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JAC Advance Access originally published online on August 27, 2008
Journal of Antimicrobial Chemotherapy 2008 62(5):1118-1121; doi:10.1093/jac/dkn333
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Platelet–leucocyte adhesion markers before and after the initiation of antiretroviral therapy with HIV protease inhibitors

Nils von Hentig1,*, Ann-Kristin Förster1, Karina Kuczka1, Ute Klinkhardt1, Stefan Klauke2, Peter Gute2, Schlomo Staszewski3, Sebastian Harder1 and Jochen Graff1

1 Pharmazentrum Frankfurt, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Frankfurt, Germany 2 Infektiologikum Frankfurt, Frankfurt, Germany 3 HIVCENTER, Medical HIV Treatment and Research Unit, Johann Wolfgang Goethe University, Frankfurt, Germany

* Corresponding author. Tel: +49-69-63016956; Fax: +49-69-63017636; E-mail: hentig{at}em.uni-frankfurt.de

Received 9 June 2008; returned 6 July 2008; revised 18 July 2008; accepted 19 July 2008


   Abstract
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Introduction: Thromboembolic complications under antiretroviral therapy (ART) have been described in the past. In particular, the influence of protease inhibitors (PIs) on platelet activation and coagulation is currently under discussion.

Methods: HIV-1-infected, PI-naive adults (n = 18) were investigated before and 4 weeks after the start of the ART, consisting either of boosted PI regimens (n = 13) plus reverse transcriptase inhibitors (RTIs) or a double PI regimen (n = 5) without RTI co-medication. Administered PIs were saquinavir (n = 15), lopinavir (n = 4), fosamprenavir (n = 2) and atazanavir (n = 2). Platelet CD62P, CD40L (%+ cells) and PAC-1 binding [mean fluorescence intensity (MFI)] as well as monocyte CD11b (MFI) and monocyte-associated CD41 (%+ cells and MFI) expression were assessed by flow cytometry with or without platelet stimulation. To investigate the influence of platelets on coagulation, the endogenous thrombin potential (ETP) [render fluorescence units (RFI)] was determined.

Results: CD62P, PAC-1 binding and CD11b expression remained unchanged. In contrast, the mean±SD MFI of CD40L (from 18.2±9.0 to 25.5±10.4, P = 0.038) and CD41 (from 446.1±213.8 to 605.0±183.8, P = 0.010) as markers for increased platelet–leucocyte interaction increased significantly. The collagen-induced ETP time-to-peak was altered significantly from 23.8±11.4 to 17.0±4.2 min (P = 0.028), although the ETP RFI peak showed no evidence for increased procoagulatory capacity (47.1±18.6 to 57.3±19.9, P = 0.085).

Conclusions: Effects of the evaluated PI HIV therapy on platelet function assessed under field conditions seem to be minor, not affecting all investigated parameters. We found no evidence for increased platelet activation under PI-containing ART. However, CD41 as a marker for increased platelet–leucocyte interaction and CD40L, which can contribute to atherosclerosis, increased significantly.

Keywords: HIV PIs , platelets , leucocytes , CD40L , CD41


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The possible relevance of platelets in propagating the inflammatory process is increasingly being recognized. A constituent of the {alpha}-granule membrane CD62P or sP-selectin is expressed on the platelet surface upon activation, although CD62P is capable of interacting with different partners such as neutrophils and monocytes and its ligand P-selectin glycoprotein ligand-1. This results in the formation of platelet–leucocyte aggregates (PLAs)1 and the up-regulation of the MAC-1 endothelial cell ligand (CD11b/CD18) leading to interaction with the partners. Also, CD40L2 is expressed on platelets and various tissues linked to atherosclerosis, such as vascular endothelial cells, smooth muscle cells and monocytes. CD40L activation is capable of initiating various inflammatory responses such as the production of chemokines (e.g. MCP-1), cytokines or matrix metalloproteinases (e.g. MMP9). Its soluble form sCD40L, of which platelets are the major source, has been recognized as an independent risk factor of cardiovascular disease. The formation of PLAs increases on the surface of diseased vessels throughout lesion development, thus promoting the progression of atherosclerosis.3,4

HIV infection is known to trigger thrombocytopenia.5 In contrast, HIV patients show enhanced platelet activation with increased CD62P expression and release of chemokines (e.g. RANTES) from the thrombocyte granula.6 Also, platelet-independent enhanced activation of procoagulatory factors such as D-dimers and thromboembolic complications in HIV patients have been described.7,8 Usually, the reduction of the viral load under antiretroviral therapy (ART) is accompanied by a decrease in thrombocytopenia9 and is able to at least decrease the release of RANTES and of further endothelium activation markers [e.g. von Willebrand factor (vWF)] and D-dimers.

Although the endothelial part of this complication has been discussed sufficiently,10,11 data regarding PLA formation, CD62 expression and platelet-induced thrombin generation in HIV patients under ART are scarce.

We therefore evaluated markers for thrombocyte activation (CD62P and PAC-1 binding) and for leucocyte and platelet–monocyte interaction (CD11b, CD41 and CD40L) and the influence of platelets on coagulation [endogenous thrombin potential (ETP)] in 18 adult HIV-1-infected patients naive to therapy with protease inhibitors (PIs), before and 4 weeks after the initiation of highly active ART.


   Methods
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Patients and study protocol

The study was approved by the Medical Faculty Ethics Review Board of the Johann Wolfgang Goethe University Frankfurt am Main, Germany, and patients provided written informed consent prior to enrolment.

Blood samples were drawn from HIV PI therapy-naive patients immediately before starting a new therapy with HIV PIs. Baseline laboratory parameters obtained were red blood cell count, white blood cells count, platelet count, haemoglobin, haematocrit, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase and creatinine. Furthermore, the expression of CD62P and CD40L, PAC-1 binding, PLA formation (via CD41-positive monocytes), CD11b expression and thrombin generation (ETP) were determined. After 4 weeks on therapy, all analyses described earlier were repeated.

Patients on a current therapy with platelet aggregation inhibitors (e.g. acetylic salicylic acid) or patients with cardiovascular events in their anamnesis were not included in this study.

Laboratory

Citrated whole blood was assessed from patients and transferred to the laboratory within 20 min, avoiding platelet activation. The following sections provide a summary of the laboratory methods applied; a more comprehensive description is given in the Supplementary data, as well as a list of further reading [available at JAC Online (http://jac.oxfordjournals.org/)].

Flow cytometry. Platelet activation and PLA formation were measured by whole-blood flow cytometry assay. Preparation of blood samples was adapted from a consensus protocol,12 and blood was diluted with modified HEPES buffer and processed immediately for activation and immunofluorescence staining.

CD62, PAC-1 binding and CD40L. In the flow cytometric determination of platelet activation status, we focused on parameters that represented different platelet functions: CD62P expression is a marker of platelet degranulation, whereas PAC-1 reflects the activation-induced conformational change of the GPIIb/IIIa receptor, leading to fibrinogen binding and subsequent aggregation. Furthermore, activated platelets express the CD40L antigene.

To estimate platelet activation markers, double (CD40L)- or triple (CD62P/PAC-1)-colour immunofluorescence staining was used. Activated or non-activated samples were stained with saturating concentrations of anti-CD41-PC7, anti-CD62P-PE and PAC-1-FITC or anti-CD41-PC7 and anti-CD40L-PE. Data on 10 000 platelets in a log side-scatter versus log CD41-PC7 dot plot were acquired, the percentages of platelets expressing CD62P and CD40L were specified and the mean fluorescence intensity (MFI, given in arbitrary units) of PAC-1 binding was analysed and corrected for non-specific binding of isotype control.

CD41 and CD11b. CD41 represents the platelet mass attached to monocytes, and CD11b is an activation marker for the monocytes. To measure PLA formation, activated or non-activated samples were stained with saturating concentrations of anti-CD14-FITC, anti-CD11b-PE and anti-CD 41-PC7. Erythrocytes were lysed, and PLA formation was estimated by gating on leucocyte subpopulations. Percentages of CD41-positive monocytes corrected for non-specific binding and FL3-MFI of CD41-positive cells were analysed. Expression of CD11b (MFI) was specified in the total monocyte population. Data on 2000 monocytes were processed.

ETP. Platelet-rich plasma and a buffer containing ADP, collagen or thrombin receptor activating peptide (TRAP) were added to each well of a microtitre plate. The reaction was started by adding calcium chloride. Fluorescence was measured in each well at 20 s intervals for at least 66 min.

The course of the fluorescent signal from each well was retrieved from four parallel experiments to evaluate the mean fluorescence–time curve, which reflects the course of thrombin generation. Parameters of interest were the maximum generation rate (ETP peak) and the time to reach the maximum generation rate (time to peak).13

Statistics

The patients' baseline parameters were subject to descriptive statistics. The thrombocyte–leucocyte aggregation markers were analysed by means of paired sample T-test, comparing baseline with week 4 values, using SPSS® 15.0.1. Due to the explorative nature of this study, a sample size calculation was not applied, and a sample size of n = 18 was estimated as being feasible during project initiation.


   Results
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Patients

Eighteen HIV-1-infected adults aged between 28 and 75 were enrolled to the study consecutively according to their attendance in the primary HIV care unit between December 2005 and November 2006 if they met the inclusion criteria. Thirteen patients were therapy-naive and five patients had had a structured treatment interruption of 4–8 weeks prior to study enrolment. Nine patients were self-reported frequent smokers of 5–10 cigarettes per day. Baseline demographic and laboratory parameters are shown in Table 1.


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  		Table 1. Baseline parameters of 16 men and 2 women, previously HIV PI therapy-naive, starting with HIV PI-containing ART

 
Five patients received ART with two boosted HIV PIs at standard doses (saquinavir/lopinavir/ritonavir, n = 4; fosamprenavir/lopinavir/ritonavir, n = 1) due to previously experienced toxicity and/or resistance of the individual HIV strains to nucleoside RTIs (NRTIs). Thirteen patients took standard therapy with a boosted HIV PI (saquinavir/ritonavir, n = 11; fosamprenavir, n = 2) and two or three NRTIs.

Platelet and monocyte activation and formation of platelet–monocyte aggregates

After the initiation of ART, we detected significantly enhanced CD40L and CD41 activity on platelets, which are markers for increased formation of PLAs, in TRAP-activated samples. CD40L was increased from 18.2±9.0 at visit 1 to 25.5±10.4 at visit 2 (paired sample T-test P = 0.038), and CD41 was found to be altered from 446.1±213.8 at visit 1 to 605.0±183.8 at visit 2 (P = 0.010). CD11b and CD62 did not show significant changes (Table 2). The maximal thrombin generation rate (ETP peak) did not show significant alterations, but the ETP time to peak significantly decreased from 23.8±11.4 at baseline to 17.0±4.2 after 4 weeks of therapy (Table 2), indicating a faster onset of platelet-induced thrombin generation.


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  		Table 2. Platelet–leucocyte adhesion and platelet and leucocyte activation markers in 18 adult HIV-1-infected patients taking HIV PI-containing ART, before and 4 weeks after therapy onset

 

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It has been shown previously that platelet aggregation activated before therapy onset decreased after 4 weeks of therapy with the HIV PI indinavir.6 ART could also decrease previously enhanced endothelial activation markers such as VCAM-1, ICAM-1 and vWF. However, a reduction of soluble CD62 (sP-selectin) as a marker for platelet activation could not be shown in the same study.14

As platelet aggregation itself is considered a relatively crude marker for platelet activation, we used a more sensitive approach for platelet testing. In this study, we found some evidence for enhanced platelet activation during a short-course of ART with HIV PIs by determining the surface expression of activation markers and generation of thrombin at the platelet surface. Furthermore, we detected increased CD40L activation, which is capable of initiating various inflammatory responses, e.g. the production of chemokines, cytokines or MMPs that promote the development and progression of atherosclerosis.4 Also, the enhanced CD41 activation is a marker for increased formation of PLAs contributing to these processes.

Furthermore, the significantly decreased time to peak of thrombin generation indicates alterations in the coagulation system under ART, which is influenced by platelets. However, the corresponding increase in the maximum thrombin generation rate was not statistically significant.

The subgroup of five patients who received a boosted double PI regimen did not show marked differences in platelet activation compared with those who were on NRTI-containing therapy.

It has been shown that HIV-infected subjects under ART have a cumulative risk of developing atherosclerosis, myocardial infarction and other thromboembolic events. As the reasons for this are supposed to be multifactorial, e.g. a combination of metabolic syndrome, hyperlipidaemia and lipodystrophy,15–17 the increased platelet–leucocyte aggregation may further contribute to the genesis of cardiovascular diseases.

Thus, this pilot study provides evidence for an enhanced formation of PLAs under therapy with HIV PIs, and the changes detected in platelet activation deserve further attention. However, as the number of participants in this study was small and the observation period short, long-term evaluations are warranted of the effects detected in larger patient populations. Furthermore, it remains to be seen whether patients with enhanced platelet activation but no presence of cardiovascular disease might benefit from anti-platelet therapy, especially when it affects platelet degranulation and CD40l formation, as has been demonstrated for P2Y12 antagonists such as clopidogrel.18


   Funding
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This study was supported by the Fritz und Heinrich Riese Foundation.


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None to declare.


   Supplementary data
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A more comprehensive description of the applied laboratory methods is provided as Supplementary data, as well as a list of further reading, available at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We are grateful for the support of this study provided by the Fritz und Heinrich Riese Foundation.


   References
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1 Rinder HM, Bonan JL, Rinder CS, et al. Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood (1991) 78:1760–9.[Abstract/Free Full Text]

2 Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature (1998) 391:591–4.[CrossRef][Web of Science][Medline]

3 Neumann FJ, Marx N, Gawaz M, et al. Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. Circulation (1997) 95:2387–94.[Abstract/Free Full Text]

4 Ross R. Atherosclerosis is an inflammatory disease. Am Heart J (1999) 138:S419–20.[CrossRef][Web of Science][Medline]

5 Cole JL, Marzec UM, Gunthel CJ, et al. Ineffective platelet production in thrombocytopenic human immunodeficiency virus-infected patients. Blood (1998) 91:3239–46.[Abstract/Free Full Text]

6 Holme PA, Muller F, Solum NO, et al. Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection. FASEB J (1998) 12:79–89.[Abstract/Free Full Text]

7 Barbaro G, Barbarini G. Highly active antiretroviral therapy-associated metabolic syndrome and cardiovascular risk. Chemotherapy (2006) 52:161–5.[CrossRef][Web of Science][Medline]

8 Barbaro G. Vascular injury, hypertension and coronary artery disease in human immunodeficiency virus infection. Clin Ter (2008) 159:51–5.[Web of Science][Medline]

9 Aboulafia DM, Bundow D, Waide S, et al. Initial observations on the efficacy of highly active antiretroviral therapy in the treatment of HIV-associated autoimmune thrombocytopenia. Am J Med Sci (2000) 320:117–23.[CrossRef][Web of Science][Medline]

10 Maggi P, Maserati R, Antonelli G. Atherosclerosis in HIV patients: a new face for an old disease? AIDS Rev (2008) 8:204–9.[Web of Science]

11 Wang X, Chai H, Yao Q, et al. Molecular mechanisms of HIV protease inhibitor-induced endothelial dysfunction. J Acquir Immune Defic Syndr (2007) 44:493–9.[CrossRef][Web of Science][Medline]

12 Schmitz G, Rothe G, Ruf A, et al. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost (1998) 79:885–96.[Web of Science][Medline]

13 Hemker HC, Giesen PL, Ramjee M, et al. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost (2000) 83:589–91.[Web of Science][Medline]

14 Wolf K, Tsakiris DA, Weber R, et al. Antiretroviral therapy reduces markers of endothelial and coagulation activation in patients infected with human immunodeficiency virus type 1. J Infect Dis (2002) 185:456–62.[CrossRef][Web of Science][Medline]

15 Cotter BR. Endothelial dysfunction in HIV infection. Curr HIV/AIDS Rep (2006) 3:126–31.[CrossRef]

16 Carr A. Cardiovascular risk factors in HIV-infected patients. J Acquir Immune Defic Syndr (2003) 34(Suppl_1):S73–8.[CrossRef][Web of Science][Medline]

17 Friis-Moller N, Weber R, Reiss P, et al. Cardiovascular disease risk factors in HIV patients—association with antiretroviral therapy. Results from the DAD study. AIDS (2003) 17:1179–93.[CrossRef][Web of Science][Medline]

18 Graff J, Harder S, Wahl O, et al. Anti-inflammatory effects of clopidogrel intake in renal transplant patients: effects on platelet–leukocyte interactions, platelet CD40 ligand expression, and proinflammatory biomarkers. Clin Pharmacol Ther (2005) 78:468–76.[CrossRef][Web of Science][Medline]


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