The use of the new ReFacto AF Laboratory Standard allows reliable measurement of FVIII:C levels in ReFacto AF mock plasma samples by a one-stage clotting assay.

Factor VIII coagulant (FVIII:C) levels measured in patients receiving ReFacto® (B-domain-deleted recombinant FVIII) using chromogenic substrate assay (CSA) and one-stage clotting assay (OSA) have frequently shown discrepancies, and the use of the ReFacto Laboratory Standard (RLS) has therefore been recommended to minimize these differences. The potency of ReFacto AF®, the albumin-free successor of ReFacto®, is determined using CSA for the titration of vials, and a new standard (RLS-AF) was developed to measure its biological efficacy using OSA. This multicentre study therefore evaluated the efficacy of this new RLS in minimizing differences between OSA and CSA when measuring FVIII:C levels in plasma. Mock plasma samples were prepared by diluting ReFacto AF® in FVIII-deficient plasma to obtain four concentrations ranging from 15 to 90 IU dL⁻¹ . FVIII:C levels were then measured in six laboratories on four separate days using three different procedures, i.e. OSA with a plasma standard (PS) as reference, OSA with RLS-AF and CSA with PS. The inter-centre standard deviation ranged from 1.4 to 5.5 IU dL⁻¹. However, FVIII:C levels measured with OSA were closer to the expected values when RLS-AF was used. In addition, the uncertainty of measurement, reflecting the inter-method discrepancy was greatly reduced when RLS-AF was employed in OSA (15%) in place of PS (33%). This study demonstrates that the OSA performed with RLS-AF to establish calibration curves provides a valuable alternative to CSA to measure FVIII:C in ReFacto-AF-treated patients.

Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

INTRODUCTION: Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. METHODS: The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. RESULTS: The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay® with the Berichrom® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom® FXIII activity but normal antigen level and clot solubility as expected. CONCLUSIONS: The measurement of FXIII antigen using the K-Assay® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available.

Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe.

The thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently described inhibitor of fibrinolysis that decreases plasminogen binding to the fibrin surface. The plasma TAFI concentration is almost entirely genetically determined. We investigated whether plasma TAFI levels and polymorphisms located in the TAFI gene could constitute risk markers of myocardial infarction (MI). Plasma TAFI antigen (Ag) levels were assayed by ELISA and 2 TAFI gene polymorphisms (Ala147Thr and C+1542G in the 3' untranslated region) were determined in a large European case-control study. This study compared 598 men recruited 3 to 6 months after MI with 653 age-matched controls from North Europe (Stockholm, Sweden, and London, England) and South Europe (Marseilles, France, and San Giovanni Rotondo, Italy). A TAFI Ag value above the 90th percentile was associated with a significantly lower risk of MI (odds ratio 0.55, P<0.02), indicating that elevated TAFI may be protective against MI. As previously shown, the 2 TAFI gene polymorphisms were in strong linkage disequilibrium and were associated with the TAFI Ag concentration, with carriers of the Thr147 and 1542C alleles having higher levels (P<0.0005). These effects were similar in controls and cases and in each center. There was a difference in allele frequency between cases and controls for the Ala147Thr polymorphism, with Thr147 allele carriers being more frequent in controls than in cases in 2 centers, Stockholm (P=0.03) and San Giovanni Rotondo (P=0.03); the odds ratio for the entire cohort was 0.78 (P<0.05). In conclusion, patients with a recent MI presented lower values of TAFI Ag and higher frequencies of the "TAFI-decreasing" alleles. The geographical differences observed do not contribute to explaining the North-South gradient in MI risk in Europe.

Does the presence of von Willebrand factor in FVIII-deficient plasma influences the measurement of FVIII inhibitor titres in haemophilia A patients?

INTRODUCTION: Reliable measurement of FVIII inhibitor is critical in the follow-up of haemophilia A patients. We performed a multicentre study to evaluate whether the presence of von Willebrand factor (VWF) in FVIII-deficient plasma (FVIII-DP) influences FVIII inhibitor titres. METHODS: Six French haematology laboratories participated in this study. Three samples with varying FVIII inhibitor titres (1, 5 and 15 BU/mL) and one sample without any detectable FVIII inhibitor were tested using four different procedures for FVIII inhibitor assay. The Nijmegen method and a modified assay with imidazole were performed using FVIII-DP with and without VWF in the control mixture and as substrate plasma in the FVIII one stage assay (OSA). Each mixture (reference and test) was incubated for two hours at 37 °C with buffered normal pool plasma. RESULTS: Higher inhibitor titres were measured in 5 and 15 BU/mL samples when assays were performed with the Nijmegen method and FVIII-DP without VWF. When samples were diluted in imidazole buffer, similar inhibitor titres, close to expected values, were measured whether VWF was present in the FVIII-DP or not. The data obtained were also more accurate when residual FVIII activity levels between 40% and 60% were used to calculate inhibitor titres, despite a linear type I reaction kinetics. CONCLUSION: These results support the hypothesis that reliable FVIII inhibitor titres can be measured without the use of FVIII-DP containing VWF when an imidazole-modified assay is used.

Weak and non-independent association between plasma TAFI antigen levels and the insulin resistance syndrome.

Increased plasma thrombin-activatable fibrinolysis inhibitor (TAFI) levels were recently shown to be a part of the insulin resistance syndrome. We investigated the relationship between plasma TAFI antigen levels and insulin resistance markers and compared these results with those obtained for PAI-1 and fibrinogen which are known to be closely related to insulin resistance syndrome and fat mass, respectively. Eighty-nine obese females had 1.3-, 1.2-, and 3-fold higher circulating TAFI, fibrinogen and PAI-1, respectively, compared with 64 lean females. Univariate analysis showed that the significance level for association between TAFI or fibrinogen concentrations and insulin resistance markers was lower than the significance level for association between PAI-1 and insulin resistance markers. Nevertheless, TAFI, fibrinogen, and PAI-1 plasma levels were significantly associated to each other. In linear stepwise ascendant analysis, insulin resistance markers accounted for 50% of the interindividual variability of plasma PAI-1 and only for 10% of plasma TAFI and 13% of fibrinogen variability. The contribution of insulin resistance markers to plasma TAFI antigen levels variability disappeared when PAI-1 or fibrinogen was entered in the statistical model. TAFI mRNA was detected in the liver but not in adipose tissue and endothelial cells. No TAFI mRNA was detected in normal or atherosclerotic vessels either. These results suggest that elevated TAFI antigen levels found in obese subjects are not independently associated with the metabolic markers of the insulin resistance syndrome. Increased plasma TAFI antigen levels in obesity might reflect a specific pathway of regulation at the liver level.

The plasminogen activator inhibitor-1 -675 4G/5G genotype influences the risk of myocardial infarction associated with elevated plasma proinsulin and insulin concentrations in men from Europe: the HIFMECH study.

UNLABELLED: Although the potential role of plasminogen activator inhibitor-1 (PAI-1) in the development of coronary artery disease is strongly supported by its biological characteristics, results of clinical studies remain controversial. OBJECTIVES: To investigate whether plasma PAI-1 concentrations and the -675 4G/5G polymorphism located in the PAI-1 gene could constitute risk markers for myocardial infarction (MI). PATIENTS AND METHODS: We used a European case-control study, the HIFMECH study, comparing 598 men with MI and 653 age-matched controls. RESULTS: Insulin resistance explained a major part of the variation in PAI-1 (24%) whereas inflammation had only a minor contribution (0.01%). For both cases and controls plasma PAI-1 concentrations were significantly higher in the North than the South, and in both regions were higher in individuals with MI compared with control subjects [overall odds ratio (OR) for a 1 SD increase=1.54, 95% confidence interval (CI) 1.34, 1.77]. This difference was observed in all the centers studied. Overall, the difference between cases and control subjects remained significant after controlling for inflammation variables (OR=1.30, 95% CI 1.08, 1.57), but lost significance after controlling for insulin resistance variables (OR=1.17, 95% CI 0.98, 1.40). The 4G allele was associated with significantly higher PAI-1 levels in cases but not controls and, taken independently, did not modify the risk of MI (P=0.9). However, a significant interaction was observed with both insulin or proinsulin and the risk of MI (P=0.05 and 0.02, respectively), but not with triglycerides or body mass index (BMI). The insulin or proinsulin effect on risk was observed only in the carriers of the 4G/4G genotype. This interaction appeared not to be mediated by plasma PAI-1 antigen concentrations (P=0.01 and 0.02 after adjustment for PAI-1 plasma levels). The interaction with proinsulin but not insulin remained statistically significant after further adjustment for other factors associated with insulin resistance (triglycerides and BMI) and C-reactive protein (P=0.01). CONCLUSION: This study suggests that PAI-1 has a role in risk of MI in the presence of underlying insulin resistance. A significant interaction between insulin or proinsulin and the -675 4G/5G polymorphism was observed in risk for MI. The mechanisms for these interactions remain to be determined.

Anticoagulant response to Agkistrodon contortrix venom (ACV test): a new global test to screen for defects in the anticoagulant protein C pathway.

As specific assays used to identify defects in the protein C (PC) anticoagulant pathway are laborious and expensive, we describe here a global test to screen for these defects. This assay is expressed as the ratio of two activated partial thromboplastin times, one in the absence and one in the presence of 0,125 U/ml of the PC activator of Agkistrodon contortrix venom (ACV). Eight of the 168 healthy volunteers of the control group exhibited an ACV ratio below the lower normal limit of 3.37 [6 subjects with the mutation Arg 506 to Gln in their factor V gene (FV R506Q) and one with PS deficiency]. 128 patients who have had at least one episode of deep-vein thrombosis were retrospectively studied. All patients carrying FV Q506R (n = 48), PC deficiency (n = 14) or combined defects, i.e. FV Q506R and PC deficiency (n = 4) or FV Q506R and PS deficiency (n = 3), had ACV ratios < 3.37. PS deficient plasmas (n = 20) exhibited ACV ratios which overlapped normal range. ACV ratios of one out of seven patients with antithrombin deficiency, and 10% of patients without identified defect in the PC anticoagulant pathway (n = 30) were < 3.37. An ACV ratio raised to 3.70 could lead to a test identifying all patients with a defect in the PC anticoagulant pathway.

Effect of 24 hours of normoglycaemia on tissue-type plasminogen activator plasma levels in insulin-dependent diabetes.

We have previously demonstrated that a short period of normoglycaemia obtained through an artificial pancreas in uncontrolled insulin-dependent diabetics improves parameters of the functional microangiopathy such as erythrocyte deformability and platelet aggregation. Because recently an immunoradiometric assay for tissue plasminogen activator (t-PA) was developed we measured t-PA levels in 18 uncontrolled insulin-dependent diabetics before and after 24 hr of normoglycaemia induced by insulin to look for a modification of endothelial cells function. After 24 hr of strict control, plasma free insulin levels rose significantly, total t-PA R-Ag, its active fibrin binding fraction and euglobulin fibrinolytic activity were significantly decreased. These results suggest a responsibility for insulin in the decrease in t-PA blood level and could explain at least partially the relation between hyperinsulinism, thrombosis and atherogenesis.

Influence of six mutations of the protein C gene on the Gla domain conformation and calcium affinity.

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluvastatin decreases soluble thrombomodulin in cardiac transplant recipients.

We conducted a randomized, placebo controlled, double-blind, cross-over study, to assess the effects of a 4-week fluvastatin therapy on plasma markers of endothelial activation or injury in 20 transplanted heart recipients. The levels of thrombomodulin and von Willebrand factor antigen were higher at baseline in cardiac transplant recipients than in age and sex-matched healthy controls. Plasma total cholesterol showed a 21% reduction on fluvastatin therapy (p = 0.0001). Fluvastatin treatment had no significant effect on creatininemia, plasma cyclosporine, PAI-1 antigen, PAI-1 activity, tPA antigen, and Von Willebrand factor. However, fluvastatin produced a significant decrease of plasma thrombomodulin (66.7 ng/ml on placebo versus 58.8 ng/ml on fluvastatin, p <0.001), suggesting a rapid improvement of endothelial injury in these patients.

Increase in plasma concentration of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII:C and in erythrocyte sedimentation rate with age.

Elderly patients have previously been shown to have an increased plasma concentration of tissue plasminogen activator (t-PA) antigen (t-PA Ag). Since the concentration of t-PA Ag depends on both free t-PA and t-PA complexed with inhibitors, mainly plasminogen activator inhibitor (PA inhibitor), we have investigated the relationship between the plasma concentration of PA inhibitor and age in 20 elderly and 20 young individuals. Elderly individuals showed a slight increase in PA inhibitor, in parallel with increase on others, acute-phase proteins, fibrinogen, von Willebrand factor, factor VIII:C, and the erythrocyte sedimentation rate. The increase in PA inhibitor as well as other acute-phase proteins in the elderly may be significant in relation to the increased incidence of thrombotic disease.

Increased PA-inhibitor levels in the postoperative period--no cause-effect relation with increased cortisol.

It has been reported that the level of PA-inhibitor increases in postoperative patients and on the other hand that glucocorticoids increase the PA-inhibitor level in cell culture. Because surgery is associated with increased plasma cortisol level, a relation between the postoperative increase in plasma cortisol and PA-inhibitor levels was looked for. Blood samples were collected from 8 patients undergoing extensive abdominal surgery, before operation and postoperatively at 2 hr, 4 hr, 24 hr and daily for 7 days. Plasma cortisol and PA-inhibitor were increased 2 hr after surgery, when there was a significant correlation (p less than 0.05). The maximum increase was at 24 hr and the values fell to normal on day 6. An increase in t-PA related antigen (t-PA R:Ag) and a decrease in euglobulin fibrinolytic activity (EFA) also occurred. In 7 controls 0.25 mg ACTH was given intravenously and blood was collected after 1/2, 1, 2, 4, 6 hr. Although the increase in plasma cortisol level following ACTH was comparable to that observed after surgery the increase was not associated with significant change in PA-inhibitor level, t-PA R:Ag or EFA. A cause-effect relationship between the increased plasma cortisol and PA-inhibitor level could not be shown. The mechanism of the postoperative increase in PA-inhibitor thus remains unknown.

Associations of fibrinogen, factor VII and PAI-1 with baseline findings among 10,500 male participants in a prospective study of myocardial infarction--the PRIME Study. Prospective Epidemiological Study of Myocardial Infarction.

The contribution of coagulation factors and fibrinolytic variables to the development of ischaemic arterial disease is still not clearly established. The PRIME study is a prospective cohort study of myocardial infarction in men aged 50-59 years and recruited from three MONICA field centers in France (Lille, Strasbourg and Toulouse) and the center in Northern Ireland (Belfast). Baseline examination included measurement of plasma fibrinogen, factor VII, and PAI-1 activity in over 10,500 participants. We investigated the associations of these haemostatic variables with cardiovascular risk factors, prevalent atherosclerotic disease and geographical area. Fibrinogen level increased with age, smoking, waist-to-hip ratio, LDL-cholesterol, and it decreased with educational level, leisure physical activity, alcohol intake and HDL-cholesterol. Factor VII activity increased with body mass index, waist-to-hip ratio, triglycerides. HDL- and LDL-cholesterol. PAI-1 activity increased with body mass index, waist-to-hip ratio, triglycerides, alcohol intake, smoking, and decreased with leisure physical activity. PAI-1 level was higher in diabetic subjects than in subjects without diabetes. Cardiovascular risk factors explained 8%, 9%, and 26% of the total variance in fibrinogen, factor VII, and PAI-1, respectively. Compared with participants without prevalent cardiovascular disease, those with previous myocardial infarction (n = 280), angina pectoris (n = 230), or peripheral vascular disease (n = 19) had significantly higher levels of fibrinogen. but those with stroke (n = 67) had not. PAI-1 activity showed a similar pattern of association. The odds ratio for cardiovascular disease associated with a rise of a one standard deviation in fibrinogen and PAI-1 was 1.31 (95% confidence interval: 1.20 to 1.42, p <0.001) and 1.38 (95% confidence interval: 1.27 to 1.49, p<0.001), respectively. After adjustment for cardiovascular risk factors, these associations were attenuated but remained highly significant. There was no significant association between factor VII activity and prevalent cardiovascular disease. Fibrinogen level and, to a lesser extent, factor VII and PAI-1 activity were higher in Northern Ireland than France after adjustment for the main cardiovascular risk factors. These geographical variations are consistent with the 2 to 3-fold higher incidence of myocardial infarction in Northern Ireland than France. Our results provide further epidemiological evidence for a possible role of fibrinogen and PAI-1 in the pathogenesis of coronary heart disease.

Antibodies to phosphatidylethanolamine as the only antiphospholipid antibodies found in patients with unexplained thromboses.

The objective of this study was to assess the interest of antiphosphatidylethanolamine antibodies (aPE) in unexplained thrombosis (UT) defined as thrombotic episode without any of the main autoimmune and hereditary thrombophilic defects. Results from 98 UT were compared to those of (I) 142 patients with thrombophilia: 67 antiphospholipid syndrome (APS) and 75 hereditary hemostatic defects (HHD); (II) 110 patients without thrombosis: 60 with systemic lupus erythematosus (SLE) and 50 with infectious diseases (ID). As compared to controls (100 blood donors), aPE prevalence was significantly higher in both autoimmune contexts (APS: 43%; SLE: 40%, p<0.0001) and among non-autoimmune pathologies, only in UT (18%, p = 0.001) conversely to HHD (8%) or ID (10%). aPE prevalence in UT was not statistically different from that found in Primary APS (32%, p = 0.076) but lower than in Secondary APS (65%, p <0.005). In UT, aPE were mainly of IgM isotype like in Primary APS and they were found alone whereas in SLE they were always associated with classical antiphospholipid antibodies. No significant association was found between any isotype of aPE and a site of thrombosis in UT as well as in APS. In conclusion, this study demonstrates an increase of the prevalence of aPE in patients with unexplained thrombosis. Thus, aPE investigation appears to be of interest in UT and their persistent presence could define a biological variant of APS.

Daytime fluctuations of plasminogen activator inhibitor 1 (PAI-1) in populations with high PAI-1 levels.

The mechanism underlying diurnal variations in PAI-1 as well as the cellular origin of PAI-1 in subjects with high PAI-1 levels are unknown. We evaluated diurnal changes (8:00 am vs 4:00 pm) in PAI-1 (functional and immunological assays), t-PA Ag and t-PA/PAI-1 complex levels in controls and subjects with high PAI-1 levels. Three test groups were recruited among obese hyperinsulinemic subjects, emergency care unit patients with inflammatory syndrome or infection and pregnant women. The classical afternoon decrease of PAI-1 level was observed in controls and obese subjects but its amplitude was greater in the latter. The decrease in t-PA Ag and t-PA/PAI-1 complex levels was the same in controls and in obese. As, in previous studies, elevated PAI-1 levels have been correlated with insulin resistance and a decrease in insulin sensibility has been described in the early morning, it is proposed that this "dawn phenomenon" could be implicated in the circadian variations of PAI-1 in controls and could be amplified in obese subjects. Great variability in PAI-1, t-PA Ag or t-PA/PAI-1 complex levels was observed in patients with acute inflammatory syndrome or infection for whom classical biorhythms are suppressed. No diurnal changes in PAI-1 and other fibrinolytic parameters were observed in patients with inflammatory syndrome or in pregnant women suggesting that other sources and/or other regulatory mechanisms of PAI-1 production are involved.

New direct assay of free protein S antigen applied to diagnosis of protein S deficiency.

Congenital deficiencies of protein S (PS) are associated with thrombophilia. Their characterization and classification have been hampered by the complex physiology of the protein C-protein S system and the poor standardization and reliability of laboratory assays. The free active form of protein S is usually determined by immunoassay using polyclonal antibodies in the plasma supernate after polyethyleneglycol (PEG) precipitation. A new one step ELISA using two monoclonal antibodies specific for distinct epitopes of the free form of protein S has been developed for the direct measurement of free PS in untreated plasma. We have tested two ELISA assays for free PS. One assay was based on the PEG precipitation (Asserachrom PS, Stago, Asnières, France) whereas the other was a one step ELISA assay (Asserachrom free PS, Stago). Values were obtained in 35 PS deficient patients recruited among 500 consecutive patients evaluated by the laboratory for diagnosis of congenital disorders of coagulation. Values were compared to those obtained in 50 patients with no PS deficiency matched for age and sex with the PS deficient patients as well as in 33 normal subjects and in 12 pregnant women. Strong correlation was found between the two tests (r = 0.81, p < 10(-5)) in the entire population (n = 130), as well as in the separate groups. The new one step ELISA was more accurate than the PEG free PS determination. Determination of PS activity and antigens allowed us to separate quantitative and qualitative deficiencies. Among the qualitative deficiencies, isolated decrease in PS activity was the most frequent defect observed (66%). This fact questions the substitution of PS activity assays by the one step antigenic free PS ELISA assay.

Plasma levels of free and total TFPI, relationship with cardiovascular risk factors and endothelial cell markers.

Free-TFPI (f-TFPI) presents high anticoagulant activity and its plasma level correlates with unfavorable outcomes in unstable angina. Total TFPI (t-TFPI) represents mainly the lipid-bound form which seems to have a poor anticoagulant activity. Until now, it is not known whether the variations of f-TFPI plasma levels are determined by environmental factors. The aim of our study was to evaluate the influence of cardiovascular risk factors on plasma levels of f-TFPI and relations with other endothelial derived molecules in a population of 626 patients (277 men and 349 women) attending a metabolic ward for primary prevention of coronary disease. Free and total TFPI plasma levels were poorly correlated. f-TFPI levels increased with age in both sexes, t-TFPI in women only. Age-adjusted correlations of TFPI levels with conventional cardiovascular risk factors and endothelial cell markers showed different patterns for f-TFPI and t-TFPI. f-TFPI correlated with parameters associated with insulin resistance, particularly in females. f-TFPI was also positively associated in both genders with fibrinogen and endothelial cell markers: t-PA, thrombomodulin (TM) and von Willebrand factor (vWF). t-TFPI correlated strongly with LDL-C in both sexes. It also correlated negatively with parameters of the insulin resistance syndrome. t-TFPI also correlated with TM but not with other endothelial cell markers. The results of the multivariate step by step analysis showed that cardiovascular risk factors poorly explained the f-TFPI variability (7% and 4% in men and women, respectively), whereas they explained 16 and 20% of t-TFPI variability in men and women respectively (mostly related to LDL-C). In conclusion, this study showed that free- and total-TFPI are regulated differently. f-TFPI strongly correlates with endothelial cell markers and t-TFPI is more related to conventional cardiovascular risk factors. The strong gender effect on f-TFPI levels remains to be explained.

Deficient t-PA release and elevated PA inhibitor levels in patients with spontaneous or recurrent deep venous thrombosis.

The fibrinolytic system was investigated in 120 patients with spontaneous or recurrent deep vein thrombosis (DVT) without any known organic disease able to explain by itself the occurrence of a thrombosis and without any known defect of antithrombin III, Heparin Cofactor II, Protein C, or Protein S. The assays included: Euglobulin fibrinolytic activity (EFA), tissue-type plasminogen activator related antigen (t-PA-Ag) and plasminogen activator inhibitor activity (PA inhibitor), which were measured before and after 10 min of venous occlusion (V.O.). On the basis of the results, the patients could be classified in 3 groups: good responders with an at least two-fold increase of EFA after venous occlusion (n = 76), poor responders with a lesser increase of EFA due to deficient release of t-PA (n = 12), and poor responders with a normal t-PA release but an increased level of PA-Inhibitor (n = 32). The poor responders due to deficient t-PA release (10% of total) had a higher incidence of recurrence of deep vein thrombosis, than the other groups (p less than 0.01). An overall correlation was found between the level of PA-Inhibitor activity and the triglyceride level (r = 0.40, p less than 0.01), suggesting that these elevations may be due to a common cause, at least in some of the patients. It is concluded that a poor fibrinolytic response to venous occlusion occurs in 35 percent of DVT patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of low molecular weight heparin ex vivo activities in clinical laboratories using various anti-Xa assays: interlaboratory variability and requirement for an agreed low molecular weight heparin standard.

The only sensitive and convenient assay to assess the biological activity of low molecular weight heparins (LMWHs) is based on the potentiation of activated factor Xa inhibition. Several procedures for measuring the socalled anti Xa activity have been proposed. In this collaborative study including eight laboratories, we have used four different assays (three amidolytic and one clotting based methods) for measuring the anti Xa activity of ex vivo samples obtained after injecting three different LMWHs. The dispersion of the results obtained by calibration against standard heparin could be reduced by using any of the three LMWHs for calibration. A coefficient of variation less than 0.20 between values obtained in different laboratories using a variety of methods seems acceptable. However it is necessary to refer to a common international standard for expressing the results in units and to define, for each of the three products, the therapeutic range.