August 9, 2008

Relationship Between Progression to AIDS and Thrombophilic Abnormalities in HIV Infection

By Lijfering, Willem M Sprenger, Herman G; Georg, Rita R; van der Meulen, Piet A; van der Meer, Jan

BACKGROUND: HIV-infected patients are at increased risk of venous and arterial thrombosis. We hypothesized that acquired thrombophilic abnormalities that could predispose to thrombosis are most pronounced in patients in advanced stages of HIV infection. METHODS: We included 109 consecutive HIV-infected patients in the study and tested them twice for currently known thrombophilic abnormalities at an interval of at least 3 months (median, 3 months; range, 3-12 months). Detailed information was collected about the date of diagnosis of HIV infection, HIV treatment, and previous episodes of venous and arterial thrombosis.

RESULTS: After HIV infection was diagnosed, 16% of the patients experienced symptomatic thrombosis (venous, 10%; arterial, 6%). Repeated measurements established protein C deficiency in 9% of the patients, increased factor VIII concentrations in 41%, high fibrinogen concentrations in 22%, and free protein S deficiency in 60%. Median factor VIII concentrations were higher in patients with AIDS (CD4 cell counts

CONCLUSIONS: Multiple acquired and persistent thrombophilic abnormalities are more frequently observed in HIV-infected patients than in the healthy population. The frequencies of these thrombophilic abnormalities increase with the progression to AIDS. These findings may contribute to the high prevalence of venous and arterial thrombosis in HIV-infected patients.

(c) 2008 American Association for Clinical Chemistry

Several reports have documented an increased risk of venous and arterial thrombosis in HIV-infected patients (1-4). Patients with AIDS, as documented by CD4 cell counts

We studied a group of HIV-infected patients primarily to ascertain whether progression to AIDS was associated with an increased frequency and/or severity of thrombophilic abnormalities and secondarily to determine the overall risk of venous and arterial thrombosis in HIV-infected patients.

Materials and Methods


Between May 2006 and December 2006, we asked 120 consecutive HIV- infected patients treated at the outpatient clinic of our university hospital to participate in our study. The study was approved by the institutional review board of our hospital, and informed consent was obtained from all of the participants. Detailed information about the date of HIV diagnosis, HIV status, HIV treatment, previous episodes of venous and arterial thrombosis, exposure to risk factors for thrombosis, and anticoagulant treatment was retrospectively collected by physicians at the outpatient clinic via a questionnaire and review of medical records. In women, the use of oral contraceptives and their obstetric histories were also documented, considering that oral contraceptives and pregnancy are risk factors for venous thrombosis and may be associated with thrombophilic abnormalities. Clinical data were collected before laboratory testing to avoid bias in assessing clinical outcome events. To determine whether HIV status was correlated with thrombophilic abnormalities, we simultaneously collected blood samples for measurements of CD4 cell counts and HIV RNA, and for thrombophilia testing. Thrombophilia tests included those for the following: deficiencies in antithrombin, protein C, total protein S, and free protein S; factor V Leiden; the prothrombin G20210A mutation; increased concentrations of fibrinogen and factor VIII; and lupus anticoagulant. We also measured anticardiolipin antibodies and measured C-reactive protein (CRP)3 to assess the effects of acute- phase inflammatory reactions. We repeated all tests with a second blood sample collected after an interval of at least 3 months (median, 3 months; range, 3-12 months) to confirm the concentrations of proteins and CD4 cell counts obtained in the first set of measurements.


Lymphocyte subsets (CD3, CD4, CDS) were analyzed within 24 h of collection with standard flow cytometry techniques. Plasma concentrations of HIV RNA were measured with the NucliSENS HIV RNA assay (detection limit, 4 X 104 copies/L; Organon Teknika). CRP was measured by nephelometry (BN II; Dade Behring); CRP concentrations s 5 mg/L were used to identify acute-phase inflammatory reactions. We measured the activities of antithrombin (COATEST; Chromogenix) and protein C (Berichrom Protein C; Behring) with chromogenic-substrate assays and measured the concentrations of protein C and protein S with ELISAs (Dako). We defined antithrombin deficiency as a decreased antithrombin activity (3.5 g/L. Reference intervals were determined from measurements in healthy volunteers who had no personal or family history of venous thrombosis, were not pregnant, and had not used oral contraceptives during the preceding 3 months. PCR analyses were used to demonstrate factor V Leiden and prothrombin G20210A (25,26). We used 3 different phospholipid-dependent coagulation tests to screen for lupus anticoagulant: the dilute Russell viper venom time, the activated partial thromboplastin time, and tissue thromboplastin inhibition (27). Tests that produced abnormal results were repeated with a 1:1 mixture of patient plasma to plasma from a healthy individual to exclude deficiencies in coagulation factors. If the test result remained abnormal, we confirmed phospholipid dependence with a phospholipid-neutralization test. We used Gradipore reagents (LA-screen and LA-confirm) for the dilute Russell viper venom time test, Actin FSL (Dade Behring) to measure the activated partial thromboplastin time, and Thromboplastin IS (Dade Behring) in 2 dilutions (1 part reagent plus 49 parts diluent and 1 part reagent plus 499 parts diluent) to measure tissue thromboplastin inhibition. The anticardiolipin ELISA was carried out with samples diluted 1 part reagent plus 99 parts diluent in PBS (140.0 mmol/L NaCl, 9.0 mmol/L Na^sub 2^HPO^sub 4^, and 1.3 mmol/L NaH^sub 2^PO^sub 4^, pH 7.4) containing 100 mL/L fetal calf serum. We performed duplicate measurements of 9 calibrators for IgG and IgM anticardiolipin antibodies (Louisville APL Diagnostics) according to the manufacturer's instructions to prepare a calibration curve. Concentrations >/=4 x 104 IU/L were considered positive (27). Blood samples were taken from patients undergoing long-term anticoagulant treatment with vitamin K antagonists after treatment had been interrupted; nadroparin was administered subcutaneously in the meantime. DEFINITIONS

Patients were classified into 3 groups according to their HIV status (28). Patients with CD4 cell counts >5 x 10^sup 8^/L after repeated measurement were classified as having asymptomatic HIV infection; patients with CD4 cell counts between 2 x 10^sup 8^/ Land 5 x 10^sup 8^/Lwere classified as having early symptomatic HIV disease; and patients with CD4 cell counts of


Data for continuous variables are expressed as medians and ranges, and categorical data are expressed as counts and percentages. Differences between groups were evaluated with the Student t-test or the Mann-Whitney U-test, depending on whether the data were normally distributed, for continuous data and with the Fisher exact test for categorical data. A 2-tailed P value of

Because 2 blood samples were collected, we categorized our results as "single abnormality," indicating a specific result in at least one blood sample, and as "confirmed abnormality," indicating a specific result in both blood samples.

Statistical analyses were performed with SAS software (version 9.1; SAS Institute).


We asked 120 consecutive HIV-infected patients to participate in the study. Two of these patients failed to provide informed consent, 4 refused collection of a second blood sample, 2 died (one from liver cell carcinoma and another from non-Hodgkin lymphoma in combination with deep vein thrombosis), and 3 were lost to follow- up for geographic reasons. The data for the remaining 109 patients were analyzed. The median interval between HIV diagnosis and the date of study entry was 5 years (range, 0-20 years). The clinical characteristics of the patient population are summarized in Table 1. Sixty-six percent were men, and the median age at HIV diagnosis was 34 years (range, 16-73 years). Eleven patients ( 10%) were discovered to have venous thrombosis while they were HIV positive, and arterial thrombosis was found in 6 patients (6%). Seventy-five percent of the patients received highly active antiretroviral therapy (HAART). The median CD4 cell count was 4.3 x 10^sup 8^/L (range, 0.2-12.2 x 10^sup 8^/L) for the first blood sampling and 4.0 x 10^sup 8^/L (range, 0.3-12.2 x 10^sup 8^/L) for the second blood sampling, with an individual median difference of 0.6 x 10^sup 8^/L (range, 0-3.4 x 10^sup 8^/L) indicating stable CD4 cell counts during the study period. The median interval between the blood collections was 3 months (range, 3-12 months).

Table 2 summarizes the results of the thrombophilia tests. We confirmed protein C deficiency in 9% of the patients, increased factor VIII concentrations in 41%, and increased fibrinogen concentrations in 22%. After excluding patients with CRP concentrations >5 mg/L, we confirmed increased factor VIII and fibrinogen concentrations in 31% and 15% of the patients, respectively. Free protein S deficiency was demonstrated in 74% of the patients and confirmed in 60%. This result was not confounded by oral contraceptive use or pregnancy. None of the female patients used oral contraceptives, were pregnant, or were within 6 months of delivery. The use of oral contraceptives was discouraged because their interactions with antiretroviral therapy make oral contraceptives less reliable. The most frequent thrombophilk abnormalities were analyzed further. Over the entire study period, the median fibrinogen, factor VIII, and free protein S concentrations were 3.6 g/L (range, 1.9-5.5 g/L), 2260 IU/L (range, 1160-3700 IU/L), and 450 IU/L (range, 200610 IU/L), respectively, in patients with AIDS-defining illness (CD4 cell count /=2 x 10^sup 8^/L) (Table 3). The prevalences of persistent increased fibrinogen concentrations (P = 0.006), increased factor VIII concentrations (P

Table 1. Clinical characteristics of 109 HIV-infected patients.(a)

Table 2. Thrombophilic abnormalities in 109 HIV-infected patients.(a)

At more advanced stages of HIV infection (CD4 cell counts >5 x 10^sup 8^/L vs > 2-5 x 10^sup 8^/L), factor VIII concentrations were significantly higher, whereas free protein S concentrations were significantly lower (Fig. 1). The difference was less pronounced for fibrinogen concentrations and was statistically significant only when asymptomatic HIV patients were compared with AIDS patients (P = 0.027). A positive relationship was observed between increasing factor VIII and fibrinogen concentrations, whereas an inverse relationship was observed between increasing factor VIII concentrations and decreasing free protein S concentrations (Fie. 2).

Table 3. Thrombophilic abnormalities related to HIV status.(a)

The overall annual incidences of venous thrombosis and arterial thrombosis were 1.61% (95% CI, 0.81%-2.89%) and 0.87% (95% CI, 0.32%- 1.88%), respectively. The median age at the time of the first event was 45 years (range, 22-56 years) for venous thrombosis and 53 years (range, 44 -59 years) for arterial thrombosis. The median intervals between the onset of venous thrombosis and arterial thrombosis and the time of collection of the first blood sample were 3.2 years (range, 0-11 years) and 5.0 years (range, 1.7-14.1 years), respectively. In a univariate analysis, smoking, hyperlipidemia, hypertension, and diabetes mellitus were not associated with thrombophilic abnormalities.

Fig. 1. The relationship of factor VIIl, fibrinogen, and free protein S concentrations to CD4 cell counts in HIV-infected patients.

Data are presented as box-and-whisker plots. The horizontal line within each box represents the median value; the lower and the upper sides of each box represent the first and third quartile, respectively; whiskers are extended to 1.5 times the box width (the interquartile range) and connect the values outside the box within 1.5 interquartile ranges. Dashed horizontal lines represent mean reference concentrations bracketed by the upper and lower limits of reference intervals. The exclusion of results from patients with thrombosis (an asterisk indicates the median concentration) did not alter the results.

Fig. 2. Fibrinogen and free protein S concentrations in HIV- infected patients related to factor VUI (FVIII) concentrations.

Data are presented as box-and-whiskef plots. The horizontal line within each box represents the median value; the lower and the upper sides of each box represent the first and third quartile, respectively; whiskers are extended to 1,5 times the box width (the interquartile range) and connect the values outside the box within 1.5 interquartile ranges. Dashed lines indicate the mean concentration in the reference (i.e., healthy) population. Excluding patients with thrombosis did not alter the results.


We confirmed protein C deficiency in 9% of the HIV-infected patients, increased factor VIII concentrations in 41%, increased fibrinogen concentrations in 22%, and free protein S deficiency in 60%. After excluding patients with high CRP concentrations, these prevalences remained high compared with those in the healthy population, which exhibits a

Sixteen percent of our cohort of 109 consecutive patients with HIV experienced thrombosis during a median follow-up period of 5 years; venous events were documented in 10% of the patients, arterial events in 6%. The annual incidences of venous thrombosis (1.61%) and arterial thrombosis (0.87%) were 5- to 16-fold higher and 2- to 8-fold higher, respectively, than in the healthy population (i.e., 0.1%-0.3% and 0.1%~0.4%) (36-38). The median age at the onset of venous thrombosis was 45 years, 17 years earlier than the median age of onset for venous thrombosis in nonHIV- infected patients (39), and the median age for arterial thrombosis onset was 53 years, a decade earlier than that documented in the Framingham study (38). Although these results should be interpreted cautiously because of the small size of the study population, they do suggest that HIV -infected patients aie at high risk of venous and arterial thrombosis, as has also been demonstrated in other studies (1-4).

Although our study was of a relatively small number of patients, it is the largest to date that has analyzed acquired thrombophilic abnormalities in HIV-infected patients. Our finding that the development of AIDS was associated with increasing thrombophilic abnormalities may have clinical relevance. HAART is used for (long- term) immunologie reconstitution, which may improve these thrombophilic abnormalities and lead to a decreased risk of venous and arterial thrombosis. Indeed, one study has shown a decreased risk of arterial thrombosis or death in more than 36 000 HIV- infected patients who received HAART (4). Another study reported a decrease in the concentrations of von Willebrand factor, a carrier of factor VIII, after HIV-infected patients began HAART (40). Larger prospective studies that address endothelial activation markers and thrombophilic abnormalities in HW-infected patients may clarify the relationship between HIV infection and venous thrombosis and the association between venous and arterial thrombosis. Our data suggest that such a link in HIV-infected patients is plausible. Because it is not common practice to screen for thrombophilia in HIV-infected patients, the medical charts often did not provide sufficient information about CD4 cell counts in patients at the time of thrombosis, and therefore we are unable to comment on CD4 cell counts and/or thrombophilic abnormalities at the time of thrombosis in our study. Our small numbers did not enable us to compare the risk of thrombosis in subgroups. Other studies, however, have shown the risk of venous thrombosis to be highest in patients with AIDS, with an odds ratio of 29.9 (95% CI, 3.6-246.3) in patients with AIDS vs patients with non-AIDS-defining illness (5-7), whereas free protein S deficiency and increased factor VIII concentrations have been reported in 78%-100% of HIV-infected patients at the time of venous thrombosis (3 ), in apparent agreement with our findings. A further limitation of our data is that patients infected with HIV are a heterogeneous group who have higher rates of coinfections compared with the healthy population, and this factor might have contributed to thrombophilic abnormalities as well. An analysis of these variables was beyond the scope of our study.

We conclude that HiV-infected patients have a higher prevalence of thrombophilic abnormalities and more persistent thrombophilic abnormalities than the healthy population. These abnormalities increase with the development of AIDS and may contribute to the high prevalence of venous and arterial thrombosis in HIV-infected patients.

Grant/Funding Support: None declared. Financial Disclosures: None declared.

3 Nonstandard abbreviations: CRP1 C-reactive protein; Cl, confidence interval; HAART, highly active antiretroviral therapy.


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Willem M. Lijfering,1* Herman G. Sprenger,2 Rita R. Georg,2 Piet A. van der Meulen,2 and Jan van der Meer1

1 Division of Haemostasis, Thrombosis and Rheology and 2 Division of Infectious Diseases, Department of Internal Medicine, University Medical Center Groningen (UMCG), Groningen, The Netherlands.

* Address correspondence to this author at: Division of Haemostasis, Thrombosis and Rheology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Fax 31- 50-3611790; e-mail [email protected]

Received January 21, 2008; accepted March 18, 2008.

Previously published online at DOI: 10.1373/dinchem.2008.103614

Copyright American Association for Clinical Chemistry Jul 2008

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