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Comparison of pathological clotting using haematological, functional and morphological investigations in HIV-positive and HIV-negative patients with deep vein thrombosis

Abstract

Background

Patients infected with the human immunodeficiency virus (HIV) are more prone to systemic inflammation and pathological clotting, and many may develop deep vein thrombosis (DVT) as a result of this dysregulated inflammatory profile. Coagulation tests are not routinely performed unless there is a specific reason.

Methods

We recruited ten healthy control subjects, 35 HIV negative patients with deep vein thrombosis (HIV negative-DVT), and 13 HIV patients with DVT (HIV positive-DVT) on the primary antiretroviral therapy (ARV) regimen-emtricitabine, tenofovir and efavirenz. Serum inflammatory markers, haematological results, viscoelastic properties using thromboelastography (TEG) and scanning electron microscopy (SEM) of whole blood (WB) were used to compare the groups.

Results

The DVT patients (HIV positive and HIV negative) had raised inflammatory markers. The HIV positive-DVT group had anaemia in keeping with anaemia of chronic disorders. DVT patients had a hypercoagulable profile on the TEG but no significant difference between HIV negative-DVT and HIV positive-DVT groups. The TEG analysis compared well and supported our ultrastructural results. Scanning electron microscopy of DVT patient’s red blood cells (RBCs) and platelets demonstrated inflammatory changes including abnormal cell shapes, irregular membranes and microparticle formation. All the ultrastructural changes were more prominent in the HIV positive-DVT patients.

Conclusions

Although there were trends that HIV-positive patients were more hypercoagulable on functional tests (viscoelastic profile) compared to HIV-negative patients, there were no significant differences between the 2 groups. The sample size was, however, small in number. Morphologically there were inflammatory changes in patients with DVT. These ultrastructural changes, specifically with regard to platelets, appear more pronounced in HIV-positive patients which may contribute to increased risk for hypercoagulability and deep vein thrombosis.

Clinical implications

  • HIV-positive patients do have a hypercoagulable profile compared to HIV-negative patients.

  • Increased inflammation is present in patients with DVT.

  • Ultrastructural analysis, using the scanning electron microscope, allows a more detailed coagulation profile, especially in HIV-positive patients.

Background

The prevalence of HIV and Acquired Immune Deficiency Syndrome (AIDS) is a world-wide pandemic. The Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates that 1 million AIDS-related deaths occurred during 2016 [1] and 1.7 million (1.4 million–2.3 million) people were newly infected with HIV by the end of 2018 (http://www.unaids.org/en/resources/fact-sheet). Although treatment of the infection with antiretroviral regimes (ARVs) is essential to addressing the pandemic, the condition is characterized by a large plethora of additional conditions associated, and also co-existing with the HIV infection, including the presence of systemic inflammation. Systemic inflammation is associated with an increase in circulating pro-inflammatory biomarkers, and is closely associated with an amplified propensity to form pathological blood clots (which is associated with hypercoagulability or an over-activated coagulation system) [2,3,4,5,6,7,8].

During HIV infection, various circulating inflammatory biomarkers, including cytokines interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF)-α and also other pro-inflammatory biomarkers are present [9]. An increase in these biomarkers are also present in cardiovascular disease [10, 11] and it is therefore not surprizing that HIV positive individuals are known to have an increased presence of cardiovascular complications [12, 13], including an increased risk to develop atherosclerosis and venous thromboembolic disease [14] and also DVT [15,16,17]. The presence of DVT is also classified as a systemic inflammatory process [18], and associated with pathological clotting and upregulated circulating inflammatory biomarkers [19].

The prevalence of developing a DVT in HIV positive individuals is increased two to ten times compared to the general population [20]. HIV positive individuals also have a 43% increase in age-adjusted odds ratio for pulmonary embolism, a common complication of DVT, compared to HIV negative individuals [16]. Multiple coagulation abnormalities have been reported in HIV positive patients such as decreased levels of protein C and S; and increased levels of von Willebrand factor [21,22,23]. However, the association of these abnormalities with DVT is not always consistent [21, 24]. Coagulation investigations are therefore not performed routinely in patients with HIV infection. Standard coagulation investigations are also not performed routinely as part of the management in patients with DVT, with the exception of a D-dimer which is used to assist with the diagnosis [25,26,27].

In the current paper, we therefore study the haematological profiles, including clotting and various inflammatory markers, in the presence of DVT in HIV positive and HIV negative individuals and compare the results to that of healthy individuals. We compare inflammatory markers for iron (iron saturation, transferrin and serum ferritin), fibrinogen, high-sensitive C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and haematology analyser results, together with viscoelastic properties of whole blood (WB) and platelet poor plasma (PPP). We also looked at ultrastructure of platelets and erythrocytes/red blood cells (RBCs) (using whole blood smears) with the SEM, as well as after thrombin was added to whole blood, to study clot structure.

Materials and methods

The aim was to compare the inflammatory and haematological profile of HIV patients with DVT to HIV negative patients with DVT. An analytical and descriptive prospective case control study was used from 2 Pretoria academic hospitals, Kalafong Provincial Tertiary and Steve Biko Academic Hospital, both from urban South Africa. Ten healthy control subjects, 35 HIV negative patients with DVT (HIV negative-DVT), and 13 HIV patients with DVT (HIV positive-DVT) on the primary ARV regimen-emtricitabine, tenofovir and efavirenz—were recruited for the study. For each individual, five blood tubes of venous blood were drawn (this included ethylenediamine tetraacetic acid, buffered tri-sodium citrate and serum separator tubes). The Research Ethic Committee, Faculty of Health Sciences, University of Pretoria, South Africa approved the study (Ethics reference number: 547/2015). Inclusion criteria for the healthy individuals were known HIV negative status and no medical history of any chronic diseases. Patients were included in the research groups if they had confirmed HIV status and confirmed symptomatic DVT on doppler ultrasound or comparative imaging, such as a venogram or computerised tomography scan. In order to minimise opportunistic infections as a confounding factor, HIV positive patients were only included with a World Health Organization Clinical stage 1 and CD4+ cells greater than 170 absolute number (per mm3). Exclusion criteria for the healthy individuals and for DVT patients include smoking, pregnancy‚ or the use of any inflammatory-, anticoagulant-, antiplatelet-, hormone replacement- or oral contraceptive- medication.

Inflammatory marker analysis

Serum iron (total iron in blood) levels were measured together with iron saturation, transferrin (iron binding protein) and serum ferritin (iron storage form). Serum iron levels were measured by a modification of the automated AAII-25 colorimetric method. Fibrinogen (quantitative measurement of functional fibrinogen by automated coagulation analysers), CRP (measured by latex-enhanced nephelometry) and ESR (measured by an automated ESR analyser) levels were also assessed.

Haematology analysis

A haematology analyser (Advia 2120i, Siemens Healthcare) was used to do full blood counts, and the analysis included white cell count (and its differential count), RBC count, haemoglobin level, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC), as well as platelet count and mean platelet volume (MPV).

Viscoelastic properties of whole blood and platelet poor plasma using thromboelastography

Citrated WB, as well as PPP were used. Whole blood, collected in a citrate tube, was centrifuged to obtain PPP (15 min at 3000g). Whole blood was used to assess the full coagulation process, while PPP was used to assess coagulation without the influence of platelets and RBCs on the viscoelastic properties of the clot. Calcium chloride was added to either WB or PPP and 7 different parameters measured, which included: reaction time (R-time), kinetic time (K-time), alpha angle, maximum amplitude (MA), maximum rate of thrombus generation (MRTG), time to maximum rate of thrombus generation (TMRTG) and total thrombus generation (TTG).

Ultrastructure of platelets and red blood cells (RBCs)

The ultrastructure of platelets and RBCs were studied after preparing whole blood smears for scanning electron microscopy (SEM). 10 µl of WB was placed directly on a glass microscope slide, followed by fixing in 2.5% glutaraldehyde, dehydration (as per usual SEM preparation) [28] and mounting. Micrographs were taken with Zeiss Crossbeam 540 Field Emission Gun Scanning Electron Microscopy.

Statistical analysis

Statistical analyses were performed on GraphPad Prism 5. All data were subjected to one-way ANOVA analysis. A post-test to compare groups was performed using Tukey’s multiple comparison test. Values of significance stated at P < 0.05.

Results

Table 1 demonstrates the demographics of the study.

Table 1 Demographics

Inflammatory marker and haematological parameter analysis

Inflammatory marker analyses are shown in Table 2 and haematology analysis are shown in Table 3. Markers with no available results were excluded. The HIV negative-DVT group appeared to have anaemia when compared to the control group, but when adjusted for gender the HIV negative-DVT group still had haemoglobin mean values within the normal reference ranges for male and females, respectively‚ and only the females had decreased serum iron, transferrin, and ferritin levels. The HIV positive-DVT group had anaemia and‚ when adjusted for gender‚ demonstrated low haemoglobin levels for both males and females (furthermore the females also had decreased red cell count, haematocrit, mean cell volume and mean cell haemoglobin concentration), as well as decreased levels of serum iron and transferrin in both genders. The changes in serum iron, transferrin and ferritin in the HIV positive-DVT group reflects low systemic iron status, but the raised serum ferritin (although not statistically significant) may be due to the inflammatory status of the individuals.

Table 2 Analysis of inflammatory markers using one-way ANOVA with Tukey’s multiple comparison test
Table 3 Analysis of haematological markers using one-way ANOVA with Tukey’s multiple comparison test

Inflammation is reflected, whether from the DVT or the HIV infection, by the raised CRP and ESR. Surprisingly, the platelet count was not decreased in the HIV positive-DVT group. We expected this parameter, as well as the MPV to be markedly decreased, due to, e.g. HIV thrombocytopaenia, which is usually common amongst HIV patients, but in our sample this was not the case.

Thromboelastography

Table 4 shows a comparison of the WB and PPP TEG results between the various groups. The WB and PPP, in the HIV negative-DVT and HIV positive-DVT groups, are suggestive of clot hypercoagulability and it is reflected by a rapid R-time, K-time, MRTG and TMRTG. However, with regard to the WB, only the R-time and TMRTG in the HIV-DVT group compared to the control group; and only the TMRTG in the HIV negative-DVT group compared to the control group were statistically significant. The PPP only demonstrated a statistically significant difference with the K-time in both the DVT groups (HIV positive and HIV negative) compared to the control group. Interestingly, there were no significant differences in hypercoagulability between the HIV positive-DVT and the HIV negative-DVT groups.

Table 4 TEG results of WB and PPP using one-way ANOVA with Tukey’s multiple comparison test

Scanning electron microscopy

SEM micrographs of representative healthy RBCs and platelets are shown in Fig. 1, while Figs. 2 and 3 show SEM of RBCs and platelets in HIV negative-DVT and HIV positive-DVT patients.

Fig. 1
figure1

Scanning electron microscopy micrographs of a comparison with representative healthy RBCs from other studies [8], b representative healthy platelets (see arrows) and RBCs from the current study, c, d after addition of thrombin to whole blood, where fibrin fibres are formed over the discoid RBCs with no cellular distortion (from the current study)

Fig. 2
figure2

Representative scanning electron microscopy micrographs of RBCs from HIV negative-DVT patients. a Whole blood with thrombin, showing RBC entrapped in fibrin matter, b RBCs agglutinated to each other (no thrombin), c higher magnification showing agglutinated plasma proteins attached to the RBC membrane and d a hyperactivated platelet

Fig. 3
figure3

Representative scanning electron microscopy micrographs of RBCs and platelets from HIV positive-DVT patients. a RBC with pathological membrane; b, c whole blood with thrombin, showing RBCs trapped in dense matted fibrin deposits and d a hyperactivated platelet

Discussion

Both DVT groups (HIV negative and HIV positive groups) had parameters suggesting anaemia compared to the control group (Table 3). However, the RBC count, Hb and Hct levels in the HIV negative-DVT group, even though lower than the control group, are still within the normal reference ranges [29]. The Hb and Hct in the HIV positive-DVT group were significantly lower than both the HIV negative-DVT and control groups; and lower than the normal reference ranges indicating an anaemia. Anaemia is commonly found in HIV positive patients but the cause of the anaemia is not always clear [30,31,32,33,34,35]. An inadequate erythropoietin feedback mechanism is suspected to be a major contributor in HIV-related anaemia [30]. A low reticulocyte count is commonly found with associated polychromasia (abnormally high number of immature RBCs), indicating a possible under-producing bone marrow [30, 36,37,38]. Other factors that contribute to HIV-associated anaemia, includes intestinal malabsorption, autoimmune haemolysis, bone marrow malignancies, blood loss and opportunistic complications [30, 32, 38, 39]. Even with the decreased RBC count, Hb and Hct levels in the HIV negative-DVT group (as compared to the control group), there were no significant differences with MCV, MCH as well as MCHC (Table 3). The changes in the HIV negative-DVT group may support an anaemia typically associated with inflammation, also known as anaemia of chronic disorders [30, 40].

The RCDW is the coefficient of variation of RBC volume. The higher the value, the more anisocytosis (unequal RBC sizes) present. The RCDW of the HIV positive-DVT group was greater than the control and HIV negative-DVT groups (Table 3). A raised RCDW is commonly associated with a decrease in haemoglobin and MCV concentration; but with a raised CRP, fibrinogen and white cell count [41]. This correlates with the haematological and inflammatory markers found in the HIV positive-DVT group (Tables 2, 3). RCDW is strongly associated with mortality. Patel and colleagues reported the all-cause mortality risk increases by 22% for every 1% increase in RCDW. Furthermore, the physiological association between RCDW and mortality has been hypothesised to be related to the systemic factors involved in inflammatory conditions and oxidative stress which affects erythrocyte maturation and degradation [41,42,43].

The ESR is the extent in which erythrocytes sediment in 1 h [44]. The ESR in both the HIV negative-DVT and HIV positive-DVT groups were raised compared to the control group (Table 2). In inflammatory conditions the ESR rises as the erythrocytes become sticky and adhere to each other which can be seen as rouleaux formation [45,46,47].

Fibrinogen, a high molecular weight plasma protein, is a crucial factor in the coagulation pathway (factor I). Increased fibrinogen levels are associated with thrombo-embolic events. Fibrinogen also has a role in inflammation as it tends to adhere to the membrane receptors of cells involved with inflammation. Fibrinogen can adhere to the RBCs, which becomes “heavier” resulting in an increased ESR and blood viscosity [48,49,50,51,52,53,54,55]. The fibrinogen levels were greater (but not statistically significant) in the HIV negative-DVT and HIV positive-DVT groups compared to the control group (Table 2) which correlates with the raised ESR levels seen in both groups. Increase fibrinogen concentration in the inflammatory response can explain the raised fibrinogen concentration in the HIV positive-DVT group which contributed to the DVT. The concurrent use of ARV medication has not been shown to affect the fibrinogen concentration [56]. The HIV negative-DVT group with a raised fibrinogen concentration is either due to the DVT resulting in the inflammatory response (and the raised fibrinogen concentration) or the raised fibrinogen concentration contributing to the DVT.

The WCC in the HIV positive-DVT group was statistically significantly raised compared to the control group (Table 2), although still within the normal reference range. Similar to patients with ischemic strokes, thromboembolism results in an inflammatory reaction with raised leukocyte count and CRP [57, 58]. CRP, like the leukocyte count, is an important indicator of inflammatory conditions [59]. HIV is usually associated with a decreased immune function. The elevated leukocyte count in these HIV positive-DVT patients may be due to the HIV infection itself or to opportunistic infections, regardless of whether the patient has a DVT or not. Also, with the ARV treatment HIV is suppressed and the lymphocytes, particularly the CD4 lymphocytes, increase in turnover [60,61,62]. The concentration of CRP is increased by proinflammatory cytokines, interleukin 1 and 6 [63]. The HIV negative-DVT group has an inflammatory response to the DVT which is reflected by the statistically significantly raised CRP levels compared to the control group (Table 2). The same argument can be made for the raised CRP in the HIV positive-DVT group, however the CRP concentration (as well as fibrinogen) is commonly raised in HIV positive patients compared to the general population even without a DVT [64,65,66,67,68]. The raised CRP in HIV positive-DVT patients (Table 2) indicates a sustained acute phase response [66]. This was statistically significant in the HIV positive-DVT group compared to the control group. The CRP in the HIV positive-DVT group was almost double compared to the HIV negative-DVT group. Increasing CRP concentrations has been reported with HIV disease progression, and this increase does not appear to be affected by ARV treatment [63]. Previously it was noted that increased levels of CRP and fibrinogen are independently highly predictive of 5 year mortality risk in HIV positive patients, especially where the CD4 count is low [69, 70].

Considering all the inflammatory markers (WCC, fibrinogen, CRP and ESR), each marker was statistically significantly raised in the HIV positive-DVT group compared to the control group, with the exception of fibrinogen (Table 2). In the HIV negative-DVT group compared to the control group, only CRP was statistically significantly raised. CRP may therefore be a more sensitive acute phase marker to differentiate an inflammatory condition between DVT patients (HIV negative and HIV positive) compared to healthy subjects. Interestingly, no inflammatory marker was statistically significantly raised in the HIV positive-DVT group compared to the HIV negative-DVT group.

The transferrin, serum iron and iron saturation levels reflects the amount of iron in the body. Transferrin is a plasma protein that transports iron in the blood [62], whereas ferritin is an intracellular structure capable of storing iron atoms. The concentration of serum ferritin is related to the reticuloendothelial iron stores [71]. Serum ferritin and iron concentrations are also indicators for acute phase responses to inflammation [71], although serum ferritin appears to be a better marker of inflammation than iron status [72].

Iron deficiency may be a contributor to anaemia in the HIV positive-DVT group in keeping with a low MCH and MCHC, although this is not reflected with the MCV which was within the normal reference range (Table 3) [73, 74]. A low serum iron and transferrin level seen in the HIV positive-DVT group, but with a raised ferritin level (as compared to the control group), can be explained by an immunologically altered iron metabolism where the body has adequate or increased iron stores but is unable to utilize those stores [32, 37, 60, 61, 71, 75,76,77]. This functional iron deficiency can be considered a host defence mechanism by withholding iron from possible pathogens [78, 79]. However, as iron is required for normal immune function, iron deficiency can also increase the risk of infection [79].

Although the inflammatory RBC changes have been documented in non-communicable diseases, there are only a few reports of communicable diseases, specifically HIV, and the effect on RBCs and the coagulation system [80,81,82,83,84,85]. Multiple abnormal RBC shape changes and membrane abnormalities were noted in the patients with DVT (HIV negative and HIV positive groups) (Fig. 1 to 3). During inflammatory diseases, RBCs exposed to oxidative stress and inflammatory molecules undergoes biochemical membrane changes which can result in biophysical shape changes and eryptotic cells [28, 86,87,88,89,90,91,92,93]. Eryptosis is a co-ordinated suicidal death of the red blood cells, similar to apoptosis, that allows for the removal of defective, infected or potentially harmful cells before they undergo haemolysis [94,95,96,97,98]. The abnormal RBCs present with an abnormal expression of phosphatidylserine, a cell membrane lipid, on the external membrane layer. RBCs that display phosphatidylserine also contribute to the hypercoagulation state and they provide a prothrombotic surface for the formation of thrombin [40, 96, 99,100,101,102,103,104,105,106,107,108,109,110]. Membrane vesicle formation and microparticle shedding (microscopic extracellular membranous structures) were also seen in both DVT groups. RBC-derived microvesicles or microparticles, is known to be associated with the expression of phosphatidylserine [111]. RBC-derived microparticles appear to enhance thrombin generation resulting in a hypercoagulable state, such as in post transfusion DVT, sickle cell disease and haemolytic anaemia [112, 113]. As the microparticle presence might also be associated with increased thrombin presence, the complement system can therefore also be activated and thereby enhance the systemic inflammatory response which is also a hypercoagulable state [114]. Microparticles are also thought to originate from CD4 lymphocytes [115]. As the HIV virus infects CD4 lymphocytes, HIV positive patients may be more prone to developing microparticles and therefore enhancing the hypercoagulable state.

Whole blood with thrombin SEM analysis showed the incorporation of RBCs into the fibrin network. The incorporation of RBCs into the fibrin network stabilises and strengthens the clot by decreasing the permeability of fibrin and increasing the resistance to fibrinolysis [4, 116, 117]. Healthy (discoid) RBCs in netted fibrin fibers are shown Fig. 1c, d. However, in our HIV negative-DVT and HIV positive-DVT groups, the RBCs are trapped in a matted fibrin fiber network. During inflammation, fibrin fibres tend to increase in diameter and assume a matted rather than a netted appearance; while their viscoelasticity may also be influenced by the RBC inclusion in the fibrin network [99]. Also, under conditions of low partial pressure of oxygen, acidosis and in response to mechanical deformation, RBCs release ATP and ADP activating platelets and promoting aggregation and release of platelet granules [113]. This can happen as part of the HIV and DVT pathology. The (hyper) activation of platelets, together with an abnormal matted fibrin network, contracts the clot containing the trapped pathological RBCs into a tight package (Fig. 3b, c). The result is the formation of polyhedrocytes, which is commonly found in DVT [118].

Platelet functioning depends on the quality and the quantity of the platelets [119]. Platelet count is a measure of the number of platelets in a volume of blood. Thrombocytopenia (low platelet count) is a common finding in HIV positive patients, whether it be due to increased destruction or decrease production of platelet cells [56]. However, in this study both the HIV negative-DVT and HIV positive-DVT groups had a non-statistically significant increase in the platelet count (Table 3). It should be noted that platelet count is not always associated with an increased risk of DVT [120]. The mean platelet volume measures the average size of platelets in the blood and is a common platelet activation marker [120,121,122,123,124,125,126,127]. An elevated MPV is associated with low-grade inflammation as well as thrombosis [128]. However, both HIV negative-DVT and HIV positive-DVT groups had a decrease in the mean platelet volume compared to the control group (Table 3). These results may be in keeping with a venous thrombosis where the thrombus is due to activation of the coagulation cascade instead of platelets [129]. It should also be kept in mind that platelets shape and volume do vary, resulting in changes in MPV, even in healthy persons [127]. Together with these results, the ultrastructure of platelets in the HIV positive-DVT group also have features different to that of the control group and the HIV negative-DVT group (Figs. 1b, d, 3d). The control group and the HIV negative-DVT group have (hyper) activated platelet aggregates with smooth intact membranes, pseudopodia formation, openings of the open canalicular channels and membrane blebbing interspersed among smooth intact membranes. These are the typical morphological features of activated platelets seen in healthy individuals [130]. The HIV positive-DVT patients have activated platelet aggregates which are also seen to have the same features; but with the addition of shrivelled aggregates with irregular membranes, torn membrane surface and shedding of procoagulant vesicles. These features are suggestive of apoptosis, cell death, as was seen in the red blood cells. Similar ultrastructural changes in HIV patients were documented by Pretorius et al. in 2008 [80]. These ultrastructural changes may be due to altered viral infected megakaryocyte morphology or due to direct infection and damage by the HIV virus. The HIV virus may gain entry into the platelets by undergoing phagocytosis or through the openings of the open canalicular system channels [80, 131, 132]. Regardless of the way of entry, platelets containing the HIV virus are activated. It is not clear if the platelets containing the virus facilitates viral replication and spreading; or assists in clearance of the virus [131]. With the latter, the ultrastructural changes may be due to the immune response of the body resulting in antibody-induced destruction of the platelets [80]. Antiretroviral medication has been shown to have platelet related effects such as a decreased prevalence of HIV associated thrombocytopaenia but may have an increased bleeding risk [133]. This increased bleeding risk may be explained by the ultrastructural changes seen on the SEM. It is possible that the use of different ARV combinations may result in different morphological changes observed, however to standardize the results, only patients on the primary regimen were recruited for this study.

Our TEG analysis compared well and supported our ultrastructural results (Table 4). The HIV negative-DVT group compared to the control group showed significant differences with regards to TMRTG, while the HIV positive-DVT group compared to the control group, showed significant differences with regards to R-time, and the TMRTG. According to Pretorius and colleagues not all the parameters need be abnormal to indicate pathological coagulability and the degree of coagulability can be related to the number of parameters that are abnormal [5].

Both DVT groups (HIV negative and HIV positive), using whole blood, indicate a hypercoagulable profile that has a rapid initiation and amplification, resulting in the rapid formation of thrombin. The TEG parameters in the HIV positive-DVT group compared to the HIV negative-DVT group indicate a hypercoagulable profile but there were no statistical significance in any of the parameters. Both the HIV negative-DVT and HIV positive-DVT groups have rapid R-times and K-times (Table 4) and trapped RBCs between a matted (hypercoagulable) fibrin network were noted. Considering that pulmonary embolism can result in up to 10% mortality [134] and half of the patients with DVT may have non-symptomatic (silent) pulmonary embolism [135], the changes found on the TEG and SEM may provide for a risk of assessment of the DVT complicating to pulmonary embolism. [136].

Conclusion

Although there were trends that HIV-positive patients were more hypercoagulable on functional tests (viscoelastic profile) compared to HIV-negative patients, there were no significant differences between the 2 groups. The sample size was, however, small in number. Morphologically there were inflammatory changes in patients with DVT. These ultrastructural changes, specifically with regard to platelets, appear more pronounced in HIV-positive patients which may contribute to an increased risk for hypercoagulability and deep vein thrombosis.

Availability of data and materials

The dataset(s) supporting the conclusions of this article are available from the authors.

Abbreviations

ARV:

Antiretroviral therapy

AIDS:

Acquired immune deficiency syndrome

CRP:

C-reactive protein

DVT:

Deep vein thrombosis

ESR:

Erythrocyte sedimentation rate

Haem:

Haematological

Hb:

Haemoglobin

HIV:

Human immunodeficiency virus

IL:

Interleukin

K-time:

Kinetic time

MA:

Maximum amplitude

MCH:

Mean corpuscular haemoglobin

MCHC:

Mean corpuscular haemoglobin concentration

MCV:

Mean corpuscular volume

MPV:

Mean platelet volume

MRTG:

Maximum rate of thrombus generation

Plt:

Platelet count

PPP:

Platelet poor plasma

RBCs:

Red blood cells

RCC:

Red cell count

RCDW:

Red cell distribution width

R-time:

Reaction time

SEM:

Scanning electron microscopy

TEG:

Thromboelastography

TMRTG:

Time to maximum rate of thrombus generation

TNF:

Tumor necrosis factor

TTG:

Total thrombus generation

UNAIDS:

Joint United Nations Programme on HIV/Acquired Immune Deficiency Syndrome

WB:

Whole blood

WCC:

White cell count

References

  1. 1.

    Harding R. Palliative care as an essential component of the HIV care continuum. Lancet HIV. 2018;5(9):e524–30.

    PubMed  Google Scholar 

  2. 2.

    de Villiers S, Swanepoel A, Bester J, Pretorius E. Novel diagnostic and monitoring tools in stroke: an individualized patient-centered precision medicine approach. J Atheroscler Thromb. 2015;23:493–504.

    PubMed  Google Scholar 

  3. 3.

    Page MJ, Bester J, Pretorius E. The inflammatory effects of TNF-alpha and complement component 3 on coagulation. Sci Rep. 2018;8(1):1812.

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Pretorius E, Lipinski B. Thromboembolic ischemic stroke changes red blood cell morphology. Cardiovasc Pathol. 2013;22(3):241–2.

    PubMed  Google Scholar 

  5. 5.

    Pretorius E, Swanepoel AC, DeVilliers S, Bester J. Blood clot parameters: thromboelastography and scanning electron microscopy in research and clinical practice. Thromb Res. 2017;154:59–63.

    CAS  PubMed  Google Scholar 

  6. 6.

    van Rooy MJ, Duim W, Ehlers R, Buys AV, Pretorius E. Platelet hyperactivity and fibrin clot structure in transient ischemic attack individuals in the presence of metabolic syndrome: a microscopy and thromboelastography study. Cardiovasc Diabetol. 2015;14:86.

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Bester J, Matshailwe C, Pretorius E. Simultaneous presence of hypercoagulation and increased clot lysis time due to IL-1β, IL-6 and IL-8. Cytokine. 2018;S1043–4666(18):30007–13.

    Google Scholar 

  8. 8.

    Bester J, Pretorius E. Effects of IL-1β, IL-6 and IL-8 on erythrocytes, platelets and clot viscoelasticity. Sci Rep. 2016;6:32188.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Wada NI, Jacobson LP, Margolick JB, Breen EC, Macatangay B, Penugonda S, et al. The effect of HAART-induced HIV suppression on circulating markers of inflammation and immune activation. AIDS (London, England). 2015;29(4):463–71.

    CAS  PubMed Central  Google Scholar 

  10. 10.

    Siti HN, Kamisah Y, Kamsiah J. The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review). Vascul Pharmacol. 2015;71:40–56.

    CAS  PubMed  Google Scholar 

  11. 11.

    Khambhati J, Engels M, Allard-Ratick M, Sandesara PB, Quyyumi AA, Sperling L. Immunotherapy for the prevention of atherosclerotic cardiovascular disease: promise and possibilities. Atherosclerosis. 2018;276:1–9.

    CAS  PubMed  Google Scholar 

  12. 12.

    Teer E, Essop MF. HIV and cardiovascular disease: role of immunometabolic perturbations. Physiology (Bethesda, Md). 2018;33(1):74–82.

    CAS  Google Scholar 

  13. 13.

    Deeks SG, Tracy R, Douek DC. Systemic effects of inflammation on health during chronic HIV infection. Immunity. 2013;39(4):633–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Baker JV, Brummel-Ziedins K, Neuhaus J, Duprez D, Cummins N, Dalmau D, et al. HIV replication alters the composition of extrinsic pathway coagulation factors and increases thrombin generation. J Am Heart Assoc. 2013;2(4):e000264.

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Vululi ST, Bugeza S, Zeridah M, Ddungu H, Openy AB, Frank M, et al. Prevalence of lower limb deep venous thrombosis among adult HIV positive patients attending an outpatient clinic at Mulago Hospital. AIDS Res Ther. 2018;15(1):3.

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Malek J, Rogers R, Kufera J, Hirshon JM. Venous thromboembolic disease in the HIV-infected patient. Am J Emerg Med. 2011;29(3):278–82.

    PubMed  Google Scholar 

  17. 17.

    Louw S, Jacobson BF, Buller H. Human immunodeficiency virus infection and acute deep vein thromboses. Clin Appl Thromb Hemost. 2008;14(3):352–5.

    PubMed  Google Scholar 

  18. 18.

    Reiter M, Bucek RA, Koca N, Dirisamer A, Minar E. Deep vein thrombosis and systemic inflammatory response: a pilot trial. Wien Klin Wochenschr. 2003;115(3–4):111–4.

    CAS  PubMed  Google Scholar 

  19. 19.

    Mosevoll KA, Johansen S, Wendelbo Ø, Nepstad I, Bruserud Ø, Reikvam H. Cytokines, adhesion molecules, and matrix metalloproteases as predisposing, diagnostic, and prognostic factors in venous thrombosis. Front Med. 2018;5:147.

    Google Scholar 

  20. 20.

    Aquila AM. Deep venous thrombosis. J Cardiovasc Nurs. 2001;15(4):25–44.

    CAS  PubMed  Google Scholar 

  21. 21.

    Kiser KL, Badowski ME. Risk factors for venous thromboembolism in patients with human immunodeficiency virus infection. Pharmacotherapy. 2010;30(12):1292–302.

    CAS  PubMed  Google Scholar 

  22. 22.

    Jacobson MC, Dezube BJ, Aboulafia DM. Thrombotic complications in patients infected with HIV in the era of highly active antiretroviral therapy: a case series. Clin Infect Dis. 2004;39(8):1214–22.

    PubMed  Google Scholar 

  23. 23.

    Jong E, Louw S, Meijers JC. The hemostatic balance in HIV-infected patients with and without antiretroviral therapy: partial restoration with antiretroviral therapy. AIDS Patient Care STDS. 2009;23(12):1001–7.

    PubMed  Google Scholar 

  24. 24.

    Aboulafia D. An update on HIV-associated venous thromboembolism in the era of highly active antiretroviral therapy. J Coagul Disord. 2010;2:49–56.

    Google Scholar 

  25. 25.

    Goldhaber SZ, Bounameaux H. Pulmonary embolism and deep vein thrombosis. Lancet. 2012;379(9828):1835–46.

    PubMed  Google Scholar 

  26. 26.

    Di Nisio M, van Es N, Büller HR. Deep vein thrombosis and pulmonary embolism. Lancet. 2016;388(10063):3060–73.

    PubMed  Google Scholar 

  27. 27.

    Büller HR, ten Cate-Hoek AJ, Hoes AW, et al. SAfely ruling out deep venous thrombosis in primary care. Ann Intern Med. 2009;150(4):229–35.

    PubMed  Google Scholar 

  28. 28.

    Pretorius E, Bester J, Vermeulen N, Alummoottil S, Soma P, Buys AV, et al. Poorly controlled type 2 diabetes is accompanied by significant morphological and ultrastructural changes in both erythrocytes and in thrombin-generated fibrin: implications for diagnostics. Cardiovasc Diabetol. 2015;14:30.

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Lawrie D, Coetzee LM, Becker P, Mahlangu J, Stevens W, Glencross DK. Local reference ranges for full blood count and CD4 lymphocyte count testing. SAMJ. 2009;99(4):243–8.

    PubMed  Google Scholar 

  30. 30.

    Kreuzer KA, Rockstroh JK. Pathogenesis and pathophysiology of anemia in HIV infection. Ann Hematol. 1997;75(5–6):179–87.

    CAS  PubMed  Google Scholar 

  31. 31.

    Bain BJ. The haematological features of HIV infection. Br J Haematol. 1997;99(1):1–8.

    CAS  PubMed  Google Scholar 

  32. 32.

    Castella A, Croxson TS, Mildvan D, Witt DH, Zalusky R. The bone marrow in AIDS A histologic, hematologic, and microbiologic study. Am J Clin Pathol. 1985;84(4):425–32.

    CAS  PubMed  Google Scholar 

  33. 33.

    Stanley SK, Kessler SW, Justement JS, Schnittman SM, Greenhouse JJ, Brown CC, et al. CD34+ bone-marrow cells are infected with HIV in a subset of seropositive individuals. J Immunol. 1992;149(2):689–97.

    CAS  PubMed  Google Scholar 

  34. 34.

    Kaczmarski RS, Davison F, Blair E, Sutherland S, Moxham J, McManus T, et al. Detection of HIV in hematopoietic progenitors. Br J Haematol. 1992;82(4):764–9.

    CAS  PubMed  Google Scholar 

  35. 35.

    Moses AV, Williams S, Heneveld ML, Strussenberg J, Rarick M, Loveless M, et al. Human immunodeficiency virus infection of bone marrow endothelium reduces induction of stromal hematopoietic growth factors. Blood. 1996;87(3):919–25.

    CAS  PubMed  Google Scholar 

  36. 36.

    Frontiera M, Myers AM. Peripheral-blood and bone-marrow abnormalities in the acquired-immunodeficiency-syndrome. West J Med. 1987;147(2):157–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mir N, Costello C, Luckit J, Lindley R. HIV-disease and bone-marrow changes—a study of 60 cases. Eur J Haematol. 1989;42(4):339–43.

    CAS  PubMed  Google Scholar 

  38. 38.

    Spivak JL, Bender BS, Quinn TC. Hematologic abnormalities in the acquired immune-deficiency syndrome. Am J Med. 1984;77(2):224–8.

    CAS  PubMed  Google Scholar 

  39. 39.

    Camaschella C. How I manage patients with atypical microcytic anaemia. Br J Haematol. 2013;160(1):12–24.

    PubMed  Google Scholar 

  40. 40.

    Straat M, van Bruggen R, de Korte D, Juffermans NP. Red blood cell clearance in inflammation. Trans Med Hemother. 2012;39(5):353–60.

    Google Scholar 

  41. 41.

    Patel KV, Ferrucci L, Ershler WB, Longo DL, Guralnik JM. Red blood cell distribution width and the risk of death in middle-aged and older adults. Arch Intern Med. 2009;169(5):515–23.

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Douglas SW, Adamson JW. Anemia of chronic disorders—studies of marrow regulation and iron-metabolism. Blood. 1975;45(1):55–65.

    CAS  PubMed  Google Scholar 

  43. 43.

    Weiss G, Goodnough LT. Medical progress: anemia of chronic disease. N Engl J Med. 2005;352(10):1011–23.

    CAS  PubMed  Google Scholar 

  44. 44.

    Miller A, Green M, Robinson D. Simple rule for calculating normal erythrocyte sedimentation-rate. Br Med J. 1983;286(6361):266.

    CAS  Google Scholar 

  45. 45.

    Skalak R, Zarda PR, Jan KM, Chien S. Mechanics of rouleau formation. Biophys J. 1981;35(3):771–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Brigden ML. Clinical utility of the erythrocyte sedimentation rate. Am Fam Physician. 1999;60(5):1443–50.

    CAS  PubMed  Google Scholar 

  47. 47.

    Andresdottir MB, Sigfusson N, Sigvaldason H, Gudnason V. Erythrocyte sedimentation rate, an independent predictor of coronary heart disease in men and women—The Reykjavik study. Am J Epidemiol. 2003;158(9):844–51.

    PubMed  Google Scholar 

  48. 48.

    Pretorius E, Kell DB. Diagnostic morphology: biophysical indicators for iron-driven inflammatory diseases. Integr Biol. 2014;6(5):486–510.

    CAS  Google Scholar 

  49. 49.

    Cho Y-I, Cho DJ. Hemorheology and microvascular disorders. Kor Circ J. 2011;41(6):287–95.

    Google Scholar 

  50. 50.

    Kesmarky G, Kenyeres P, Rabai M, Toth K. Plasma viscosity: a forgotten variable. Clin Hemorheol Microcirc. 2008;39(1–4):243–6.

    PubMed  Google Scholar 

  51. 51.

    Rampling MW, Meiselman HJ, Neu B, Baskurt OK. Influence of cell-specific factors on red blood cell aggregation. Biorheology. 2004;41(2):91–112.

    CAS  PubMed  Google Scholar 

  52. 52.

    Walton BL, Byrnes JR, Wolberg AS. Fibrinogen, red blood cells, and factor XIII in venous thrombosis. J Thromb Haemost. 2015;13:S208–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Lominadze D, Dean WL. Involvement of fibrinogen specific binding in erythrocyte aggregation. FEBS Lett. 2002;517(1–3):41–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Carvalho FA, Connell S, Miltenberger-Miltenyi G, Pereira SV, Tavares A, Ariens RAS, et al. Atomic force microscopy-based molecular recognition of a fibrinogen receptor on human erythrocytes. ACS Nano. 2010;4(8):4609–20.

    CAS  PubMed  Google Scholar 

  55. 55.

    Oliveira S, Vitorino de Almeida V, Calado A, Rosario HS, Saldanha C. Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane. Biochimica Et Biophysica Acta-Biomembranes. 2012;1818(3):481–90.

    Google Scholar 

  56. 56.

    Osime EO, Oresanja OO, Okwara BU. Packed cell volume Platelet count, PT, PTTK and Fibrinogen concentration of HIV positive patients on antiretroviral drugs. Pak J Med Sci. 2015;31(6):1533–6.

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Fox EA, Kahn SR. The relationship between inflammation and venous thrombosis—a systematic review of clinical studies. Thromb Haemost. 2005;94(2):362–5.

    CAS  PubMed  Google Scholar 

  58. 58.

    Chirinos JA, Heresi GA, Velasquez H, Jy W, Jimenez JJ, Ahn E, et al. Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism. J Am Coll Cardiol. 2005;45(9):1467–71.

    CAS  PubMed  Google Scholar 

  59. 59.

    Gabay C. Mechanisms of disease—acute-phase proteins and other systemic responses to inflammation. New Engl J Med. 1999;340(17):1376.

    Google Scholar 

  60. 60.

    Zon LI, Groopman JE. Hematologic manifestations of the human immune-deficiency virus (HIV). Semin Hematol. 1988;25(3):208–18.

    CAS  PubMed  Google Scholar 

  61. 61.

    Boelaert JR, Weinberg GA, Weinberg ED. Altered iron metabolism in HIV infection: mechanisms, possible consequences, and proposals for management. Infect Agents Dis Rev Issues Comment. 1996;5(1):36–46.

    CAS  Google Scholar 

  62. 62.

    Waldvogel-Abramowski S, Waeber G, Gassner C, Buser A, Frey BM, Favrat B, et al. Physiology of iron metabolism. Transf Med Hemother. 2014;41(3):213–21.

    Google Scholar 

  63. 63.

    Lau B, Sharrett AR, Kingsley LA, Post W, Palella FJ, Visscher B, et al. C-reactive protein is a marker for human immunodeficiency virus disease progression. Arch Intern Med. 2006;166(1):64–70.

    CAS  PubMed  Google Scholar 

  64. 64.

    Dolan SE, Hadigan C, Killilea KM, Sullivan MP, Hemphill L, Lees RS, et al. Increased cardiovascular disease risk indices in HIV-infected women. JAIDS. 2005;39(1):44–54.

    PubMed  Google Scholar 

  65. 65.

    Arinola OG, Adedapo KS, Kehinde AO, Olaniyi JA, Akiibinu MO. Acute phase proteins, trace elements in asymptomatic human immunodeficiency virus infection in Nigerians. Afr J Med Med Sci. 2004;33(4):317–22.

    CAS  PubMed  Google Scholar 

  66. 66.

    Noursadeghi M, Miller RF. Clinical value of C-reactive protein measurements in HIV-positive patients. Int J STD AIDS. 2005;16(6):438–41.

    PubMed  Google Scholar 

  67. 67.

    Madden E, Lee G, Kotler DP, Wanke C, Lewis CE, Tracy R, et al. Association of antiretroviral therapy with fibrinogen levels in HIV-infection. Aids. 2008;22(6):707–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Reingold JS, Wanke C, Kotler DP, Lewis CE, Tracy R, Heymsfield S, et al. Association of HIV infection and HIV/HCV coinfection with C-reactive protein levels—the fat redistribution and metabolic change in HIV Infection (FRAM) study. JAIDS. 2008;48(2):142–8.

    CAS  PubMed  Google Scholar 

  69. 69.

    Tien PC, Choi AI, Zolopa AR, Benson C, Tracy R, Scherzer R, et al. Inflammation and mortality in HIV-infected adults: analysis of the FRAM study cohort. JAIDS. 2010;55(3):316–22.

    CAS  PubMed  Google Scholar 

  70. 70.

    Chaudhary M, Kashyap B, Gautam H, Saini S, Bhalla P. Role of C-reactive protein in HIV infection: a pilot study. Viral Immunol. 2008;21(2):263–6.

    CAS  PubMed  Google Scholar 

  71. 71.

    Fitzsimons EJ, Brock JH. The anaemia of chronic disease—remains hard to distinguish from iron deficiency anaemia in some cases. BMJ. 2001;322(7290):811–2.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Kell DB, Pretorius E. Serum ferritin is an important inflammatory disease marker, as it is mainly a leakage product from damaged cells. Metallomics. 2014;6(4):748–73.

    CAS  PubMed  Google Scholar 

  73. 73.

    Urrechaga E, Hoffmann JJML, Izquierdo S, Escanero JF. Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes. Int J Lab Hematol. 2015;37(3):334–40.

    CAS  PubMed  Google Scholar 

  74. 74.

    Stoltzfus RJ. Defining iron-deficiency anemia in public health terms: a time for reflection. J Nutr. 2001;131(2):565S–7S.

    CAS  PubMed  Google Scholar 

  75. 75.

    Fuchs D, Hausen A, Reibnegger G, Werner ER, Wernerfelmayer G, Dierich MP, et al. Immune activation and the anemia associated with chronic inflammatory disorders. Eur J Haematol. 1991;46(2):65–70.

    CAS  PubMed  Google Scholar 

  76. 76.

    Perkocha LA, Rodgers GM. Hematologic aspects of human immunodeficiency virus-infection—laboratory and clinical considerations. Am J Hematol. 1988;29(2):94–105.

    CAS  PubMed  Google Scholar 

  77. 77.

    Minchella PA, Armitage AE, Darboe B, Jallow MW, Drakesmith H, Jaye A, et al. Elevated hepcidin is part of a complex relation that links mortality with iron homeostasis and anemia in men and women with HIV infection. J Nutr. 2015;145(6):1194–201.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306(5704):2090–3.

    CAS  PubMed  Google Scholar 

  79. 79.

    Jonker FAM, van Hensbroek MB. Anaemia, iron deficiency and susceptibility to infections. J Infect. 2014;69:S23–7.

    PubMed  Google Scholar 

  80. 80.

    Pretorius E, Oberholzer HM, Smit E, Steyn E, Briedenhann S, Franz CR. Ultrastructural changes in platelet aggregates of HIV patients: a scanning electron microscopy study. Ultrastruct Pathol. 2008;32(3):75–9.

    PubMed  Google Scholar 

  81. 81.

    Pretorius E, Smit E, Oberholzer HM, Steyn E, Briedenhann S, Franz RC. Investigating the ultrastructure of platelets of HIV patients treated with the immuno-regulator, Canova: a qualitative scanning electron microscopy study. Histol Histopathol. 2009;24(4):399–405.

    CAS  PubMed  Google Scholar 

  82. 82.

    Jackson BS, Mokoena T. Comparison of the therapeutic dose of warfarin in HIV-infected and HIV-uninfected patients: a study of clinical practice. BMJ Open. 2017;7:2.

    Google Scholar 

  83. 83.

    Majluf-Cruz A, Silva-Estrada M, Sanchez-Barboza R, Montiel-Manzano G, Trevino-Perez S, Santoscoy-Gomez M, et al. Venous thrombosis among patients with AIDS. Clin Appl Thromb Hemost. 2004;10(1):19–25.

    PubMed  Google Scholar 

  84. 84.

    Haugaard AK, Lund TT, Birch C, Ronsholt F, Troseid M, Ullum H, et al. Discrepant coagulation profile in HIV infection: elevated D-dimer but impaired platelet aggregation and clot initiation. Aids. 2013;27(17):2749–58.

    CAS  PubMed  Google Scholar 

  85. 85.

    Ronsholt FF, Gerstoft J, Ullum H, Johansson PI, Katzenstein TL, Ostrowski SR. Thromboelastography on plasma reveals delayed clot formation and accelerated clot lyses in HIV-1 infected persons compared with healthy controls. BMC Infect Dis. 2015;15:388.

    PubMed  PubMed Central  Google Scholar 

  86. 86.

    Nordell AD, McKenna M, Borges AH, Duprez D, Neuhaus J, Neaton JD, et al. Severity of cardiovascular disease outcomes among patients with HIV Is related to markers of inflammation and coagulation. J Am Heart Assoc. 2014;3:3.

    Google Scholar 

  87. 87.

    Pretorius E, Olumuyiwa-Akeredolu O, Mbotwe S, Bester J. Erythrocytes and their role as health indicator: using structure in a patient-orientated precision medicine approach. Blood Rev. 2016;30(4):263–74.

    PubMed  Google Scholar 

  88. 88.

    Swanepoel AC, Pretorius E. Scanning electron microscopy analysis of erythrocytes in thromboembolic ischemic stroke. Int J Lab Hematol. 2012;34(2):185–91.

    CAS  PubMed  Google Scholar 

  89. 89.

    Santos-Silva A, Rebelo I, Castro E, Belo L, Catarino C, Monteiro I, et al. Erythrocyte damage and leukocyte activation in ischemic stroke. Clin Chim Acta. 2002;320(1–2):29–35.

    CAS  PubMed  Google Scholar 

  90. 90.

    Robinson JM. Reactive oxygen species in phagocytic leukocytes. Histochem Cell Biol. 2008;130(2):281–97.

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Pretorius E, du Plooy J, Soma P, Gasparyan AY. An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus. Rheumatol Int. 2014;34(7):1005–9.

    CAS  PubMed  Google Scholar 

  92. 92.

    Pretorius E, Swanepoel AC, Buys AV, Vermeulen N, Duim W, Kell DB. Eryptosis as a marker of Parkinson’s disease. Aging US. 2014;6(10):788–819.

    CAS  Google Scholar 

  93. 93.

    Bester J, Soma P, Kell DB, Pretorius E. Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS). Oncotarget. 2015;6(34):35284–303.

    PubMed  PubMed Central  Google Scholar 

  94. 94.

    Lang KS, Myssina S, Brand V, Sandu C, Lang PA, Berchtold S, et al. Involvement of ceramide in hyperosmotic shock-induced death of erythrocytes. Cell Death Differ. 2004;11(2):231–43.

    CAS  PubMed  Google Scholar 

  95. 95.

    Sirachainan N, Thongsad J, Pakakasama S, Hongeng S, Chuansumrit A, Kadegasem P, et al. Normalized coagulation markers and anticoagulation proteins in children with severe beta-thalassemia disease after stem cell transplantation. Thromb Res. 2012;129(6):765–70.

    CAS  PubMed  Google Scholar 

  96. 96.

    Kempe DS, Akel A, Lang PA, Hermle T, Biswas R, Muresanu J, et al. Suicidal erythrocyte death in sepsis. J Mol Med. 2007;85(3):269–77.

    Google Scholar 

  97. 97.

    Lang F, Lang E, Foeller M. Physiology and pathophysiology of eryptosis. Trans Med Hemother. 2012;39(5):308–14.

    Google Scholar 

  98. 98.

    Lang F, Qadri SM. Mechanisms and significance of eryptosis, the suicidal death of erythrocytes. Blood Purif. 2012;33(1–3):125–30.

    PubMed  Google Scholar 

  99. 99.

    Litvinov RI, Weisel JW. Role of red blood cells in haemostasis and thrombosis. ISBT Sci Series. 2017;12(1):176–83.

    CAS  Google Scholar 

  100. 100.

    Schroit AJ, Zwaal RFA. Transbilayer movement of phospholipids in red-cell and platelet membranes. Biochem Biophys Acta. 1991;1071(3):313–29.

    CAS  PubMed  Google Scholar 

  101. 101.

    Pajic-Lijakovic I. Erythrocytes under osmotic stress—modeling considerations. Prog Biophys Mol Biol. 2015;117(1):113–24.

    CAS  PubMed  Google Scholar 

  102. 102.

    Thevenin BJM, Low PS. Kinetics and regulation of the ankyrin-band-3 interaction of the human red-blood-cell membrane. J Biol Chem. 1990;265(27):16166–72.

    CAS  PubMed  Google Scholar 

  103. 103.

    Davis LH, Otto E, Bennett V. Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion-exchanger. J Biol Chem. 1991;266(17):11163–9.

    CAS  PubMed  Google Scholar 

  104. 104.

    Burton NM, Bruce LJ. Modelling the structure of the red cell membrane. Biochem Cell Biol. 2011;89(2):200–15.

    CAS  PubMed  Google Scholar 

  105. 105.

    Grey JL, Kodippili GC, Simon K, Low PS. Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane. Biochemistry. 2012;51(34):6838–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Czogalla A, Sikorski AF. Do we already know how spectrin attracts ankyrin? Cell Mol Life Sci. 2010;67(16):2679–83.

    CAS  PubMed  Google Scholar 

  107. 107.

    Cunha SR, Mohler PJ. Ankyrin protein networks in membrane formation and stabilization. J Cell Mol Med. 2009;13(11–12):4364–76.

    CAS  PubMed  PubMed Central  Google Scholar 

  108. 108.

    Devaux PF, Herrmann A, Ohlwein N, Koziov MM. How lipid flippases can modulate membrane structure. Biochim Et Biophys Acta-Biomemb. 2008;1778(7–8):1591–600.

    CAS  Google Scholar 

  109. 109.

    Clark MR. Flippin’ lipids. Nat Immunol. 2011;12(5):373–5.

    CAS  PubMed  Google Scholar 

  110. 110.

    Powell RJ, Machiedo GW, Rush BF, Dikdan G. Oxygen free-radicals—effect on red-cell deformability in sepsis. Crit Care Med. 1991;19(5):732–5.

    CAS  PubMed  Google Scholar 

  111. 111.

    Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freyssinet JM, et al. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation. 2000;101(8):841–3.

    CAS  PubMed  Google Scholar 

  112. 112.

    Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med. 2008;358(12):1229–39.

    CAS  PubMed  Google Scholar 

  113. 113.

    van Beers EJ, Schaap MCL, Berckmans RJ, Nieuwland R, Sturk A, van Doormaal FF, et al. Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematol Hematol J. 2009;94(11):1513–9.

    Google Scholar 

  114. 114.

    Zecher D, Cumpelik A, Schifferli JA. Erythrocyte-derived microvesicles amplify systemic inflammation by thrombin-dependent activation of complement. Arterioscler Thromb Vasc Biol. 2014;34(2):313–20.

    CAS  PubMed  Google Scholar 

  115. 115.

    Eyal A, Veller M. HIV and venous thrombotic events. S Afr J Surg. 2009;47(2):54–6.

    CAS  PubMed  Google Scholar 

  116. 116.

    Wohner N, Sotonyi P, Machovich R, Szabo L, Tenekedjiev K, Silva MMCG, et al. Lytic resistance of fibrin containing red blood cells. Arterioscler Thromb Vasc Biol. 2011;31(10):2306–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Varin R, Mirshahi S, Mirshahi P, Klein C, Jamshedov J, Chidiac J, et al. Whole blood clots are more resistant to lysis than plasma clots—greater efficacy of rivaroxaban. Thromb Res. 2013;131(3):E100–9.

    CAS  PubMed  Google Scholar 

  118. 118.

    Cines DB, Lebedeva T, Nagaswami C, Hayes V, Massefski W, Litvinov RI, et al. Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin. Blood. 2014;123(10):1596–603.

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119.

    Reikvam H, Steien E, Hauge B, Liseth K, Hagen KG, Storkson R, et al. Thrombelastography. Transfus Apheres Sci. 2009;40(2):119–23.

    Google Scholar 

  120. 120.

    Braekkan SK, Mathiesen EB, Njolstad I, Wilsgaard T, Stormer J, Hansen JB. Mean platelet volume is a risk factor for venous thromboembolism: the Troms circle divide study. J Thromb Haemost. 2010;8(1):157–62.

    CAS  PubMed  Google Scholar 

  121. 121.

    Coban E, Yazicioglu G, Avci AB, Akcit F. The mean platelet volume in patients with essential and white coat hypertension. Platelets. 2005;16(7):435–8.

    CAS  PubMed  Google Scholar 

  122. 122.

    Nadar SK, Blann AD, Kamath S, Beevers DG, Lip GYH. Platelet indexes in relation to target organ damage in high-risk hypertensive patients—a substudy of the anglo-scandinavian cardiac outcomes trial (ASCOT). J Am Coll Cardiol. 2004;44(2):415–22.

    PubMed  Google Scholar 

  123. 123.

    Papanas N, Symeonidis G, Maltezos E, Mavridis G, Karavageli E, Vosnakidis T, et al. Mean platelet volume in patients with type 2 diabetes mellitus. Platelets. 2004;15(8):475–8.

    CAS  PubMed  Google Scholar 

  124. 124.

    Nadar SK, Lip GY, Blann AD. Platelet morphology, soluble P selectin and platelet P-selectin in acute ischaemic stroke—the West Birmingham Stroke Project. Thromb Haemost. 2004;92(6):1342–8.

    CAS  PubMed  Google Scholar 

  125. 125.

    Greisenegger S, Endler G, Hsieh K, Tentschert S, Mannhalter C, Lalouschek W. Is elevated mean platelet volume associated with a worse outcome in patients with acute ischemic cerebrovascular events? Stroke. 2004;35(7):1688–91.

    CAS  PubMed  Google Scholar 

  126. 126.

    Martin JF, Bath PMW, Burr ML. Influence of platelet size on outcome after myocardial-infarction. Lancet. 1991;338(8780):1409–11.

    CAS  PubMed  Google Scholar 

  127. 127.

    Vagdatli E, Gounari E, Lazaridou E, Katsibourlia E, Tsikopoulou F, Labrianou I. Platelet distribution width: a simple, practical and specific marker of activation of coagulation. Hippokratia. 2010;14(1):28–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  128. 128.

    Gasparyan AY, Ayvazyan L, Mikhailidis DP, Kitas GD. Mean platelet volume: a link between thrombosis and inflammation? Curr Pharm Des. 2011;17(1):47–58.

    CAS  PubMed  Google Scholar 

  129. 129.

    Prandoni P. Venous and arterial thrombosis: two aspects of the same disease? Eur J Int Med. 2009;20(6):660–1.

    Google Scholar 

  130. 130.

    Swanepoel AC, Nielsen VG, Pretorius E. Viscoelasticity and ultrastructure in coagulation and inflammation: two diverse techniques, one conclusion. Inflammation. 2015;38(4):1707–26.

    CAS  PubMed  Google Scholar 

  131. 131.

    Gresele P, Falcinelli E, Sebastiano M, Baldelli F. Endothelial and platelet function alterations in HIV-infected patients. Thromb Res. 2012;129(3):301–8.

    CAS  PubMed  Google Scholar 

  132. 132.

    Youssefian T, Drouin A, Masse JM, Guichard J, Cramer EM. Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation. Blood. 2002;99(11):4021–9.

    CAS  PubMed  Google Scholar 

  133. 133.

    Metcalf Pate KA, Mankowski JL. HIV and SIV associated thrombocytopenia: an expanding role for platelets in the pathogenesis of HIV. Drug Discov Today Dis Mech. 2011;8(1–2):e25–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  134. 134.

    Sandler DA, Martin JF. Autopsy proven pulmonary embolism in hospital patients: are we detecting enough deep vein thrombosis? J R Soc Med. 1989;82(4):203–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  135. 135.

    Meignan M, Rosso J, Gauthier H, Brunengo F, Claudel S, Sagnard L, et al. Systematic lung scans reveal a high frequency of silent pulmonary embolism in patients with proximal deep venous thrombosis. Arch Intern Med. 2000;160(2):159–64.

    CAS  PubMed  Google Scholar 

  136. 136.

    Jackson BS, Pretorius E. Pathological clotting and deep vein thrombosis in patients with HIV. Semin Thromb Hemost. 2019;45(2):132–40.

    PubMed  Google Scholar 

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Acknowledgements

We thank the National Research Foundation (NRF) of South Africa (E Pretorius: Rated researcher grant), as well as the Medical Research Council (MRC of South Africa (E Pretorius: grant holder)) for supporting this research. The funding organizations did not have any involvement in the research. We thank Professor T Mokoena and the personnel (especially Mr CJ Meintjes) from the Department of Surgery, University of Pretoria. We thank the Ultrasound department, Kalafong Provincial Tertiary Hospital (specifically Doctors Rowaida A. Ahmed, Amelia Venter and Shaakera M.H. Ismail) for assisting with the recruitment of patients for the study.

Funding

National Research Foundation (NRF) of South Africa (E Pretorius: Rated researcher grant), as well as the Medical Research Council (MRC of South Africa (E Pretorius: grant holder)). The funding organizations did not have any involvement in the research.

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BSJ and EP conceived the experiments; BSJ and JNG conducted the experiments; BSJ and EP analysed the results. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Etheresia Pretorius.

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The Human Research Ethics Committee, Faculty of Health Sciences, University of Pretoria, South Africa approved the study (reference number: 547/2015). Consent was obtained from each participant. Consent was also obtained from the Chief Executive Office of each hospital.

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Consent obtained from each patient/person.

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The authors declare no competing interests.

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Jackson, B.S., Nunes Goncalves, J. & Pretorius, E. Comparison of pathological clotting using haematological, functional and morphological investigations in HIV-positive and HIV-negative patients with deep vein thrombosis. Retrovirology 17, 14 (2020). https://doi.org/10.1186/s12977-020-00523-3

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Keywords

  • Hypercoagulability
  • Human immunodeficiency virus (HIV)
  • Deep vein thrombosis (DVT)
  • Inflammation