Plasminogen Concentration Research Articles

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

Fribrinolysis kinetics and its application

A new, convenient, and rapid method for kinetic measurement of human fibrinolysis was established. The alteration of absorbance (A) in the process of blood coagulation and lyses was automatically scanned and recorded using a UV2000 spectrophotometer connected to a computer. The parameters of human fibrinolysis kinetics were established. Urokinase at 20 U/mL was the optimal concentration used. There was significant difference in fibrinolysis kinetics and plasma plasminogen concentration between 22 normal subjects and 27 patients with acute myeloblastic leukemia (P<0.05 and <0.01 respectively). The coefficience of variation was (5.24+/-1.51)%. This method could also be used to measure the plasma fibrinogen concentration at the same time. It was concluded that this method was stable and was capable of providing dynamic, direct experimental data and multiparemeters for clinicians. It was also valuable in evaluating the anti-and pro-fibrinolytic capcity of patients' plasmas, allowing for monitoring of therapy, choice of drugs and adjustment of drug concentrations.

The Plasminogen-Binding Group A Streptococcal M Protein-Related Protein Prp Binds Plasminogen via Arginine and Histidine Residues

The migration of the human pathogen Streptococcus pyogenes (group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the plasminogen-binding group A streptococcal M protein (PAM)-related protein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 microM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (K(d) = 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys(96) and Lys(101) reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg(107) and His(108) to alanine. Furthermore, mutagenesis of Arg(107) and His(108) abolished plasminogen binding by Prp despite the presence of Lys(96) and Lys(101) in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.

The Maintenance of High Affinity Plasminogen Binding by Group A Streptococcal Plasminogen-binding M-like Protein Is Mediated by Arginine and Histidine Residues within the a1 and a2 Repeat Domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.

Plasminogen inhibits TNFα-induced apoptosis in monocytes

AbstractMonocytes are major mediators of inflammation, and apoptosis provides a mechanism for regulating the inflammatory response by eliminating activated macrophages. Furthermore, as a consequence of apoptosis, plasminogen binding is markedly increased on monocytoid cells. Therefore, we investigated the ability of plasminogen to modulate monocyte apoptosis. Apoptosis of monocytoid cells (human monocytes and U937 cells) was induced with either TNFα or cycloheximide. When apoptosis was induced in the presence of increasing concentrations of plasminogen, apoptosis was inhibited in a dose-dependent manner with full inhibition achieved at 2 μM plasminogen. Plasminogen treatment also markedly reduced internucleosomal DNA fragmentation and reduced levels of active caspase 3, caspase 8, and caspase 9 induced by TNFα or by cycloheximide. We examined the requirement for plasmin proteolytic activity in the cytoprotective function of plasminogen. A plasminogen active site mutant, [D(646)E]-Plg, failed to recapitulate the cytoprotective effect of wild-type plasminogen. Furthermore, antibodies against PAR1 blocked the antiapoptotic effect of plasminogen. Our results suggest that plasminogen inhibits monocyte apoptosis. The cytoprotective effect of plasminogen requires plasmin proteolytic activity and requires PAR1. Because apoptosis of monocytes plays a key role in inflammation and atherosclerosis, these results provide insight into a novel role of plasminogen in these processes.

The Influence of Age on in vitro Plasmin Generation in the Presence of Fibrin Monomer

Background and Objectives: The components of the fibrinolytic system interact to generate plasmin from its zymogen form, plasminogen. At birth, all the components of the fibrinolytic system are present but with differing plasma concentrations. The present study was undertaken to explore the effect of physiological, age-dependent factors of the fibrinolytic system during childhood on the capacity to generate plasmin. Design and Methods: Total plasmin generation was measured in venous plasma from umbilical cords and adults, on plastic and cell surfaces, in the presence of fibrin monomer, Desafib. Plasminogen, its inhibitors α₂-antiplasmin and plasminogen activator inhibitor type 1, and plasmin-α₂-antiplasmin complex in the time samples were assayed by enzyme-linked immunosorbent assay. The effect of addition of plasminogen on the plasmin generation in cord plasma and the effect of lipoprotein on adult and cord plasmin generation were measured. Results: On the surface of human umbilical vein endothelial cells, onset of plasmin generation was earlier (40 min) compared to plastic (60 min) but total plasmin generation was similar on both surfaces. The addition of plasminogen to cord plasma increased plasmin generation. Supplementation of lipoprotein in adult plasma had an inhibitory effect, but there was no significant effect in cord plasma. Interpretations and Conclusions: Plasmin generation is reduced in newborn compared to adult plasma. Decreased plasmin generation in cord plasma is likely due to decreased plasminogen concentration. The antifibrinolytic effect of lipoprotein is more pronounced in adults as compared to newborns due to the presence of higher plasminogen concentration.

Fluorescent Labeling Study of Plasminogen Concentration and Location in Simulated Bovine Milk Systems

A fluorescent labeling method was developed to study plasminogen (PG) concentration and location in simulated bovine milk. Activity and stability of PG labeled with Alexa Fluor 594 (PG-594) were comparable to those of native PG. The fluorescent signal of PG-594 exhibited pH, temperature, and storage stability, and remained stable throughout typical sample treatments (stirring, heating, and ultracentrifugation). These characteristics indicate broad applicability of the fluorescent labeling technique for milk protease characterization. In an example application, PG-594 was added to simulated milk samples to study effects of heat and β-lactoglobulin (β-LG) on the distribution of PG. Before heating, about one-third of the PG-594 remained soluble in the whey fraction (supernatant) whereas the rest became associated with the casein micelle. Addition of β-LG to the system slightly shifted PG-594 distribution toward the whey fraction. Heat-induced PG-594 binding to micelles in whey-protein-free systems was evidenced by a decrease of PG-594 from 31 to 15% in the whey fraction accompanied by an increase of PG-594 from 69 to 85% in casein micelle fractions. When β-LG was present during heating, more than 95% of PG-594 became associated with the micelle. A comparison with the distribution pattern of PG-derived activities revealed that heat-induced PG binding to micelles accompanies heat-induced PG inactivation in the micelle fraction. Incubation of the casein micelles with the reducing agent β-mercaptoethanol revealed that disulfide bonds formed between PG and casein or between PG and casein-bound β-LG are the mechanisms for heat-induced PG binding to casein micelles. Western blotting and zymography results correlated well with fluorescent labeling studies and activity studies, respectively. Theoretically important findings are: 1) when heated, serum PG is capable of covalently binding to micellar casein or complexing with β-LG in whey and then coadhering to micelles, and 2) PG that associated with micellar casein through lysine binding sites before heating is capable of developing heat-induced disulfide bonds with casein. The overall results are PG covalently binding to micelles and inactivation thereafter. Our results suggest that, instead of thermal denaturation through irreversible unfolding, covalent bond formation between PG and other milk proteins is the mechanism of PG inhibition during thermal processing.

Plasminogen activator inhibitor-1 impairs plasminogen activationmediated vascular smooth muscle cell apoptosis

The role of plasminogen activator inhibitor-1 (PAI-1) in vascular smooth muscle cell (VSMC) apoptosis mediated by plasminogen activation was studied with the use of aorticVSMC derived from mice with deficiency of PAI-1 (PAI-1 (-/-) ), tissue-type (t-PA (-/-) ) or urokinase-type (u-PA (-/-) ) plasminogen activator or from wildtype (WT) mice with corresponding genetic background. Plasminogen incubated with confluent VSMC was activated in a concentration-dependent and saturable manner for all four cell types, with maximal activation rates that were comparable for WT, u-PA (-/-) and t-PA (-/-) cells, but about two-fold higher for PAI-1 (-/-) cells. Plasminogen activation was impaired by addition of the lysine analogue 6-aminohexanoic acid, and by addition of t-PA and u-PA neutralizing antibodies, suggesting that it depends on binding to cell surface COOH-terminal lysine residues, and on plasminogen activator activity. Morphological alterations consistent with apoptosis were observed much earlier in PAI-1 (-/-) than in WT VSMC. Without addition of plasminogen, the apoptotic index was similar for all four cell types, whereas after incubation with physiological plasminogen concentrations, it was greater in PAI-1 (-/-) VSMC, as compared to WT, t-PA (-/-) or u-PA (-/-) VSMC. Furthermore, the apoptotic rate paralleled the release of plasmin. Thus, plasmin-mediated apoptosis of VSMC occurs via plasminogen activation by either t-PA or u-PA and is impaired by PAI-1.

The effect of 40 kHz ultrasound on tissue plasminogen activator-induced clot lysis in three in vitro models

In previous work from the same laboratory, high-frequency ultrasound (US) (3 MHz) was shown to promote in vitro fibrinolysis through enhanced supply of plasminogen to the clot surface. The application of high-frequency US is limited in vivo due to tissue heating. Low-frequency US, however, has less tissue heating and improved penetration. Internal plasma clot lysis and external lysis with compacted and non-compacted plasma clots were used to determine the magnitude of the effect of low-frequency US (40 kHz; 0.5 W/cm2) on tissue plasminogen activator-induced lysis and to elucidate the mechanisms behind the effect. Ultrasound enhanced lysis in all three models, with the largest effects (4-fold) in the external lysis model with compacted plasminogen-poor clots. The acceleration effect of ultrasound in this model decreased with increasing t-PA—and decreasing plasminogen concentrations. Ultrasound had a much smaller effect in this model when compacted plasminogen-rich clots were used. In the external lysis, non-compacted clot model, ultrasound resulted in consistently higher lysis rates. The acceleration effect of lysis, increased slightly (1.3 to 1.8-fold) with increasing t-PA—and decreasing plasminogen concentrations. Plasminogen supply to the clot surface was again shown to be an important contributor to ultrasound-enhanced lysis. [M. Pieters, R. T. Hekkenberg, M. Barrett-Bergshoeff, and D. C. Rijken. Ultrasound in Med &amp; Biol 30, 1545–1552 (2004).]

Plasminogen interaction with platelets: The importance of carboxyterminal lysines

Introduction Thrombin stimulation enhances plasminogen binding to platelets and promotes platelet-dependent plasmin generation. The objective of this study was to determine whether carboxyterminal lysines (C-lysines) are important for these processes, as they are in other cell types. Materials and methods 125I-plasminogen and varying concentrations of unlabeled plasminogen were added to washed platelets that were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. In some experiments the platelets were digested with carboxypeptidase B to remove C-lysines. Platelet-dependent plasmin generation was also studied by adding plasminogen and tissue plasminogen activator to platelet suspensions and monitoring the conversion of a plasmin specific chromogenic substrate. The cells were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. The effect of the thrombin inhibitor lepirudin and the plasmin inhibitor aprotinin on plasminogen binding and the appearance of C-lysines was also investigated. Results Thrombin, but not thrombin receptor activating peptide or ADP, stimulated high-affinity binding of plasminogen and greatly promoted platelet-dependent plasmin generation. Digestion with carboxypeptidase B eliminated thrombin-induced high-affinity binding and reduced thrombin-induced plasmin generation by increasing the Michaelis constant. Lepirudin, but not aprotinin, inhibited thrombin-stimulated plasminogen binding to platelets. Conclusion C-terminal lysines are necessary for high-affinity binding of plasminogen to platelets and for platelet-supported plasmin generation. The origin of the C-lysines is not clear, but they may result from a direct effect of thrombin, rather than an intermediate enzyme such as plasmin.

Plasminogen activities and concentrations in patients with sporadic Creutzfeldt–Jakob disease

Human plasminogen has been shown to interact with the abnormal disease-specific prion protein. Till now, no data are available for patients with Creutzfeldt–Jakob disease (CJD). Therefore, we compared plasminogen concentrations and plasminogen activities in patients with sporadic CJD and controls with other dementia, which were collected in the framework of the German CJD Surveillance study. Patients with CJD had significantly higher plasminogen concentrations than patients with other forms of dementia and plasminogen specific activities were lower in CJD patients. The reasons for these abnormalities are not clear at the moment. The results may reflect a disease-specific prion protein and plasminogen interaction in patients with CJD. Other possible explanations are plasminogen polymorphisms and genotypes with distinct plasminogen activity levels in CJD than in controls, which should be a subject for further studies.

Inhibition of Plasminogen Activation by Lipoprotein(a): CRITICAL DOMAINS IN APOLIPOPROTEIN(a) AND MECHANISM OF INHIBITION ON FIBRIN AND DEGRADED FIBRIN SURFACES

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).

Measurement of plasminogen concentration and differentiation of plasmin and plasminogen using Fourier-transform infrared spectroscopy

Abstract Methods using Fourier-transform infrared (FT-IR) spectroscopy in combination with multivariate statistical analysis were developed to determine the plasminogen concentration in various protein solutions and to differentiate between plasmin and plasminogen. FT-IR spectra of plasminogen and plasmin in water and solutions containing sodium caseinate, whey protein concentrate, and a mixture of β -lactoglobulin and sodium caseinate were obtained using attenuated total reflectance (ATR) FT-IR. Discriminant analysis and partial least-squares analysis were used to analyze data. Standard curves to determine plasminogen and plasmin concentrations in water and protein solutions were constructed successfully using PLS ( R 2 >0.9). FT-IR combined with discriminant analysis was also able to differentiate between plasminogen and plasmin. Developed methods will allow further study of the factors that influence plasminogen and plasmin in the presence of casein and whey proteins.

The fibrinolytic system in children.

The fibrinolytic system comprises a cascade of serine proteinase activation events that culminate in the generation of plasmin and, subsequently, degradation of fibrin. Although all components of the fibrinolytic system are present at birth, plasma concentrations of key components in infants and children differ physiologically from those in adults. Until the age of 6 months, plasma concentrations of plasminogen and alpha (2)-antiplasmin are decreased to 50% and 80% of adult values, respectively, while plasma concentrations of tissue-type plasminogen activator are decreased and plasminogen activator inhibitor-1 are increased throughout childhood. In addition, the rate of plasmin generation in newborns and the overall fibrinolytic activity during childhood are decreased compared with adults. Strong evidence suggests that age-dependent differences in the fibrinolytic system critically influence the effectiveness and safety of thrombolytic agents. In addition, recent studies suggest that impaired fibrinolysis may play an important role in the pathogenesis of several diseases such as venous and arterial diseases and vasculitis, which are associated with both endothelial cell damage and increased thrombotic risk. This article will discuss the ontogenic features of the fibrinolytic system in children and summarize the available information on the effect of developmental fibrinolysis on both the course of specific disease states and the response to thrombolytic therapy in children.

Plasminogen and tissue-type plasminogen activator deficiency as risk factors for thromboembolic disease.

To review the published evidence for an association between a deficiency of plasminogen or tissue-type plasminogen activator (tPA) and the risk of thrombosis. Review of the medical literature, with an emphasis on the last 10 years. After an initial assessment of the literature, including review of clinical study design and laboratory methods, a draft manuscript summarizing the findings was prepared and circulated to participants in the College of American Pathologists Conference on Diagnostic Issues in Thrombophilia. The key findings and each recommendation were presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form. The consensus of the conference was that routine laboratory assessment of plasminogen and tPA concentration in patients with thrombophilia is not warranted at this time. Analysis of plasminogen and tPA gene alterations in patients with thrombophilia is also not warranted at this time. Determination of plasminogen concentration should be performed in patients suspected of having ligneous conjunctivitis.

The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion.

Chromaffin cells express components of the plasminogen/plasmin system, including its major activator, tissue plasminogen activator (t-PA), and high-affinity cellular receptors for plasminogen, which promote local concentration and activation of plasminogen at the cell surface. Our studies suggest that plasmin participates in local neuroendocrine prohormone processing and that perturbation of this system profoundly affects the secretory characteristics of the cells. These results suggest the presence of a local, functionally active, chromaffin cell plasminogen/plasmin system that plays a major role in the regulation of catecholamine release from catecholaminergic cells.

Differential regulation of collagen degradation by rabbit keratocytes and polymorphonuclear leukocytes

Purpose. Both activated keratocytes and infiltrated polymor-phonuclear leukocytes (PMNs) contribute to corneal ulceration by degrading stromal collagen. The regulation of such collagen degradation by inflammatory cytokines was investigated with rabbit keratocytes and PMNs cultured in three-dimensional collagen gels. Methods. Rabbit keratocytes or PMNs were cultured for 24 h in three-dimensional gels of type I collagen in the presence of plasminogen and various concentrations of either interleukin (IL)-1a, IL-6, IL-8, or tumor necrosis factor-a (TNF-a). Degradation of collagen during culture was assessed by measurement of released hydroxyproline. Results. IL-1a increased the amount of collagen degraded by keratocytes or PMNs in a dose-dependent manner, whereas IL-6 had no effect on collagen degradation by either cell type. IL-8 increased the extent of collagen degradation by PMNs but not that by keratocytes, and TNF-a promoted collagen degradation by keratocytes but not that by PMNs. Conclusion. Inflammatory cytokines regulate collagen degradation by rabbit keratocytes and PMNs in culture in a differential manner, and therefore may contribute to the roles of these cells in corneal ulceration.

Activation of coagulation and fibrinolysis during coronary surgery: on-pump versus off-pump techniques.

The authors studied the changes in selected hemostatic variables in patients undergoing coronary surgery with on-pump coronary artery bypass grafting (CABG) or off-pump coronary artery bypass surgery (OPCAB) techniques. Platelet counts and plasma concentrations of antithrombin, fibrinogen, D dimer, alpha(2) antiplasmin, and plasminogen were measured preoperatively, 5 min after administration of heparin, 10 min after arrival in the intensive care unit, and 24 h after surgery in patients scheduled to undergo OPCAB (n = 15) or CABG (n = 15). To correct for dilution, hemostatic variables and platelet counts were adjusted for the changes in immunoglobulin G plasma concentrations and hematocrit, respectively. Adjusting for dilution, antithrombin and fibrinogen concentrations decreased to a similar extent in patients undergoing OPCAB or CABG (pooled means and 95% confidence limits of the mean: 95.5% of baseline, 93-98%, P = 0.002, and 91.7% of baseline, 88-95%, P = 0.0001), respectively, whereas alpha(2)-antiplasmin concentrations were unchanged. Only CABG was associated with a reduction in platelet counts (76% of baseline, 66-85%, P = 0.0001), plasminogen concentrations (96% of baseline, 91-99%, P = 0.011), and increased D-dimer formation (476%, 309-741%, P = 0.004). Twenty-four hours after surgery, platelet counts were still lower in patients undergoing CABG (P = 0.049), but all the investigated variables adjusted for dilution were similar in the two groups. Coronary surgery causes a net consumption of antithrombin and fibrinogen. A transient decrease in platelet counts, with plasminogen activation and increased D-dimer formation, however, is only observed with CABG. Twenty-four hours after surgery, the hemostatic profiles of patients in both groups are similar.

Basic Principles in Thrombolysis: Regulatory Role of Plasminogen

During thrombolytic therapy, patients are treated with a plasminogen activator in order to stimulate the fibrinolytic system by converting the precursor plasminogen into the active enzyme plasmin. The fibrinolytic process can be divided into two phases. In the first phase, plasminogen binds to intact fibrin and initial fibrinolysis takes place. As a result, carboxyterminal lysine residues are generated, which represent new binding sites for plasminogen. In the second phase, plasminogen binds to these sites and fibrinolysis is accelerated because the local plasminogen concentration is strongly enhanced and because this plasminogen has a higher reactivity. For instance, both single-chain urokinase-type plasminogen activator (scu-PA) and staphylokinase have a high preference for this type of plasminogen, which explains their fibrin-selective action. A recently discovered thrombin-activatable fibrinolysis inhibitor (TAFI) eliminates carboxyterminal lysine residues from partially degraded fibrin and, thus, inhibits the second phase of fibrinolysis. These mechanisms show that plasminogen plays an important regulatory role in fibrinolysis and thrombolysis.

Enzyme-Linked Immunosorbent Assay for the Specific Detection of Angiostatin-Like Plasminogen Moieties in Biological Samples

An enzyme-linked immunosorbent assay (ELISA) was developed for the specific detection of human angiostatin-like plasminogen moieties (comprising kringles 1–4) in biological samples. The assay involves prior removal of all other plasminogen moieties by immunoadsorption of diluted samples (to about 10 ng/ml plasminogen) with a mixture of insolubilized MA-42B12 (directed against kringle 5) and MA-31E9 (directed against the proteinase domain). The recovery of angiostatin during this procedure is ≥95%. Subsequently, angiostatin-like fragments are detected in an ELISA, based on two monoclonal antibodies reacting with nonoverlapping epitopes in the kringle 1–3 domain: MA-36E6 for capture and MA-34D3 for tagging. The assay has a lower detection limit of about 0.1 ng/ml and is performed with intra- and interassay coefficient of variation of 2.4% and 15%. In tumor fluids obtained from cancer patients ( n=10), angiostatin levels ranged between 0.24 and 6.7 μg/ml (1.62±0.60 μg/ml; mean±S.E.M.) The identity of angiostatin was confirmed by immunoblotting using specific monoclonal antibodies. A weak correlation ( r=.66) was observed with the total plasminogen concentration in these samples. This ELISA thus appears suitable for the specific quantitation of angiostatin-like plasminogen moieties in biological samples, and may be useful to study its (patho)physiological relevance.