*2.1.2. Hypercoagulate state - Primary prothrombotic abnormalities boosted by direct effect of HIV*


especially in the setting of ART. It is not sufficient alone to cause thrombosis, but it may add an additional risk among patients with other risk factors for venous clots.

222 Immunodeficiency

*of HIV* 

surgery, DIC).

found with thromboses.

arterial smooth muscle cells [**2**].

apoptotic CD4 lymphocytes.

than that documented in the Framingham study.

In a Nederland cohort [**4**] of 109 patients with HIV, the annual incidences of venous and arterial thrombosis were 5- to 16-fold higher and 2- to 8-fold higher, respectively, than in the healthy population. 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 non-HIV- infected patients; and the median age for arterial thrombosis onset was 53 years, a decade earlier

*2.1.2. Hypercoagulate state - Primary prothrombotic abnormalities boosted by direct effect* 

• Protein S (PS) deficiency (27-76%): decreased synthesis by the endothelial cells, hepatocytes and megakariocytes injured in HIV infection; antibodies to PS and low levels of circulating free antigen; tumor necrosis-factor alpha (TNF α) can lower the levels of active protein S, down-regulating the protein S synthesis in the endothelial

• Protein C (PC) deficiency (0-14%): altered synthesis and metabolism of PC, as well as low-grade disseminated intravascular coagulation (DIC) with consumptive coagulopathy, in the setting of HIV infection with severe immunosuppression[**5**].

• Antithrombin deficiency: decreased protein synthesis (liver diseases and malnutrition), protein-losing nephropathies or enteropathies, consumptive states (malignancy,

• Antiphospholipid antibodies: Anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) have been reported in HIV-infected patients with a prevalence ranging from 7 to 94% and 0 to 72%, respectively. They have been implicated with antiphospholipid syndrome features (mainly avascular bone and cutaneous necrosis) or playing a limited role beside the multifactorial origin of HIV-related thrombosis [**7**]. HIV infection induces destruction of CD4 lymphocytes, which leads to polyclonal stimulation of B cells and hypergammaglobulinemia, resulting in antibodies against damaged endothelial cells (phospholipids exposed) and inhibition of protein S synthesis [**5**]. It is thought that LA activity in these patients might be an epiphenomenon secondary to chronic immune stimulation in HIV infection and no pathogenic correlation has been

• Tissue Factor (TF): TF expression on circulating monocytes has been postulated in the setting of driving immune activation by translocation of microbial products through the damaged gut. In addition, HIV- associated gp120 causes release of TF via activation of

• Microparticles (MP): The MP are generated from endothelial cells, platelets and

• HIV-associated autoimmune haemolytic anemia has been related with an increased risk

• Homocysteine: Mild to moderate hyperhomocysteinemia (11-29%), related to a homozygous C677T mutation of the methylenetetrahydrofolate reductase gene [**8**],

of thromboembolism during the acute phase of hemolysis [**5**].

cells; and loss of protein S in urine in HIV-related nephropathy[**5**].

• Presence of Factor V Leiden (activated protein C resistance)[**6**].

• Endothelial dysfunction: A dysfunctional venous endothelium, induced by HIV, may express heparin cofactor II deficiency and increased amounts of P-selectin, von Willebrand factor, TF, plasminogen activator inhibitor-1 and factor V, all of which may promote blood clotting. Biomarkers of endothelial dysfunction [**9**] (P-selectin, D-dimer and hyaluronic acid), coagulation, and tissue fibrosis may help identify HIV-infected patients at elevated risk of venous thromboembolism (VTE). Other endothelial dysfunction biomarker is asymmetric dimethylarginine (ADMA), a competitive inhibitor for endothelial nitric oxide synthase (eNOS) which seems to be related to an increased immune activation pathways in HIV-1 infection [**10**].

In addition, intravenous drug use (IDU) can induce endothelial injury [**11**]. From a Danish HIV cohort [**12**], the 5-year risk of VTE was 8.0% [95% confidence interval (CI) 5.78-10.74%] in IDU HIV-infected patients, 1.5% (95% CI 1.14-1.95%) in non-IDU HIVinfected patients and 0.3% (95% CI 0.29-0.41%) in the population comparison cohort. In non-IDU HIV-infected patients, adjusted incidence rate ratios (IRRs) for unprovoked and provoked VTE were 3.42 (95% CI 2.58-4.54) and 5.51 (95% CI 3.29- 9.23), respectively, compared with the population comparison cohort. In IDU HIVinfected patients, the adjusted IRRs were 12.66 (95% CI 6.03-26.59) for unprovoked VTE and 9.38 (95% CI 1.61-54.50) for provoked VTE. Low CD4 cell count had a minor impact on these risk estimates, while ART increased the overall risk (IRR 1.93; 95% CI 1.00-3.72).

• High density lipoprotein (HDL). A prospective study from South Africa [**13**] compared thrombotic profiles of 30 HIV-positive and 30 HIV-negative patients with acute coronary syndrome (ACS). Patients with HIV were younger; and besides smoking (73% vs 33%) and low HDL (0.8 ± 0.3 vs 1.1 ± 0.4), they had fewer traditional risk factors. Thrombophilia was more common in HIV-positive patients with lower protein C (PC; 82 ± 22 vs 108 ± 20) and higher factor VIII levels (201 ± 87 vs 136 ± 45). Patients with HIV had higher frequencies of aCL 47% vs 10%) and antiprothrombin antibodies (87% vs 21%).

In addition to promoting cholesterol efflux from lipid-filled macrophages (foam cells) and reverse cholesterol transport, HDL protects low density lipoprotein (LDL) from oxidation and decreases expression of adhesion molecules on endothelial cells (including E-selectin and sICAM-1). HDL also improves endothelial function via stimulation of nitric oxide synthase activity, increases prostacyclin production by endothelial cells and inhibits endothelial TF expression, all of which enhance endothelium-dependent vasodilation and down-regulate thrombotic pathways. In this way, HDL possesses anti-inflammatory and antithrombotic properties. A crosssectional study [**14**] was designed to assess large and small high density lipoprotein particle (HDLp) concentrations in persons with untreated HIV infection. Lower small HDLp (primarily responsible for HDL's anti-inflammatory properties and inhibition of endothelial activation) concentrations and higher IL-6, sICAM-1 and D-dimer levels were found. The relationship of these markers to HIV-mediated atherosclerotic risk requires further study. The Strategies for Management of AntiRetroviral Therapy (SMART) trial demonstrated a 60% increased relative risk for cardiovascular disease (CVD) with a strategy of CD4+ cell count–guided interruption of ART, and adverse changes in HDL after stopping ART may explain some of the excess CVD risk. IL-6 and D-dimer levels increased after discontinuation of ART, and this was associated with increases in HIV RNA levels. In addition, baseline HDLp, but not low density lipoprotein particle (LDLp), predicted CVD risk in SMART.

HIV-Infected Patients and Potential Impact on Thrombotic Events 225

and beta2-glycoprotein I). Furthermore, cytomegalovirus induces thrombotic events via endo- thelial damage [**5**]. In a cross-sectional study [**16**] of 104 consecutive HIV- infected patients, active cytomegalovirus infection (defined as patients who had anti cytomegalovirus antibody levels above the 75th percentile, > 209IU/ml), was associated with hypercoagulability independently of stage of HIV disease. It was observed higher levels of anticoagulant factors (antithrombin and total protein S levels) and higher procoagulant factors (factor VIII and fibrinogen levels), with a balance shifted to a procoagu- lant state. The majority of thrombosis have been associated with gastroin-

Kaposi´s sarcoma and non-Hodgkin lymphoma (B cell), via abnormalities of lymphatic flow and stasis; and anal and cervical carcinoma, via production of procoagulants, invasion of

Hospitalization in the past 3 months, was the risk factor more strongly associated with





The Nederland cohort of 109 patients with HIV, previously referred above [**4**], showed that ART may improve thrombophilic abnormalities and lead to a decreased risk of

thrombosis in patients with HIV/AIDS, in a case-control study by Ahonkhai et al. [**1**].

vascular space or secretion of vascular permeability factors from tumor cells [**5**].

testinal-related disease [**3**].

**2.4. Hospitalization** 

**2.5. Therapy** 

[**2**].

deficiency.

infections.

introduction of the ART.

venous and arterial thrombosis.

*2.3.2. HIV-associated malignancy* 

#### **2.2. HIV disease state**

#### *2.2.1. CD4 cell count*

CD4 cell count at the time of the thrombotic event, is the strongest predictor in some multivariate models. Protein C and protein S deficiencies [**5**] and increased von Willebrand factor and fibrinogen concentrations [**4**], have been correlated with immunossuppresion, evidenced by reduced CD4 cell counts. Although the frequency of thrombosis is higher in the presence of lower CD4, there are reports of thrombosis occurring with higher CD4 [**15**].

#### *2.2.2. Viral load*

High HIV RNA level is predictive of progression of HIV infection and higher risk of thrombosis.

*The frequencies of thrombophilic abnormalities (described above), increases with the progression to AIDS* [**2**].

#### **2.3. Comorbidities - Secondary prothrombotic abnormalities**

#### *2.3.1. Infections*

A link between infection and thrombosis via endothelial activation [**2**] has been suggested, through up-regulation of some cytokines (the same ones that activate the coagulation system and appear during HIV infection), such as TNFα, IL-1, IL-6, factor VIII and fibrinogen, as well as down-regulation of fibrinolytic proteins, protein C (consumption as an antiinflammatory mediator) and free protein S concentrations. Deficiency of free protein S appears during an acute inflammatory process, such as opportunistic infections: C4b binding protein is increased up to 400% of its typical concentration [**4**] and binds free protein S, which is the active component as anticoagulant[**5**].

HIV-associated infections, including syphilis, *Pneumocystis jiroveci, Mycobacterium tuberculosis and avium intracellulare* [**3**] and hepatitis C [**5**], have been related to the induction of antiphospholipid antibodies (molecular mimicry between infectious agent and beta2-glycoprotein I). Furthermore, cytomegalovirus induces thrombotic events via endo- thelial damage [**5**]. In a cross-sectional study [**16**] of 104 consecutive HIV- infected patients, active cytomegalovirus infection (defined as patients who had anti cytomegalovirus antibody levels above the 75th percentile, > 209IU/ml), was associated with hypercoagulability independently of stage of HIV disease. It was observed higher levels of anticoagulant factors (antithrombin and total protein S levels) and higher procoagulant factors (factor VIII and fibrinogen levels), with a balance shifted to a procoagu- lant state. The majority of thrombosis have been associated with gastrointestinal-related disease [**3**].

#### *2.3.2. HIV-associated malignancy*

Kaposi´s sarcoma and non-Hodgkin lymphoma (B cell), via abnormalities of lymphatic flow and stasis; and anal and cervical carcinoma, via production of procoagulants, invasion of vascular space or secretion of vascular permeability factors from tumor cells [**5**].

#### **2.4. Hospitalization**

Hospitalization in the past 3 months, was the risk factor more strongly associated with thrombosis in patients with HIV/AIDS, in a case-control study by Ahonkhai et al. [**1**].

#### **2.5. Therapy**

224 Immunodeficiency

**2.2. HIV disease state** 

occurring with higher CD4 [**15**].

*2.2.1. CD4 cell count* 

*2.2.2. Viral load* 

*2.3.1. Infections* 

thrombosis.

*AIDS* [**2**].

endothelial activation) concentrations and higher IL-6, sICAM-1 and D-dimer levels were found. The relationship of these markers to HIV-mediated atherosclerotic risk requires further study. The Strategies for Management of AntiRetroviral Therapy (SMART) trial demonstrated a 60% increased relative risk for cardiovascular disease (CVD) with a strategy of CD4+ cell count–guided interruption of ART, and adverse changes in HDL after stopping ART may explain some of the excess CVD risk. IL-6 and D-dimer levels increased after discontinuation of ART, and this was associated with increases in HIV RNA levels. In addition, baseline HDLp, but not low density

CD4 cell count at the time of the thrombotic event, is the strongest predictor in some multivariate models. Protein C and protein S deficiencies [**5**] and increased von Willebrand factor and fibrinogen concentrations [**4**], have been correlated with immunossuppresion, evidenced by reduced CD4 cell counts. Although the frequency of thrombosis is higher in the presence of lower CD4, there are reports of thrombosis

High HIV RNA level is predictive of progression of HIV infection and higher risk of

*The frequencies of thrombophilic abnormalities (described above), increases with the progression to* 

A link between infection and thrombosis via endothelial activation [**2**] has been suggested, through up-regulation of some cytokines (the same ones that activate the coagulation system and appear during HIV infection), such as TNFα, IL-1, IL-6, factor VIII and fibrinogen, as well as down-regulation of fibrinolytic proteins, protein C (consumption as an antiinflammatory mediator) and free protein S concentrations. Deficiency of free protein S appears during an acute inflammatory process, such as opportunistic infections: C4b binding protein is increased up to 400% of its typical concentration [**4**] and binds free

HIV-associated infections, including syphilis, *Pneumocystis jiroveci, Mycobacterium tuberculosis and avium intracellulare* [**3**] and hepatitis C [**5**], have been related to the induction of antiphospholipid antibodies (molecular mimicry between infectious agent

lipoprotein particle (LDLp), predicted CVD risk in SMART.

**2.3. Comorbidities - Secondary prothrombotic abnormalities** 

protein S, which is the active component as anticoagulant[**5**].


The Nederland cohort of 109 patients with HIV, previously referred above [**4**], showed that ART may improve thrombophilic abnormalities and lead to a decreased risk of venous and arterial thrombosis.

In other study [**19**], the incidence of VTE in patients with HIV in the post - protease inhibitor era (after 1996) was higher than in HIV patients before 1996. However, the higher incidence since 1996 is small, probably not clinically significant, and not necessarily because of protease inhibitors.

HIV-Infected Patients and Potential Impact on Thrombotic Events 227

[25] In a previous published letter, we described 9 patients with HIV infection and thrombotic event, defined as thrombosis involving an artery or a deep or splanchnic vein.

All of them were heterosexual patients, and one of them was an intravenous drug user. Median age was 38 years (range, 35-58 years). Seven of them were male. Only one patient had a family history of thrombosis (stroke). Four patients were coinfected with hepatitis C virus. The most frequent Centers for Disease Control and Prevention (CDC) HIV stage was 3 [**26**]. There were neither AIDS-related malignancies or autoimmune disorders nor concurrent opportunistic infections at the time of the thrombotic event. Six patients were on ART, and only two patients were undergoing protease inhibitor therapy. Basic coagulation assays were unremarkable and only one patient was evaluated for hypercoagulate state and







**3. Case reports** 

thrombosis.

high viral load, without ART.

Here, we included 2 patients more as follows.

was found to have low protein C and S values.

thrombosis associated with a catheter placement.

antithrombin-III (enteral loss); she was not receiving ART.

shunt was performed and the patient survived and went well.

probability ventilation/perfusion lung scanning, in another one.

Moreover, another study [**20**] analyzed levels of von Willebrand factor, D-dimer and factor VIII, in 160 HIV-infected and homosexual patients with a median age of 46 years, of whom 92% were male, 70% using ART, 74% Caucasian, 11% African American, 9% Hispanic, and 6% Asian. Significant lower levels of these parameters were observed in HIV-infected patients on ART compared to patients not on ART. Significant lower levels of protein C and free protein S, and increased activated protein C sensitivity ratio (APCsr) were found in the HIV-infected patients not on ART. However, *although the prevalence of coagulation abnormalities was lower in HIV- infected patients using ART, a considerable proportion of HIV - infected patients on ART showed endothelial cell activation and increased APCsr, suggestive of a persistent procoagulant state.* 

Immune depletion contributes to HIV – related inflammation, and ADMA concentrations decrease in patients with HIV infection under ART, slowing down endothelial activation. However, ADMA levels (or changes) did not consistently correlate with HIV RNA levels (or changes) [**21**].

Otherwise, some drugs from ART show more favourable profile related to decrease immune activation. Switching from ritonavir - boosted protease inhibitors to raltegravir (integrase inhibitor), appeared to de- crease biomarkers of inflammation (high sensitivity C reactive protein, osteoprotegerin, IL-6, TNFα, insulin and Ddimer) [**22**].


The data referred above, support the hypothesis that HIV-infected individuals are more likely to have clinically detected thromboembolic disease as opposed to non-HIV-infected individuals. One study performed by Malek et al. [**23**] revealed up to a 43% increase in developing a pulmonary embolism (PE), 10% increase in developing a deep venous thrombosis (DVT), and 40% increase in developing PE or DVT, in an HIV-infected individual over the 9-year study period. This increase differed by age group, with age group 21 to 50 years having the highest odds for PE among HIV+ individuals (OR, 1.58; 95% CI, 1.54-1.63).

Also, other study by Kiser et al. [**24**], showed patients with HIV had higher rate of VTE, being younger than 50 years (3.31% vs 0.53% in age-matched healthy controls, p < 0.0001), had a CD4(+) cell count less than 200 cells/mm³, or a diagnosis of acquired immunodeficiency syndrome.

Anyway, many authors propose the need to perform long-term, prospective studies assessing the factors associated with thrombotic events in patients with HIV.

#### **3. Case reports**

226 Immunodeficiency

dimer) [**22**].

[**3**].

1.54-1.63).

immunodeficiency syndrome.

necessarily because of protease inhibitors.

*increased APCsr, suggestive of a persistent procoagulant state.* 

correlate with HIV RNA levels (or changes) [**21**].

In other study [**19**], the incidence of VTE in patients with HIV in the post - protease inhibitor era (after 1996) was higher than in HIV patients before 1996. However, the higher incidence since 1996 is small, probably not clinically significant, and not

Moreover, another study [**20**] analyzed levels of von Willebrand factor, D-dimer and factor VIII, in 160 HIV-infected and homosexual patients with a median age of 46 years, of whom 92% were male, 70% using ART, 74% Caucasian, 11% African American, 9% Hispanic, and 6% Asian. Significant lower levels of these parameters were observed in HIV-infected patients on ART compared to patients not on ART. Significant lower levels of protein C and free protein S, and increased activated protein C sensitivity ratio (APCsr) were found in the HIV-infected patients not on ART. However, *although the prevalence of coagulation abnormalities was lower in HIV- infected patients using ART, a considerable proportion of HIV - infected patients on ART showed endothelial cell activation and* 

Immune depletion contributes to HIV – related inflammation, and ADMA concentrations decrease in patients with HIV infection under ART, slowing down endothelial activation. However, ADMA levels (or changes) did not consistently

Otherwise, some drugs from ART show more favourable profile related to decrease immune activation. Switching from ritonavir - boosted protease inhibitors to raltegravir (integrase inhibitor), appeared to de- crease biomarkers of inflammation (high sensitivity C reactive protein, osteoprotegerin, IL-6, TNFα, insulin and D-


The data referred above, support the hypothesis that HIV-infected individuals are more likely to have clinically detected thromboembolic disease as opposed to non-HIV-infected individuals. One study performed by Malek et al. [**23**] revealed up to a 43% increase in developing a pulmonary embolism (PE), 10% increase in developing a deep venous thrombosis (DVT), and 40% increase in developing PE or DVT, in an HIV-infected individual over the 9-year study period. This increase differed by age group, with age group 21 to 50 years having the highest odds for PE among HIV+ individuals (OR, 1.58; 95% CI,

Also, other study by Kiser et al. [**24**], showed patients with HIV had higher rate of VTE, being younger than 50 years (3.31% vs 0.53% in age-matched healthy controls, p < 0.0001), had a CD4(+) cell count less than 200 cells/mm³, or a diagnosis of acquired

Anyway, many authors propose the need to perform long-term, prospective studies

assessing the factors associated with thrombotic events in patients with HIV.

[25] In a previous published letter, we described 9 patients with HIV infection and thrombotic event, defined as thrombosis involving an artery or a deep or splanchnic vein. Here, we included 2 patients more as follows.

All of them were heterosexual patients, and one of them was an intravenous drug user. Median age was 38 years (range, 35-58 years). Seven of them were male. Only one patient had a family history of thrombosis (stroke). Four patients were coinfected with hepatitis C virus. The most frequent Centers for Disease Control and Prevention (CDC) HIV stage was 3 [**26**]. There were neither AIDS-related malignancies or autoimmune disorders nor concurrent opportunistic infections at the time of the thrombotic event. Six patients were on ART, and only two patients were undergoing protease inhibitor therapy. Basic coagulation assays were unremarkable and only one patient was evaluated for hypercoagulate state and was found to have low protein C and S values.

