the main purpose of antiretroviral therapy ( art ) is to improve the prognosis of patients infected with human immunodeficiency virus ( hiv ) by decreasing the incidence of opportunistic infection and subsequent hospitalization and mortality.1 however , several studies have suggested that highly active antiretroviral therapy ( haart ) results in a higher incidence of cardiovascular events.2,3 in the normal fibrinolytic system , thrombin converts fibrinogen to active fibrin . plasmin then degrades the cross - linked fibrin into soluble degradation products by the tissue type ( t - pa ) and the urokinase - type plasminogen . it is the t - pa that is mainly responsible for the dissolution of fibrin formed in the circulation,4 and the normal process of fibrinolysis can be inhibited either by antagonizing plasmin through alpha-2-antiplasmin or by specific plasminogen activators ( pai ) of which there are three types . pai type 1 ( pai-1 ) , which is synthesized in endothelial cells , adipocytes , and the liver , is physiologically the most important . 4 elevated plasma t - pa indicates inhibited endogenous fibrinolysis , and occurs when free t - pa released into the blood from endothelial cells forms a complex with circulating pai-1 . as a result , assays of t - pa antigen predominantly measure inactive t - pa / pai-1 complexes . concentration of the t - pa/ pai-1 complex levels in plasma correlate strongly with levels of t - pa antigen and pai-1 activity.5,6 there is evidence that elevated t - pa levels predict cardiovascular disease in apparently healthy individuals,7 and this has been reported to account for more than 20% of deaths in some studies of hiv - infected patients.8 to date , most studies of the effects of art involve monitoring hiv - infected patients undergoing therapy for 6 months to 1 year , the general assumption being that the effects of art on hematological and hemostatic parameters will be noticed after a prolonged period . the null hypothesis for the current study is that short - term therapy of hiv - infected patients will have no significant effect on fibrinolytic markers . the main objective of a start study is to find out whether changes other than hematological changes occur during short - term art , which could form the basis of further monitoring and improvement in the management of hiv - infected patients . this start study aimed to determine the values of fibrinolytic parameters within the first 3 months of art , determine the period before changes in these parameters are noticeable , and assess the dangers associated with late commencement or noncommencement of art . the study participants comprised cohorts of 20 patients aged from 18 to 60 years who were receiving health care at the asokoro district hospital in the federal capital territory , abuja , nigeria . the asokoro district hospital has a center where special care such as voluntary counseling and testing , art , and follow - up are provided . a total of 40 participants were enrolled in the start study with each group consisting of 20 participants . the inclusion criteria for patients were as follows : group 1 : aged 1860 years ; must have tested positive for hiv and not previously been on any antiretroviral drug , and must not present any symptom suggestive of a systemic disease . group 2 : aged 1860 years ; must have tested positive for hiv but not be willing to commence art for at least 3 months , and must not present with any symptoms suggestive of a systemic disease . participants with sickle cell disease or hepatitis , who were pregnant , or who were taking oral contraceptives were excluded from the study . the start study received institutional ethical approval from the management of the asokoro general hospital . patients signed a written informed consent form , and were enrolled into the study before blood samples were collected . a repeated measures design comprising an experimental or treatment group and one control group was adopted for this study . this design allowed for a longitudinal study of individual subjects in which fibrinolytic parameters were measured multiple times under different conditions , thereby monitoring trends during the 3-month study period . the experimental group comprised hiv - positive individuals ( art nave subjects ) who registered for art ( group 1 ) , and the positive control group which consisted of hiv positive individuals but who were not on art ( group 2 ) . a total of 7 ml of blood was collected by clean venous puncture from participants , with 4.5 ml blood delivered into a bottle containing 0.5 ml sodium citrate and 2.5 ml into a bottle containing edta anticoagulant . principle and sample preparation : when red blood cells are mixed with cd4 monoclonal antibody in the presence of phycoerythrin , the cells are coated in phycoerythrin . when passed through the measuring area , cells or particles twenty microliters of well mixed whole blood ( with edta added as an anticoagulant ) was pipetted into a partec test tube . twenty microliters of cd4 mab pe was then added , mixed gently , and this was then incubated for 15 minutes at room temperature , protected from light , and 800 l of no lyse buffer was then added and mixed gently . cd4-pe fluorescence was analyzed on a partec flow cytometer with an excitation light source with a wavelength of 488 nm . following this , 840 l of the prepared blood sample was transferred to the cytometer for cd4 determination . d - dimer concentrations in plasma were determined using elisa kits ( technozym d - dimer , lot # rh02b00 ) purchased from technoclone ( vienna , austria ) . one hundred microliters of calibrators , control plasmas , and test samples was pipetted into test wells , covered with film , and incubated in a hot air oven at 37c for 60 minutes . the test wells were then washed three times with 200 l washing buffer , using an automated elisa washer . one hundred microliters of conjugate working solution was pipetted into wells , covered with strips of film , and then incubated at 37c for 60 minutes . following this , one hundred microliters of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature for 10 minutes . one hundred microliters of stopping solution was then added into wells , these were shaken for 10 seconds , and measured within 10 minutes using an elisa reader at a wavelength of 450 nm . concentrations of test samples were read off from the calibration curve and also checked with the controls . citrated plasma and twenty - five microliters of calibrators , control plasmas , and test samples were pipetted into test wells , 75 l of incubation buffer was then added , and wells were covered test strips with film and incubated in a hot air oven at 37c for 60 minutes . wells were emptied thoroughly , 100 l of conjugate working solution was pipetted into wells , and these were covered with film . the test wells were incubated at 37c for 60 minutes and washed three times with 200 l of washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells ; these were shaken for 10 seconds , and then measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of test samples were read off from the calibration curve and also cross checked with the controls . citrated plasma and all required components in the kit were brought to room temperature . twenty - five l of calibrators , control plasmas , and test samples was pipetted into test wells , 75 l of incubation buffer was added , and then covered test strips with film and incubated in hot air oven at 37c for 60 minutes . wells were emptied , and 100 l of conjugate working solution was pipetted into wells which were then covered with film . the wells were further incubated at 37c for 60 minutes and washed three times with 200 l washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells , shaken for 10 seconds , and measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of the test samples were read off from the calibration curve , and checked with the controls . data obtained from the study were subjected to descriptive statistics ( such as calculation of means and standard deviations ) to allow a general evaluation of all study variables . inferential analyses were conducted using repeated measures analysis of covariance ( ancova ) with between - subject variable conditions ( experimental vs control ) , demographic characteristics , and therapy duration . the measured parameter values within each group at baseline were used as covariates , while the interaction effect of associated independent factors was included in the analytical models of all study outcomes . analysis to determine the existence or otherwise of relationships within and between fibrinolytic parameters over the start study duration was carried out by conducting correlation analyses . all analyses were performed using spss software version 16.0 ( spss inc , chicago , il ) . the study participants comprised cohorts of 20 patients aged from 18 to 60 years who were receiving health care at the asokoro district hospital in the federal capital territory , abuja , nigeria . the asokoro district hospital has a center where special care such as voluntary counseling and testing , art , and follow - up are provided . a total of 40 participants were enrolled in the start study with each group consisting of 20 participants . the inclusion criteria for patients were as follows : group 1 : aged 1860 years ; must have tested positive for hiv and not previously been on any antiretroviral drug , and must not present any symptom suggestive of a systemic disease . group 2 : aged 1860 years ; must have tested positive for hiv but not be willing to commence art for at least 3 months , and must not present with any symptoms suggestive of a systemic disease . participants with sickle cell disease or hepatitis , who were pregnant , or who were taking oral contraceptives were excluded from the study . the start study received institutional ethical approval from the management of the asokoro general hospital . patients signed a written informed consent form , and were enrolled into the study before blood samples were collected . a repeated measures design comprising an experimental or treatment group and one control group was adopted for this study . this design allowed for a longitudinal study of individual subjects in which fibrinolytic parameters were measured multiple times under different conditions , thereby monitoring trends during the 3-month study period . the experimental group comprised hiv - positive individuals ( art nave subjects ) who registered for art ( group 1 ) , and the positive control group which consisted of hiv positive individuals but who were not on art ( group 2 ) . a total of 7 ml of blood was collected by clean venous puncture from participants , with 4.5 ml blood delivered into a bottle containing 0.5 ml sodium citrate and 2.5 ml into a bottle containing edta anticoagulant . principle and sample preparation : when red blood cells are mixed with cd4 monoclonal antibody in the presence of phycoerythrin , the cells are coated in phycoerythrin . when passed through the measuring area , cells or particles twenty microliters of well mixed whole blood ( with edta added as an anticoagulant ) was pipetted into a partec test tube . twenty microliters of cd4 mab pe was then added , mixed gently , and this was then incubated for 15 minutes at room temperature , protected from light , and 800 l of no lyse buffer was then added and mixed gently . cd4-pe fluorescence was analyzed on a partec flow cytometer with an excitation light source with a wavelength of 488 nm . following this , 840 l of the prepared blood sample was transferred to the cytometer for cd4 determination . d - dimer concentrations in plasma were determined using elisa kits ( technozym d - dimer , lot # rh02b00 ) purchased from technoclone ( vienna , austria ) . citrated plasma and one hundred microliters of calibrators , control plasmas , and test samples was pipetted into test wells , covered with film , and incubated in a hot air oven at 37c for 60 minutes . the test wells were then washed three times with 200 l washing buffer , using an automated elisa washer . one hundred microliters of conjugate working solution was pipetted into wells , covered with strips of film , and then incubated at 37c for 60 minutes . following this , the wells were washed three times with 200 l of washing buffer . one hundred microliters of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature for 10 minutes . one hundred microliters of stopping solution was then added into wells , these were shaken for 10 seconds , and measured within 10 minutes using an elisa reader at a wavelength of 450 nm . concentrations of test samples were read off from the calibration curve and also checked with the controls . citrated plasma and twenty - five microliters of calibrators , control plasmas , and test samples were pipetted into test wells , 75 l of incubation buffer was then added , and wells were covered test strips with film and incubated in a hot air oven at 37c for 60 minutes . wells were emptied thoroughly , 100 l of conjugate working solution was pipetted into wells , and these were covered with film . the test wells were incubated at 37c for 60 minutes and washed three times with 200 l of washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells ; these were shaken for 10 seconds , and then measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of test samples were read off from the calibration curve and also cross checked with the controls . citrated plasma and all required components in the kit were brought to room temperature . twenty - five l of calibrators , control plasmas , and test samples was pipetted into test wells , 75 l of incubation buffer was added , and then covered test strips with film and incubated in hot air oven at 37c for 60 minutes . wells were emptied , and 100 l of conjugate working solution was pipetted into wells which were then covered with film . the wells were further incubated at 37c for 60 minutes and washed three times with 200 l washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells , shaken for 10 seconds , and measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of the test samples were read off from the calibration curve , and checked with the controls . a total of 7 ml of blood was collected by clean venous puncture from participants , with 4.5 ml blood delivered into a bottle containing 0.5 ml sodium citrate and 2.5 ml into a bottle containing edta anticoagulant . principle and sample preparation : when red blood cells are mixed with cd4 monoclonal antibody in the presence of phycoerythrin , the cells are coated in phycoerythrin . when passed through the measuring area , cells or particles twenty microliters of well mixed whole blood ( with edta added as an anticoagulant ) was pipetted into a partec test tube . twenty microliters of cd4 mab pe was then added , mixed gently , and this was then incubated for 15 minutes at room temperature , protected from light , and 800 l of no lyse buffer was then added and mixed gently . cd4-pe fluorescence was analyzed on a partec flow cytometer with an excitation light source with a wavelength of 488 nm . following this , 840 l of the prepared blood sample was transferred to the cytometer for cd4 determination . d - dimer concentrations in plasma were determined using elisa kits ( technozym d - dimer , lot # rh02b00 ) purchased from technoclone ( vienna , austria ) . citrated plasma and all required components in the kit were brought to room temperature . one hundred microliters of calibrators , control plasmas , and test samples was pipetted into test wells , covered with film , and incubated in a hot air oven at 37c for 60 minutes . the test wells were then washed three times with 200 l washing buffer , using an automated elisa washer . one hundred microliters of conjugate working solution was pipetted into wells , covered with strips of film , and then incubated at 37c for 60 minutes . following this , one hundred microliters of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature for 10 minutes . one hundred microliters of stopping solution was then added into wells , these were shaken for 10 seconds , and measured within 10 minutes using an elisa reader at a wavelength of 450 nm . concentrations of test samples were read off from the calibration curve and also checked with the controls . twenty - five microliters of calibrators , control plasmas , and test samples were pipetted into test wells , 75 l of incubation buffer was then added , and wells were covered test strips with film and incubated in a hot air oven at 37c for 60 minutes . wells were emptied thoroughly , 100 l of conjugate working solution was pipetted into wells , and these were covered with film . the test wells were incubated at 37c for 60 minutes and washed three times with 200 l of washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film , and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells ; these were shaken for 10 seconds , and then measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of test samples were read off from the calibration curve and also cross checked with the controls . twenty - five l of calibrators , control plasmas , and test samples was pipetted into test wells , 75 l of incubation buffer was added , and then covered test strips with film and incubated in hot air oven at 37c for 60 minutes . wells were emptied , and 100 l of conjugate working solution was pipetted into wells which were then covered with film . the wells were further incubated at 37c for 60 minutes and washed three times with 200 l washing buffer . one hundred l of substrate solution was then pipetted into test wells , covered test strip with film and then incubated at room temperature ( 20c25c ) for 20 minutes . one hundred l of stopping solution was pipetted into wells , shaken for 10 seconds , and measured using an elisa reader at a wavelength of 450 nm within 10 minutes . concentrations of the test samples were read off from the calibration curve , and checked with the controls . data obtained from the study were subjected to descriptive statistics ( such as calculation of means and standard deviations ) to allow a general evaluation of all study variables . inferential analyses were conducted using repeated measures analysis of covariance ( ancova ) with between - subject variable conditions ( experimental vs control ) , demographic characteristics , and therapy duration . the measured parameter values within each group at baseline were used as covariates , while the interaction effect of associated independent factors was included in the analytical models of all study outcomes . analysis to determine the existence or otherwise of relationships within and between fibrinolytic parameters over the start study duration was carried out by conducting correlation analyses . all analyses were performed using spss software version 16.0 ( spss inc , chicago , il ) . there were more males ( 60% ) than females ( 40% ) , and 60% of the study participants were between 26 and 36 years of age . d - dimer was positively and significantly correlated with cd4 t lymphocyte counts in both aids - associated retrovirus ( arv ) and non - arv patients ( r = 0.304 , p < 0.01 vs r = 0.477 , p < 0.001 ) . t - pa was negatively and significantly correlated with cd4 t lymphocytes only in those undergoing art ( r = 0.294 , p < 0.01 ) . age was positively correlated with cd4 t lymphocytes and negatively correlated with d - dimer ( r = 0.244 , p < 0.001 vs r = 0.446 , p < 0.001 ) . pai-1 was not significantly correlated with any of the parameters among patients undergoing art but was weakly correlated with d - dimer among patients not receiving art ( r = 0.261 , p < 0.05 ) . when different durations of therapy were examined , pai-1 correlated negatively and significantly with t - pa at baseline and at month 1 ( r = 0.543 , p < 0.01 ) , while pai-1 correlated positively and strongly with d - dimer values at month 2 ( r = 0.550 , p < 0.01 ) . at month 3 , cd4 correlated negatively and significantly with d - dimer and t - pa ( r = 0.551 , p < 0.01 , and r = 0.583 , p < 0.01 , respectively ) , while pai-1 correlated positively with cd4 cell count ( r = 0.447 , p < 0.05 ) . the correlations of these parameters at baseline , month 1 , 2 , and 3 are presented in table 3 . among the hiv patients undergoing art , a progressive increase in cd4 cell count was observed from 192 l / ml at baseline to 323 l / ml after 3 months of therapy . in contrast , there was a significant drop in the cd4 count from 329 l / ml at baseline to 310 l / ml at the end of the third month among non - arv patients . figure 1 shows a reciprocal trend in cd4 t cell count between arv and non - arv subjects . in arv patients , d - dimer values dropped from 301.0 l / ml at baseline to 172.0 l / ml at month 2 , and then increased again to 226.0 l / ml at the end of the third month . the reverse was seen in patients who were not on art , as illustrated in figure 2 . the median baseline value of pai-1 at the beginning of therapy was 14.0 g / ml , which increased progressively to 18.2 g / ml at the end of the third month . the baseline value of t - pa at the beginning of therapy was 5.15 g / ml . this value progressively declined to 1.10 g / ml at the end of the first month . the value increased slightly to 1.45 g / ml and 1.5 g / ml at the end of the second and third months , respectively . the trend among the non - arv patients was reciprocal in nature when compared to those on therapy , and is illustrated in figure 3 . the major finding from this preliminary start study is that there is interplay between t - pa , d - dimer , and pai-1 resulting from the impact of art . it appears that haart increases the cd4 t cell count while also promoting the release of plasminogen activator inhibitor , which suppresses the action of t - pa . this induces a hypofibrinolytic state , with a reduction in d - dimer values able to be observed by the end of the first month . by the second month , the pai-1 level drops considerably , allowing the t - pa level to start rising . a further increase in pai-1 level occurred at the end of the second month with t - pa activity remaining low , maintaining a hypofibrinolytic state in the first 2 months of treatment . as the t - pa values increased moderately from the second month , there was a corresponding increase in the d - dimer concentrations , but these remained within the normal range . a positive correlation seems to exist , however , between t - pa and d - dimer , even though this was not statistically significant . a significant negative correlation exists between cd4 t cell count and d - dimer in haart patients . it follows that with haart therapy , the cd4 t cell count increases while t - pa value decreases . the hypofibrinolytic state observed in the current study is corroborated by earlier reports that art induces a hypofibrinolytic state as a result of the increase in pai-1 , which is reported to be marker of atherothrombotic risk.1416 several other cases of thrombosis associated with protease inhibitors ( pis ) containing haart have been reported.17,18 the pis containing haart are believed to promote thrombosis through pleiotropic effects , including alterations in lipid , pai-1 , and fibrogen levels . crum - cianflone et al reported that the progression of hiv is associated with t cell proliferation and activation , as well as high levels of circulating inflammatory markers , with immune activation persisting long after initiation of haart rather than art.19 the inflammatory responses caused by advanced hiv disease play a pivotal role in the pathogenesis of atherosclerosis inflammation , endothelial dysfunction , and impaired fibrinolysis , which may further contribute to the increased cardiovascular risk observed in the hiv - infected population.911 the pro - inflammatory state associated with hiv infection may also give rise to atherogenic lipid changes . the lipid profile in antiretroviral - nave hiv - infected subjects is characterized by low total cholesterol , hdl cholesterol , and ldl cholesterol , as well as elevated triglyceride levels ( mainly appearing as low density lipoprotein).1215 substantial evidence exists that haart induces a hypofibrinolytic state in hiv - infected individuals , as reflected in increased plasma levels of plasminogen activator and homocystein , which are thought to be markers of atherothrombotic risk.1416 in this study , a negative correlation was observed between cd4 t lymphocytes and d - dimer concentrations . the implication is that an increase in cd4 t lymphocytes will lead to a decrease in the d - dimer concentrations , and vice versa . this observation appears to agree with an earlier report by crum - cianflone et al,19 who found that venous thromboembolisms are associated with low cd4 cell counts . this appears to be a result of immune / inflammatory activation causing increased d - dimer concentrations , which induces a hypercoagulable state . art may play a major role in promoting arterial and venous thrombosis among hiv - infected persons as early as 3 months into the treatment . prethrombotic assessment through measurement of fibrinolytic parameters should be introduced early in the course of art .