14eha 451-P Thrombosis and vascular biology. Philip Badenhorst DOI: 10.3252/pso.eu.14eha.2009 Poster presented at EHA 14 on: 5th June 2009 ADAMTS13 LEVELS IN HIV INFECTED PATIENTS WITH AND WITHOUT TTP P Badenhorst 2 , B van Staden 2 , M Meiring 2 , W Janse van Rensburg 1 & H Deckmyn 3 1 University of the Free State & 2 NHLS, Bloemfontein, South Africa and 3 KU Leuven, Kortrijk, Belgium INTRODUCTION Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disease characterised by microvascular platelet deposition and thrombus formation in selected organs with resulting microangiopathic haemolytic anaemia, renal failure, neurological symptoms and thrombocytopenia. Typically a very rare disorder, TTP is being seen with increased frequency in patients infected with the human immunodeficiency virus (HIV). Deficiency of the Von Willebrand factor cleavage protease, also known as ADAMTS13, has been implicated as a major aetiological factor in TTP. However, before studying the role of ADAMTS13 in HIV related TTP, it is necessary to know the normal values of ADAMTS13 levels in the population group in question because lower ADAMTS13 levels had been reported in other populations such as the Chinese. It is also necessary to know whether HIV infection in the absence of TTP has any effect on ADAMTS13 levels. Proteolysis of ultra-large von Willebrand disease (ULVWF) by ADAMTS-13 Deficiency in ADAMTS13 AIM The aim of the study was threefold: 1. To compare the ADAMTS13 levels in the local Caucasian and African populations; 2. To study the effect of HIV infection on ADAMTS13 levels by correlating CD4 counts and viral loads with ADAMTS13 levels in HIV positive patients without TTP; and 3. To measure ADAMTS13 levels in patients with HIV associated TTP. METHODS Ethical Approval: Ethical approval was obtained from the Ethics Committee of the Faculty of Health Sciences, University of the Free State. Study populations: Group 1 (n=40): Normal group - subdivided into Caucasian (n=19) and African (n=21) participants respectively. No history of chronic disease or use of chronic medication; no symptom(s) of disease and a normal full blood count. Group 2 (n=36): HIV positive patients from a local HIV-clinic were randomly selected on a number of clinic days (patients were aware of their HIV status). Three Sub-groups: CD4<200 not on HAART; CD4<100 not on HAART; patients with a viral load of 3400 to 700 000 RNA copies/ml. Group 3 (n=20): Patients with HIV associated TTP were also tested. Sample collection and preparation: EDTA and citrated venous blood were collected, processed and analysed. Plasma was aliquoted and stored at -80˚C until ADAMTS13 & viral load assays were done. CD4 counts were determined with a Beckman Coulter flow cytometer (Epics XL.MCL) and viral loads with a Roche Amplicor HIV-1 (RT-PCR) kit according to the manufacturer’s instructions. ELISA for ADAMTS13-antigen levels: An ELISA was done using three antibodies 20A5, 5C11 and 13F7 that were received from the laboratory of Prof Deckmyn, Belgium. A 96-well ELISA plate was coated overnight with the 20A5 anti- ADAMTS13 monoclonal antibody. After washing with PBS containing 0.1 % Tween-20, the plate was blocked with 4% skimmed milk in PBS for 2 hours at room temperature. Plasma samples of each normal subject or patient were diluted in (1:10 and 1:20 dilutions) and added in duplicate. Normal pooled plasma was calibrated against a standard plasma form American Diagnostica and added in serial dilutions, i.e. 100ng/ml, 50, 25, 12.5, 6.25, 3.125 and 1.56 ng/ml. A blank and a plasma control with a known ADAMTS13 level (American Diagnostica, USA) were added to the plate and incubated for 1.5 hours at 37°C. The 2 biotinylated anti-ADAMTS13 antibodies 5C11and 13F7 were added and incubated after which peroxidase-conjugated streptavidine was added. The colour was developed and read at 490 nm with the SYNERGY HT microplate bio-kinetics reader (Bio- tek Instruments, Vermont, USA). An eight-point standard curve was drawn with the known ADAMTS13 levels on the X-axis and the optical density values on the Y-axis. The ADAMTS13 level value for each subject/patient was read off the standard curve. Example of ELISA assay Statistical analysis: The mean values, standard deviations and reference ranges were determined. The p-value (parametric) between the two normal groups was also determined. With a p-value < 0.05 seen as a statistically significant difference. RESULTS (Fig. 1) ADAMTS13 levels in healthy, normal subjects: Caucasian group: Mean = 739 ng/ml, range = 585 - 893 ng/ml, SD = 77. African group: Mean = 714 ng/ml, range = 620 - 808 ng/ml, SD = 47. P-value = 0.1498 (no significant difference). ADAMTS13 levels and CD4 counts: Sub-group CD4 <200 – not on HAART: Mean = 696 ng/ml, SD = 81. Sub-group CD4 <100 - not on HAART: Mean = 710ng/ml, SD = 109. There was no significant difference between these two groups and between them and the normal control group. ADAMTS13 levels and viral loads: Viral loads were also measured to determine whether the viral load had any effect on ADAMTS13 levels. Viral loads varying from 3 400 to 700 000 had no effect on ADAMTS13 levels. HIV-associated TTP group: The ADAMTS13 levels were also measured in 20 HIV associated TTP patients. The values were all low (144 ± 109 ng/ml) when compared with the normal reference value of 585 – 893 ng/ml. Figure 1: ADAMTS13 levels of different patient groups DISCUSSION Although Feys et al [1] found lower normal ADAMTS13 levels in a Chinese population, we found no statistical or clinical significant difference in the South African Caucasian and African population groups. The incidence of TTP is higher in HIV-infected individuals. In South-Africa it is by far the most common cause of TTP (up to 80% of cases). The precise pathophysiology of HIV-related TTP is not yet clear. It is also not known whether the severity of HIV infection as reflected by he CD4 counts and viral loads has any effect on ADAMTS13 levels. However, our results showed no difference in ADAMTS13 levels regardless the CD4 count and viral load when comparing it to normal, healthy subjects. We have measured the ADAMTS13-antigen levels in 20 HIV+ patients with the typical clinical picture of TTP. In all these cases the ADAMTS13 levels were significantly reduced. However, Gunther et al. [2] found that some typical HIV+ TTP cases showed normal ADAMTS13 activity. They postulated two explanations. First, in some cases of HIV-related TTP, endothelial injury with release of VWF and the associated loss of endothelial thromboresistance is sufficient to cause an overt thrombotic microangiopathy and precipitate an acute episode of TTP even in the presence of normal cleaving protease activity. Second, in other cases the release of VWF may be sufficient to overwhelm the capacity of the cleaving protease and result in a “consumptive deficiency” . They found that only a minority of patients with severely reduced protease activity had antibodies against ADAMTS13. HIV-related TTP appears to be the result of an interaction of diverse pathogenic mechanisms as stated above. CONCLUSION 1. We have standardised an ELISA for measuring ADAMTS13- antigen levels and established reference ranges for the local Caucasian and African population groups. We found no statistical difference in the ADAMTS13-antigen levels between Caucasians and Africans. The reference range for ADAMTS13-antigen levels in our laboratory is 585 – 893 ng/ml 2. We could also show that neither HIV infection, nor the severity of the disease as reflected by CD4 count and viral load had any effect on ADAMTS13 levels, making it a useful diagnostic tool in the setting of HIV positive patients with TTP. 3. We found low ADAMTS13 levels in all 20 patients with HIV associated TTP patients (244 ± 121 ng/ml). References 1. Feys HB, LIU F, Dong N, et al. ADAMTS13 plasma level determination uncovers antigen absence in acquired TTP and ethnic differences. J Thromb Haemost. 2006; 4: 955-962. 2. Gunther K, Garizio D, Dhlamini B. The pathogenesis of HIV-related TTP – is it different? ISBT Science Series. 2006; 1: 246-250. 0 100 200 300 400 500 600 700 800 900 Normal Caucasians Normal Africans HIV+: CD4 < 200 HIV+: CD4 < 100 HIV+: Viral load 3 400 - 700 000 TTP patients ADAMTS13 Levels Group ADAMTS13 levels of different patient groups