIX• FX• Fibrinogen
Anticoagulation:• Antithrombin (AT)• TFPI• Protein C• Protein S
HIV Viremia and Coagulation Factor Composition
OFF ARTON ART with
HIV RNA <400
Start ART(VS)
Defer ART(DC)
Stop ART(DC)
Continue ART(VS)
B) Study the Effect of Starting ART
A) Baseline Comparison
of Untreated vs. Treated
RandomizeRandomize
3 Complimentary Comparisons to Study the Effects of HIV Replication
C) Study the Effect of Stopping ART
Follow-up Follow-up
J. Baker, K. Brummel-Ziedins. et. al. JAHA 2013;2:e000264
Coagulation FactorsProcoagulation:
• FII (prothrombin)• FV• FVII• FVIII• FIX• FX• Fibrinogen
Anticoagulation:• Antithrombin (AT)• TFPI• Protein C• Protein S
JAHA 2013;2:e000264
-25
0
25
50
Factors Used for Modeling Thrombin Kinetics
% D
iff.
at B
aselin
e B
etw
een P
art
icip
ants
Off
vs. O
n A
RT
Fibrinogen
0.13
AT
<0.0001
Protein C
0.001
Protein S(Total)
<0.0001
Protein S(Free)
<0.00010.79
FVFII
<0.0001
F VII
<0.0001
FVIII
<0.0001
FIX
0.94
FX
0.0002
vWF
<0.0001
TFPI
0.03
A) Baseline Comparison: Off vs. On ART
✓ Inhibits protein synthesis• transcription inhibition
• RNA cleavage
✓ Inhibits cell growth
✓ Promotes T-cell responses• CD8+ T-cell CMI
• CD4+ Th2 differentiation
✓ B-cell development
✓ Macrophage activation
1-Baker, Brummel-Ziedines et. al. JAHA 2013;2
2-Start et. al. Ann Rev Biochem 1998;67:227
Computational Modeling Indicates HIV Viremia is Associated with
Hepatocyte Protein Production and Thrombin Generation
0
10
20
30
40
50
60
70
80
90
100
0 200 400 600 800 1000 1200
Thro
mb
in (
nM
)
Time (s)
Untreated n=197
ART-treated n=475
J Bio Chem 1994;269:23367, Throm Haem 2008;6:104
Baker, Brummel-Ziedins et. al. JAHA 2013;
HIV Changes in
Plasma Factors:
f-II (prothrombin)
-- f-V f-VII f-VIII-- f-IX f-X TFPI AT-III Protein C
Mortality Risk and Imbalance of Coagulation Factor Composition Cases (n=134) and Controls (n=388) SMART/ESPRIT on ART with Viral Suppression
Coagulation Marker
D-dimer
Factor II (%)
Factor V (%)
Factor VII (%)
Factor VIII (%)
Factor IX (%)
Factor X (%)
TFPI ng/mL
Antithrombin (%)
Protein C (%)
OR (95% CI) for Mortality
4th vs. 1st Quartile
0.1 1 10
Hepatocyte
Dependent
Factors
Brummel-Ziedins et. al. Res Prac Throm Heam 2018; 2:708
✓ Mortality risk was associated with greater
estimates of thrombin generation
✓ Excess thrombin generation was accounted
for by FVIII, AT and Protein C
Adjusted for age,
gender, race, study,
and enrollment date
and country
Altered Coagulation as a Consequence of Aging
Aging Alterations in
Coagulation Biology
Clinical Risk• Arterial Thrombosis
• Venous Thrombosis
• Frailty Phenotype?
Inflammation
✓ Fibrinogen
✓ hsCRP
✓ IL-6
Procoagulant Factors
✓ FV
✓ FVIII
✓ FIX
✓ PAI-1
✓ vWF
Lack of Anticoagnt
Response
(inconsistent or no change)
✓ Protein C
✓ Protein S
✓Antithrombin
1-Wilkerson & Sane, Seminars in Throm and Hemo 2002;28(6):555
2-Walston et. al. Arch Intern Med 2002; 162:2333
Frailty Phenotype and Associations with Coagulation Activity Independent of CVD:
• D-dimer*• FVIII*• CRP*
CV Heath Study (n=4735)*Independent of CVD, DM, age, sex, race
Brenchley et. al. JCI 2004;200(6):749, Brenchley et. al. Nat Med 2006;12(12):1365, Estes,
Baker, Brenchley et. al. JID 2008;198:456, Sandler & Douek Nat Rev Micro 2012;10;655-666
Hypothesis: HIV infection damages mucosal integrity, leading to microbial
translocation (endotoxemia), activation of coagulation, and tissue injury
Am J Phy Lung Cell Mol Phy 2006;291:L307-L311 (Fig. 1)
r=0.36 P=0.002 r=0.27 P=0.025
HIV viremia and Endotoxemia Associated with TF Expression on Monocytes
Slide c/o N. Funderburg;
BLOOD 2010;115(2):161
TF Expression on Macrophages is Associated with:✓ HIV viral load
✓ LPS levels
✓ D-dimer levels
✓ sCD14 levels
Patrolling Monocytes (CD14dim/CD16+) Increase Surface Tissue Factor (TF) Expression in Response to HIV-1 Exposure
%Ti
ssu
e F
acto
r+
CD14+CD16- CD14+CD16+ CD14dimCD16+
Traditional Inflammatory Patrolling
Slide c/o N. Funderburg; BLOOD 2012;120(23):4599 LPS HIV
Altered Coagulation as a Pathophysiologic Mechanism
HIV Infection Coagulopathy End-Organ
Diseases
✓ Mortality (all-cause)
✓ Ischemic CVD
❑ Kidney, Liver,
Brain, other organs
❑ Frailty PhenotypeMEDIATORS
✓ Viral Replication
?
?
✓ Damage to Mucosal
Immunity/Integrity
Altered Coagulation
Factor Composition
Thrombin
Generation
EndotoxemiaTissue Factor Activity
(Extrinsic Pathway)
?
INF
LA
MM
AT
ION
Conceptual Model for Targeting Coagulation to Reduce Inflammation
TF activity / MΦ
Thrombin (IIa)
D-dimers
Factor Xa
TF/VIIa
Fibrin (Clot)Fibrinogen
PAR-2
PAR-1
Vascular inflammation
PAR-4
InflammationCellular/Vascular Activation
Cytokines (e.g., IL-6, TNFα)
Tissue Damage
Platelet Activation
Edoxaban
XIa
Fibrinolysis
Vorapaxar
‘ADVICE trial’
‘TACTICAL-HIV’
Key References:
Cirino & Vergnolle. Cur Opin in Pharm 2006;6:428-434
Shpacovitch et. al. J of Leuk Biology 2008;83:1309-1322
Krupiczojc, et. al. Int J of Biochem Cell Bio 2008;40:1228-1237
Delvaeye & Conway. Blood 2009;114(12):2367-2374
Baker et. al. CROI 2019 abstract
Kent et. al. Lancet HIV 2018
Coagulation activity
drives immune activation
via ’protease-activated
receptors’ (PARs)
Factor Xa is a key
activator of PARs 1 & 2
Inhibition of PAR activity
may reduce inflammation
along vascular surfaces
IXa
Screen Baseline 1 2 3 4 5 6 7 8 9 10 11 12
Blood Specimens
X X X X X X X X X X X X
Pre-Tx Follow-up Pre-Tx Follow-up
Timeline
Edoxaban 30mg daily
N=20
Edoxaban 30mg daily
N=20Placebo daily
N=20
Placebo daily
N=20
Randomize
WASH OUT
WASH OUT
Target Population: HIV+ on ART with viral suppression, and D-dimer >100 ng/mL (in FEU)
Screened
and Eligible
N=44
TACTICAL-HIV: Targeted AntiCoagulation Therapy to Reduce
Inflammation and Cellular Activation in Long-term HIV Disease
Outcomes:
Inflammation: • IL-6 (primary)
• IL-1𝛃• TNFR-1
• sCD14
• sCD163
Coagulation: • D-dimer
• TAT
Adherence and
tolerability
Bleeding and
bruising events
Percent Difference (95% CI) in Edoxaban vs. Placebo Treatment Effect
IL-6
TNF-RI
IL-1β
sCD163
sCD14
D-dimer
TAT
0.2 0.4 0.6 0.8 1 1.2 1.4
Percent Change in Biomarker on Edoxaban vs. on Placebo
TACTICAL-HIV: Inflammation and Coagulation Biomarkers
Edoxaban did not lower
inflammation or monocyte
activation biomarkers
Edoxaban significantly
reduced coagulation activity
✓ D-dimer lowered by 42%
✓ Thrombin Antithrombin (TAT)
lowered by 26%
TACTICAL-HIV: Adverse Events
Types of Bleeding or Bruising Events (n=50): bruising (n=12), bleeding gums (n=13), epistaxis (n=10), blood in stool (n=7), hematuria (n=1), lacerations (n=7)
Event
Grade
1 or 2
(N)
Grade
3 or 4
(N)
Receiving
Edoxaban
(N)
Receiving Placebo or
During Washout
(N)
E vs. P
# Events
p-value
Bleeding/Bruising 50 0 33 17 0.03
Non-Bleeding/Bruising 0 20 10 10 0.99
Total Adverse Events 50 20 43 27 0.06
Serious Adverse Events -- 2 1 1 --
DEATHS 0 -- -- -- --
ADVICE: Attenuation of D-dimer using Vorapaxar to target Inflammatory and Coagulation Endpoints
Study DesignA randomized, double-blind clinical trial of
vorapaxar (2.5mg daily) versus placebo (n=65)
Patient Population (key eligibility)
• HIV-infected adults ≥40yrs
• HIV-1 RNA <50 copies/mL for 6 months
• No protease inhibitors and/or NNRTIs
• Plasma d-dimer >200ng/mL
Outcomes (over 12 weeks)
• Safety
• Reduction in D-dimer levels
• Reduction in inflammation and immune activation (IL-6, Hs-CRP, sCD163, sCD14)
• PAR-1 expression (subset)
Randomized
N=65
Total screened
N=125
Not randomised (N=60)
Ineligible N=55
Lost to FUp prior to Rand N=4
Withdrew consent prior to
Rand N=1
placebo N=31 vorapaxar N=34
Lost to follow up
(N=1)
immediately post
randomisation prior to
any study drug
Withdrawn
consent (N=1)
Week 4
Completed week 18
N=30
Completed week 18
N=33
Primary endpoint: d-dimer
Endpoint Mean % [95%CI] change Log10 transformed
data
placebo vorapaxarDifference between
treatment groups† p-value
Baseline to week 8-12
d-dimer (ng/mL) -8·5 [-18·4, 2·5] -10·8 [-23·1, 3·4] -0·02 [-0·10, 0·05] 0·56
† Linear regression for change (log10) modelled against treatment and baseline outcome variable
0
500
1,000
1,500
d-di
mer
(ng
/mL)
0 1 4 8 12 18
Placebo Vorapaxar
Week of trial
Vorapaxar did not lower
inflammation or immune
activation measures:
✓ IL-6
✓ hsCRP
✓ sCD163
✓ sCD14
✓ PAR-1 expression on T-cells
Vorapaxar was safe with no
increase in bleeding risk
O’brien et. al.
OFID 2017;4(1): ofw278
Study Population
• HIV-infected participants on
suppressive ART for >48 weeks
Outcomes
• Change in sCD14 (primary)
• Secondary outcomes➢ Safety
➢ Serum thromboxane (estimate of
cyclooxygenase inhibition)
➢ Endothelial function, by FMD
➢ D-dimer
➢ Other immune activation markers
RESULTS: RCT of Aspirin in ART-treated persons with HIV
n=121; 1:1:1 (300mg, 100mg, placebo)
O’brien et. al. OFID 2017;4(1):ofw278
Reduction in
Thromboxane B2
No Tx Effect on:✓ sCD14
✓ sCD163
✓ IL-6
✓ D-dimer
✓ KT ratio
✓ T-cell activation
✓ T-cell exhaustion
✓ Monocyte
phenotypes
12 week follow-up
Altered Coagulation as a Pathophysiologic Mechanism?
HIV Infection Coagulopathy End-Organ
Diseases
?
→ How can we address the limitations in the epidemiologic
data, including the potential for reverse causality
INFLAMMATION✓ Profile of Coag. Factors
✓ Tissue Factor Activity
Thrombin
Generation
Utilizing Genetic Variants and Mendelian Randomization (MR) Methods to Support Causal Inference
Gene Variant Biomarker Level Clinical Disease
Rationale: Inheritance of a genetic variant is independent from (or random with respect to)
inheritance of other traits that may confound associations with clinical risk.
Thus, addressing:
✓Unresolved confounding
✓Selection Bias
✓Reverse Causality
CONFOUNDERS
e.g., D-dimer e.g., CVD & non-CVD outcomese.g., ‘SNP’
Mendelian Randomization Methods Applied to Coagulation Markers
Circ. Genom. Precis. Med. 2018;11(1):e001956
Age (Dordr). 2015;37(4):9820
Genetic
Variant
Genetic
Variant
ETP*
*ETP = Endogenous Thrombin Potential
vWF
FVIII
D-dimer
PAI-1
tPA
Genetic
Variant
Genetic
Variant
Genetic
Variant
Genetic
Variant
Ischemic HD
Ischemic HD
Ischemic HD
Not supportive
of causality for
Ischemic HD,
but still predict
clinical risk
LDLc
LDLc
Independent
causality not
supported
✓ Causal inferenceD-dimer
Fibrinogen
PAI-1
Genetic
variant
Genetic
variant
Genetic
variant
Not supportive
of causality for
neurocognitive
dysfunction
vWFGenetic
variant
Lack of significance persisted when
restricted to those age >50
Causal Pathways for Diseases may Differ between Different Populations, and
Experimental Designs are Essential to Study Multiple Pathways
Population-C
Intervention-2
Candidate
Surrogate
MarkerPopulation-A
Intervention-1
Clinical
Endpoint
Candidate
Surrogate
MarkerPopulation-BClinical
Endpoint
Candidate
Surrogate
Marker
Clinical
Endpoint
1-Fleming & DeMets. Annals Internal Medicine 1996
2-Patel et. al. NEJM 2011;365:883
3-Eikelboom et. al. NEJM 2017;377:1319
4-Zannad, et. al. NEJM 2018;379(14):1332
EXAMPLE: Rivaroxaban
A) Prevents stroke among those with atrial fibrillation2
B) Prevents MI/stroke among those with stable CAD3
C) Does not prevent MI/stroke among those with HF4
→ Thrombin-mediated events are not the major
driver of events among those with HF (below)
Primary Outcome:
myocardial infarction, stroke, death from any cause
Intervention-1
Kidney Lung Brain
Extensive Clot Formation in Multiple Organs from SIV+ Macaques Consistent
with Thrombotic Microangiopathy (TMA) as a Mechanisms for Disease
Blood. 120: 1357-1366, 2012
In Summary: Is Altered Coagulation a Pathophysiologic Mechanism?
HIV Infection Coagulopathy End-Organ
Diseases?
What’s Needed?:
1) Basic science on mechanisms and the connection to end-organ pathology
2) Clinical science on phenotype of coagulopathy in setting of contemporary ART regimens
✓ Identify target populations at excess risk, and candidate surrogate markers
✓ Determine the influence of age (e.g., ‘interaction’) on HIV-associated alterations in coagulation
3) Interrogate the causal pathway from mechanisms of coagulopathy through clinical risk
✓ experimental designs (i.e., RCTs)
✓ Interventions with established safety profiles
✓ leverage HIV cohort/trials with genomic and proteomic data
→ The causal relationship between HIV-related coagulopathy and disease risk remains unclear
✓ Profile of Coag. Factors
✓ Tissue Factor ActivityThrombin
Generation
Acknowledgments:
Hennepin Healthcare Research Institute• Jon Klaphake• Kelly Garcia-Myers• Bill Lundberg• Hill Wang• Keith Henry
University of Minnesota• Jim Neaton• Birgit Grund• Shweta (Sharma) Mistry• Tim Schacker• Julian Wolfson• Tess Peterson
Colleagues• Andrew Phillips• Jens Lundgren• Sean Emery• Tony Kelleher• Stephen Kent• Matthew Freiberg
Funding• R01AG045032, NIA• R01HL126542, NHLBI• UM1AI120197, NIAID• R21HL137435, NHLBI• 17IRG33350064, AHA
Research Participants, Our Program, and Collaborators
NIH/NIAID• Irini Sereti
University of Vermont• Russ Tracy• Kathleen Brummel-Ziedins
University of North Carolina• Nigel Key• Micah Moobery
University of Cape Town• Mpiko Ntseke• Ntobeko Ntusi• Graeme Meintjes