Plasminogen Concentration Research Articles

Microparticle‐enhanced nephelometric immunoassay of plasminogen in bovine milk

A sensitive microparticle‐enhanced nephelometric immunoassay of bovine plasminogen (detection limit in reaction mixture, 4 fig l‐1) is described. It is based on nephelometric measurement of the light scattered by microparticle clusters formed during a sandwich assay that uses microparticle‐anti‐plasminogen conjugate and free antibodies from antiplasminogen rabbit antiserum. This immunoassay was performed in 200‐fold diluted bovine milk, after release of plasminogen from caseins by ε‐amino‐n‐caproic acid treatment and elimination of the caseins. It allowed the quantification of plasminogen in milk for a wide range of concentrations (0.9–14.4 mg l‐1), with accuracy (linear recovery in overloading assays) and precision (within‐ and between‐run coefficients of variation lower than 5%). When it was applied to 106 herd milk samples, this microparticle‐enhanced nephelometric immunoassay yielded milk plasminogen concentrations ranging between 1.1 and 4.9 mg l‐1, with a mean of 2.0 mg l‐1, and disclosed possible seasonal variations.

Interactions between insulin-like growth factor-I (IGF-I) and the system of plasminogen activators and their inhibitors in the control of IGF-binding protein-3 production and proteolysis in human osteosarcoma cells

Limited proteolysis in vivo of insulin-like growth factor-binding protein-3 (IGFBP-3) by as yet unidentified serine proteases plays a key role in controlling the bioavailability of IGFBP-3-associated insulin-like growth factors (IGFs). Both the IGF system and the system of plasminogen activators (PAs) and their inhibitors (PAIs) are involved in bone remodeling, and plasmin has been shown to provoke dissociation of IGFBP-IGF complexes in cultured MG-63 human osteoblasts. The aim of this work was to investigate interactions between IGF-I and the PA/PAI system and their influence on IGFBP-3 production and proteolysis in this cell model. At confluency, MG-63 cells maintained for 3 days in serum-free medium constitutively secreted IGFBP-2 and small amounts of IGFBP-3 and IGFBP-4. As shown by Western ligand and immunoblot analyses of the culture medium and Northern blot analysis of IGFBP-3 messenger RNA, production of these IGFBPs, and of IGFBP-3 in particular, was dose dependently stimulated by the addition of 12.5-100 ng/ml recombinant human (rh) IGF-I. Increasing concentrations of plasminogen (0.05-3.5 micrograms/ml) added during the final 12 h of culture reduced the amounts of IGFBP detectable by Western ligand blotting, especially IGFBP-3. This reduction reflected proteolysis, as shown by immunoblotting, which revealed 30-, 20-, and 16-kilodalton fragments of IGFBP-3. In the presence of 25 ng/ml IGF-I, which stimulated IGFBP-3 production, proteolysis was reduced by approximately half. Incubation of glycosylated [125I]rh-IGFBP-3 as substrate in cell-free conditioned medium gave the same results. Addition of 50 ng/ml rhIGF-I to conditioned medium (to promote IGFBP-3-rhIGF-I complex formation) failed to diminish plasmin-induced proteolysis of IGFBP-3. Urokinase PA activity in the conditioned medium decreased significantly when the cells were cultured with rhIGF-I, indicating a direct action of IGF-I on urokinase PA and/or PAI production. Our results support the notion of a regulation loop whereby IGF-I controls its own bioavailability via its action on both IGFBP-3 production and the PA/PAI system, which regulates IGFBP-3 proteolysis. The proteolytic cleavages of IGFBP-3 caused by plasmin were the same as those caused in vivo by serine protease acting on this IGFBP.

Quantitative Characterization of Bovine Plasminogen Binding to Caseins

The binding of bovine plasminogen to whole casein, α s-casein, β-casein, and κ-casein is responsible for the progressive proteolysis of milk and dairy products. A sensitive and accurate microparticle-enhanced nephelometric immunoassay was developed to measure free plasminogen after interaction between bovine plasminogen and caseins and the quantitative parameters of plasminogen/casein binding were established. Two classes of binding sites for plasminogen were found in this study on each of the investigated caseins. Their dissociation constants ( K d ) were determined by varying the plasminogen concentration at pH 6.6 and performing Scatchard analysis. The two binding sites appeared to be one of high affinity ( K d = 32 nM) and the other of lower affinity ( K d > 370 nM). The number of both binding sites per casein monomer was low (0.04 to 0.53). The great propensity of casein monomers to self-associate in homopolymers where plasminogen binding sites could be hidden and in copolymers present in bovine milk in the form of whole casein micelles accounts for calculated binding sites <1 per monomer.

Kinetics of activation of variant plasminogens in a noncrosslinked fibrin clot: Abnormal plasmin generation with the plasminogen.Streptokinase complex, urokinase and tissue plasminogen activator

A purified clot lysis system (noncrosslinked) was developed to evaluate and study variant plasminogens from patients with a history of thrombophilia. Plasmin generation was studied in the soluble phase and in the clot lysis system with different activators, streptokinase and its derivatives, urokinase and tissue plasminogen activator. Five of the variant plasminogens (Dl, D2, K, S, and F) generated low levels of plasmin in the soluble phase, whereas two (M and Y) generated near normal levels of plasmin. Plasmin generation in the clot lysis system divided the variants into three groups: one variant (Dl) had a very prolonged lysis time or a negligible plasmin generation rate with three direct activators, three variants (D2, K and S) had moderately prolonged lysis times or low plasmin generation rates with these activators, and three variants (F, M, and Y) had normal lysis times and normal plasmin generation rates with these activators. Kinetics of activation parameters with three direct plasminogen activators were determined from a non-linear regression analysis of plots of velocity versus plasminogen concentration and were verified by Lineweaver-Buck plots where possible. With plasminogen.streptokinase, the four variants with prolonged lysis times, had normal apparent Michaelis constants and low catalytic rate constants. Three variants with normal lysis times had high apparent Michaelis constants showing decreased affinity for the plasminogen.streptokinase activator complex, and high catalytic rate constants. With urokinase, the variant with the longest clot lysis time had a high apparent Michaelis constant and a low catalytic rate constant, three variants with prolonged lysis times gave low catalytic rate constants; two of them gave normal apparent Michaelis constants and one gave a high apparent Michaelis constant. Of the three variants with normal lysis times, one gave normal kinetic constants and two had high apparent Michaelis constants with high catalytic rate constants. With tissue plasminogen activator, the variant with the longest clot lysis time gave a high apparent Michaelis constant and a high catalytic rate constant. The three variants with prolonged lysis times gave variable results, one had a slightly higher apparent Michaelis constant with a low catalytic rate constant, the second one had a very low apparent Michaelis constant with a low catalytic rate constant and the third one had a high apparent Michaelis constant with a normal catalytic rate constant. Three variants with normal lysis times had slightly higher Michaelis constants and near normal catalytic rate constants. With the noncrosslinked fibrin substrate, all of the variant plasminogens have lowered catalytic efficiencies with two or three plasminogen activators. The theoretical aspects of these plasmin generation data are discussed.

Fibrinolysis potentiating activity following endothelial cells-platelet interaction.

When porcine endothelial cells in culture are incubated in the presence of human platelets, a 90kDa neutral proteinase activity is generated on casein gel (PECAP-Platelet Endothelial Cell Activated Protease). This activity was undetected when platelet extract or serum free EC conditioned medium were analysed under similar conditions. The optimum pH, isoelectric point, molecular weight and inhibitory profile of this activity were similar to Glu-plasmin. However, the low plasminogen content (less than 50ng/ml) in the conditioned medium of endothelial cells incubated with platelet could not contribute alone to this activity and the presence of a plasmin potentiating factor was suggested. This factor was separated from plasminogen by lysine-Sepharose chromatography.

Relationship of fibrinolysis and platelet function to bleeding after cardiopulmonary bypass.

In order to study the effects of cardiopulmonary bypass (CPB) on fibrinolysis and platelet function and the possible relationship of these effects on post-operative blood loss, 127 patients undergoing CPB were examined. There was a significant reduction in the median levels of fibrinogen, plasminogen, alpha 2-antiplasmin, fibrinolytic potential and platelet aggregation during CPB (P < or = 0.001). Median levels of soluble fibrin, fibrinogen degradation products, D-dimer and PAI-1 were increased, while the level of t-PA activity remained constant. Post-CPB levels of fibrinogen and plasminogen correlated negatively with blood loss (P = 0.003 and P < 0.001, respectively) and interestingly, lower levels of alpha 2-antiplasmin and higher levels of t-PA activity before CPB were associated with greater blood loss after CPB (P < 0.001 and P = 0.004, respectively). Better pre-CPB platelet function correlated with lower levels of D-dimer before and after CPB. As expected, haemodilution had a significant effect on fibrinolytic and coagulation parameters post-CPB; the greater the haemodilution, the more the concentration of fibrinogen, plasminogen and alpha 2-antiplasmin fell post-CPB and the greater the blood loss. The increase in PAI-1 levels intra-CPB appeared to result in mean t-PA activity remaining unchanged 1 h post-CPB. Post-CPB increases in soluble fibrin were paralleled by increases in fibrinogen degradation products and D-dimer, suggesting that intra-operative contact activation is related to activation of the fibrinolytic system. The present findings indicate the greater the fibrinolytic activation, the greater the post-CPB blood loss.

An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation.

In the preceding paper (Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197), we identified a M(r) = 40,000 endothelial cell receptor for tissue plasminogen activator (t-PA) and plasminogen (PLG) as the calcium- and phospholipid-binding protein, annexin II (Ann-II). Here, we examined the effect of Ann-II on t-PA-dependent plasminogen activation in a purified system. Purified native Ann-II bound t-PA, plasminogen, and plasmin with high affinity (Kd = 25 nM, 161 nM, and 75 nM, respectively). At fixed plasminogen concentrations, preincubation with purified native Ann-II was associated with an approximately 21-fold increase in the rate of Glu-PLG activation and an approximately 14-fold increase in activation of Lys-PLG. Three irrelevant proteins had no effect on plasmin formation, while fibrinogen increased the rate of Glu-PLG activation by approximately 4-fold. Annexin-II-mediated enhancement of t-PA-dependent plasminogen activation was 90-95% inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B, indicating a carboxyl-terminal lysine-dependent interaction. Kinetic analyses revealed that Ann-II conferred an approximately 60-fold increase in catalytic efficiency upon t-PA-dependent activation of either Glu-PLG or Lys-PLG. Thus, Ann-II-mediated assembly of plasminogen and t-PA may promote and localize constitutive plasmin generation on the surface of the blood vessel wall.

Binding of tissue-type plasminogen activator (t-PA) toNeisseria meningitidisandHaemophilus influenzae

Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20-46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10,000 receptors per bacterium for t-PA with a Kd value of about 20 nmol l-1. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l-1. t-PA binding could be reduced about 40% by the addition of 10 mmol l-1 of the lysine analogue epsilon-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 mumol l-1 of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.

Influence of intranasal cocaine on plasma constituents associated with endogenous thrombosis and thrombolysis

purpose: As cocaine abuse has become widespread, catastrophic cocaine-associated cardiovascular events have been noted with increasing frequency. Although these incidents are thought to be caused by drug-induced vasoconstriction and/or arterial thrombosis, the influence of cocaine on the plasma constituents involved in endogenous thrombosis and thrombolysis has not been characterized. patients and methods: In 22 patients (8 men, 14 women, ages 32 to 62 years) undergoing cardiac catheterization, blood samples were procured before and 15 minutes after the administration of intranasal saline (n = 8, controls) or cocaine, 2 mg/kg (n = 14), and the plasma concentrations of fibrinogen, plasminogen, and lipoprotein(a), as well as tissue plasminogen activator activity and plasminogen activator inhibitor (PAI-1) activity, were measured. results: No variable changed with the use of intranasal saline, whereas the use of cocaine resulted in an increase in PAI-1 activity (0.48 + 0.06 [mean + SD] nmol/L at baseline, 0.53 + 0.05 nmol/L after cocaine, P = 0.011). conclusion: Intranasal cocaine administration is associated with an increase in plasma PAI-1 activity. This may be important in recreational users of cocaine who experience vascular thrombosis.

Artificial ultraviolet whole-body radiation does not modify serum lipoprotein, plasma fibrinogen, plasminogen or antithrombin III concentrations in post-myocardial infarction patients

The relationship of ischaemic heart disease (IHD) to seasonal and latitude variation has prompted speculation that exposure to the ultraviolet component of solar radiation may reduce IHD risk. This hypothesis was partially tested by exposing 14 post-myocardial infarction patients to a 6 week course of artificial whole-body ultraviolet radiation (UVR). Serum lipoprotein and plasma coagulation factor concentrations were measured before and after the course of UVR. Results were compared with similar measurements from a placebo-controlled group of 13 post-myocardial patients. Despite a more than two-fold rise in mean serum 25-OHD, serum lipoprotein and plasma fibrinogen, anti-thrombin III and plasminogen concentrations did not change significantly in the UVR group. Significant but minor change in prothrombin time and thrombin time in the placebo group appear unlikely to be of biological significance. Seasonal and latitude variation in these IHD risk factors appear unrelated to corresponding variation in solar UVR exposure.

Protein C, antithrombin III and plasminogen: effect of age, sex and blood group

We conducted a cross-sectional study of antithrombin III (ATIII), protein C (PC) and plasminogen (Plg) concentrations in a population of healthy plasma donors in the Trent Region. The distribution of values for protein C was log normal whereas for ATIII and Plg the distributions were positively skewed and differed significantly from normal and log normal. Males had higher antithrombin III concentrations (mean 1.10 iu/ml, range 0.72-1.65) than females (mean 1.07 iu/ml, range 0.75-1.69) (P = 0.001) and levels increased with age in women. Younger women aged 25-34 had significantly lower plasma concentrations of ATIII compared to males of similar age. For protein C, concentrations were higher in males (mean 1.07 u/ml, range 0.37-2.11) than in females (mean 1.01 u/ml, range 0.59-1.61) (P < 0.001) and levels increased with age in both sexes P < 0.001). In women, a novel difference in protein C concentration between ABO blood groups was noted. There was no significant difference in plasminogen concentration between males and females, and in women plasminogen decreased with age (r = -0.205, P < 0.001). We conclude that these variations in ATIII and protein C with age and sex are important considerations in the determination of reference ranges for these proteins.

Fibrin-bound lipoprotein(a) promotes plasminogen binding but inhibits fibrin degradation by plasmin.

Conflicting results have been obtained from studies of the effects of lipoprotein(a) [Lp(a)] on plasminogen binding to fibrin and fibrin-dependent activation by tissue plasminogen activation (t-PA). We performed binding studies of Glu-plasminogen (0-16 microM) to immobilized D-dimer +/- Lp(a) (0.20 microM). In the absence of Lp(a), Scatchard analysis revealed a binding constant of KD = 1.01 +/- 0.18 microM, with two plasminogen binding sites per D-dimer. In the presence of Lp(a), a lower affinity (KD 3.10 +/- 0.23 microM) was found, but five binding sites were present, suggesting that plasminogen bound to fibrin-bound Lp(a) rather than to D-dimer. Consistent with this explanation was the finding that when D-dimer-coated plates were first precoated with Lp(a) before plasminogen was added, similar lower affinity plasminogen binding was found. This binding to Lp(a) was fibrin-dependent since, in its absence, plasminogen failed to bind to Lp(a). Therefore, a conformational change in Lp(a) appeared to be required for plasminogen binding to occur. This finding of two types of binding sites of different affinities helps to explain why Lp(a) has been reported to inhibit plasminogen binding to fibrin in studies in which only low concentrations of plasminogen (< 0.4 microM) were used. At these concentrations, few of the low-affinity binding sites on fibrin-bound Lp(a) will be occupied by plasminogen, an effect that was found to be exaggerated by the omission of NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of staphylokinase by enzyme-linked immunosorbent assay using monoclonal antibodies.

Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated. A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups. Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase. This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector". The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml. The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively. Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen. Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.

Binding of bovine plasminogen to immobilized casein and its activation thereon

Abstract Binding and activation of plasminogen to immobilized casein was studied using a type of ELISA performed as follows: casein was adsorbed to the wells of a microtiter plate. Plasminogen was then poured into the wells and its binding assessed by adding successively monoclonal antibody specific for plasminogen, phosphatase conjugate goat anti-mouse antibodies and phosphatase substrate. It was found that the amount of plasminogen bound depended on the casein type. Lysine binding sites, as well as electrostatic forces, were involved in plasminogen binding to casein and the amount of plasminogen bound depended on casein type. The role of electrostatic forces in binding to immobilized casein seemed to be more important than in binding to casein micelles of milk. Bound plasminogen was converted to plasmin by urokinase without dissociation from the matrix and the plasmin thus generated was active while bound. However, it was not possible to activate more than approximately 80% of bound plasminogen, and only 65% of plasminogen in milk, suggesting that the plasminogen concentration enzymatically determined in milk after activation by urokinase may be underestimated when compared to the immunological results. It was possible to inhibit the plasmin activity by aprotinin, but the enzyme was protected from inhibition by SBTI. Dissociation of plasminogen from the immobilized casein was obtained only at pH values below 4.6. No dissociation was observed with either high concentration of e-amino caproic acid or 2 m NaCl. The conditions for activation of plasminogen and inhibition of plasmin seemed to be similar to the conditions encountered in milk. It was concluded that the method developed may be a useful tool to study the various pathways involved in activation and inhibition of the plasminogen-plasmin complex.

Characterization of plasminogen activation by glycosylphosphatidylinositol-anchored urokinase.

The characteristics of plasminogen activation by glycosylphosphatidylinositol (GPI)-anchored urokinase were evaluated and compared with those reported previously for receptor-bound urokinase. When expressed in cultured bovine aortic endothelial cells, GPI anchoring of single-chain urokinase plasminogen activator (scu-PA) potentiated plasmin generation as compared with GPI-anchored scu-PA that had been released into solution from the cell surface by enzymatic cleavage of the GPI anchor ("released" scu-PA). The potentiation of plasmin generation by GPI-anchored scu-PA was inhibited in a dose-dependent manner by 6-aminohexanoic acid, a lysine analog, suggesting that the augmentation of plasmin generation by GPI-anchored scu-PA was dependent on simultaneous binding of plasminogen to the cell surface. GPI-anchored two-chain urokinase (tcu)-PA cleaved a peptide substrate at a rate equivalent to that of released urokinase. However, at a plasminogen concentration of 0.5 microM, GPI-anchored tcu-PA activated plasminogen less rapidly than did released urokinase. Modeling of kinetics of individual reactions revealed that cell-associated plasminogen activation by GPI-anchored tcu-PA was characterized by a Km of approximately 0.15 microM. This value of Km was 70-fold below that for activation of solution plasminogen by GPI-anchored urokinase. There was a concomitant decrease in Vmax for plasminogen activation by anchored tcu-PA. These alterations in kinetic parameters are similar to those reported previously for the activation of plasminogen by receptor-bound tcu-PA. In addition, GPI-anchored tcu-PA exhibited a modest resistance to plasminogen activator inhibitor 1 inactivation. The enzymatic characteristics of GPI-anchored urokinase reported here resemble closely those reported previously for receptor-bound urokinase. These data suggest that the urokinase receptor may regulate plasmin generation through a relatively nonspecific localization of urokinase to the cell surface rather than through any intrinsic property of the urokinase receptor.

Lipoprotein(a): A kinetic study of its influence on fibrin-dependent plasminogen activation by prourokinase or tissue plasminogen activator

Lipoprotein(a) [Lp(a)] has been postulated to inhibit fibrinolysis due to its structural homology to plasminogen. Indeed, it has been reported that Lp(a) competitively inhibits the promotion by fibrin of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation. However, it has also been reported that this inhibition is uncompetitive. No studies have been published, to our knowledge, of the effect of Lp(a) on prourokinase (pro-UK)-catalyzed plasminogen activation. Plasminogen activation by pro-UK or a plasmin-resistant mutant pro-UK was previously shown to be promoted by fibrin fragment E2, whereas that by t-PA is promoted by fragment D. Therefore, the influence of Lp(a) on the kinetics of these two reactions was examined. When Lp(a) was added (90-600 nM), no change in the rate of plasmin generation by Ala158-pro-UK was observed. Consistent with this, immobilized Lp(a) also failed to bind to fragment E2, whereas it did bind to D dimer. When t-PA-catalyzed plasminogen activation in the presence of D dimer was measured, uncompetitive inhibition by Lp(a) was found, but only at low concentrations of D dimer (< 0.5 microM) or t-PA (0.05 nM). At higher concentrations of D dimer and t-PA, instead of inhibition, Lp(a) induced a 2.4-fold promotion of plasminogen activation. Similarly, Lp(a) enhanced (up to 2.5-fold) plasminogen binding to immobilized fibrin in both buffer and plasma milieus at the physiological concentration of plasminogen (2.0 microM). In conclusion, Lp(a) had no effect on plasminogen activation by pro-UK and induced only limited inhibition of activation by t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation of plasma lipoprotein(a) to infarct artery patency in survivors of myocardial infarction.

In the minutes to days after myocardial infarction, endogenous lysis of an occlusive coronary arterial thrombus occurs in most subjects. Compared with those in whom thrombolysis does not occur, those with antegrade flow in the infarct artery have improved left ventricular performance, less left ventricular dilatation, and improved survival. This study was performed to assess intrinsic hemostasis and fibrinolysis in survivors of myocardial infarction with or without antegrade perfusion of the infarct artery. In 105 survivors of infarction (75 men, 30 women; age, 30 to 80 years) not given thrombolytic therapy, coronary angiography revealed a patent (group 1, n = 52) or occluded (group 2, n = 53) infarct artery. Plasma concentrations of plasminogen, fibrinogen, tissue plasminogen activator activity, infarct artery. Plasma concentrations of plasminogen, fibrinogen, tissue plasminogen activator activity, plasminogen activator inhibitor activity, cholesterol, triglycerides, and lipoproteins, including lipoprotein(a) (Lp[a]), were measured in blood procured 23 +/- 13 (mean +/- SD) months after infarction. Groups 1 and 2 were similar in age, sex, race, cardioactive medications, infarct artery, extent of coronary artery disease, and left ventricular performance. Of the plasma constituents assayed, the groups were similar except that Lp(a) averaged 18.5 +/- 21.7 mg/dL in group 1 and 49.1 +/- 44.8 mg/dL in group 2 (P < .001). This difference was evident in both Caucasian (n = 65) (P = .009) and African American (n = 40) (P = .01) subjects. Survivors of myocardial infarction who failed to recanalize the infarct artery have higher plasma Lp(a) concentrations than those with a patent infarct artery. Lp(a) may inhibit intrinsic fibrinolysis.

Molecular assembly of plasminogen and tissue‐type plasminogen activator on an evolving fibrin surface

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.

Fibrinolytic Activity in Blood and Lungs of Rats Treated with Monocrotaline Pyrrole

Monocrotaline pyrrole (MCTP), a putative toxic metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary vascular thrombi that are associated with vascular remodeling, pulmonary hypertension, and right cardioventricular hypertrophy in rats. It is possible that such thrombi contribute to the lung injury and pulmonary hypertension in this model. A previous study indicated that rats treated with MCTP did not have excessive procoagulant activity in the peripheral blood. Since thrombosis may also result from insufficient fibrinolytic activity in the systemic circulation, we evaluated the fibrinolytic system of rats given MCTP. Male Sprague-Dawley rats were given a single injection of MCTP (3.5 mg/kg) or an equal volume of N, N-dimethylformamide vehicle in the tail vein and were killed at 1, 3, 5, 8, 11, or 14 days after toxin administration. Several markers of lung injury and fibrinolysis were measured. Lung injury was evident 3 days after administration of MCTP and became more pronounced with time. In MCTP-treated rats, right heart hypertrophy was observed at 11 days and became more pronounced at 14 days. There was no change in the plasminogen concentration or in the activities of tissue plasminogen activator or α 2-antiplasmin in blood throughout the time course. Beginning at Day 8 and continuing through Day 14, there was an increase in the activity of plasminogen activator inhibitor in blood of rats that received MCTP. In addition, we evaluated the fibrinolytic activity of lung tissue slices. Rats treated with MCTP had a significant decrease in fibrinolytic activity of lung tissue at Day 3. A more pronounced decrease was evident by Day 8, and this continued through Day 14. In summary, MCTP treatment of rats decreases the fibrinolytic activity of lung tissue relatively early after exposure to the toxicant and before the onset of pulmonary hypertension. The change is reflected slightly later in plasma. These alterations in fibrinolytic activity may explain why fibrin thrombi form in the lungs of rats treated with MCTP.

Substrate inhibition of fibrin-dependent plasminogen activation by tissue-type plasminogen activator

Tissue plasminogen activator (t-PA) is a poor activator of plasminogen, but its catalytic efficiency is greatly enhanced in the presence of fibrin or certain fibrin derivatives. These two very different rates of plasminogen activation provide a basis for the phenomenon of substrate inhibition, which was the subject of the present study. The reaction mixture contained Glu-plasminogen (0-4.0 microM), +/- D-dimer (0.06-1.0 microM), t-PA (0.05-50.0 nM), and S2251 (1.5 mM). Inhibition of the reaction at higher plasminogen concentrations was seen at lower concentrations of t-PA (< 1.0 nM) and D-dimer (< 0.5 microM). The maximum rate of plasminogen activation occurred at 0.2 microM Glu-plasminogen but then decreased progressively above this concentration. At a physiological concentration of plasminogen (2.0 microM), activation was about half that at 0.2 microM. This phenomenon was shown not to be related to Glu-plasminogen aggregation or to a non-competitive inhibiting contaminant. Instead, the non-linear Lineweaver-Burk curve in the presence of D-dimer was consistent with substrate inhibition. Increasing the concentration of D-dimer (1.0 microM) and/or t-PA (50 nM) overcame the inhibition and reestablished linearity in the Lineweaver-Burk plots. However, at the higher concentrations of t-PA, the catalytic efficiency was reduced by 50-120-fold, suggesting that t-PA may be less efficient at pharmacological than at physiological concentrations. In conclusion, substrate inhibition in t-PA-induced plasminogen activation is reported for the first time. This phenomenon was confined to low concentrations of t-PA and D-dimer in the presence of physiological concentrations of plasminogen. The observation suggests that small fibrin clots (hemostatic plugs) may be more resistant to t-PA-induced lysis than an occlusive thrombus.