Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility.

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P

Seven lipoprotein lipase gene polymorphisms, lipid fractions, and coronary disease: a HuGE association review and meta-analysis.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism and a major candidate gene for coronary heart disease (CHD). The authors assessed associations between 7 LPL polymorphisms and lipid fractions and CHD risk in population-based cohort, case-control, and cross-sectional studies published by January 2007. Meta-analyses of 22,734 CHD cases and 50,177 controls in 89 association studies focused on the relations of the T-93G (rs1800590), D9N (rs1801177), G188E, N291S (rs268), PvuII (rs285), HindIII (rs320), and S447X (rs328) polymorphisms to high density lipoprotein cholesterol, triglycerides, myocardial infarction, or coronary stenosis. Carriers of 9N or 291S had modestly adverse lipid profiles. Carriers of the less common allele of HindIII or of 447X had modestly advantageous profiles. The combined odds ratio for CHD among carriers was 1.33 (95% confidence interval (CI): 1.14, 1.56) for 9N, 1.07 (95% CI: 0.96, 1.20) for 291S, 0.89 (95% CI: 0.81, 0.98) for the less common HindIII allele, and 0.84 (95% CI: 0.75, 0.94) for 447X. For T-93G (odds ratio (OR) = 1.22, 95% CI: 0.98, 1.52) and PvuII (OR = 0.96, 95% CI: 0.89, 1.04), there were null associations with lipid levels or CHD risk; information on G188E was limited (OR = 2.80, 95% CI: 0.88, 8.87). The study of LPL genotypes confirms the existence of close interrelations between high density lipoprotein cholesterol and triglyceride pathways. The influence of these genotypes on CHD risk warrants further investigation.

The interaction between plasminogen and antiplasmin variants as studied by surface plasmon resonance.

The interaction between immobilized plasminogen or an elastase-degradation product from plasminogen, constituting "kringles" 1-3 and different purified variants of antiplasmin has been studied by surface plasmon resonance utilising a BIAcore. The antiplasmin variants studied are wild-type, K429E, K436E, E443G, D444G, K452E and K452T. It is shown that the two mutants K452T and K452E react in quite a similar way as wt-antiplasmin, suggesting that Lys452 is not involved in the lysine-binding site interaction between plasminogen and antiplasmin. On the other hand, the mutant K436E displays a much lower k(a). The affinity between plasminogen or the fragment constituting "kringles" 1-3 and K436E were also much lower than with wt-antiplasmin. Thus, also the data obtained with surface plasmon resonance show that Lys436 indeed is very important in the lysine-binding site mediated interaction between plasminogen and antiplasmin.

Laboratory evidence of hyperfibrinolysis in association with low plasminogen activator inhibitor type 1 activity.

Low activity of plasminogen activator inhibitor type 1 (PAI-1) has been associated with bleeding complications in surgery. We earlier reported a higher prevalence of low PAI-1 activity among patients with bleeding tendency as compared with normal control individuals. The present study evaluated whether low PAI-1 activity actually is associated with markers of increased fibrinolytic activity in plasma from patients with a history of bleeding. PAI-1 activity, plasmin-antiplasmin complex (PAP) and D-dimer were analyzed in plasma samples from 424 consecutive patients referred to the Coagulation Unit for investigation of bleeding symptoms. The median PAI-1 activity was 4.0 U/ml [interquartile range (IQR), 1-10 U/ml], the median PAP level was 1.59 mg/l (IQR, 1.40-1.91 mg/l) and the median D-dimer level was 71 microg/l (IQR, 46-111 microg/l). The median PAP concentration for patients with PAI-1 less than 1.0 U/ml was 1.73 mg/l (IQR, 1.53-2.30 mg/l), and that for PAI-1 of at least 1.0 U/ml was 1.54 mg/l (IQR, 1.36-1.83 mg/l) (P < 0.0001). There was also a significant difference between the PAP levels in patients with normal PAI-1 (1-15 U/ml) versus elevated PAI-1 (> 15 U/ml) (P = 0.024). The level of D-dimer did not correlate with PAI-1 activity. In conclusion, the activation of plasminogen measured as PAP was higher in patients with bleeding symptoms in combination with PAI-1 activity less than 1.0 U/ml than in those with PAI-1 activity of at least 1.0 U/ml. The coagulation activity under normal conditions, as measured by D-dimer, did not differ between the two patient subsets. The results support our previous definition of low PAI-1 as activity below 1.0 U/ml.

Association of apolipoprotein E genotypes with lipid levels and coronary risk.

CONTEXT: Previous reviews of associations of apolipoprotein E (apoE) genotype and coronary disease have been dominated by smaller studies that are liable to biases. OBJECTIVE: To reassess associations of apoE genotypes with circulating lipid levels and with coronary risk. DATA SOURCES: We conducted an updated meta-analysis including both published and previously unreported studies, using MEDLINE, EMBASE, BIOSIS, Science Citation Index, and the Chinese National Knowledge Infrastructure Database published between January 1970 and January 2007, reference lists of articles retrieved, and a registry of relevant studies. STUDY SELECTION: Eighty-two studies of lipid levels (86,067 healthy participants) and 121 studies of coronary outcomes (37,850 cases and 82,727 controls) were identified, with prespecified principal focus on studies with at least 1000 healthy participants for lipids and those with at least 500 coronary outcomes. DATA EXTRACTION: Information on genotype frequencies, lipid levels, coronary outcomes, and laboratory and population characteristics were recorded independently by 2 investigators and/or supplied by study investigators. RESULTS: In the most extreme comparison, people with the epsilon2/epsilon2 genotype had 1.14 mmol/L (95% confidence interval [CI], 0.87-1.40 mmol/L [44.0 mg/dL; 95% CI; 33.6-51.1 mg/dL]) or about 31% (95% CI, 23%-38%) lower mean low-density lipoprotein cholesterol (LDL-C) values than those with the epsilon4/epsilon4 genotype. There were approximately linear relationships of apoE genotypes (when ordered epsilon2/epsilon2, epsilon2/epsilon3, epsilon2/epsilon4, epsilon3/epsilon3, epsilon3/epsilon4, epsilon4/epsilon4) with LDL-C and with coronary risk. The relationship with high-density lipoprotein cholesterol was inverse and shallow and that with triglycerides was nonlinear and largely confined to the epsilon2/epsilon2 genotype. Compared with epsilon3/epsilon3, the odds ratio for coronary disease was 0.80 (95% CI, 0.70-0.90) in epsilon2 carriers and was 1.06 (95% CI, 0.99-1.13) in epsilon4 carriers. CONCLUSIONS: There are approximately linear relationships of apoE genotypes with both LDL-C levels and coronary risk. Compared with individuals with the epsilon3/epsilon3 genotype, epsilon2 carriers have a 20% lower risk of coronary heart disease and epsilon4 carriers have a slightly higher risk.

Decreased risk for myocardial infarction and lower tumor necrosis factor-alpha levels in carriers of variants of the PDCD1 gene.

Increasing interest has been directed toward the inflammatory mechanisms involved in the pathogenesis of myocardial infarction (MI). In the search for genetic mechanisms underlying these inflammatory components, we studied variants of programmed cell death-1 (PDCD1), an immunoinhibitory receptor that inhibits lymphocyte activation and cytokine production, previously shown to be associated with several autoimmune disorders. The PD1.1, PD1.3, and PD1.6 polymorphisms of the PDCD1 gene were typed in the Stockholm Heart Epidemiology Program, a population-based clinical material consisting of 1179 first-time MI case patients and 1528 unaffected control subjects. Individual alleles and haplotypes were studied for association with levels of the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6, and C-reactive protein and risk for MI. We observed a weak protective effect of PD1.3A allele for MI (odds ratio: 0.78, 95% confidence interval: 0.61-0.98). We also observed decreased levels of TNF-alpha in carriers of the PD1.1A/PD1.3G/PD1.6A haplotype, which is consistent with our previous observation that this haplotype may be protective from autoimmune conditions. Carriers of variants of the PDCD1 gene exhibit a decreased risk for nonfatal myocardial infarction, and PDCD1 mediates variation in TNF-alpha levels.

Structures of importance for the stability of antiplasmin as studied by site-directed mutagenesis.

Human antiplasmin, a fast-acting inhibitor of plasmin in plasma, belongs to the serpin super-family of proteins. Like other members of this family, antiplasmin has a scissile peptide bond exposed within a reactive centre loop, typically present at the surface of the molecule. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated. Data regarding "native" antiplasmin have demonstrated that both polymerization processes and formation of latent molecules are important in this respect. In this work we used site-directed mutagenesis to produce 11 single-site mutants (mainly within Abeta-sheet, Bbeta-sheet and reactive centre loop), which were expressed in Drosophila S2 cells, purified and characterized. Five of the 11 mutants were found to have a deviating stability at decreased pH. Glu346Thr was the only mutant with a lesser stability as compared to wt-antiplasmin, but the other 4 were more stable. The most stable mutant, His341Thr, was 7-fold more stable at pH 4.9 as compared to wt-antiplasmin. The wt-antiplasmin had a much more pronounced tendency to polymerize at decreased pH, as compared to "native" antiplasmin. However, many of the mutants clearly rather formed latent molecules, as judged both from PAGE-analysis at non-denaturing condition and reactivation experiments.

The complex between tPA and PAI-1: risk factor for myocardial infarction as studied in the SHEEP project.

INTRODUCTION: The tPA/PAI-1 complex seems to be an important biochemical marker for myocardial reinfarction. Therefore we explored the distribution, correlation and interaction of plasma concentrations of tPA/PAI-1 complex in all available patients and matched controls in the Stockholm Heart Epidemiology Program (SHEEP). METHODS AND PATIENTS: The SHEEP study is a case control study of 2246 patients with a first myocardial infarction (MI), of which 1267 surviving patients were subjected to blood sampling about 3 months after MI and compared with a control group, matched for age, sex and living area within the Stockholm County. The study consists of 886 (591 men and 295 women) patients and 1198 (753 men and 445 women) matched controls, who were all analysed for plasma tPA/PAI-1 complex. RESULTS: The plasma concentration of tPA/PAI-1 complex was significantly associated with the risk for MI, for both genders. Synergistic interactions were observed in men for the co exposure to high plasma tPA/PAI-1 complex concentrations in combination with smoking (OR=4.6) or diabetes mellitus (OR=7.9). Synergism was also seen in combination with exposure to serum hypercholesterolemia or increased levels of apolipoprotein B. An antagonistic effect of the co exposure to high tPA/PAI-1 complex and hypertension was found among men with a similar tendency among women, but an antagonistic effect of increased waist/hip ratio was only observed among the women. CONCLUSIONS: Measuring the plasma concentration of tPA/PAI-1 complex might be of practical value in assessing risk of MI for both genders, especially in those who are smokers or in patients with manifest diabetes mellitus.

Allelic variants of cytochromes P450 2C modify the risk for acute myocardial infarction.

SUMMARY: Cytochromes P450 (CYP) 2C8 and 2C9 are polymorphic enzymes responsible for the biosynthesis of vasoactive substances from arachidonic acid including endothelium-derived hyperpolarizing factor. Inter-individual differences in the action of these substances might be important in the pathogenesis of cardiovascular diseases such as acute myocardial infarction (AMI) and hypertension. This study describes the relationship between genetic variants of CYP2C8 and CYP2C9, and morbidity in myocardial infarction in a large Swedish patient material. The study included 1172 AMI patients and 1503 control subjects (matched by age, sex and residential area) who participated in the Stockholm Heart Epidemiology Program (SHEEP). Genotyping was performed by allelic discrimination using a 5'-nuclease assay for the CYP2C8 and CYP2C9 variants. To estimate associations to AMI risks, odds ratios (OR) with 95% confidence intervals (CI) were calculated. The frequencies of CYP2C8*1, 2C8*3, 2C9*1, 2C9*2 and 2C9*3 variants in the control group were 0.91, 0.095, 0.83, 0.11 and 0.065, respectively. The risk of AMI in the female individuals carrying the *2 or *3 variant alleles of CYP2C9 and that of all individuals carrying the *3 variant of CYP2C8 was higher [OR (95% CI): 1.3 (1.0-1.9), P = 0.09; 1.5 (1.0-2.2), P = 0.06 and 1.2 (1.0-1.5), P = 0.07, respectively] compared to the groups with CYP2C8*1 and CYP2C9*1. Possession of rare genetic variants of the CYP2C8 and CYP2C9 genes in females is associated with a modest increase in risk of AMI. This might be related to genetic differences in the formation of endogenous vasoregulating eicosanoids.

Interleukin-6 serum levels and genotypes influence the risk for myocardial infarction.

OBJECTIVE: Several studies show that the inflammatory component in atherosclerosis may contribute to increased risk for cardiovascular disease (CVD). Interleukin-6 (IL-6) is a key pro-inflammatory and immune-stimulatory cytokine of presumed importance for CVD and the metabolic syndrome. METHODS AND RESULTS: In this case-control study, 1179 surviving myocardial infarction (MI) cases and 1528 healthy controls were genotyped for three IL-6 promoter SNPs, and serum concentrations of IL-6 and C-reactive protein (CRP) were measured. In men, MI risk assessed as odds ratios (OR) was higher with increasing IL-6 levels, with the highest compared to the lowest IL-6 quartiles giving an OR of 2.7 [95% CI 1.7-4.4]. The ORs were independent from the effects of elevated CRP which were associated with modest MI risks (OR = 1.6 [95% CI 1.0-2.5]). Also, synergistic interactions between high IL-6 levels and hypercholesterolaemia further increased MI risk estimates. The -174C allele was associated with lower serum-insulin levels among male controls but did not significantly influence MI risk or IL-6 levels. CONCLUSIONS: Elevated IL-6 levels are important risk markers for MI in men, the risk being further enhanced through synergistic interaction with hypercholesterolaemia. The data provide no clear evidence that polymorphisms in the IL-6 promotor region play a significant role in the pathogenesis of MI, and it remains to be further evaluated whether or not the -174C allele is of relevance for insulin resistance.

PAI-1 level and the PAI-1 4G/5G polymorphism in relation to risk of non-fatal myocardial infarction: results from the Stockholm Heart Epidemiology Program (SHEEP).

This study examines the relationship between plasma Plasminogen Activator Inhibitor-1 (PAI-1) activity and the PAI-1 4G/5G polymorphism, and their association with the risk of myocardial infarction (MI). Furthermore, this study explores whether a high level of PAI-1 or whether the PAI-1 4G/5G polymorphism synergistically interacts with any established environmental risk factor for MI. This case-referent study of subjects aged 45-70 and living in Stockholm includes 851 men and 361 women with first-time MI and 1051 men and 505 women who were randomly chosen as referents from a population register. The adjusted odds ratio (OR) of MI was 1.9 (95% confidence interval [CI] 1.4-2.8) for men with a plasma PAI-1 activity level greater than the 90 th percentile value of referents. The corresponding OR for women was 1.5 (95% CI 0.9-2.5). A strong indication of synergistic interaction was found in men for the co-exposure to high plasma PAI-1 activity and current smoking, an adjusted synergy index score of 3.9 (95% CI 1.2-13.2). In women, the 4G allele was associated slightly with an increased risk of MI, OR 1.4 (95% CI 1.0-1.9). This association was not found in men. There were no clear indications of synergistic interaction effects involving the PAI-1 4G/5G polymorphism and the environmental exposures considered (cigarette smoking, physical inactivity, overweight, diabetes mellitus, hypercholesterolaemia, hypertension, high C-reactive protein and hypertriglyceridaemia).

Inactivation of antiplasmin at low pH: evidence for the formation of latent molecules.

Several serine proteinase inhibitors (serpins) are metastable proteins which under certain conditions may undergo conformational changes resulting in the insertion of the reactive centre loop into the so-called Abeta-sheet and hence forming latent molecules. Here we have studied the inactivation of antiplasmin as a function of pH and temperature with time. At decreased pH (4.9-5.8) and at room temperature, antiplasmin activity decreased following first-order kinetics. Analysis by polyacrylamide gel electrophoresis under non-denaturing conditions demonstrated that only minor amounts of polymerized material formed after extensive incubation (4 days) at room temperature. However, on incubation at elevated temperatures (45 or 55 degrees C), a rapid formation of polymerized material was observed. We also demonstrated that antiplasmin inactivated by treatment at pH approximately 5 at room temperature spontaneously slowly regained some activity if incubated in a buffer of neutral pH. Furthermore, by treatment with 4 M guanidinium chloride for about 30 min, followed by dialysis against a neutral phosphate buffer, considerable activity (almost 40%) was regained. Thus, we conclude that antiplasmin, at least partially, at lower temperatures is transformed into a latent form, which could be reactivated, in a similar manner as PAI-1. At increased temperature, however, polymerization seems to be the predominant reason for inactivation.

Increased incorporation of antiplasmin into the fibrin network in patients with type 1 diabetes.

OBJECTIVE: Diabetes is associated with various vascular complications and is suggested to induce a prothrombotic state. In the current study, we characterized antiplasmin incorporation into fibrin in relation to other fibrinolytic compounds in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 236 patients with type 1 diabetes and 78 control subjects were investigated. The incorporation of antiplasmin into the fibrin network and the plasma levels of plasminogen activator inhibitor type 1 (PAI-1) activity, tissue plasminogen activator (tPA) activity, tPA/PAI-1 complex, plasmin-antiplasmin complex, antiplasmin, factor XIII, and d-dimer were measured. In addition, we used global assays to study fibrinolysis. RESULTS: The incorporation of antiplasmin into the fibrin network was significantly higher in patients with type 1 diabetes than in control subjects without diabetes (1.65 ± 0.25 vs. 1.35 ± 0.18 mg/L, respectively; P < 0.0001). The patients also had lower PAI-1 activity (2.19 units/mL [interquartile range 0.96-5.42] vs. 4.25 units/mL [1.95-9.0]; P = 0.0012) and antiplasmin level in plasma (78.5 ± 13.3 vs. 83.2 ± 15.4 mg/L; P < 0.05), resulting in a higher fibrinolytic capacity (shorter clot lysis time; P = 0.0090). We did not find any important sex differences regarding fibrinolysis in the patients or in the control subjects. CONCLUSIONS: Patients with type 1 diabetes incorporate more antiplasmin into the fibrin network than control subjects without diabetes do and have a reduced PAI-1 activity and a shorter clot lysis time. These results suggest that patients with type 1 diabetes produce a fibrin clot that is more resistant to fibrinolysis, which, however, may be counteracted by an increased fibrinolytic potential in plasma.

A new method measuring the interaction between von Willebrand factor and coagulation factor VIII.

INTRODUCTION: There is a need for more reliable methods measuring the binding of coagulation factor VIII (FVIII) to von Willebrand factor (VWF) in plasma samples, for use in the clinical routine. We have developed such a method measuring FVIII binding in plasma, utilizing an ELISA system. MATERIALS AND METHODS: Microtiter plates were coated with a monoclonal antibody (ESH-8), reacting with the C2 domain in FVIII. Thereafter the wells were treated with recombinant FVIII (Kogenate Bayer®). After washing, diluted plasma samples were added and incubated for 1h. Then HRP-conjugated antibodies against VWF were added and used for quantification of bound VWF. RESULTS: A strong signal to VWF concentration response was obtained. Plasma from patients with different types of von Willebrand disease gave frequently diminished responses. However, after correction for the VWF antigen levels, by calculation of FVIII binding/VWF antigen ratio, only the patients with known von Willebrand disease type 2N (n = 4) had clearly abnormal results. The FVIII binding in 40 healthy individuals was determined as 1.08 ± 0.48 U/mL (SD). After correction for the VWF antigen levels the result was 0.94 ± 0.15. Thus, the SD declined substantially by this correction. The within-series CV and between-series CV were determined as 6.8 and 11.3%, respectively. CONCLUSIONS: We have established a simple and reliable method to detect decreased binding of FVIII to von Willebrand factor in plasma samples. The method can conveniently be used to study large populations, as well as finding minor binding defects in patients.

Primary risk factors influence risk of recurrent myocardial infarction/death from coronary heart disease: results from the Stockholm Heart Epidemiology Program (SHEEP).

BACKGROUND: Prognosis after a first myocardial infarction (MI) is influenced by primary risk factors as well as secondary risk factors. There is still a lack of follow-up studies of well-characterized patient cohorts assessing the relative importance of these factors. DESIGN: A cohort of 1635 patients (aged 45-70 years) surviving at least 28 days after a first MI were followed for 6-9 years with regard to recurrent MI/fatal coronary heart disease (CHD). Data were collected through questionnaires, physical examinations, and medical records. METHODS: Hazard ratios (HR) with 95% confidence intervals (CI) for different risk factors were calculated using the Cox proportional hazard model. RESULTS: Of the primary risk factors, diabetes in both sexes was the most important predictor of recurrent MI/fatal CHD, multivariate-adjusted HR in men 1.6 (95% CI; 1.0-2.4) and in women 2.5 (95% CI; 0.9-6.9). Other primary risk factors with prognostic influence were job strain, HR 1.5 (95% CI; 1.0-2.1), and central obesity, HR 1.4 (95% CI; 1.0-2.0), in men and a low level of apolipoprotein A1, HR 2.3 (95% CI; 1.1-5.0), and high-density lipoprotein cholesterol, HR 1.9 (95% CI; 0.9-4.1), in women. The secondary risk factors most detrimental for prognosis were heart failure in men, HR 2.2 (95% CI; 1.2-4.0), and a high peak acute cardiac enzyme level in women, HR 4.4 (95% CI; 2.0-9.7). CONCLUSIONS: Long-term follow-up of patients who survived at least 28 days after a first MI shows that several primary cardiovascular risk factors, particularly diabetes, contribute to the increased risk of recurrent MI/fatal CHD.

Quantitative trait loci in ABCA1 modify cerebrospinal fluid amyloid-beta 1-42 and plasma apolipoprotein levels.

The ATP-binding cassette transporter A1 encoded by ABCA1 plays an integral role in the efflux of cellular cholesterol and phospholipids, but may also be a central mediator of beta-amyloid (Abeta) processing. Here, genetic association of the common R219K variant of ABCA1 is shown with cerebrospinal fluid (CSF) Abeta 1-42 levels, reinforcing emerging evidence of a connection between lipid and Abeta metabolism. In support of this finding we demonstrate for the first time that CSF cholesterol and Abeta 1-42 are correlated. To affirm the plausible impact of ABCA1 variation on cholesterol and related traits as well as to empower a survey of possible interactions (e.g. age, gender, and smoking), a large Swedish population consisting of over 2,700 individuals was enlisted and extensive measures of plasma lipid parameters carried out. These analyses revealed that R219K has a strong effect on apolipoprotein B (APOB) and LDL-cholesterol (LDL-C) among smokers (P = 0.000055 and P = 0.00059, respectively), but not among non-smokers. In contrast, no effect was evident with apolipoprotein A (APOA1) or HDL-cholesterol (HDL-C) levels. Plasma APOB and LDL-C, but not APOA1 and HDL-C, were shown to be markedly elevated in smokers versus non-smokers, affirming that smoking may selectively impact the former pathway. No other genetic markers in ABCA1 exhibit effects as large as R219K, although a modest independent effect of R1587K was observed. Our data illuminate a possible genetic link between Abeta and cholesterol metabolism, but also provide an intriguing example of an environmental exposure that may modify a genotype-phenotype relationship.

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.

Criteria for the Specific Measurement of Plasmin Inhibitor Activity Using an Enzymatic Procedure.

There is a lack of well-established criteria for the specific measurement of fibrinolytic parameters. On behalf of the SSC, the subcommittee on Fibrinolysis started a process to develop criteria for the specific measurement of fibrinolytic variables. This report describes the criteria for the specific measurement of plasmin inhibitor activity. In summary, a plasma deficient in plasmin inhibitor should show an activity close to 0%. Plasma containing only the non-plasminogen binding form of plasmin inhibitor should show an activity nearby the activity of a plasma deficient for plasmin inhibitor. Other inhibitors of plasmin, like a 2 -macroglobulin, antithrombin in the presence of heparin, and C1-esterase inhibitor should not interfere in the assay at the level usually found in pathological conditions or at the higher normal level.