Major Inhibitor Of Fibrinolysis Research Articles

PAI1: a novel PP1-interacting protein that mediates human plasma's anti-apoptotic effect in endothelial cells.

Activation of apoptotic signalling in endothelial cells contributes to the detrimental effects of a variety of pathological stimuli. In investigating the molecular events underlying the anti‐apoptotic effect of human plasma in cultured human endothelial cells, we unexpectedly uncovered a novel mechanism of apoptosis suppression by human plasma through an interaction between two previously unrelated proteins. Human plasma inhibited hypoxia–serum deprivation‐induced apoptosis and stimulated BADS136 and AktS473 phosphorylation. Akt1 silencing reversed part (~52%) of the anti‐apoptotic effect of human plasma, suggesting the existence of additional mechanisms mediating the anti‐apoptotic effect other than Akt signalling. Human plasma disrupted the interaction of BAD with protein phosphatase 1 (PP1). Mass spectrometry identified fourteen PP1‐interacting proteins induced by human plasma. Notably, a group of serine protease inhibitors including plasminogen activator inhibitor 1 (PAI1), a major inhibitor of fibrinolysis, were involved. Silencing of PAI1 attenuated the anti‐apoptotic effect of human plasma. Furthermore, combined Akt1 and PAI1 silencing attenuated the majority of the anti‐apoptotic effect of human plasma. We conclude that human plasma protects against endothelial cell apoptosis through sustained BAD phosphorylation, which is achieved by, at least in part, a novel interaction between PP1 with PAI1.

Plasminogen activator inhibitor-1 is elevated in patients with COPD independent of metabolic and cardiovascular function.

IntroductionPlasminogen activator inhibitor-1 (PAI-1), a major inhibitor of fibrinolysis, is associated with thrombosis, obesity, insulin resistance, dyslipidemia, and premature aging, which all are coexisting conditions of chronic obstructive pulmonary disease (COPD). The role of PAI-1 in COPD with respect to metabolic and cardiovascular functions is unclear.MethodsIn this study, which was nested within a prospective cohort study, the serum levels of PAI-1 were cross-sectionally measured in 74 stable COPD patients (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stages I–IV) and 18 controls without lung disease. In addition, triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, waist circumference, blood pressure, smoking status, high-sensitive C-reactive protein (hs-CRP), adiponectin, ankle–brachial index, N-terminal pro-B-type natriuretic peptide, and history of comorbidities were also determined.ResultsThe serum levels of PAI-1 were significantly higher in COPD patients than in controls, independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction. A multivariate regression analysis revealed triglyceride and hs-CRP levels to be the best predictors of PAI-1 within COPD. GOLD Stages II and III remained independently associated with higher PAI-1 levels in a final regression analysis.ConclusionThe data from the present study showed that the serum levels of PAI-1 are higher in patients with COPD and that moderate-to-severe airflow limitation, hypertriglyceridemia, and systemic inflammation are independent predictors of an elevated PAI-1 level. PAI-1 may be a potential biomarker candidate for COPD-specific and extra-pulmonary manifestations.

Endogenous α2-Antiplasmin Is Protective during Severe Gram-Negative Sepsis (Melioidosis)

α2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit plasmin. Although the fibrinolytic system is strongly affected by infection, the functional role of A2AP in the host response to sepsis is unknown. To study the role of A2AP in melioidosis, a common form of community-acquired sepsis in Southeast Asia and Northern Australia caused by the gram-negative bacterium Burkholderia pseudomallei. In a single-center observational study A2AP was measured in patients with culture-proven septic melioidosis. Wild-type and A2AP-deficient (A2AP(-/-)) mice were intranasally infected with B. pseudomallei to induce severe pneumosepsis (melioidosis). Parameters of inflammation and coagulation were measured, and survival studies were performed. Patients with melioidosis showed elevated A2AP plasma levels. Likewise, A2AP levels in plasma and lung homogenates were elevated in mice infected with B. pseudomallei. A2AP-deficient (A2AP(-/-)) mice had a strongly disturbed host response during experimental melioidosis as reflected by enhanced bacterial growth at the primary site of infection accompanied by increased dissemination to distant organs. In addition, A2AP(-/-) mice showed more severe lung pathology and injury together with an increased accumulation of neutrophils and higher cytokine levels in lung tissue. A2AP deficiency further was associated with exaggerated systemic inflammation and coagulation, increased distant organ injury, and enhanced lethality. This study is the first to identify A2AP as a protective mediator during gram-negative (pneumo)sepsis by limiting bacterial growth, inflammation, tissue injury, and coagulation.

The Plasminogen Activator Inhibitor-1 Gene Polymorphism in Determining the Risk of Pediatric Ischemic Stroke − Case Control and Family-Based Study

Pediatric ischemic stroke, though relatively rare, remains an important medical problem since 20-40% of patients have recurrent strokes and 50-85% of them suffer from long-term neurological deficits. Approximately 20-50% of the affected children have prothrombotic disorders, therefore upon looking for possible genetic causes of the disease we focused on the plasminogen activator inhibitor (PAI-1)--the major inhibitor of fibrinolysis. The aim of the present study was to investigate a possible association between the -675_-674insG PAI-1 gene polymorphism and pediatric ischemic stroke. The study population consisted of 343 individuals: 70 children with ischemic stroke, 140 their biological parents and 133 control children. The PAI-1 gene polymorphism was genotyped using the restriction fragment length polymorphism and was visualized by AgNO₃ staining. The transmission/disequilibrium test showed exactly the same transmission of alleles from parents to the affected children (37:37). The case-control model also did not reveal any statistical significance in alleles and genotypes distribution between patients and control children. The obtained results suggest that the 4 G/5 G polymorphism of the PAI-I gene is not a risk factor of ischemic stroke in Polish children.

A-G-4G haplotype ofPAI-1gene polymorphisms −844 G/A,HindIIIG/C, and −675 4G/5G is associated with increased risk of ischemic stroke caused by small vessel disease

Plasminogen activator inhibitor type 1 (PAI-1) is the major inhibitor of fibrinolysis. It was reported that PAI-1 gene polymorphisms affected PAI-1 level and might therefore influence the risk of vascular diseases, including stroke. We studied the association of three common polymorphisms in PAI-1 gene (-844 G/A, -675 4G/5G, and HindIII G/C) with the odds of different causes of ischemic stroke. We studied 390 patients with ischemic stroke due to large vessel disease (n = 117), small vessel disease (n = 121), and cardioembolism (n = 152) as well as 291 controls. The etiology of ischemic stroke was established using TOAST criteria. PAI-1 polymorphisms were genotyped with restriction fragment length polymorphism and single strand conformation polymorphism method. A-G-4G haplotype of PAI-1 gene was found more frequently in stroke patients with small vessel disease than in control subjects (44.9% vs 35.7%; P = 0.02). No association was found between investigated genotype or allele frequencies and distinct causes of ischemic stroke. Our results demonstrate that A-G-4G PAI-1 gene haplotype is associated with increased risk of small vessel disease stroke, but this study does not support an association of -844 G/A, -675 4G/5G, and HindIII G/C PAI-1 gene polymorphisms with particular etiology of ischemic stroke.

Plasminogen activator inhibitor-1 is associated with coronary artery calcium in Type 1 diabetes

Background Elevated levels of plasminogen activator inhibitor-1 (PAI-1), the major inhibitor of fibrinolysis, is associated with coronary artery disease (CAD). This association may not be independent of factors related to insulin resistance (IR). Patients with Type 1 diabetes mellitus have increased CAD and an increase in sub-clinical CAD which develops earlier in life. It is not known if PAI-1 is associated with sub-clinical CAD in Type 1 diabetes or if this association is independent of IR. Methods and Results Type 1 diabetes patients ( n=560) and participants without diabetes ( n=693) were assessed for coronary artery calcium (CAC), a surrogate for subclinical CAD, by electron-beam computed tomography. PAI-1 was associated with CAC in both Type 1 diabetes (OR=1.32, 95% CI=1.12-1.58) and non-diabetes (OR=1.34, 95% CI=1.13–1.58), after controlling for traditional risk factors not associated with IR. In Type 1 diabetes, the relationship between PAI-1 and CAC was strongest for younger participants ( P=.02 for PAI-1-by-age interaction) after controlling for factors related to IR. PAI-1 was positively associated with CAC for Type 1 diabetes participants younger than 45 years of age. Conclusion PAI-1 levels are independently related to CAC in younger Type 1 diabetes participants. PAI-1 levels were not independently related to CAC in non-diabetes participants.

The 4G/5G polymorphism in the plasminogen activator inhibitor-1 gene is not associated with HELLP syndrome

Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of fibrinolysis, and a single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene has been identified. Subjects homozygous for the 4G allele have the highest PAI-levels due to increased PAI-1 gene transcription. Pre-eclampsia, and one of its most severe forms, the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, are characterized by increased placental thrombosis based on a procoagulatory state in the mother. Several studies have investigated the role of the PAI-1 4G/5G polymorphism in pre-eclampsia, but no study has focused especially on HELLP syndrome. Therefore we aimed to assess the association between HELLP syndrome and the 4G/5G polymorphism in the PAI-1 gene. Genotyping of the PAI-1 4G/5G promoter polymorphism was performed in 102 Caucasian women with HELLP syndrome and 102 Caucasian women with uncomplicated pregnancies. The 4G/4G genotype was more frequent in women with HELLP syndrome than in controls (35.3% vs. 22.5%, respectively) but this difference was not significantly different (P = 0.129). The frequency of the 4G allele was 0.588 in patients and 0.515 in controls. These data suggest that women carrying a 4G/4G genotype of the PAI-1 gene are not at increased risk for developing HELLP syndrome and are thus in line with the majority of previous studies on the association between the PAI-1 4G/5G polymorphism and pre-eclampsia.

The -675 4G/5G polymorphism at the Plasminogen Activator Inhibitor 1 (PAI-1) gene modulates plasma Plasminogen Activator Inhibitor 1 concentrations in response to dietary fat consumption

The objective of the study was to determine whether Plasminogen Activator Inhibitor Type 1 (PAI-1) -675 4G/5G polymorphism is associated with the response of functional plasma PAI-1 concentrations to changes in the amount and quality of dietary fat in healthy subjects. PAI-1 is the major inhibitor of fibrinolysis, and a lower level of fibrinolytic activity could be implicated in an increased risk of IHD. Fifty-nine healthy Spanish volunteers (ten 4G/4G homozygotes, twenty-eight heterozygotes 4G/5G and twenty-one 5G/5G homozygotes) consumed three diets for periods of 4 weeks each: a SFA-rich diet (38 % fat, 20 % SFA), followed by a carbohydrate-rich diet (30 % fat, 55 % carbohydrate) and a MUFA-rich diet (38 % fat, 22 % MUFA) according to a randomized crossover design. At the end of each dietary period plasma lipid and functional plasma PAI-1 concentrations were determined. Subjects carrying the 4G allele (4G/4G and 4G/5G) showed a significant decrease in PAI-1 concentrations after the MUFA diet, compared with the SFA-rich and carbohydrate-rich diets (genotype x diet interaction: P = 0.028). 5G/5G homozygotes had the lowest plasma PAI-1 concentrations compared with 4G/4G and 4G/5G subjects (genotype: P = 0.002), without any changes as a result of the amount and the quality of the dietary fat. In summary, no differences in plasma PAI-1 concentration response were found after changes in dietary fat intake in 5G/5G homozygotes, although these subjects displayed the lowest concentrations of PAI-1. On the other hand, carriers of the 4G allele are more likely to hyper-respond to the presence of MUFA in the diet because of a greater decrease in PAI-1 concentrations.

Plasminogen Activator Inhibitor Type-1-Deficient Mice Have an Enhanced IFN-γ Response to Lipopolysaccharide and Staphylococcal Enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.

Plasminogen-activator inhibitor-1 polymorphisms are associated with obesity and fat distribution in the Qu??bec Family Study: evidence of interactions with menopause

Obesity is associated with increased plasma levels of plasminogen-activator inhibitor-1 (PAI1), the major fibrinolysis inhibitor. PAI1 levels are also increased at menopause, a condition that is associated with fat mass gain, especially in the abdominal area. We hypothesized that genetic variations within PAI1 gene are related to the amount of body fat and its regional distribution. We genotyped 666 subjects of the Quebec Family Study for five PAI1 gene polymorphisms. Stratified analyses were performed with analysis of covariance in men (n = 280) and women (n = 386) separately. PAI1-675 4G/5G polymorphism was strongly associated with body mass index (P < or = 0.01) and fat mass (P < or = 0.05) in women. The PAI1-675 4G/5G promoter polymorphism and the c.43G<A (p.A15T, rs6092) variant within the exon 1 were associated with abdominal visceral fat but only in postmenopausal women (P < or = 0.05). More specifically, homozygotes for the -675 5G and the 43A alleles had about 50% more visceral fat compared to carriers of the -675 4G allele as well as carriers of the 43G allele. No association was observed in men. Taken together, these results suggest that the PAI1 gene is associated with obesity and may modulate the changes in adipose tissue distribution generally observed at menopause.

Purification of recombinant plasminogen activator inhibitor-1 in the active conformation by refolding from inclusion bodies

Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators. Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions. Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity. In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells. The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction. Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1. The purified protein yielded a single, 43 kDa protein band upon SDS–polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources. Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions. Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation.

The 4G/4G genotype of the 4G/5G polymorphism of the type-1 plasminogen activator inhibitor (PAI-1) gene is a determinant of penetrating behaviour in patients with Crohn's disease.

Crohn's disease is a heterogeneous disorder with polygenic inheritance. To assess the effect of the 4G/5G polymorphism of the type-1 plasminogen activator inhibitor (PAI-1) gene, the major inhibitor of fibrinolysis, on Crohn's disease susceptibility and phenotype. One hundred and fifty-seven patients with Crohn's disease and 350 controls were included prospectively. Medical records were reviewed to determine changes in the Crohn's disease phenotype. The 4G/5G polymorphism was assessed by polymerase chain reaction techniques. The frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes were similar in patients with Crohn's disease and controls. The 4G/4G genotype (P < 0.0001; odds ratio, 4.84) and male sex (P = 0.009; odds ratio, 2.63) were independent risk factors for penetrating behaviour in Crohn's disease. Most Crohn's disease patients had a non-penetrating phenotype at diagnosis. The probability of development of a penetrating phenotype within 5 years of diagnosis was higher in patients with the 4G/4G genotype (72% vs. 19%, P < 0.0001). The 4G/4G genotype of the PAI-1 gene does not influence Crohn's disease susceptibility, but increases by five-fold the probability of penetrating behaviour. Most patients with the 4G/4G genotype have a non-penetrating phenotype at diagnosis, but develop a penetrating behaviour within 5 years. Genotyping the 4G/5G polymorphism may be useful for the identification of a sub-group of patients with aggressive Crohn's disease, who might benefit from specific therapy.

A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.

Metformin therapy is associated with a decrease in plasma plasminogen activator inhibitor-1, lipoprotein(a), and immunoreactive insulin levels in patients with the polycystic ovary syndrome

Sixteen nondiabetic women with polycystic ovary syndrome (PCOS) aged 18 to 33 years were studied before and after 8 weeks on metformin (1.5 g/d) therapy to assess whether reducing hyperinsulinemia would reduce the levels of the major inhibitor of fibrinolysis, antigenic plasminogen activator inhibitor type 1 (PAI-1). Compared with six normal control women, PCOS women had a higher body mass index (BMI), waist to hip ratio, fasting insulin (I 0), insulin area under the curve during oral glucose tolerance testing (IA), glucose area under the curve during oral glucose tolerance testing (GA), IA GA ratio, PAI-1, luteinizing hormone (LH) and ratio of LH to follicle-stimulating hormone (FSH), and free testosterone, and lower high-density lipoprotein (HDL) cholesterol (all P < .025). On metformin, BMI decreased 1.3% ( P = .04), I 0 43% ( P = .002), IA 31% ( P = .03), GA 11% ( P = .02), PAI-1 16% ( P = .01), lipoprotein(a) [Lp(a)] 42% ( P = .004), free testosterone 46% ( P = .0006), LH 44% ( P = .004), and the LH FSH ratio 41% ( P = .0001). On metformin, absolute and percent reductions in I 0 correlated with absolute and percent reductions in PAI-1 ( r = .60, P = .015 and r = .64, P = .008). On metformin, by stepwise multiple regression, the absolute reduction in I 0 was a significant determinant of the absolute reduction in PAI-1 (partial R 2 = 35%, P = .02), and the percent reduction in I 0 was a significant determinant of the percent reduction in PAI-1 (partial R 2 = 52%, P = .003). Metformin decreases I 0 in hyperinsulinemic PCOS patients, reverses the hyperinsulinemia-driven endocrinopathy, decreases PAI-1, and decreases Lp(a), and should thus reduce the increased risk of atherothrombosis in PCOS.

Inheritance conjointly contributing to fibrinolysis and hyperlipidemia

Our specific aim was to assess within-family relationships of basal fibrinolytic activity and its determinants in hyperlipidemic probands (n = 34) with high lipoprotein (a) [Lp(a)] levels (>35 mg/dL) and their first-degree relatives (n = 74) and in hyperlipidemic probands (n = 19) with Lp(a) <35 and their first-degree relatives (n = 23). Probands' plasminogen activator inhibitor activity (PAI-Fx), the major fibrinolysis inhibitor, correlated with first-degree relatives' PAI-Fx in high-Lp(a) kindreds ( r = .30, P = .06) and in Lp(a) <35 kindreds ( r = .43, P ≤ .05). Probands' tissue plasminogen activator activity (tPA-Fx), the major fibrinolysis activator, was inversely associated with first-degree relatives' PAI-Fx in high-Lp(a) kindreds ( r = −.30, P = .06) and in Lp(a) <35 kindreds ( r = −.49, P ≤ .025). These correlations [irrespective of probands' Lp(a)] pointed to within-family heritability of the major fibrinolysis inhibitor, PAI-Fx, and the fibrinolysis stimulator, tPA-Fx. There were many other within-family correlations. High-Lp(a) probands' tPA-Fx, the stimulator of fibrinolysis, correlated with first-degree relatives' tPA-Fx ( r = .32, P ≤ .05). High-Lp(a) probands' plasminogen was inversely correlated with first-degree relatives' α 2-antiplasmin, a major fibrinolytic inhibitor ( r = −.41, P ≤ .01), and with their Lp(a) [ r = −.24, P ≤ .05]. High-Lp(a) probands' tPA-Fx correlated inversely with first-degree relatives' apolipoprotein (apo) B ( r = −.28) and triglyceride ([TG] r = −.41), and positively with their high-density lipoprotein cholesterol ([HDLC] r = .40) and apo A-1 ( r = .33; all P ≤ .025). High-Lp(a) probands' PAI-Fx correlated positively with first-degree relatives' apo B ( r = .34) and TG ( r = .47), and inversely with HDLC ( r = −.34) and apo A-1 ( r = −.30; all P ≤ .01). By stepwise regression, the Quetelet index (a measure of relative ponderosity) was independently inversely associated with tPA-Fx ( P ≤ .05) and positively associated with tPA-Ag and PAI-Fx ( P ≤ .05). TG was a positive independent determinant of PAI-Fx ( P ≤ .05), α 2-antiplasmin ( P ≤ .05), and plasminogen ( P ≤ .05). Lp(a) was a positive, independent determinant of fibrinogen ( P ≤ .05). Thus, relative ponderosity, high TG level, and high Lp(a) level, which would promote atherosclerosis, would simultaneously decrease fibrinolysis. In hyperlipidemic probands' kindreds with and without high Lp(a) levels, significant multigenerational within-family relationships of PAI-Fx, tPA-Fx, α 2-antiplasmin, TG, HDLC, apo B, apo A-1, and Lp(a) suggest that fibrinolytic activity and its determinants may be a heritable complex, which conjointly contributes to and promotes atherosclerosis and thrombosis.

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.

The Mutual Relationship Between the Two Molecular Forms of the Major Fibrinolysis Inhibitor Alpha-2-Antiplasmin in Blood

Alpha-2-antiplasmin, a major inhibitor of fibrinolysis, is synthesized in the liver and occurs in blood in two molecular forms: a very active plasminogen-binding (PB) form and a less active nonplasminogen-binding (NPB) form. This study investigates the origin and mutual relationship of these two forms in vivo and in vitro. Despite wide variation in plasma concentration of the inhibitor (16% to 138%), the ratio between the two forms in vivo was found to be, in the main, constant among healthy volunteers, heterozygotes for a congenital deficiency of alpha-2-antiplasmin, and patients with a stable liver cirrhosis: PB/NPB = 2.41 ± 0.34 (SD). Resynthesis after depletion or increased synthesis in the acute-phase reaction showed a specific increase of the PB form of the molecule in blood after discontinuation of L-asparaginase or streptokinase therapy and after myocardial infarction. In vitro studies demonstrated that only the PB form was present after one day in the culture medium of the human cell line Hep G2, while the NPB form appeared after 11 days. Clearance after inhibition of synthesis by L-asparaginase therapy revealed a more rapid decrease in the PB form relative to the NPB form in blood, demonstrated by a change in the PB-NPB ratio from 2.86 ± 0.55 to 1.74 ± 0.24 (mean of 6, SD). An apparently spontaneous first order conversion from the PB to NPB form, with an apparent half-life of about eight days, was demonstrated at 37 °C in plasma and serum in vitro. The conversion was found to be temperature dependent and uninfluenced by the fibrinolytic components fibrinogen, fibrin, and plasminogen. Additions of a variety of enzymes or inhibitors did not interfere with the process. These results demonstrate that the PB form of alpha-2-antiplasmin is produced by the liver and that the NPB form is formed in the circulation.

The mutual relationship between the two molecular forms of the major fibrinolysis inhibitor alpha-2-antiplasmin in blood

Alpha-2-antiplasmin, a major inhibitor of fibrinolysis, is synthesized in the liver and occurs in blood in two molecular forms: a very active plasminogen-binding (PB) form and a less active nonplasminogen-binding (NPB) form. This study investigates the origin and mutual relationship of these two forms in vivo and in vitro. Despite wide variation in plasma concentration of the inhibitor (16% to 138%), the ratio between the two forms in vivo was found to be, in the main, constant among healthy volunteers, heterozygotes for a congenital deficiency of alpha-2-antiplasmin, and patients with a stable liver cirrhosis: PB/NPB = 2.41 +/- 0.34 (SD). Resynthesis after depletion or increased synthesis in the acute-phase reaction showed a specific increase of the PB form of the molecule in blood after discontinuation of L-asparaginase or streptokinase therapy and after myocardial infarction. In vitro studies demonstrated that only the PB form was present after one day in the culture medium of the human cell line Hep G2, while the NPB form appeared after 11 days. Clearance after inhibition of synthesis by L-asparaginase therapy revealed a more rapid decrease in the PB form relative to the NPB form in blood, demonstrated by a change in the PB-NPB ratio from 2.86 +/- 0.55 to 1.74 +/- 0.24 (mean of 6, SD). An apparently spontaneous first order conversion from the PB to NPB form, with an apparent half-life of about eight days, was demonstrated at 37 degrees C in plasma and serum in vitro. The conversion was found to be temperature dependent and uninfluenced by the fibrinolytic components fibrinogen, fibrin, and plasminogen. Additions of a variety of enzymes or inhibitors did not interfere with the process. These results demonstrate that the PB form of alpha-2-antiplasmin is produced by the liver and that the NPB form is formed in the circulation.

INCREASED BLOOD VISCOSITY AND FIBRINOLYTIC INHIBITOR IN TYPE II HYPERLIPOPROTEINAEMIA

Blood rheology and several haemostatic factors were studied in patients with type II hyperlipoproteinaemia (HLP) and matched controls. HLP patients had increased blood viscosity (p<0·01), the mean level being 18% higher at a low shear-rate (0·94 s -1) and 13% higher at a high shear-rate (94 s -1). The increased viscosity was due partly to a raised haematocrit (p<0·05), and partly to increased plasma viscosity (p<0·01) associated with increased plasma fibrinogen (p<0·02). Red cell deformability was normal, and viscosity was unrelated to either lipid or lipoprotein concentrations. Levels of the major fibrinolytic inhibitor, α 2-antiplasmin, measured by both functional and immunological techniques were higher in HLP patients (mean increase 30-32%). Plasminogen activator levels were normal in HLP patients and the ratio of fibrinolytic inhibitor to activator was therefore increased. Plasminogen concentrations were also increased. Levels of factor VIII activity and antigen, antithrombin III and anti-factor Xa activity, α 2-macroglobulin, platelet count, and platelet aggregation by adenosine diphosphate and adrenaline did not differ significantly in HLP patients and controls. These results suggest that the premature arterial disease associated with HLP may be related to increased blood viscosity, which reduces arterial blood flow, and increased α 2-antiplasmin, the major inhibitor of fibrinolysis.