THROMBOSIS RESEARCH 64; 445453,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
POOR FIBRINOLYI’IC RESPONSE TO VENOUS OCCLUSION BY DIFFERENT CRITERIA IN PATIENTS WITH DEEP VEIN THROMBOSIS
Mojca Stegnar, Polona Petemel, Dugan Keber and Nina Vene From the University Medical Centre, Trnovo Hospital of Internal Medicine, Ljubljana, Yugoslavia (Received 18.6.1991; accepted in revised form 3.9.1991 by Editor F. Haverkate) ABSTRACT Five criteria for poor response to a 20 min venous occlusion test were applied to 58 patients 3 months or more after acute deep vein thrombosis (DVT). The criteria were arbitrarily defined as the last 5 percentiles of response distributions in an age- and sex-matched healthy control group of 51 subjects. The criteria were: 1. euglobulin clot lysis time after venous occlusion 1 140 min; 2. t-PA activity after venous occlusion 5 0.04 IU/ml; 3. increase in t-PA antigen above resting value 5 2-fold, 4. ratio between t-PA antigen increase and resting PAI activity 5 0.5 ng/IU; 5. PAI activity after venous occlusion 2 6 IU/ml. The last criterion of poor response was the only one that was significantly more frequently reached by patients than by controls: 28% (pcO.005) of all DVT patients and 35% (pcO.005) of the subgroup with idiopathic DVT (N=34) were found to be poor responders. The percentage of poor responders according to the other four criteria was 7-11% in all patients and 9-15% in the subgroup with idiopathic DVT and thus was not significantly higher than in controls (5% by definition). It was concluded that residual PA1 activity after venous occlusion might be a useful criterion for prospective studies on recurrence of DVT. INTRODUCTION Poor fibrinolytic response to venous occlusion is defined as the absence of, or low increase in, fibrinolytic activity after this stimulus. Lack of increase in activity is caused either by a high level of basal plasminogen activator inhibitor 1 (PAI-l), or by a low increase in tissuetype plasminogen activator (t-PA) antigen after venous occlusion, or both (I-4). Key words: deep vein thrombosis, venous occlusion , fibrinolysis 445
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Poor fibrinolytic response to venous occlusion is considered to be one of the most common pathological findings in patients with deep vein trombosis (DVT), since about l/3 of them are classified as poor responders (3,5,6). Poor response to venous occlusion has also been shown to be associated with a higher recurrence of thrombotic episodes (67) indicating that impaired fibrinolysis might be involved in the pathogenesis of the disease. However, the criteria used to define poor response to venous occlusion vary from one investigator to another. The diversity of assays used to measure fibrinolytic activity (e.g. global and specific methods), the lack of their standardization, the use of different durations of venous occlusion (from 5 to 20 min) and the lack of established normal ranges with clear cut-off points make it difficult to summarize data from different studies in order to enable clinical interpretation of results in individual patients. In several studies dealing with poor response to venous occlusion, definitions of poor responses were derived from small control groups or were arbitrarily established without control groups. Due to the fact that t-PA and PAI- show wide physiological ranges, because they vary with sex, age and body weight (8-U), it has been suspected that some differences between DVT patients and healthy subjects in responses to venous occlusion might stem from unmatched study groups. The present study was, therefore, undertaken in order to establish if the distribution of responses by several different criteria varies in a population of patients with DVT from the distribution in a well matched healthy population.
SUBJECTS, MATERIALS AND METHODS
All subjects gave informed consent before enrolment into the study which was approved by the State Ethical Committee and carried out according to the principles of the Declaration of Helsinki. Controls and patients were studied in parallel. Patients with DVT
Fifty-eight patients (26 women and 32 men, age 17-61, mean+SD: 43~14 years) admitted because of acute DVT were enrolled in the study 3 months or more (24+13 months) after discharge from hospital. Most of the patients (N=41, 70%) had suffered DVT of the femoral and/or iliacal veins, 13 (22%) DVT of the calf and 4 (8%) DVT of the arm. At the time of blood sampling 19 (33%) patients were being treated with vitamin K antagonists. In 34 (59%) patients no underlying cause for DVT (immobilization, surgery, trauma, delivery or abortion within one month before DVT, pregnancy, hormonal treatment including oral contraceptives) could be found (idiopathic DVT). In the others (N=24, 41%) at least one of the above possible causes was identified (“secondary” DVT). All patients had antithrombin III, protein C and plasminogen in the range of normal reference values. Healthy subjects
Fifty-one apparently healthy subjects (26 men and 25 women) served as controls. They were 19 to 58 years old, with a mean age for all subjects of 40+11 years and a similar age
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distribution for both sexes. None of them had history of superficial thrombophlebitis, DVT or any other condition known to predispose to venous thrombosis. None of the female controls was taking contraceptive drugs. Eight subjects were moderate smokers (up to 20 cigarettes per day). Blood sampling
Resting blood samples were obtained between 7 a.m. and 9 a.m. after a 30 min rest. Blood was sampled from an antecubital vein, in most cases without application of a tourniquet. After obtaining resting blood samples a sphygmomanometer cuff was wrapped around the other arm and inflated for 20 min to a level midway between the systolic and diastolic blood pressure. The post-occlusion blood samples were obtained from an antecubital vein in the last minute of occlusion before the cuff was deflated. The blood flowed directly into precooled siliconized glass tubes with 0.13 mol/l trisodium citrate (1 volume of citrate to 9 volumes of blood), placed in ice water and was then centrifuged for 10 min at 2000 g and 4”C. Plasma was transferred to small plastic vials, frozen in liquid nitrogen and stored at -700C until analyzed. Laboratory methods
Euglobulin clot lysis time (12) was determined as follows: 1 volume of plasma was diluted with 19 volumes of distilled water and acidified to pH 5.9 with acetic acid. Precipitated euglobulins were collected by centrifugation, dissolved to the original plasma volume with sodium borate buffer (0.15 M/l NaCl, 2.6 mM/l sodium tetraborate, pH 9.0) and clotted with an equal volume of 0.025 M/l CaCl2 Clot lysis was observed visually and recorded in minutes. t-PA activity (13), t-PA antigen (8) and PAI activity (14)were determined as described elsewhere (15). Their values after venous occlusion were corrected for hemoconcentration (16), calculated from the hematocrit obtained in a blood cell counter (Coulter ZF, Luton, England). Triglycerides (17), total cholesterol (18) and glucose (19) were also determined. The body mass index was calculated from the ratio: (body weight in g)/(body height in cm)2 (20). Criteria for poor response to venous occlusion
Five different criteria were used to define poor responders to venous occlusion. All were derived from the distribution of responses in the control group. Since distributions of responses were not normal, the highest or the lowest fifth percentiles were considered as the limit between good and poor response. By such a definition the percentage of poor responders among healthy controls was 5% by all criteria. Poor responses were thus defined as: 1. euglobulin clot lysis time after venous occlusion 2 140 min; 2. t-PA activity after venous occlusion 5 0.04 III/ml; 3. increase in t-PA antigen over resting value 5 2-fold; 4. ratio: (absolute increase in t-PA antigen) / (resting PAI activity) ( 0.5 ng/IU, 5. PAI activity after venous occlusion 16 IU/ml.
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Vol. 84, No. 4
Statistical methods Fibrinolytic parameters were described by medians and ranges and biochemical parameters by means and standard deviations. Differences between groups were tested by the Mann-Whitney U-test (fibrinolytic parameters) and Student’s t-test (biochemical parameters). Differences in frequencies of poor responders were evaluated by the chisquare test, and associations between variables by linear and multiple regression (21). All calculations were made using the SPSS/PC+ computer programme (SPSS inc., Chicago, 11,USA).
RESULTS In resting plasma samples, as compared to healthy controls, patients with DVT had significantly longer euglobulin clot lysis time (247 vs 235 min, p=O.O5), higher t-PA antigen (8.2 vs 6.1 ng/ml, p
POOR FIBRINOLYI’IC RESPONSE TO VENOUS OCCLUSION BY DIFFERENT CRITERIA IN PATIENTS WITH DEEP VEIN THROMBOSIS
Mojca Stegnar, Polona Petemel, Dugan Keber and Nina Vene From the University Medical Centre, Trnovo Hospital of Internal Medicine, Ljubljana, Yugoslavia (Received 18.6.1991; accepted in revised form 3.9.1991 by Editor F. Haverkate) ABSTRACT Five criteria for poor response to a 20 min venous occlusion test were applied to 58 patients 3 months or more after acute deep vein thrombosis (DVT). The criteria were arbitrarily defined as the last 5 percentiles of response distributions in an age- and sex-matched healthy control group of 51 subjects. The criteria were: 1. euglobulin clot lysis time after venous occlusion 1 140 min; 2. t-PA activity after venous occlusion 5 0.04 IU/ml; 3. increase in t-PA antigen above resting value 5 2-fold, 4. ratio between t-PA antigen increase and resting PAI activity 5 0.5 ng/IU; 5. PAI activity after venous occlusion 2 6 IU/ml. The last criterion of poor response was the only one that was significantly more frequently reached by patients than by controls: 28% (pcO.005) of all DVT patients and 35% (pcO.005) of the subgroup with idiopathic DVT (N=34) were found to be poor responders. The percentage of poor responders according to the other four criteria was 7-11% in all patients and 9-15% in the subgroup with idiopathic DVT and thus was not significantly higher than in controls (5% by definition). It was concluded that residual PA1 activity after venous occlusion might be a useful criterion for prospective studies on recurrence of DVT. INTRODUCTION Poor fibrinolytic response to venous occlusion is defined as the absence of, or low increase in, fibrinolytic activity after this stimulus. Lack of increase in activity is caused either by a high level of basal plasminogen activator inhibitor 1 (PAI-l), or by a low increase in tissuetype plasminogen activator (t-PA) antigen after venous occlusion, or both (I-4). Key words: deep vein thrombosis, venous occlusion , fibrinolysis 445
446
POOR RESPONSE IN VEIN THROMBOSIS
Vol. 64, No. 4
Poor fibrinolytic response to venous occlusion is considered to be one of the most common pathological findings in patients with deep vein trombosis (DVT), since about l/3 of them are classified as poor responders (3,5,6). Poor response to venous occlusion has also been shown to be associated with a higher recurrence of thrombotic episodes (67) indicating that impaired fibrinolysis might be involved in the pathogenesis of the disease. However, the criteria used to define poor response to venous occlusion vary from one investigator to another. The diversity of assays used to measure fibrinolytic activity (e.g. global and specific methods), the lack of their standardization, the use of different durations of venous occlusion (from 5 to 20 min) and the lack of established normal ranges with clear cut-off points make it difficult to summarize data from different studies in order to enable clinical interpretation of results in individual patients. In several studies dealing with poor response to venous occlusion, definitions of poor responses were derived from small control groups or were arbitrarily established without control groups. Due to the fact that t-PA and PAI- show wide physiological ranges, because they vary with sex, age and body weight (8-U), it has been suspected that some differences between DVT patients and healthy subjects in responses to venous occlusion might stem from unmatched study groups. The present study was, therefore, undertaken in order to establish if the distribution of responses by several different criteria varies in a population of patients with DVT from the distribution in a well matched healthy population.
SUBJECTS, MATERIALS AND METHODS
All subjects gave informed consent before enrolment into the study which was approved by the State Ethical Committee and carried out according to the principles of the Declaration of Helsinki. Controls and patients were studied in parallel. Patients with DVT
Fifty-eight patients (26 women and 32 men, age 17-61, mean+SD: 43~14 years) admitted because of acute DVT were enrolled in the study 3 months or more (24+13 months) after discharge from hospital. Most of the patients (N=41, 70%) had suffered DVT of the femoral and/or iliacal veins, 13 (22%) DVT of the calf and 4 (8%) DVT of the arm. At the time of blood sampling 19 (33%) patients were being treated with vitamin K antagonists. In 34 (59%) patients no underlying cause for DVT (immobilization, surgery, trauma, delivery or abortion within one month before DVT, pregnancy, hormonal treatment including oral contraceptives) could be found (idiopathic DVT). In the others (N=24, 41%) at least one of the above possible causes was identified (“secondary” DVT). All patients had antithrombin III, protein C and plasminogen in the range of normal reference values. Healthy subjects
Fifty-one apparently healthy subjects (26 men and 25 women) served as controls. They were 19 to 58 years old, with a mean age for all subjects of 40+11 years and a similar age
Vol. 64, No. 4
POOR RESPONSE IN VEIN THROMBOSIS
447
distribution for both sexes. None of them had history of superficial thrombophlebitis, DVT or any other condition known to predispose to venous thrombosis. None of the female controls was taking contraceptive drugs. Eight subjects were moderate smokers (up to 20 cigarettes per day). Blood sampling
Resting blood samples were obtained between 7 a.m. and 9 a.m. after a 30 min rest. Blood was sampled from an antecubital vein, in most cases without application of a tourniquet. After obtaining resting blood samples a sphygmomanometer cuff was wrapped around the other arm and inflated for 20 min to a level midway between the systolic and diastolic blood pressure. The post-occlusion blood samples were obtained from an antecubital vein in the last minute of occlusion before the cuff was deflated. The blood flowed directly into precooled siliconized glass tubes with 0.13 mol/l trisodium citrate (1 volume of citrate to 9 volumes of blood), placed in ice water and was then centrifuged for 10 min at 2000 g and 4”C. Plasma was transferred to small plastic vials, frozen in liquid nitrogen and stored at -700C until analyzed. Laboratory methods
Euglobulin clot lysis time (12) was determined as follows: 1 volume of plasma was diluted with 19 volumes of distilled water and acidified to pH 5.9 with acetic acid. Precipitated euglobulins were collected by centrifugation, dissolved to the original plasma volume with sodium borate buffer (0.15 M/l NaCl, 2.6 mM/l sodium tetraborate, pH 9.0) and clotted with an equal volume of 0.025 M/l CaCl2 Clot lysis was observed visually and recorded in minutes. t-PA activity (13), t-PA antigen (8) and PAI activity (14)were determined as described elsewhere (15). Their values after venous occlusion were corrected for hemoconcentration (16), calculated from the hematocrit obtained in a blood cell counter (Coulter ZF, Luton, England). Triglycerides (17), total cholesterol (18) and glucose (19) were also determined. The body mass index was calculated from the ratio: (body weight in g)/(body height in cm)2 (20). Criteria for poor response to venous occlusion
Five different criteria were used to define poor responders to venous occlusion. All were derived from the distribution of responses in the control group. Since distributions of responses were not normal, the highest or the lowest fifth percentiles were considered as the limit between good and poor response. By such a definition the percentage of poor responders among healthy controls was 5% by all criteria. Poor responses were thus defined as: 1. euglobulin clot lysis time after venous occlusion 2 140 min; 2. t-PA activity after venous occlusion 5 0.04 III/ml; 3. increase in t-PA antigen over resting value 5 2-fold; 4. ratio: (absolute increase in t-PA antigen) / (resting PAI activity) ( 0.5 ng/IU, 5. PAI activity after venous occlusion 16 IU/ml.
448
POOR RESPONSE IN VEIN THROMBOSIS
Vol. 84, No. 4
Statistical methods Fibrinolytic parameters were described by medians and ranges and biochemical parameters by means and standard deviations. Differences between groups were tested by the Mann-Whitney U-test (fibrinolytic parameters) and Student’s t-test (biochemical parameters). Differences in frequencies of poor responders were evaluated by the chisquare test, and associations between variables by linear and multiple regression (21). All calculations were made using the SPSS/PC+ computer programme (SPSS inc., Chicago, 11,USA).
RESULTS In resting plasma samples, as compared to healthy controls, patients with DVT had significantly longer euglobulin clot lysis time (247 vs 235 min, p=O.O5), higher t-PA antigen (8.2 vs 6.1 ng/ml, p
