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