1. Center for Vaccine Research, University of Pittsburgh
Anticoagulant therapies alleviate SIV-associated hypercoagulation, immune activation and inflammation
Presenting Author: Kevin Raehtz
Primary Author: Dr. Tianyu He
Center for Vaccine Research, University of Pittsburgh

2. Background
Cardiovascular disease remains one of the leading non-AIDS causes of death among chronically HIV-infected subjects, fueled by a hypercoagulable state along with persistent immune activation and inflammation (IA/INFL).
Ixolaris (N=5)
20 ug/kg/day, s.q., SID
Vorapaxar (N=5)
1/2 tablet (1.04 mg)/animal, oral, SID
80 days
112 days
Dabigatran (N=5)
150 mg/animal, oral, BID
Necropsy
Control (N=8)
No treatment
Dabigatran
Cardiovascular disease remains one of the leading non-AIDS causes of death among chronically HIV-infected subjects, fueled by a hypercoagulable state along with persistent immune activation and inflammation (IA/INFL). We previously demonstrated that in vivo treatment with the tissue factor (TF) inhibitor (Ixolaris) alleviates both IA/INFL and hypercoagulation. We also showed in vitro that TF and its induced downstream thrombin production tie IA/INFL and hypercoagulation via protease-activated receptor (PAR) signaling. We hypothesized that anticoagulant therapies targeting these pathways could break the vicious cycle of hypercoagulation and IA/INFL and reduce the risk of cardiovascular comorbidities in HIV infection.
In this study, we experimentally administered a thrombin inhibitor (Dabigatran) and a PAR-1 inhibitor (Vorapaxar) to SIV-infected pigtail macaques (PTMs), and compared efficacy with prior treatment with the TF inhibitor (Ixolaris) adminisitered to the same species. Five adult male PTMs were used for each of these experimental groups, with administration of the anticoagulants starting on the day of infection. Eight untreated SIV-infected PTMs were used as controls. Although the treatments were maintained for various periods of time, all parameters assessed in this study were compared at the same timepoints (10, 42, and 80 dpi). At each of these timepoints, which we designated as acute, setpoint and chronic stages of infection, we measured coagulation markers and levels of IA/INFL.
*ixolaris was a novel drug with limited supply so we had to be conservative with treatment
*Vorapaxar was model to follow ixolaris
** Dabigatran has shown to be potentially associated with cardiovascular events, which is why we maintained treatment until necropsy
*** Different treatment periods due to different drug availibility.
Vorapaxar

3. Results
The anticoagulant therapies resulted in different extents of reduction in hypercoagulation, immune activation and inflammation markers.
All three anticoagulant therapies resulted in a different extent of reduction in hypercoagulation, marked by lower levels of D-dimer, sTF, and sP-selectin compared with controls after SIV infection. Treated PTMs also showed lower levels of CRP, and proinflammatory cytokines and chemokines. Meanwhile, treated PTMs had fewer activated CD4 T cells, as shown by reduced HLA-DR and CD38 expression, along with lower monocyte activation, as marked by reduced CD80 expression.
The complete profile of all the makers measured in this study was not fully presented here due to time constraints, but can be found on my poster. All three anticoagulant therapies had beneficial effects in reducing hypercoagulation, immune activation and inflammation, although it is clear from this data that Ixolaris has the most potent effect. Ixolaris also had the most consistent and significant effect throughout the course of SIV infection. Futhermore, Ixolaris-treated PTMs had a better survival rates than untreated PTMs, with no progression to AIDS within 100 days post infection.
*normally, 2 out of 5 PTMs progress to AIDS within 100 days; Ixolaris treatment, 0 out of 5 PTMs died within 100 days (no rapid progression)
**The other two groups did have rapid progresion

4. Results
Markers of hypercoagulation and platelet activation significantly correlate with immune activation and inflammation markers.
In this study, we also correlated biomarkers of hypercoagulation with markers of IA/INFL, and identified any significant correlations between these parameters. Here in this correlogram, each dot represents the correlation between a pair of markers, with only significant Spearman correlations (p< 0.05) being shown, and the larger, darker dots indicating stronger correlations.
Overall, multiple markers of hypercoagulation and platelet activation significantly correlate with IA/INFL markers. Some of the strongest correlations are listed in the lower left corner. For example, coagulation marker PF4 is highly correlated with the proinflammatory cytokine IL-17. The same strong correlation with IL-17 was found for sICAM-1 and sTF. These two makers also positively correlated with sCD40 ligand.

5. Conclusions
Our results confirm the strong relationship between hypercoagulation and IA/INFL, and suggest that the use of anticoagulation therapies in the context of HIV/SIV infection may be beneficial.
While all three anticoagulant treatments had positive effects in reducing IA/INFL, Ixolaris was clearly superior. This indicates that the further upstream an intervention can target, the greater the impact on both coagulation and IA/INFL.
*CD4 and VLs were not altered, but infection is highly pathogenic
*Can’t recommend translation to humans yet because don’t know long term effects of drugs and haven’t tested in parallel with ART
*Could not compare different tissues between animals because they received different experimental designs (timing, treatment duration, etc)
*Experiments were fettered by the availability of drugs

6. Acknowledgement Funding Pandrea/Apetrei lab NIAID
Dr. Ivona Pandrea
Dr. Cristian Apetrei
Dr. Tianyu He
Dr. George Haret-Richter
Tammy Dunsmore
Dr. Cuiling Xu
Dr. Egidio Brocca-Cofano
Dr. Elizabeth Falwell
Dr. Paola Sette
Gabriella Padovani
Kathryn Martin
NIAID
Dr. Irini Sereti
Dr. Ivo Francischetti
Dr. Bruno Andrade
Dr. Melissa Schechter
Dr. Dongying Ma
Dr. Alan Sher
Dr. Kevin Tosh
Dr. Amrit Singh
Dr. Virginia Sheikh
Funding
Funding for this study was provided by grants from the National Institute for Health (NIH): RR025781, DK108837, R01HL117715, R01HL123096, R01DK113919, R01AI to I. Pandrea and C. Apetrei.
University of Vermont
Dr. Russel Tracy
University of Lisbon
Dr. Ruy Ribeiro
With this, I’d like to thank everyone in the Pandrea/Apetrei lab….
And our collaborators at NIH, in particular Drs. Irini Sereti, Ivo Francischetti and Bruno Andrade, for assistance in the TF inhibitor study and providing Ixolaris for the experiments. We would also like to thank Dr. Russel Tracy from University of Vermont for his insight in the study design and helpful discussion, as well as Dr. Ruy Ribeiro from University of Lisbon for help with statistical analysis. And lastly, our funding source…