Sugammadex hypersensitivity and underlying mechanisms: a randomised study of healthy non-anaesthetised volunteers.

BACKGROUND: We investigated potential for hypersensitivity reactions after repeated sugammadex administration and explored the mechanism of hypersensitivity. METHODS: In this double-blind, placebo-controlled study (NCT00988065), 448 healthy volunteers were randomised to one of three arms to receive three repeat i.v. administrations of either sugammadex 4 mg kg-1, 16 mg kg-1, or placebo. Primary endpoint was percentage of subjects with hypersensitivity (assessed by an independent adjudication committee). Secondary endpoint of anaphylaxis was classified per Sampson and Brighton criteria. Exploratory endpoints included skin testing, serum tryptase, anti-sugammadex antibodies [immunoglobulin (Ig) E/IgG], and other immunologic parameters. RESULTS: Hypersensitivity was adjudicated for 1/148 (0.7%), 7/150 (4.7%), and 0/150 (0.0%) subjects after sugammadex 4 mg kg-1, 16 mg kg-1, and placebo, respectively. After sugammadex 16 mg kg-1, one subject met Sampson criterion 1 and Brighton level 1 (highest certainty) anaphylaxis criteria; two met Brighton level 2 criteria. After database lock it was determined that certain protocol deviations could have introduced bias in the reporting of hypersensitivity signs/symptoms in a subject subset. Objective laboratory investigations indicated that potential underlying hypersensitivity mechanisms were unlikely to have been activated; the results suggest that most of the observed hypersensitivity reactions were unlikely IgE/IgG-mediated. CONCLUSION: Dose-dependent hypersensitivity or anaphylaxis reactions to sugammadex were observed when administered without prior neuromuscular blocking agent. Laboratory investigations do not suggest prevalent allergen-specific IgE/IgG-mediated immunologic hypersensitivity. Because it could not be fully excluded that estimates of hypersensitivity/anaphylaxis incidence were unbiased, an additional study was conducted to characterise the potential for hypersensitivity reactions and is described in a companion report. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov NCT00988065; Protocol number P06042.

Heritability and GWAS Studies for Monocyte-Lymphocyte Ratio.

The monocyte-lymphocyte ratio (MLR) is a useful biomarker for disease development, but little is known about the extent to which genetic and environmental factors influence MLR variation. Here, we study the genetic architecture of MLR and determine the influence of demographic and lifestyle factors on MLR in data from a Dutch non-patient twin-family population. Data were obtained in 9,501 individuals from the Netherlands Twin Register. We used regression analyses to determine the effects of age, sex, smoking, and body mass index (BMI) on MLR and its subcomponents. Data on twins, siblings and parents (N = 7,513) were analyzed by genetic structural equation modeling to establish heritability and genome wide single nucleotide polymorphism (SNP) data from a genotyped subsample (N = 5,892) and used to estimate heritability explained by SNPs. SNP and phenotype data were also analyzed in a genome-wide association study to identify the genes involved in MLR. Linkage disequilibrium (LD) score regression and expression quantitative trait loci (eQTL) analyses were performed to further explore the genetic findings. Results showed that age, sex, and age × sex interaction effects were present for MLR and its subcomponents. Variation in MLR was not related to BMI, but smoking was positively associated with MLR. Heritability was estimated at 40% for MLR, 58% for monocyte, and 58% for lymphocyte count. The Genome-wide association study (GWAS) identified a locus on ITGA4 that was associated with MLR and only marginally significantly associated with monocyte count. For monocyte count, additional genetic variants were identified on ITPR3, LPAP1, and IRF8. For lymphocyte count, GWAS provided no significant findings. Taking all measured SNPs together, their effects accounted for 13% of the heritability of MLR, while all known and identified genetic loci explained 1.3% of variation in MLR. eQTL analyses showed that these genetic variants were unlikely to be eQTLs. In conclusion, variation in MLR level in the general population is heritable and influenced by age, sex, and smoking. We identified gene variants in the ITGA4 gene associated with variation in MLR. The significant SNP-heritability indicates that more genetic variants are likely to be involved.

Longitudinal weight differences, gene expression and blood biomarkers in BMI-discordant identical twins.

BACKGROUND: Body mass index (BMI) discordant monozygotic (MZ) twins allow an examination of the causes and consequences of adiposity in a genetically controlled design. Few studies have examined longitudinal BMI discordance in MZ pairs. OBJECTIVES: The aim of this work was to study the development over time of BMI discordance in adolescent and adult MZ twin pairs and to examine lifestyle, metabolic, inflammatory and gene expression differences associated with concurrent and long-term BMI discordance in MZ pairs. SUBJECTS/METHODS: BMI data from 2775 MZ twin pairs, collected in eight longitudinal surveys and a biobank project between 1991 and 2011, were analyzed to characterize longitudinal discordance. Lifestyle characteristics were compared within discordant pairs (ΔBMI⩾3 kg m(-2)) and biomarkers (lipids, glucose, insulin, C-reactive protein, fibrinogen, interleukin (IL)-6, tumor necrosis factor-α and soluble IL-6 receptor and liver enzymes aspartate aminotransferase, alanine aminotransferase and gamma glutamyl transferase) and gene expression were compared in peripheral blood from discordant pairs who participated in the Netherlands Twin Register biobank project. RESULTS: The prevalence of discordance ranged from 3.2% in 1991 (mean age=17, s.d.=2.4) to 17.4% (N=202 pairs) in 2009 (mean age=35, s.d.=15) and was 16.5% (N=174) among pairs participating in the biobank project (mean age=35, s.d.=12). Of the 699 MZ pairs with BMI data from 3 to 5 time points, 17 pairs (2.4%) were long-term discordant (at all available time points; mean follow-up range=6.4 years). Concurrently discordant pairs showed significant differences in self-ratings of which twin eats most (P=2.3 × 10(-13)) but not in leisure time exercise activity (P=0.28) and smoking (P>0.05). Ten out of the 14 biomarkers showed significantly more unfavorable levels in the heavier of twin of the discordant pairs (P-values <0.001); most of these biomarker differences were largest in longitudinally discordant pairs. No significant gene expression differences were identified, although high ranking genes were enriched for Gene Ontology terms highlighting metabolic gene regulation and inflammation pathways. CONCLUSIONS: BMI discordance is uncommon in adolescent identical pairs but increases with higher pair-mean of BMI at older ages, although long-term BMI discordance is rare. In discordant pairs, the heavier twin had a more unfavorable blood biomarker profile than the genetically matched leaner twin, in support of causal effects of obesity.

Alpha 2-antiplasmin Enschede: alanine insertion and abolition of plasmin inhibitory activity.

An abnormal alpha 2-antiplasmin that is associated with a serious bleeding tendency has been found in a Dutch family and is referred to as alpha 2-antiplasmin Enschede. This abnormal alpha 2-antiplasmin is converted from an inhibitor of plasmin to a substrate. The molecular defect of alpha 2-antiplasmin Enschede, as revealed by sequencing of cloned genomic DNA fragments, consists of an alanine insertion near the active site region of the molecule. Substitution of this fragment into complementary DNA for a wild-type alpha 2-antiplasmin yields a translation product with physical and functional properties typical of the abnormal alpha 2-antiplasmin Enschede. The naturally occurring mutant may serve as a model for investigating the structures that determine the properties of an inhibitor versus those of a substrate in serine protease inhibitors.

alpha 2-Antiplasmin Enschede: dysfunctional alpha 2-antiplasmin molecule associated with an autosomal recessive hemorrhagic disorder.

alpha 2-Antiplasmin (alpha 2-AP) is a major fibrinolysis inhibitor, whose complete, congenital absence has been found to be associated with a distinct hemorrhagic diathesis. We studied a 15-yr-old male with a hemorrhagic diathesis after trauma from early childhood on. This bleeding tendency was associated with a minimal alpha 2-AP level recorded functionally in the immediate plasmin inhibition test: less than or equal to 4% of normal. However, a normal plasma concentration of alpha 2-AP antigen (83%) was found. His sister (5 yr old) showed similar results (2 and 92%). In their family, eight heterozygotes could be identified by half-normal activity results and normal antigen concentrations. The inheritance pattern is autosomal recessive. On analysis, the alpha 2-AP of the propositus was homogeneous in all respects tested, suggesting a homozygous defect. We designated the abnormal alpha 2-AP as alpha 2-AP Enschede. alpha 2-AP Enschede showed the following characteristics: (a) complete immunological identity with normal alpha 2-AP; (b) normal molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) normal alpha-electrophoretic mobility; (d) presence in plasma of both molecular forms excluding an excessive conversion to the less reactive non-plasminogen-binding form; (e) quantitatively normal binding to lys-plasminogen and to immobilized plasminogen kringle 1-3; and (f) normal Factor XIII-mediated binding to fibrin. Functional abnormalities were found in: (i) no inhibition of amidolytic activities of plasmin and trypsin, even on prolonged incubation; (ii) no formation of plasmin-antiplasmin complexes in plasma with plasmin added in excess; and (iii) no inhibition of fibrinolysis by fibrin-bound alpha 2-AP. In the heterozygotes, the presence of abnormal alpha 2-AP did not interfere with several functions of the residual normal alpha 2-AP. One-dimensional peptide mapping showed an abnormal pattern of papain digestion. We conclude that in this family, abnormal antiplasmin molecules, defective in plasmin inhibition but with normal plasminogen-binding properties, have been inherited. The residual plasminogen-binding properties do not protect against a hemorrhagic diathesis.

Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times.

One possibility for bringing drugs to their specific targets is to use the drug-laden liposomes that have been made target-specific by the attachment of appropriate proteins. Such 'directed' proteoliposomes and most other particles are rapidly removed from the bloodstream, however, by the mononuclear phagocytes in the liver and spleen. This causes suboptimal drug accumulation at the target site. Coating the liposome surface with poly(ethylene glycol) (PEG) may prolong the circulation time of liposomes. Using plasminogen as a homing device we have shown that the PEG-modified liposomes with such a homing device coupled to the ends of the long PEG chains may combine long vesicle circulation times in the blood with high target binding capability. The PEG-coated proteoliposomes with homing devices attached at the very bilayer surface, on the contrary, are longlived but have only little or no capability to bind to their targets.

Development of a procedure for coupling the homing device glu-plasminogen to liposomes.

The aim of this study was to find a suitable way of coupling the homing-device glu-plasminogen to the outside of liposomes. The described procedure is based on the reaction of thiol-groups introduced in the protein with thiol-reactive groups of the liposome. Details on the thiolation of proteins with the reagent succinimidyl-S-acetylthioacetate (SATA) were studied for a model-protein, amylase. Increasing the incubation-ratio SATA: amylase resulted in a gradually growing number of introduced thiol-groups, until a maximum of about 5 mol SH per mol amylase was reached. The enzymatic activity of the derivatized protein was even higher than that of native amylase. The thiol-introduction was then applied to glu-plasminogen itself. After activation with SATA, the protein was incubated with liposomes containing the thiol-reactive anchor maleimido-4-(p-phenylbutyrate)-phosphatidylethanolamine (MPB-PE). Under the chosen conditions, incubation of 0.5-2.5 mg/ml protein with 6.0-7.5 mumol/ml phospholipid for 30-120 min resulted in coupling-ratios of 20 to 94 micrograms glu-plasminogen per mumol phospholipid. This corresponds with about 140 to 660 protein molecules per liposome. SATA-derivatization of glu-plasminogen brought about a loss of its enzymatic activity induced by streptokinase. This activity of liposomally coupled plasminogen was about 52 to 74% of the activity of native glu-plasminogen (depending on the coupling-ratio). Although this may seem a significant loss of activity, it was shown that the capacity of liposomal glu-plasminogen to bind to its target, fibrin, was not reduced but several fold higher under the used conditions than that of the free protein. Therefore, the described method for thiol-introduction is an effective way to thiolate amylase without loss of activity, and to bind the homing-device glu-plasminogen to liposomes without substantially interfering with its fibrin-binding/homing capacity.

Depression of factor XII-dependent fibrinolytic activity characterizes patients with early myocardial reinfarction after recombinant tissue-type plasminogen activator therapy.

Twenty patients with acute myocardial infarction treated with recombinant tissue-type plasminogen activator (rt-PA) had endogenous factor XII-dependent fibrinolytic activity levels measured throughout the hospital period and those levels were prospectively correlated with the incidence of recurrent myocardial infarction until 8 weeks after hospital discharge. Within the follow-up period, recurrent myocardial infarction was observed in 8 patients, whereas the remaining 12 patients showed no clinical evidence of recurrence. The patients in the reinfarction group were characterized by a more pronounced depletion of and sustained lower levels of factor XII-dependent fibrinolytic activity than were the patients with no reinfarction (p less than 0.05). The decrease in fibrinolytic activity during rt-PA therapy was significantly associated with a depletion of functional alpha 2-antiplasmin, the primary plasmin inhibitor. These results indicate that, paradoxically, coronary thrombolysis with rt-PA involves depletion of endogenous factor XII-dependent fibrinolytic activity levels, which constitutes a risk for early myocardial reinfarction.

Long-lasting depression of the factor XII-dependent fibrinolytic system in patients with myocardial infarction undergoing thrombolytic therapy with recombinant tissue-type plasminogen activator: a randomized placebo-controlled study.

In a randomized placebo-controlled study, seven patients with acute myocardial infarction allocated to intravenous treatment with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) and seven patients allocated to placebo were studied during eight sampling periods before and after treatment. Seven patients with acute myocardial infarction treated intravenously with 1.5 million U of streptokinase were later studied during two sampling periods before and after treatment. The placebo group showed no significant deviations of endogenous factor XII-dependent fibrinolytic activity (p greater than 0.05). In the rt-PA group, this activity decreased significantly (p less than 0.001) after the infusion and remained depressed throughout the 1st 4 days. A significant decrease in activity (p less than 0.05) was also found in the streptokinase-treated patients. The depletion of factor XII-dependent fibrinolytic activity was not due to generation of inhibition or a depletion of factor XII, prekallikrein and plasminogen, but could be related to the proactivator of this system. It is concluded that rt-PA (and streptokinase) treatment in patients with acute myocardial infarction causes a prolonged depletion of factor XII-dependent fibrinolytic activity. This depression of endogenous fibrinolytic activity needs to be evaluated in relation to the enhanced risk of coronary reocclusion after thrombolytic therapy.

Early spontaneous intermittent myocardial reperfusion during acute myocardial infarction is associated with augmented thrombogenic activity and less myocardial damage.

OBJECTIVES: This study investigated the influence of early spontaneous intermittent reperfusion on the extent of myocardial damage and its relation to endogenous hemostatic activity. BACKGROUND: In the early phase of acute myocardial infarction coronary occlusion is often intermittent, even before thrombolytic therapy is administered. The relation between this phenomenon, myocardial damage and hemostatic activity is unknown. METHODS: Holter ST segment recording and pretreatment plasma tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, prothrombin fragment F1 + 2 and soluble fibrin levels were measured in 57 patients with acute evolving myocardial infarction. Spontaneous intermittent myocardial reperfusion, defined as two or more episodes of transient resolution of ST segment elevation to within 0.05 mV of baseline, lasting > or = 1 min, before the start of recombinant t-PA (rt-PA) treatment was present in 28 patients (group 1) and absent in 29 (group 2). Left ventriculography and coronary angiography were performed 90 min after intravenous rt-PA administration. Plasma creatine kinase-MB fraction (CK-MB) levels were measured every 6 h for 24 h, and C-reactive protein levels were measured daily for 3 days. RESULTS: Group 1 had lower peak plasma CK-MB (141.9 +/- 28.3 vs. 203.8 +/- 23.3 IU/liter [mean +/- SEM], p < 0.014) and C-reactive protein levels (16 +/- 4 vs. 28 +/- 4 mg/liter on day 1; 26.6 +/- 5.5 vs. 61.8 +/- 14.4 mg/liter on day 2; 19.6 +/- 4.2 vs. 40.6 +/- 6.5 mg/liter on day 3, p < 0.012) and a higher left ventricular ejection fraction (62.9 +/- 4% vs. 51.1 +/- 5%, p < 0.04) than group 2. Group 1 had lower plasma t-PA antigen levels (15.6 vs. 27 micrograms/liter, p < 0.006) but higher prothrombin fragment F1 + 2 (1.8 vs. 1.1 nmol/liter, p < 0.003) and soluble fibrin levels (66.8 vs. 31 nmol/liter, p < 0.01). Coronary patency at 90 min was similar. CONCLUSIONS: Early spontaneous intermittent reperfusion during acute myocardial infarction is associated with augmented thrombogenic activity and less subsequent myocardial damage. This finding is consistent with a protective effect of intermittency on the myocardium and a procoagulant effect of spontaneous lysis on blood. It may also reflect a different rate of evolution of coronary thrombosis and myocardial infarction in patients with and those without spontaneous intermittent myocardial reperfusion.

Angiotensin I-converting enzyme and plasminogen activator inhibitor-1 gene variants: risk of mortality and fatal cardiovascular disease in an elderly population-based cohort.

OBJECTIVES: We studied the contribution of putative risk genotypes at the angiotensin I-converting enzyme inhibitor (ACE D/D) and plasminogen activator inhibitor-1 (PAI-1 4G/4G) loci to all-cause and cardiovascular mortality in a population-based cohort. BACKGROUND: The ACE D/D and PAI-1 4G/4G genotypes have been consistently associated with elevated plasma activities of the gene products. Their role in cardiovascular disease, although explored intensively, is still equivocal. METHODS: The ACE and PAI-1 genotypes were determined in 648 subjects > or =85 years old. In a cross-sectional analysis, the genotype distributions in a subset of 356 elderly subjects who were born in Leiden, The Netherlands, were compared with those in 250 young subjects whose families originated from the same geographic region. In addition, the complete cohort of elderly subjects was followed over 10 years for all-cause and cardiovascular mortality and was stratified according to genotype. RESULTS: In the cross-sectional analysis, the ACE and PAI-1 genotype distributions were similar in elderly and young subjects. In the prospective follow-up study, however, the age-adjusted risk of fatal ischemic heart disease was increased threefold (95% confidence interval [CI] 1.2 to 7.6) in elderly men carrying the PAI-1 4G/4G genotype. The risk of all-cause mortality was not increased among elderly subjects carrying the PAI-1 4G/4G (relative risk [RR] 0.9, 95% CI 0.7 to 1.1) or the ACE D/D genotype (RR 0.9, 95% CI 0.7 to 1.1), nor did we observe elevated risks of death from all cardiovascular diseases combined. There was no interaction between the genotypes. CONCLUSIONS: The PAI 4G/4G genotype may be a risk factor for fatal ischemic heart disease in elderly men. The impact of moderately increased ACE and PAI-1 activities associated with the ACE D/D and PAI-1 4G/4G genotypes is too small to affect mortality in the general population.

Plasmin inhibitors in the prevention of systemic effects during thrombolytic therapy: specific role of the plasminogen-binding form of alpha 2-antiplasmin.

To delineate the role of plasmin inhibitors, especially the two molecular forms of alpha 2-antiplasmin (that is, the plasminogen-binding and the nonplasminogen-binding forms), in the control of systemic effects during thrombolytic therapy, the consumption of plasmin inhibitors and the degree of fibrinogen breakdown were studied in 35 patients with acute myocardial infarction treated with recombinant tissue-type plasminogen activator (rt-PA) or streptokinase. At a low degree of plasminogen activation (in six patients treated with rt-PA), plasminogen-binding alpha 2-antiplasmin was consumed first. At a higher degree of plasminogen activation (in 20 patients), plasminogen-binding alpha 2-antiplasmin became exhausted (less than 20%) and other plasmin inhibitors (that is, nonplasminogen-binding alpha 2-antiplasmin and alpha 2-macroglobulin) were consumed. After extensive plasminogen activation (in nine patients treated with streptokinase), plasminogen-binding alpha 2-antiplasmin consumption was complete and nonplasminogen-binding alpha 2-antiplasmin and alpha 2-macroglobulin were consumed to about 30% to 50% of the pretreatment level. No significant C1-inactivator consumption occurred, even at extreme degrees of plasminogen activation. Fibrinogen breakdown as a marker for systemic effects correlated strongly with consumption of plasminogen-binding alpha 2-antiplasmin. Fibrinogen breakdown did occur, but only when the amount of plasminogen-binding alpha 2-antiplasmin was decreased to less than 20% of the pretreatment level. The other plasmin inhibitors could not prevent fibrinogen breakdown. These results were confirmed by in vitro studies. It is concluded that plasminogen-binding alpha 2-antiplasmin is the most important inhibitor of plasmin in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Early coronary reperfusion blunts the procoagulant response of plasminogen activator inhibitor-1 and von Willebrand factor in acute myocardial infarction.

The effects of early coronary recanalization on the plasma levels of two procoagulant acute phase proteins, the fastacting plasminogen activator inhibitor and von Willebrand factor, were investigated in 24 patients with myocardial infarction receiving intravenous recombinant tissue-type plasminogen activator (rt-PA) within 6 h of the onset of symptoms. Coronary angiography was performed before and 90 min after the start of rt-PA infusion. Continuous electrocardiographic recordings and 4 h plasma creatine kinase MB isoenzyme (CK MB) were performed over the first 24 h. Plasma plasminogen activator inhibitor activity, von Willebrand factor and C-reactive protein were measured before rt-PA infusion, daily for the first 3 days and after 90 days. In the entire group, plasminogen activator inhibitor activity peaked at 24 h (day 1), representing a significant increase over values at all other times (p = 0.03). von Willebrand factor was higher in the first 2 days of infarction compared with after 90 days (p = 0.001). C-reactive protein peaked on day 2, with an eightfold increase over values on admission (p = 0.001). In the 16 patients with a patent infarct-related artery at 90 min, infarct size estimated by integrated 24 h CK MB, time for ST segment elevation to decrease to half-maximum and peak C-reactive protein were reduced significantly by more than twofold compared with values in the 8 patients with an occluded artery at 90 min. The patients with early recanalization also had lower plasminogen activator inhibitor activity on day 2 (p = 0.05) and day 3 (p = 0.02) and lower 0 to 72 h averaged von Willebrand factor (p = 0.01). Thus, early coronary recanalization curtails the response of plasminogen activator inhibitor activity and von Willebrand factor to myocardial infarction, most likely by reducing the extent of ischemia and necrosis and the consequent acute phase reaction. By blunting the early postinfarction procoagulant state, prompt recanalization may reduce the risk of thromboembolic complications in the first days after myocardial infarction.

Report on the Dutch consensus development meeting for implementation and further development of population screening for colorectal cancer based on FOBT.

A consensus development meeting was held to evaluate whether or not in the Netherlands all requirements were fulfilled for implementation of population screening with FOBT for colorectal cancer, or whether consensus was present that fulfilment by additional research or organisational actions could be obtained within 2-3 years. There was consensus that all classical Wilson and Jungner (1968) criteria, and six additional ones added more recently, had already been fulfilled or could be fulfilled within 2-3 years. Consequently, it was concluded that a national population screening for colorectal cancer should be implemented and carried out in the Netherlands in line with current national and European cancer screening programmes. A list of organisational actions to be taken was established. Research that is needed before the actual national launch of the screening within 2-3 years has been defined. Priorities have to be set for research and organisational actions for the coming 2-3 years for the implementation of population screening. In addition, research suggestions have been defined for the next 10-15 years for evaluation and/or improvement of implemented FOBT screening, and for future screening methodology. It was considered essential that infrastructure for future research would be embedded in the screening programme. A project group to arrange this should be formed.

Tissue plasminogen activator inhibition in diabetes mellitus.

Depression of fibrinolysis may be relevant to the vascular complications of diabetes mellitus. Plasminogen activator inhibitor (PAI) is an important inhibitor of fibrinolysis in humans, and we have found basal activities of PA inhibition to be elevated in patients with diabetes compared with a reference group of healthy subjects (mean +/- SD 268 +/- 268 vs. 105 +/- 48%; P less than .0001). With a monoclonal antibody, it was shown that high inhibition values were due to PAI-1. No differences in PA inhibition were noted in relation to type of diabetes, diabetic treatment, or presence or absence of vascular complications. Basal PA inhibition did not correlate with in vivo (B beta 15-42 antigen) or ex vivo (fibrin plate) fibrinolytic activity or HbA1. In patients with non-insulin-dependent diabetes mellitus, treatment with the anabolic steroid stanozolol significantly reduced PA inhibition. These findings suggest a further abnormality of fibrinolysis in diabetes, but the lack of a relationship among other measures of fibrinolysis renders its biologic significance uncertain.

Fibrinolytic system during long-distance running in IDDM patients and in healthy subjects.

OBJECTIVE: Endurance exercise has been advocated in diabetes mellitus to improve both metabolic control and prevent atherosclerotic complications. The response of the fibrinolytic system during prolonged exercise has not been studied in diabetes. RESEARCH DESIGN AND METHODS: In seven male marathon runners with IDDM and eight healthy nondiabetic male control subjects, matched for age and degree of training, we studied fibrinolytic and coagulation parameters during a 3-h 32-km outdoor running session. Measurements included t-PA, u-PA, PAI-1, PAP (as a measure of in vivo activation of fibrinolysis), FbDPs, FGN, vWF, and VIII:C. RESULTS: In both IDDM and control subjects, levels of t-PA, u-PA, PAP, vWF, and VIII:C continued to rise throughout the exercise, whereas PAI-1 showed a similar decline in both groups. FbDP rose slightly in both groups, and FGN remained unchanged. t-PA levels during exercise correlated closely with exercise intensity. These findings indicate that continued stimulation by exercise does not deplete endothelial PA stores. Differences between IDDM and control subjects were seen only for t-PA, vWF, and u-PA. The AUC during exercise (AUC0.5-3.0) of t-PA in IDDM was insignificantly lower than in control subjects (53 +/- 19 vs. 67 +/- 31 ng.ml-1.h), but the ratio of t-PA to exercise intensity was lower in IDDM (0.24 +/- 0.11 vs. 0.31 +/- 0.13, P less than 0.05). The AUC0.5-3.0 of vWF was lower in IDDM than in control subjects (569 +/- 268 vs. 880 +/- 265%.h, P less than 0.05). The AUC0.5-3.0 of u-PA was higher in IDDM than in control subjects (15.1 +/- 3.5 vs. 11.2 +/- 1.8 ng.ml-1.h, P less than 0.05). CONCLUSIONS: Despite a defect in the exercise-induced endothelial release of vWF and t-PA, the overall potential to activate fibrinolysis is intact in IDDM, possibly by enhancement of u-PA after exercise. Our data suggest that in IDDM, as in nondiabetic subjects, long-distance running may slow the progression of atherosclerosis by stimulating fibrinolysis.

No effect of unfractioned or low molecular weight heparin treatment on markers of vascular wall and hemostatic function in incipient diabetic nephropathy.

OBJECTIVE: The high risk for cardiovascular disease in IDDM patients with nephropathy may be mediated by abnormal function of the vascular wall. We investigated whether heparin was able to modulate markers of vascular wall and hemostatic function in patients with incipient nephropathy. RESEARCH DESIGN AND METHODS: Thirty-five IDDM patients with incipient nephropathy were randomized to treatment with placebo, unfractioned heparin, or low molecular weight heparin in a double-blind trial. The treatment was given as 1 h of conventional intravenous high-dose treatment and in a conventional subcutaneous low-dose regime for 3 months. Transcapillary escape rate of albumin and plasma levels of von Willebrand factor, fibrinogen, prothrombin fragment 1 + 2, thrombin-antithrombin III complexes, tissue type plasminogen activator, tissue plasminogen activator inhibitor type 1, total cholesterol, HDL cholesterol, and triglycerides were measured before and after treatment. Of the patients, 31 completed the study. RESULTS: We found no significant effect of heparin on markers of vascular wall and hemostatic function by any of the treatments. CONCLUSIONS: Treatment with high- or low-dose heparin induced no modulation of markers of vascular wall or hemostatic function in IDDM patients with incipient diabetic nephropathy.

Consequences for the APTT due to direct action of factor XIa on factor X, resulting in bypassing factors VIII-IX.

BACK GROUND: It has recently been reported that factor XIa can activate factor X directly and can bypass factors VIII-IX. We evaluated the consequences for factor analysis with the one-stage APTT. METHODS: APTT was performed with the Actin FS reagent with ellagic acid as the standard. Silica, high lipid (PTT-A) or low lipid (PTT-LA) were also tested. Factor depleted and deficient plasma's were obtained from commercial sources. RESULTS: The APTT clotting times in factor XII, XI, High Molecular Weight Kininogen, factor X and factor V deficient plasma's were all significantly longer (>100s) than the clotting times of factor VIII- and IX-depleted or deficient plasma's (<100s). That the shorter times for factor VIII and IX deficient plasmas were due to contact activation was supported by biphasic inhibition of the clotting times with addition of Corn Trypsin Inhibitor and Trasylol. The role of factor XI and the by-passing of factor VIII/IX was shown by the use of quenching antibodies towards factor XI and VIII. Enriching factor VIII or IX depleted plasma with purified factor XI and addition of factor XIa showed a strong dependence on factor XI level. Calibration curves for factor analysis were steeper for factors FXII, HMWK, FX and FV, compared to those of both factors VIII and IX. Curves for VIII/IX were found steeper by the use of APTT-A/silica-based, 50% diluted substrate plasma and low factor XI in the substrate plasma. CONCLUSIONS: In factors VIII and IX deficient plasmas, the APTT shows an activity which can be attributed to contact activation of factor X by factor XIa. This direct activity is lower with silica reagent compared to ellagic acid, dilution of plasma and low factor XI in substrate plasma.

Plasma kallikrein-mediated activation of the renin-angiotensin system does not require prior acidification of prorenin.

Activation of prorenin in the neutral phase after pH 3.3 dialysis of human plasma depends on clotting factor XII-initiated prekallikrein to kallikrein conversion. Acid dialysis may be necessary for destroying kallikrein inhibitors or rendering prorenin susceptible to attack by kallikrein. If the latter possibility proves true, it is difficult to see how the factor XII-kallikrein pathway could activate prorenin in vivo. Plasma prorenin was therefore separated from active renin and from the protease inhibitors alpha 2-macroglobulin, C1-inactivator, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, and antithrombin III by gel filtration on Sephadex G-100 and affinity chromatography on Blue Sepharose CL-6B at neutral pH. The resulting prorenin preparation could be activated at pH 7.5 by highly purified human plasma kallikrein, which was prepared from prekallikrein by activation with active factor XII fragment beta-factor XII a. Activation proceeded at 4 and 37 C at a kallikrein concentration of 2 micrograms/ml, which is approximately 5% of the prekallikrein concentration in normal plasma. It appears that an acid-induced conformational change of the prorenin molecule is not required for its activation by plasma kallikrein.