Plasma available prior to the matching event was obtained for VTE patients and controls at a median of 34 days [interquartile range (IQR) 12–44] vs. 23 days (IQR 10–59), respectively; P-value was equal to 0.26. VTE patients had higher levels of markers of monocyte activation, sCD14; endothelial dysfunction and coagulation including P-selectin, thrombomodulin, and vWF; inflammation, SAA, CRP, IL-6, IL-8, TARC, TIMP-1, and D-dimer; and tissue fibrosis, hyaluronic acid (Fig. 1). There was a trend toward higher MCP-4 in VTE patients than in controls. Plasma levels of the remaining tested markers did not differ between the groups (Table, Supplemental Digital Content 1, http://links.lww.com/QAD/A121). There were no differences in biomarkers for patients on protease inhibitor-containing or, specifically, IDV-containing regimens. Thrombomodulin levels were higher in patients co-infected with hepatitis C virus (HCV) (3.71 vs. 2.61, P = 0.05), although other biomarker measurements were similar. Median biomarker levels in patients co-infected with CMV were not different from those without CMV. Reference values from healthy, HIV-negative participants are provided in Supplemental Digital Content 2, http://links.lww.com/QAD/A121.
The interval from specimen collection to VTE event was significantly correlated with D-dimer (r = −0.44, P = 0.03) and vWF (r = −0.48, P = 0.02) levels. There was no correlation with other biomarkers determined to be significant by univariate analyses.
A multivariate regression analysis was performed that included active non-HIV infection, provocation, nadir CD4 cell count, more than one lifetime opportunistic infection, albumin, and all biomarkers determined by univariate analyses to be associated with VTE. P-selectin (P = 0.002), D-dimer (P = 0.01), and hyaluronic acid (P = 0.009) were found to be independently associated with VTE risk. Alternative models including immunologic AIDS, HIV viral load, and CMV disease did not alter these findings.
The odds ratios of incident VTE were calculated for participants with baseline levels of P-selectin, D-dimer, or hyaluronic acid above the median. The odds ratios of VTE for participants with biomarker values exceeding the median value for all participants were 11.4 [95% confidence interval (CI) 3.1–42.3] for D-dimer, 7.6 (95% CI 2.3–24.9) for P-selectin, 1.8 (95% CI 0.7–3.8) for hyaluronic acid, and 11.2 (95% CI; 3.7–33.6) when both D-dimer and P-selectin were above the median (Table 4).
In this case–control study of HIV-infected patients, elevated plasma levels of P-selectin, D-dimer, and hyaluronic acid were strongly and independently linked to risk of subsequent VTE when measured prior to the event occurrence. HIV-specific risk factors including low nadir CD4 cell count, immunological AIDS, history of multiple opportunistic infections, and CMV viremia were associated with thromboembolic events, as well as ongoing, non-HIV infection and the presence of a provocation (recent hospitalization, surgery, or trauma).
In HIV-uninfected persons with a first VTE, up to half of all events are unprovoked . In comparison, roughly two thirds of first-time events in our HIV-infected population occurred without an identifiable provocation. The prevalence of non-HIV infection was high in VTE patients and their HIV-positive controls, although substantially higher in VTE patients, consistent with its known relationship to VTE in the general population. Contrary to other traditional clinical risk factors for VTE in patients without HIV, current malignancy, family history of hypercoagulability, and cardiovascular disease were not related to thrombosis in this study. The presence of a provocation or active infection in HIV-infected individuals, even in the absence of other traditional risk factors, has clinical implications for inpatient and outpatient management; extended-distance travel, surgery, hospitalization, and ongoing non-HIV infections necessitate consideration of venous thromboprophylaxis .
At the time of the matching event, the vast majority of patients and controls were receiving ART with a median duration of therapy since HIV diagnosis of 6.9 and 7.3 years, respectively. Most patients had normalized CD4 cell counts (>500 cells/μl) at the time of VTE diagnosis, which is above the threshold at which current DHHS guidelines recommend starting antiretroviral treatment . On the contrary, nadir CD4 cell count was significantly lower and multiple lifetime opportunistic infections were more prevalent among VTE patients than controls, despite a similar duration of HIV infection (9.3 vs. 10.5 years). Combined, these results support the hypothesis that earlier initiation of ART may modify the risk of venous thrombotic disease .
HIV viremia and plasma HIV-RNA levels were not associated with VTE risk in this cohort, although other studies have reported otherwise [7,10]. Persistent low-level viremia has been observed in a majority of individuals with less than 50 copies/μl  and chronic immune activation is evident even in patients with suppressed HIV viremia below detectable levels . The contribution of viral replication to observed biomarker levels and related coagulopathies cannot, thus, be precluded in this study .
Associations between D-dimer products of fibrinolysis and all-cause mortality as well as cardiovascular disease have been well established in HIV-infected and noninfected populations [27,35–38]. The relationship between latest pre-event levels of D-dimer and venous thrombotic disease is likely attributable, in part, to subclinical clot formation. Measurement of D-dimer has significant long-term predictive value in high-risk individuals and is a strategy already used to stratify risk of VTE recurrence. Furthermore, a recent meta-analysis found that timing of testing post-VTE did not affect this association . In a previous report, we linked risk of CVD in HIV to elevated D-dimer levels up to 2 years prior to the index event . Combined with our current findings, these data suggest that there may be clinical utility in D-dimer measurement among HIV-infected patients at elevated risk for venous and arterial thrombosis.
In addition to elevated D-dimer levels observed in HIV-infected participants with an impending VTE, several markers of inflammation (IL-6, CRP, and SAA) were also higher in patients compared with controls.
A hypercoagulable state often exists among patients with HIV, including a high prevalence of detectable anticardiolipin antibodies . In this study, we evaluated their role in VTE and determined that although anticardiolipin antibodies were detected more commonly in HIV-infected patients than in healthy, uninfected individuals, there was no association with VTE.
The relationship of immunodeficiency with increased coagulation factors may be due to the increased risk of opportunistic infections . In support of this explanation, we found that a history of multiple lifetime opportunistic infections and advanced immunodeficiency were associated with VTE risk in the studied population. Further, CMV viremia was more prevalent among VTE patients than controls. Active CMV infection was recently associated with hypercoagulability independent of stage of HIV disease . These findings suggest that CMV disease, rather than immunodeficiency alone, may result in a heightened inflammatory state leading to activation of the coagulation cascade in co-infected patients.
Another explanation for chronic inflammation in HIV may be related to the ability of HIV to cross the endothelial cell membrane, causing endothelial disruption via induction of intracellular biochemical changes and subsequent activation of inflammatory cascades and expression of endothelial cell adhesion molecules . To investigate the role of endothelial dysregulation in venous clot formation, we quantified circulating levels of P-selectin, vWF, TARC, and TIMP-1 and observed each to be significantly associated with VTE risk. A higher degree of immunodeficiency among VTE patients may in part explain these findings due to its association with impaired vascular function and endothelial inflammation in HIV disease [44,45].
P-selectin was found to be independently and most strongly associated with thrombosis in this cohort. Stored in endothelial cells and platelet granules, P-selectin interacts with its receptor to promote a hypercoagulable environment by inducing the generation of prothrombotic microparticles from leukocytes and upregulation of tissue factor expression on monocytes [46–49]. Prospective studies in HIV-uninfected participants with malignancies have demonstrated that P-selectin is significantly elevated in patients with an impending or acute VTE. Furthermore, P-selectin has been shown to have comparable diagnostic value to D-dimer in patients with confirmed DVTs .
Plasma levels of the soluble lipopolysaccharide (LPS) receptor sCD14 can be utilized as a marker of monocyte activation . In our cohort, we observed a significantly higher level of sCD14 in VTE patients compared with controls, suggesting that overstimulation of monocytes from increased peripheral exposure to microbial products may contribute to a prothrombotic state in chronic HIV infection. These findings are consistent with a recent study that showed an increased proportion of monocytes expressing surface procoagulants in HIV-infected patients following LPS stimulation . Contrary to expectations, we did not observe any appreciable differences in soluble TF plasma levels between groups. LPS-mediated activation of TF-dependent coagulation pathways may be confined specifically to monocyte surface TF expression.
Monocyte production of inflammatory cytokines may also be induced by hyaluronic acid degradation products through stimulation of the Toll-like receptors (TLR)-2 and TLR4 . A strong indicator of liver fibrosis in HCV co-infected  and mono-infected  individuals, increased hyaluronic acid levels are also predictive of AIDS or death . Although compared with controls, fewer VTE patients in our study were co-infected with HCV, overall hyaluronic acid levels were higher in VTE patients, and in a multivariate analysis, independently linked to VTE disease. These results suggest that hyaluronic acid-mediated tissue injury and inflammation may contribute to VTE risk in patients without other evidence of hepatic injury.
The limitations of our study include a relatively small number of events evaluated and the risk of increased type I error, given the considerable number of variables compared. However, in the multivariate analyses, three biomarkers retained significance: the associations of P-selectin, D-dimer, and hyaluronic acid with VTE remained strong, are biologically plausible, and relevant to other HIV-related adverse outcomes associated with their increased levels.
In summary, indices of endothelial dysfunction, coagulation, and tissue fibrosis were most strongly and independently associated with risk of venous thrombotic disease, although a history of multiple lifetime opportunistic infections, clinical CMV disease and CMV viremia, immunodeficiency, and the presence of an ongoing non-HIV infection or provocation were also related. Our findings suggest that unique risk factors in HIV-infected individuals exist that may contribute significantly to augmented risk of VTE disease. Furthermore, indices of endothelial dysfunction, coagulation, and tissue fibrosis that have been linked to morbidity and mortality in HIV infection were more strongly associated with VTE risk than other covariates examined in this study. Our results present a clinical impetus to further explore both their contributions to immunopathogenesis and the therapeutic role of modulating these biomarkers and associated inflammatory and coagulation pathways to potentially modify risk of noninfectious complications of HIV infection.
This study was funded by the Intramural Program of the NIH, NIAID, Critical Care Medicine Department, and with federal funds from the National Cancer Institute, NIH, under contract number HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
I.S. and C.H. designed the study, contributed to the analysis and interpretation of the data, and in writing the manuscript. L.W.M. contributed to the study design, clinical and laboratory data acquisition, data analysis and interpretation, and in writing the manuscript. V.S. and T.D.N. contributed to the acquisition of clinical data. A.R. performed laboratory studies. B.O.P. contributed to study conception and revision of the manuscript. S.R.P. provided plasma samples from HIV-negative volunteers. J.S. assisted with the study design and interpretation of statistical analyses. J.M.M. provided clinical management of patients enrolled in NIH protocols that were included in the study. All authors have reviewed and approved the final version of the manuscript for publication.
L.W.M. was a 2009–2010 participant in the Clinical Research Training Program, a public–private partnership supported jointly by the NIH and Pfizer Inc. via a grant to the Foundation for NIH from Pfizer Inc.
The authors would like to thank the study participants at the NIH and the staff of Outpatient Clinic 8 at NIAID, Ven Natarajan and Yunden Badralmaa for CMV testing, Manuel van Deventer and Tracey Bosworth for ACA panel testing, Aaron Richterman for technical assistance, as well as Catherine Rehm and McCamie DeArmon for sample identification.
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