Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis.

INTRODUCTION: Chronic insufficiency alters homeostasis, in part due to endothelial inflammation. Plasminogen activator inhibitor-1 (PAI-1) is increased in renal disease, contributing to vascular damage. We assessed PAI-1 activity and PAI-1 4G/5G polymorphism in hemodialysis (HD) subjects and any association between thrombotic vascular access (VA) events and PAI-1 polymorphism. METHODS: Prospective, observational study in 36 HD patients: mean age: 66.6 +/- 12.5 yr, males n=26 (72%), time on HD: 28.71 +/- 22.45 months. Vascular accesses: 10 polytetrafluoroethylene grafts (PTFEG), 22 arteriovenous fistulae (AVF), four dual lumen catheters (CAT). Control group (CG): 40 subjects; mean age: 60.0 +/- 15 yrs, males n=30 (75%). Group A (GA): thrombotic events (n=12), and group B (GB): No events (n=24). Groups were no different according to age (69.2 +/- 9.12 vs. 65.3 +/- 14.5 yrs), gender (males: 7; 58.3% vs. 18; 81.8%), time on HD (26.1 +/- 14.7 vs. 30.1 +/- 38.7 months), causes of renal failure. Time to follow-up for access thrombosis: 12 months. RESULTS: PAI-1 levels in HD: 7.21 +/- 2.13 vs. CG: 0.42 +/- 0.27 U/ml (p<0.0001). PAI-1 4G/5G polymorphic variant distribution in HD: 5G/5G: 6 (17%), 4G/5G: 23 (64%); 4G/4G: 7 (19%) and in CG: 5G/5G: 14 (35%); 4G/5G: 18 (45%); 4G/4G: 8 (20%). C-reactive protein (CRP) in HD: 24.5 +/- 15.2 mg/L vs. in CG 2.3 +/- 0.2 mg/L (p<0.0001). PAI-1 4G/5G variants: GA: 5G/5G: 3; 4G/5G: 8; 4G/4G: 1; GB: 5G/5G: 3; 4G/5G: 15; 4G/4G: 6. Thrombosis occurred in 8/10 patients (80%) with PTFEG, 3/22 (9%) in AVF, and 1/4 (25%) in CAT. Among the eight PTFEG patients with thrombosis, seven were PAI 4G/5G. CONCLUSIONS: PAI-1 levels were elevated in HD patients, independent of their polymorphic variants, 4G/5G being the most prevalent variant. Our data suggest that in patients with PTFEG the 4G/5G variant might be associated with an increased thrombosis risk.

Low anticoagulant activity of high sulphated heparan sulphates.

Two high sulphated heparin-like polysaccharides (L1, MW 16,000 and L2, MW 11,700) were isolated from rat liver tissues, after DEAE-cellulose chromatography. Heparan sulphates from heart and lung tissues were isolated for comparison and fractionated according to their molecular weight. The anticoagulant activities in vitro were studied using clotting antifactor Xa, antifactor IIa, and APTT assay methods, falling in a narrow range (5-44 IU/mg) although the wide variability in molecular weight and sulphate content. The heparan sulphate nature of fractions L1 and L2 (sulphate/disaccharide ratio 2.05 and 2.48, respectively) has been verified by: a) low iduronic/glucuronic acid ratio; b) nitrous acid degradation followed by gel chromatography; c) heparinase treatment followed by gel chromatography; d) electrophoretic behaviour. Native proteoglycans have been isolated and the glycosidic chains compared with L1 and L2. Their anticoagulant activities in vitro and the fact that anti-Xa clotting activity was not neutralized by protamine sulphate are in accordance with the results of structural studies.

Reactivity to beta 2 glycoprotein I clearly differentiates anticardiolipin antibodies from antiphospholipid syndrome and syphilis.

Anticardiolipin antibodies (aCL) detected by standard ELISA are found in association with autoimmune and infectious diseases. It is now recognized that beta 2 glycoprotein I (beta 2GPI) is a cofactor for aCL binding to cardiolipin (CL). To examine differences in cofactor requirements, aCL positive sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied. Using an ELISA with human purified beta 2GPI adsorbed onto irradiated plates, we detected high binding activity in 29 out of 35 samples from APS patients and low in only 1 out of 37 aCL positive syphilis sera. Moreover, a good correlation (r = 0.79) was also observed in the former group between aCL and anti beta 2GPI. Whole IgG and affinity purified IgG aCL from APS patients did not bind to CL in the absence of beta 2GPI, but recognized beta 2GPI on irradiated plates in the absence of phospholipids. In contrast, IgG purified from syphilis patients only bound to CL alone. Taken together, these data indicate that performing both ELISA (aCL and anti beta 2GPI) it could be possible to distinguish aCL from autoimmune or infectious diseases.

Relationship of anti beta2-glycoprotein I and anti prothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies.

The lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are clinically relevant because of their association with thrombosis and pregnancy loss. The group of antiphospholipid antibodies (aPL) includes antibodies primarily directed against various phospholipid-binding proteins, mainly beta2-glycoprotein I (beta2GPI) and prothrombin. Some studies suggest that there is an association between the presence of anti beta2GPI antibodies (alphabeta2GPI) of IgG isotype and thrombosis. Therefore, aPL defined according to the plasma protein to which they are directed appear to be more appropriate for the evaluation of their clinical importance. Using home-made ELISAs we evaluated the presence of alphabeta2GPI and antiprothrombin antibodies (anti-II) of both isotypes (IgG and IgM) in a group of 233 patients with LA and/or aCL. Forty-four women had a history of pregnancy loss, 45 patients had a history of venous thrombosis (VT) and 32 of arterial thrombosis (AT). Patients from the autoimmune group (systemic lupus erythematosus and antiphospholipid syndrome) had a higher prevalence of alphabeta2GPI and/or anti-II than those from the miscellaneous group. In the univariate analysis, a significant association was shown between the presence of alphabeta2GPI-IgG (OR 3.2; 95% CI 1.5-6.6) and previous VT, but not AT. Anti-II were related to VT but the multivariate analysis showed that alphabeta2GPI-IgG are the only independent risk factor for VT (OR 3.0; 95% CI 1.3-6.2). The presence of alphabeta2GPI-IgM correlates well with a history of pregnancy loss (OR 2.6; 95% CI 1.1-6.1). The coagulation tests profile showed that the clotting assays were more prolonged in patients having aCL, alphabeta2GPI or anti-II. But a higher prevalence of abnormal results was only found for the dilute Russell viper venom time in patients with VT, as compared to those without thrombosis (94.4% vs. 58.7%, p <0.02). The measurement of alphabeta2GPI of both isotypes could help to identify aPL-positive patients with a higher risk for thrombosis and pregnancy loss, although this association should be confirmed by prospective studies.

Plasminogen: an important hemostatic parameter in septic patients.

BACKGROUND: Previously we observed in some but not all septic patients a low plasma concentration of plasminogen. OBJECTIVES: To investigate prospectively whether plasma levels of plasminogen or the ratio of plasminogen to alpha-2-antiplasmin have a prognostic value for survival from sepsis and to study the variation of other hemostatic parameters during septicemia. PATIENTS: The study population consisted of 45 consecutive patients with septicemia, 15 non-septic patients from the same intensive care unit and 30 healthy volunteers. MEASUREMENTS AND MAIN RESULTS: Plasminogen concentrations were significantly lower (p < 0.001) in plasma of septic patients (median 0,62 IU/ml range: 0.15-1,06) than in plasma of healthy controls (median 1.00 IU/ml, range: 0.75-1.10) or of non-septic intensive care patients (median 1.00 IU/ml, range: 0.82-1.08). Among the other parameters tested, plasminogen activator inhibitor (PAI-1) antigen concentration and PAI activity were similar in septic and non-septic intensive care patients, but higher than in healthy controls. Concentrations of elastase-alpha-1-protease inhibitor or of thrombin-antithrombin complexes were higher in septic patients than in non-septic intensive care patients or healthy controls. A degraded form of plasminogen of 38 kDa was detected by Western blot analysis in the plasma of septic patients, but not in plasma of non-septic intensive care patients or controls. Plasminogen alone or the ratio of plasminogen to antiplasmin were good markers for survival from septicemia. E.g. for plasminogen at a cut off of 0.65 IU/ml, sensitivity was 90.5% and specificity 66.7%, whereas for the ratio of plasminogen over antiplasmin at a cut off ratio of 0,65 IU/ml, sensitivity was 95.2% and specificity 70.8%. CONCLUSION: Plasminogen or the ratio of plasminogen to antiplasmin are sensitive markers for survival in patients with septicemia.

Severe hemorrhagic tendency in heterozygous alpha 2-antiplasmin deficiency.

We report a case of partial deficiency of alpha 2-antiplasmin. A 45 year old Spanish man had a life long severe bleeding tendency after trauma. Routine coagulation and platelet functional tests in symptomatic and asymptomatic periods were normal. During the bleeding there was a rapid whole blood clot lysis and the concentration of alpha 2-antiplasmin in plasma was 20 to 25% of normal controls. After recovery these levels showed a slight increase (35 to 45% of normal). Addition of normal plasma to the patient's plasma increased alpha 2-plasmin inhibitor activity. Bidimensional electrophoresis of alpha 2-antiplasmin in the patient's plasma showed a normal pattern. Family studies showed that one of the proband's two sons had a mild hemorrhagic tendency and 40% of functional and antigenic levels of alpha 2-antiplasmin.

Impaired clot lysis by rt-PA catalyzed mini-plasminogen activation.

The fibrinolytic system contains a proenzyme plasminogen (Plg) which is converted to plasmin (Plm) by the action of Plg activators. Physiological Plg activators are: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plg was shown to be further cleaved by leukocyte elastase producing several fragments, one of which is called mini-plasminogen (mini-Plg) or neo-plasminogen Val442. In this paper we studied whether mini-Plg is able to produce clot lysis when it is activated by rt-PA in purified systems and in Plg depleted normal plasma. We found that mini-Plg clot lysis time was longer than that of Plg. Clot lysis times were 2.3 minutes +/- 0.06 for Plg and 9.8 minutes +/- 0.1 for mini-Plg. Mini-Plg is less efficient than Plg in producing clot lysis at all studied concentrations (0.1-1.2 microM). In Plg depleted normal human plasma mini-Plg is unable to produce complete clot lysis in presence of rt-PA. Although mini-Plg can be activated to mini-Plm by rt-PA, these results show that the activation process is insufficient to produce an efficient clot lysis.

Plasmatic homocysteine response to vitamin supplementation in elderly people.

Homocysteine (Hcy) increase is now widely accepted as a risk factor for vascular disease. The effects of folic acid (FA) and vitamins B12 and B6 in lowering Hcy have been extensively studied, but there is still little data on the response to FA dietary administration. Our purpose was to evaluate the impact of the diet and the degree of response to different doses of pharmacological FA supplementation. In a prospective, randomized, and simple blind study, 50 elderly subjects were given a 400-microg/day FA diet and were randomly assigned to one of the following treatments: Group I = placebo tablet; Group II = tablet containing 1-mg folic acid, 1-mg B12, and 25-mg B6; and Group III = tablet containing 2.5-mg folic acid and same B6 and B12 doses as Group II. Forty-four subjects completed the study, and their plasmas were evaluated. Hcy concentration significantly decreased even in patients with normal basal values, and there were no differences in the response between individuals receiving diet plus placebo and those receiving diet plus pharmacological supplementation. After the treatment, the mean decrease of plasmatic Hcy levels was 10.8 (9.4, 12.5) micromol/l, geometric mean [95% confidence interval (95% CI)], and particularly, the values for Group I were 10.6 (7.4, 14.8) micromol/l. In 31% of the subjects, the post-treatment Hcy levels were less than or = 5 micromol/l. These results show that a special diet, with or without pharmacological FA and B12 and B6 supplementation, significantly decreases the Hcy levels in elderly people. Therefore, a diet with high contents of FA might have an enormous impact on the morbidity and mortality of atherothrombosis.

Plasminogen abnormalities in patients with Argentine hemorrhagic fever.

Plasminogen, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin and fibrinogen degradation products (FDP) were studied in 45 patients with Argentine hemorrhagic fever. Patients were grouped into: 17 mild, 14 moderate and 14 severe cases. Plasminogen antigen level and functional activity were found to be reduced in the moderate and severe groups, when compared to the results obtained at recovery. The functional activity of alpha 2-antiplasmin was within the normal range, except for a slight decrease on days 10-11, alpha 2-macroglobulin remained normal during the course of illness. alpha 1-antitrypsin also remained normal except on days 10-11, when an increase in the antigen level was noted. FDP titre was normal (less than 10 micrograms/ml) in all patients during the course of disease. Plasminogen decrease was not attributable to liver insufficiency neither to a primary nor secondary fibrinolysis. The decreased antigen and reduced functionality of plasminogen in these patients we believe is related to proteolytic degradation by leukocyte enzymes.

Mini-plasminogen like molecule in septic patients.

Plasma from 7 septic patients with positive blood cultures were studied. None of them presented either clinical or laboratory evidence of Disseminated Intravascular Coagulation. The white cells count varied between 5 and 45 X 10(9)/l. In plasma functional plasminogen levels varied between 25 and 45%, while those of alpha 2-antiplasmin were normal (80-105%). The levels of elastase ranged between 250 and 750 micrograms/ml. Leukocyte elastase digests plasminogen "in vitro" and is able to produce several fragments; one of them called mini-plasminogen lacking lysine binding sites; therefore it does not bind to lysine-Sepharose 4B. Two different behaviors were observed in the plasmatic plasminogen of these patients with respect to their binding capacity to lysine-Sepharose 4 B. 3 patients had plasminogen which did not bind to lysine-Sepharose 4 B; the other 4 had two different components, one of which bound to lysine-Sepharose 4 B and another one which did not bind. Previous studies "in vitro" have shown that leukocyte elastase modifies alpha 2-antiplasmin, initially producing a non-plasminogen binding form. A free alpha 2-antiplasmin (non-plasminogen binding form) was detected in the plasma of these patients with sepsis by crossed immunoelectrophoresis with plasminogen in the first dimension. It seems tenable that high levels of leukocyte elastase could be responsible for these findings although, the possible relationships to leukocyte elastase still remain to be proven but could possibly explain this effect.

Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies.

OBJECTIVE: To evaluate plasma levels of markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with or without antiphospholipid antibodies (aPL) and to compare them to those found in patients with antiphospholipid syndrome (APS). METHODS: 42 patients with leprosy (35 lepromatous and 7 borderline): 29 aPL(+) and 13 aPL(-), as well as 26 healthy subjects as normal controls (NC) and 79 control aPL patients without leprosy (59 with and 20 without APS) were included in the study. Plasma soluble P and E selectin (sPsel and sEsel), and VCAM-1 (sVCAM-1), prothrombin F1 + 2 fragment (F1 + 2), thrombin-antithrombin complexes (TAT) and D dimer (DD) were measured by ELISA. The protein C pathway was assessed by the ProC global test. RESULTS: Leprosy patients with aPL presented increased median levels of sPsel [ng/ml (82.0 vs 36.0, p < 0.001)] and sVCAM-1 [ng/ml (495 vs 335, p < 0.001)] compared to NC, as observed in control aPL patients without leprosy. Levels of sPsel in aPL(+) patients with leprosy were significantly higher than in aPL(-) ones (52.5 ng/ml), p = 0.005. However, plasma markers of thrombin generation were increased in control aPL patients without leprosy but not in those with leprosy. ProcC global test was abnormal in 24.1% of leprosy patients with aPL compared to 4.4% of NC (p < 0.024), and to 57.2% of control patients with aPL without leprosy (p = 0.005). CONCLUSIONS: We demonstrated that although patients with leprosy present a high prevalence of aPL, and platelet and endothelial cell activation in vivo to the same extent than patients with APS, they do not show a procoagulant state.

Heparin cofactor II in diabetic patients.

Levels of heparin cofactor II (HCII) activity and antigen and electrophoretic pattern were studied both in normal subjects and in type I diabetic patients with high and normal levels of glycosylated haemoglobin. There was a significant reduction in HCII activity (83 +/- 7%) in patients with high levels of glycosylated haemoglobin compared with controls (95 +/- 17%; P < 0.001). However, plasma HCII antigen levels were not decreased in these patients.

Differences in protein S and C4b-binding protein levels in different groups of patients with antiphospholipid antibodies.

Total protein S (tPS), free protein S (fPS) and C4b-binding protein (C4b-BP) were measured by immunological assays in 73 patients with antiphospholipid (aPL) antibodies, in order to determine whether the previously reported abnormalities in PS levels in this group of patients could be related to the presence of lupus anticoagulant (LA) or anticardiolipin (aCL) antibodies. As compared with the normal controls (n = 44), the authors found a significant decrease of tPS, fPS and C4b-BP in 45 LA(+)aCL(+) patients (P < 0.001), a decrease of tPS (P < 0.001), fPS and C4b-BP (P < 0.01) in eight LA(-)aCL(+) patients and a decrease of only fPS (P < 0.05) in 20 LA(+)aCL(-) patients. There was no difference in the levels of tPS, fPS and C4b-BP between LA(+)aCL(+) and LA(-)aCL(+) patients. In contrast, the LA(+)aCL(+) patients had lower values of tPS, fPS and C4b-BP than LA(+)aCL(-) patients (P < 0.05). In some patients, protein S activity (PSact) was also measured and a high correlation was observed between fPS antigen and PSact (r = 0.93, P < 0.001). The data show that the presence of aCL antibodies is associated with a probably acquired deficiency of PS and C4b-BP. On the other hand, in LA patients without a CL antibodies, the fPS deficiency is unrelated to an increase in C4b-BP levels and may be due to abnormal binding of PS to C4b-BP.

Unsuspected hyperhomocysteinemia in chronically anticoagulated patients.

Hyperhomocysteinemia is a risk factor for arterial and venous thrombosis. The aim of this study was to evaluate plasmatic homocysteine levels in patients under chronic anticoagulant treatment with dietary restriction of green vegetables. This kind of food is a very important source not only of vitamin K but also of folates, which are involved in Hcy metabolism. It is known that the lower the folate levels, the higher the Hcy concentration, so we suspected that these patients could show hyperhomocysteinemia. A group of patients receiving oral anticoagulant treatment and a restricted diet (Group I, n = 20) was compared with a group of untreated subjects of a similar age that were not on a restricted diet (Group II, n = 35). Group I showed significantly higher levels of plasmatic Hcy and significantly lower levels of serum folate than Group II. Therefore, a diet restricted in vitamin K applied to oral anticoagulated patients could induce an unwanted increase of homocysteine levels.

Natural inhibitors of blood coagulation and fibrinolysis in patients with lupus anticoagulant.

We studied the natural inhibitors (NI) of blood coagulation and fibrinolysis in 50 patients with lupus anticoagulant (LA), in order to identify possible alterations of these NI, that could favour thrombotic manifestations. We found no statistically significant difference in antithrombin III, protein C and alpha 2-antiplasmin between controls and patients with LA, irrespective of their clinical manifestations. We found an increase of plasminogen activator inhibitor (PAI, P < 0.001) and a decrease of free protein S (PSf, P < 0.001) and total protein S (PSt, 0.01 < P < 0.05) in the patients with LA when compared with the control group. We found no difference in the levels of NI between patients with thrombosis (n = 19) and without thrombosis (n = 31) nor between patients with (n = 25) or without thrombosis and/or foetal loss (n = 25). In contrast, we observed a decrease of PSf in women with foetal loss (n = 10) as compared with women without foetal loss (n = 22, 0.01 < P < 0.05) and a decrease of PSf when comparing 19 patients with systemic lupus erythematosus (SLE) with 31 patients without SLE (0.01 < P < 0.05). These findings show that the patients with LA had several abnormalities in the NI system, but there was no significant association between levels of PAI, PSf, PSt and a history of thrombosis.

Validation of an enzyme immunoassay for the determination of total homocysteine in plasma.

In recent years, the determination of homocysteine (Hcy) has become increasingly important, since high levels of Hcy in plasma or serum represent an independent risk factor for occlusive vascular diseases. Nowadays, clinical laboratories use several analytical techniques to measure Hcy, of which high-performance liquid chromatography (HPLC) is the most popular. Recently, assays for Hcy quantification based on enzyme immunoassays (EIA) have become commercially available. Our group carried out the validation of the Axis method and compared results with those obtained by an established HPLC assay. Intra- and inter-assay coefficients of variation were < or = 8.5%. Compared with HPLC, linear regression analysis showed r=0.984, slope=0.952, intercept = 1.24 /mol/l; Bland-Altman procedure, the mean of the difference EIA-HPLC results = 0.5 micromol/l. Our results suggest that Hcy determinations by both methods are equivalent, and that the Axis assay provides reproducible and reliable data.

[Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]. / Cofactor II de la heparina (HCII), un inhibidor de trombina cuyo rol fisiológico no está completamente esclarecido.

Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.

Thrombotic events of arteriovenous fistulae in hemodialysis patients related to the C677T thermolabile variant of methylenetetrahydrofolate reductase.

BACKGROUND: Hyperhomocysteinemia is a risk factor for thrombosis, a frequent complication of vascular access (VA) in hemodialysis (HD). The enzyme methylenetetrahydrofolate reductase (MTHFR) is necessary for the remethylation of homocysteine (Hcy) to methionine. It has been postulated that patients homozygous and, to a lesser extent, heterozygous for the C677T thermolabile variant of this enzyme present a reduced catalytic activity, with secondary increases in plasmatic Hcy levels (normal: 10 +/- 5 micromol/L) and an elevated risk of vascular thromboses. METHODS: Sixty-two patients on chronic HD were divided into two groups: group A (n = 23, 37.1%) was normal for the enzyme (CC); group B (n = 39, 62.9%) was heterozygous (CT). Both groups were not different according to age, sex, time on HD, hematocrits (Hct), baseline levels of Hcy, folic acid and vitamin B12. After the 1st HD session patients were started on folic acid 10 mg/day and 500 microg/week of intravenous (i.v.) methylcobalamin. RESULTS: Two years later, thrombotic events were not different between the two groups. Group A = 5 (21.7%) vs. group B = 12 (30.7%), Hcy levels were significantly different between final and baseline measurements (group A 21.5 +/- 5.2 vs. 16.6 +/- 3.9 micromol/L, p = 0.02; group B 22.1 +/- 8.9 vs. 16.1 +/- 3.9 micromol/L, p = 0.008), folic acid (group A 22.1 vs. 346.9 ng/ml, range (r) =166-527, p < 0.001; group B 19.2 vs. 218.5 ng/ml, r = 138-298, p < 0.001) and vitamin B12 (group A 1489 vs. 3192.3 pg/ml, r = 1494-4890, p = 0.01; group B 1086 vs. 1513.8 pg/ml, r = 1092-1934, p = 0.02). CONCLUSIONS: HD patients heterozygous for the C677T variant of the enzyme MTHFR can present a similar risk of thrombotic events in arteriovenous fistulae (AVF) compared to patients normal for the enzyme at a 1-yr follow-up. These results could be explained by an adequate control of Hcy levels after folic acid and methylcobalamin replacement therapy.