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294 Activation of the fibrinolytic system in patients suffering from peripheral arterial occlusive disease and undergoing percutaneous transluminal angioplasty (PTA)
ORAL COMMUNICATIONS
292
II-8 F i b r i n o l y s i s a n d V a s c u l a r D i s e a s e s
II
87
293
O F EX-V1VO T H R O M B I DOES S U B S T A N T I A L L Y T H E I R L Y S I S IN V I T R O AGE
NOT
INFLUENCE
~abovid M. Keber D, Trnovo Hospital of btternal Medicine, Ljubljana, SIovenia The outcome of lhrombolysis in vivo is influenced by the age o f thrombosis. The poor lysability of older arterial and venous thrombi has been attributed to lhe organization of Ihrombi during whicb substitution o f fibrin with fibrous tissue takes place. In order to test tbis presumption the group o f fresh llarombi (7 arterial and 8 venous Ihrombi; age o f less than one week, median value 2 days) and the group of aged thrombi (7 arterial and 8 venous thrombi; older than one month, median value 9 weeks) were compared using bolh in vilro lysis and histological examination. Thro,nbi were obtained at autopsies or operations, ql~e probable age was estimated by Ihe lime-interval li'om the onset o f clinical symptoms and in vivo imaging (angiography, ultrasound or compuler lontography) to removal. Pieces Hf the thrombi, weighing approximately 250 rag, were exposed to lysis in 2 ml volumes of plasma with streptHkinase 2501U/lnl, urokinase 2501U/ml Hr t-PA 5001U/ml. The extent of lysis was measured by the decrease Hf Ihe wet weight Hf lhrombi Hver a period of 24 hours, q h e mullisegmental Iransverse slices of lhrombi were Irealed wifll standard Gientsa staining, Ihe exlenl of organization was delected and lhen quantified with a slandard point-count method. A nonsignificanlly better lysis (fiom 5 to 15% with different agents) was found in the fresh arterial and venous lhrombi in comparison Io aged thrombi, qlle hislological examination showed Hnly slight /H moderate organization in the aged thrombi. In conclusion, Ihe general belief in Ihe decrease of lhrombus lysability during the course of several months aging was nol confirmed by Ihis sludy. The prior clinical results ol'lrealmenl of aged Ihrombosis might be rather due to thrombi by-passing of Ihrombolylic agent via collateral circtdation and/or hindered permeation o f Ihrombolytic agent through fl~e endothelial layer of aged Ihrombi.
294
HIGHLY IMPAIRED FIBRINOLYSIS IN NON-INSULIN-DEPENDENT BUT
NOT IN INSULIN-DEPENDENT DIABETES ( T H R O M B O C H E C K STUDY) IAmiral J., 2 S e i g n e t ~ , IVissac AM.. 2Boisseau M., 3_Scarabin PY., and 4Gin H. ISerbio, Gennevilliers, 2Univiversit6 de Bordeaux, Bordeaux, 31nserm U258, H6pital Broussais, Paris, 4H6pital Pellegrin, Bordeaux (France). Elevated incidence of arterial diseases in diabetic patients may result from multifactorial parameters which may differ in tbe different types of diabetes. Two hundred and ten diabetic patients, including 121 insulin-dependent ODD), 65 non-insulin dependent (NIDD) and 22 inaugural (ID), were tested for blood activation markers, lipids and fibrinolytic variables. Patients with renal failure were excluded. When adjusted for age and sex, tPA:Ag, PAI-I activity and antigen were significantly increased in the diabetic group compared to healthy subjects (337 men, 348 women), and the global fibrinolytic capacity (GFC), evaluated with a global assay (fibrinolytic potential scored from 4 for full capacity to 0 for deficient), was decreased (p<0.0001). These modifications were confined to the NIDD sub-group who presented extremely high levels of PAl-1 activity (23.07 IU/ml versus 9.85 IU/ml for normals and 5.94 IU/ml for IDD), PAI-I:Ag (49.20 ng/ml versus 12.81 ng/ml for nonnals and 17.11 ng/ml for IDD), whereas tPA:Ag was only moderately increased (10.90 ng/m[ versus 6.90 ng/ml for normals and 7.14 ng/ml for IDD). These changes corresponded to a strong inhibition of fibrinolysis in NIDD sub-group (0.98), while the dramatic tall in GFC was not significantly different between normals, IDD and ID subgroups (3.0). Among the other parameters tested, triglycerides were elevated in all diabetic patients, but they were at a much higher concentration in NIDD subgroup (240 rng/l versus 97 mg/I for control group and 125 mg/l for IDD). For all the diabetic sub-groups, the other changes, when adjusted for age and sex, concerned yon Willebrand factor (vWF), which presented about 50% increased levels indicating the endothelial alteration, and fibrinogen and DDimer, which showed also elevated values. In addition, the body-mass-index (BMI - kg/m 2) was much higher in NIDD (30.4) as compared to controls (24.5), ID (21.1) and IDD (23.6) sub-groups. This study demonstrates that the disorders of fibrinolysis concern NIDD but not IDD or ID. While the fibrinolytic potential is within the normal range in IDD and ID, it is very deficient in NIDD and it is associated with an increase of both tPA (moderately) and PAI-I (dramatically), of triglycerides and BMI. This lack of fibrinolysis could contribute to explain the high risk of thrombotic arterial disease in NIDD.
295
Activation of the Fibrinolytic System ha Patients suffering from Peripheral arterial occlusive disease and undergoing Percutancous Transluminal
An#opt-.ty (PTA) Roller R.~. Janlseh S.. Carroll V., Lorenz M.', Nimmrtchter V.'. Pil~er E.', WoRm J.. Binder B.R.
l)el~tflmonto( VasmlarBiologyand~ ~ *Depm~ma~~"Angiology,M~;i¢~dUniversityCtinic~
Universityof Vienna AUSTRIA
Pe~ral ~ ec~lusivedisease (PAOD) an clinical mamfestation of arterinselarosis is a nmjor problena m elderly people in the civifined western world. Besides medical treatn~mt angaoplastyof occluded arteries also in aged patients tins become an accepted and world-wide performed therz~y with an increasing suc.zessrate over the past years. However, the effica~ of this therapy is limited by late regtenosisoc.currmgin up to 50% of the primarily ~ece~fully dilated arteries. As our 8~up could show recently in a large clinical trim in corotmry heart disease patients an increase of plasmmogen activatorinhibitor 1 (PAId) 48 hours after angioplasty is correlated with late restenosis in these patiellts. We were therefore i n ~ whether these findings could also be verified for PAOD petients unde~oing PTA and to look for basic differences in the fibrinolytic system of these patients versus a group of age matched healthy volunteers. 53 patients (mean age 69.3:1:1.2years) versus 50 healthy volunteers (62~4.1 years) were recruited to our study. Blood mJmples were drawn before PTA. 6, 24 and 48 hours after the intervention. Clinical outoome of the patients w~ documented by Coloured Doppler Sonography and a PARAMETER4"SEaM PAI-I-Ag (ng/ml) PAI-I~ (U/ml) t-PA-Ag(ng/mI) (% frmatnonlmu')
HEALTHY 28.1~.3.5 7,9:t:1.1 8.4z~.7 135.6:1:11.3
PATIENTS ! 36.3~.3.5 11.4+1.1 13.2:kO.g 190.8:t:8.6
Flbrln~m (ml/dl)
261.7:15.9
426.4+14.9
narrowing of the luminal vessel diameter of more than 50% compared to post PTA measures was used as indicative for resteaosis. Analysing the plo_~masamples, statistically si~pMfieamt differowam could be found in the parmnetors determined between healthy cotttrols and PAOD patients as can be seen from the table. During angioplasty, we register'ed a statistically significant morease in PAI-I antigen and activity levels (p=0.04), t-PA antigert (p=0.0001), t-PA-PAI-complex(p--0.01) and fibrinogen levels (p=0.01) 48 hou~ ~ the interventinn.Taking in comidemtion the individual clinical outcome of the tmtients 6 months ~ ansioplasty we could observe a statistically significam difference in the PAI-I ~tivity levels in plasma samples obtained 24 hours after PTA: Patients suffering from late ~ i s exhibited significantly higher levels (16.1:12.4 Ulml) them did patients with ~ u l clinical outcome (12.5+1.9 U/ml). These data are consistent with those already rcpoded for corona~ heart patients by us. However, testing these values for prognostic relevance, the elevation of PAI-I activity levels could not be used as a prognostic marker for late restenosis.
E F F E C T O F L I P O P R O T E I N ( a ) ON H U M A N B R A I N E N D O T H E L I A L
FIBR1NOLYTIC ACTIVITY Skopfil J, *Kar~idi I, V a s t a g M, **T6th M, N a g y Z National Stroke Centre, National Institute o f Psychiatry and Neurology, *lst and**3rd Dept. of Medicine, Semmelweis Unwersity of Medical School, Budapest, H u n g a r y The thromboresistanct luminal surface of the endothelial cells is important in the maintenance o f hemostatic balanceEndothelium can modulate fibrinolysis by expressing o f t - P A and PAI-I in a complex manner.In this way endothelial cells participate in the cleavage of local fibrin clot. Lipoprotein(a) (Lp(a)) is an independent risk factor for coronary artery, diseases and ischemic stroke. Lp(a) shows a close structural homology with plasminogen and may interfere with fibrinolysis by competition with plasminogen at the sites of cellular receptors and for fibrin binding. In this study the effect o f Lp(a) on the expression o f t-PA, u-PA and PAI-1 was measured in culture o f human brain microvessel endothelial cells (HBECs). H B E C s were isolated, cultured and characterised The experiments were performed on primary, confluent cell cultures. The cells were incubated in tissue culture medium with or without Lp(a) (450 tag/ml) for 72 hours. The cells incubated without Lp(a) used as control, to characterise the basal fibrinolytic activity of HBECs. Levels of t-PA, u-PA and PAI-I antigens were measured by EIL1SA Kits. In conditioned medium o f cultured HBECs 11,7 ng/ml t-PA. 10 ng/ml u-PA and 213 ng/ml PAl-1 were detected during a 72 hours period of time. The Lp(a) treatment reduced the production o f t - P A and u-PA to 63 % and 14 % o f control values, respectively. The concentration o f PAI- 1 antigen remained unchanged (102 % of control value) The effect of Lp(a) on the HBECs could be an important factor in the genesis o f ischaemic lesions in the brain. Its raised serum concentration may decrease the fibrinol~ic activity o f endothelium delaying the local clot lysis Lp(a) may influence the thrombolytic therapy of acut ischemic stroke causing unsuccessful treatment.
292
II-8 F i b r i n o l y s i s a n d V a s c u l a r D i s e a s e s
II
87
293
O F EX-V1VO T H R O M B I DOES S U B S T A N T I A L L Y T H E I R L Y S I S IN V I T R O AGE
NOT
INFLUENCE
~abovid M. Keber D, Trnovo Hospital of btternal Medicine, Ljubljana, SIovenia The outcome of lhrombolysis in vivo is influenced by the age o f thrombosis. The poor lysability of older arterial and venous thrombi has been attributed to lhe organization of Ihrombi during whicb substitution o f fibrin with fibrous tissue takes place. In order to test tbis presumption the group o f fresh llarombi (7 arterial and 8 venous Ihrombi; age o f less than one week, median value 2 days) and the group of aged thrombi (7 arterial and 8 venous thrombi; older than one month, median value 9 weeks) were compared using bolh in vilro lysis and histological examination. Thro,nbi were obtained at autopsies or operations, ql~e probable age was estimated by Ihe lime-interval li'om the onset o f clinical symptoms and in vivo imaging (angiography, ultrasound or compuler lontography) to removal. Pieces Hf the thrombi, weighing approximately 250 rag, were exposed to lysis in 2 ml volumes of plasma with streptHkinase 2501U/lnl, urokinase 2501U/ml Hr t-PA 5001U/ml. The extent of lysis was measured by the decrease Hf Ihe wet weight Hf lhrombi Hver a period of 24 hours, q h e mullisegmental Iransverse slices of lhrombi were Irealed wifll standard Gientsa staining, Ihe exlenl of organization was delected and lhen quantified with a slandard point-count method. A nonsignificanlly better lysis (fiom 5 to 15% with different agents) was found in the fresh arterial and venous lhrombi in comparison Io aged thrombi, qlle hislological examination showed Hnly slight /H moderate organization in the aged thrombi. In conclusion, Ihe general belief in Ihe decrease of lhrombus lysability during the course of several months aging was nol confirmed by Ihis sludy. The prior clinical results ol'lrealmenl of aged Ihrombosis might be rather due to thrombi by-passing of Ihrombolylic agent via collateral circtdation and/or hindered permeation o f Ihrombolytic agent through fl~e endothelial layer of aged Ihrombi.
294
HIGHLY IMPAIRED FIBRINOLYSIS IN NON-INSULIN-DEPENDENT BUT
NOT IN INSULIN-DEPENDENT DIABETES ( T H R O M B O C H E C K STUDY) IAmiral J., 2 S e i g n e t ~ , IVissac AM.. 2Boisseau M., 3_Scarabin PY., and 4Gin H. ISerbio, Gennevilliers, 2Univiversit6 de Bordeaux, Bordeaux, 31nserm U258, H6pital Broussais, Paris, 4H6pital Pellegrin, Bordeaux (France). Elevated incidence of arterial diseases in diabetic patients may result from multifactorial parameters which may differ in tbe different types of diabetes. Two hundred and ten diabetic patients, including 121 insulin-dependent ODD), 65 non-insulin dependent (NIDD) and 22 inaugural (ID), were tested for blood activation markers, lipids and fibrinolytic variables. Patients with renal failure were excluded. When adjusted for age and sex, tPA:Ag, PAI-I activity and antigen were significantly increased in the diabetic group compared to healthy subjects (337 men, 348 women), and the global fibrinolytic capacity (GFC), evaluated with a global assay (fibrinolytic potential scored from 4 for full capacity to 0 for deficient), was decreased (p<0.0001). These modifications were confined to the NIDD sub-group who presented extremely high levels of PAl-1 activity (23.07 IU/ml versus 9.85 IU/ml for normals and 5.94 IU/ml for IDD), PAI-I:Ag (49.20 ng/ml versus 12.81 ng/ml for nonnals and 17.11 ng/ml for IDD), whereas tPA:Ag was only moderately increased (10.90 ng/m[ versus 6.90 ng/ml for normals and 7.14 ng/ml for IDD). These changes corresponded to a strong inhibition of fibrinolysis in NIDD sub-group (0.98), while the dramatic tall in GFC was not significantly different between normals, IDD and ID subgroups (3.0). Among the other parameters tested, triglycerides were elevated in all diabetic patients, but they were at a much higher concentration in NIDD subgroup (240 rng/l versus 97 mg/I for control group and 125 mg/l for IDD). For all the diabetic sub-groups, the other changes, when adjusted for age and sex, concerned yon Willebrand factor (vWF), which presented about 50% increased levels indicating the endothelial alteration, and fibrinogen and DDimer, which showed also elevated values. In addition, the body-mass-index (BMI - kg/m 2) was much higher in NIDD (30.4) as compared to controls (24.5), ID (21.1) and IDD (23.6) sub-groups. This study demonstrates that the disorders of fibrinolysis concern NIDD but not IDD or ID. While the fibrinolytic potential is within the normal range in IDD and ID, it is very deficient in NIDD and it is associated with an increase of both tPA (moderately) and PAI-I (dramatically), of triglycerides and BMI. This lack of fibrinolysis could contribute to explain the high risk of thrombotic arterial disease in NIDD.
295
Activation of the Fibrinolytic System ha Patients suffering from Peripheral arterial occlusive disease and undergoing Percutancous Transluminal
An#opt-.ty (PTA) Roller R.~. Janlseh S.. Carroll V., Lorenz M.', Nimmrtchter V.'. Pil~er E.', WoRm J.. Binder B.R.
l)el~tflmonto( VasmlarBiologyand~ ~ *Depm~ma~~"Angiology,M~;i¢~dUniversityCtinic~
Universityof Vienna AUSTRIA
Pe~ral ~ ec~lusivedisease (PAOD) an clinical mamfestation of arterinselarosis is a nmjor problena m elderly people in the civifined western world. Besides medical treatn~mt angaoplastyof occluded arteries also in aged patients tins become an accepted and world-wide performed therz~y with an increasing suc.zessrate over the past years. However, the effica~ of this therapy is limited by late regtenosisoc.currmgin up to 50% of the primarily ~ece~fully dilated arteries. As our 8~up could show recently in a large clinical trim in corotmry heart disease patients an increase of plasmmogen activatorinhibitor 1 (PAId) 48 hours after angioplasty is correlated with late restenosis in these patiellts. We were therefore i n ~ whether these findings could also be verified for PAOD petients unde~oing PTA and to look for basic differences in the fibrinolytic system of these patients versus a group of age matched healthy volunteers. 53 patients (mean age 69.3:1:1.2years) versus 50 healthy volunteers (62~4.1 years) were recruited to our study. Blood mJmples were drawn before PTA. 6, 24 and 48 hours after the intervention. Clinical outoome of the patients w~ documented by Coloured Doppler Sonography and a PARAMETER4"SEaM PAI-I-Ag (ng/ml) PAI-I~ (U/ml) t-PA-Ag(ng/mI) (% frmatnonlmu')
HEALTHY 28.1~.3.5 7,9:t:1.1 8.4z~.7 135.6:1:11.3
PATIENTS ! 36.3~.3.5 11.4+1.1 13.2:kO.g 190.8:t:8.6
Flbrln~m (ml/dl)
261.7:15.9
426.4+14.9
narrowing of the luminal vessel diameter of more than 50% compared to post PTA measures was used as indicative for resteaosis. Analysing the plo_~masamples, statistically si~pMfieamt differowam could be found in the parmnetors determined between healthy cotttrols and PAOD patients as can be seen from the table. During angioplasty, we register'ed a statistically significant morease in PAI-I antigen and activity levels (p=0.04), t-PA antigert (p=0.0001), t-PA-PAI-complex(p--0.01) and fibrinogen levels (p=0.01) 48 hou~ ~ the interventinn.Taking in comidemtion the individual clinical outcome of the tmtients 6 months ~ ansioplasty we could observe a statistically significam difference in the PAI-I ~tivity levels in plasma samples obtained 24 hours after PTA: Patients suffering from late ~ i s exhibited significantly higher levels (16.1:12.4 Ulml) them did patients with ~ u l clinical outcome (12.5+1.9 U/ml). These data are consistent with those already rcpoded for corona~ heart patients by us. However, testing these values for prognostic relevance, the elevation of PAI-I activity levels could not be used as a prognostic marker for late restenosis.
E F F E C T O F L I P O P R O T E I N ( a ) ON H U M A N B R A I N E N D O T H E L I A L
FIBR1NOLYTIC ACTIVITY Skopfil J, *Kar~idi I, V a s t a g M, **T6th M, N a g y Z National Stroke Centre, National Institute o f Psychiatry and Neurology, *lst and**3rd Dept. of Medicine, Semmelweis Unwersity of Medical School, Budapest, H u n g a r y The thromboresistanct luminal surface of the endothelial cells is important in the maintenance o f hemostatic balanceEndothelium can modulate fibrinolysis by expressing o f t - P A and PAI-I in a complex manner.In this way endothelial cells participate in the cleavage of local fibrin clot. Lipoprotein(a) (Lp(a)) is an independent risk factor for coronary artery, diseases and ischemic stroke. Lp(a) shows a close structural homology with plasminogen and may interfere with fibrinolysis by competition with plasminogen at the sites of cellular receptors and for fibrin binding. In this study the effect o f Lp(a) on the expression o f t-PA, u-PA and PAI-1 was measured in culture o f human brain microvessel endothelial cells (HBECs). H B E C s were isolated, cultured and characterised The experiments were performed on primary, confluent cell cultures. The cells were incubated in tissue culture medium with or without Lp(a) (450 tag/ml) for 72 hours. The cells incubated without Lp(a) used as control, to characterise the basal fibrinolytic activity of HBECs. Levels of t-PA, u-PA and PAI-I antigens were measured by EIL1SA Kits. In conditioned medium o f cultured HBECs 11,7 ng/ml t-PA. 10 ng/ml u-PA and 213 ng/ml PAl-1 were detected during a 72 hours period of time. The Lp(a) treatment reduced the production o f t - P A and u-PA to 63 % and 14 % o f control values, respectively. The concentration o f PAI- 1 antigen remained unchanged (102 % of control value) The effect of Lp(a) on the HBECs could be an important factor in the genesis o f ischaemic lesions in the brain. Its raised serum concentration may decrease the fibrinol~ic activity o f endothelium delaying the local clot lysis Lp(a) may influence the thrombolytic therapy of acut ischemic stroke causing unsuccessful treatment.