Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of beta-sitosterol on the fibrinolytic potential in rabbits

Long-term treatment of rabbits with beta-sitosterol (40 mg/kg over 3 months) caused an increased fibrinolytic activity in blood, an increased fibrinolytic capacity and an enhanced plasminogen activator activity in tissue of lungs and kidneys. The 3-months lasting beta-sitosterol administration did not influence the content of plasminogen activator inhibitor, plasminogen, alpha 2-antiplasmin, antithrombin III and fibrinogen.     

Changes in the fibrinolytic system associated with physical conditioning

The effects of physical conditioning on plasma fibrinolytic activity were studied in two groups of subjects. Volunteers not engaged in any sport were compared with individuals having been subjected to aerobic conditioning (middle-distance runners, defined as men running more than 80 km per week). Plasma concentrations of the different components of the fibrinolytic system were evaluated before and immediately after a maximal effort treadmill protocol. Comparison of the resting parameters revealed that under basal conditions for plasma concentrations of plasminogen, fibrinogen, alpha 2-antiplasmin, protein C and protein S there were no differences between the two groups. Concentrations of the fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) were significantly higher in the runners than in the control group, indicating an increased fibrinolytic potential that seemed to be a consequence of the reduced formation of tissue plasminogen activator-plasminogen activator inhibitor (t-PA-PAI) complexes. Acute maximal exercise resulted in pronounced fibrinolysis, evidenced by the elevation of FbDP and FgDP concentrations, in both groups of subjects. The acceleration of the fibrinolytic activity was larger in conditioned individuals, which could be accounted for by a higher t-PA release and reduced formation of t-PA-PAI complexes when compared to the untrained subjects.     

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.     

Effect of exercise and oral contraceptive agents on fibrinolytic potential in trained females

It has been shown that physical exercise increases blood fibrinolytic potential, primarily by inducing a release of extrinsic plasminogen activator from the vessel wall. Synthetic estrogens have also been reported to influence fibrinolytic activity. The effect of exercise and the possible additional effect of oral contraceptive agents (OCA) on the fibronolytic system were studied in 20 competitive female rowers. Ten females used OCA (users), and 10 others did not (nonusers). All participants were subjected to standardized exhaustive exercise. Preexercise data revealed higher factor XII, total plasminogen, and free plasminogen levels together with a significantly lower C1-inactivator level in the group of users. No differences were observed in prekallikrein, high-molecular-weight kininogen, alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, and histidine-rich glycoprotein plasma levels. The factor XII-dependent fibrinolytic activator activity and the extrinsic (tissue-type) plasminogen activator were significantly higher; however, the urokinase-like fibrinolytic activator activity was significantly lower. These observations suggest a greater susceptibility to activation of the fibrinolytic pathways during OCA medication. Exercise resulted in a decrease of all factors under study but an increase in all fibrinolytic activities. No differences were observed between the two groups in the percentages of change that occurred with exercise.     

Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex

Fibrinogen-NDSK complex is a model of protofibril having some features of the fibrin polymer structure. This complex has been studied for its ability to stimulate the plasminogen activation by t-PA. The fibrinogen-NDSK complex have increased the rate of plasminogen activation by t-PA as compared to fibrinogen or NDSK taken separately. This acceleration had slow and fast phases. Lys-plasminogen was activated more effectively as compared to glu-plasminogen. The kinetic parameters of glu- and lys-plasminogen activation at fast phase were: Km--0.18 and 0.015 mu/M, Kkat--0.27 and 0.06 s-1, respectively. Fibrinogen X2--fragments, deprived of alpha C-domains and NH2-end peptides of bB-chains, formed complexes with NDSK, which however did not stimulate the plasminogen activation by t-PA. These findings have shown that the fibrinogen-NDSK complex is an effective stimulator of the plasminogen activation by t-PA. The activating ability of the complex may be due to structures formed in the course of fibrinogen and NDSK polymerization as a result of alpha C-domain interaction.     

Tissue plasminogen activator catalyzed Lys-plasminogen activation on heparin-inserted phospholipid liposomes

We prepared heparin-inserted phospholipid liposomes as a functional model of heparan sulfate present on the vascular surface and examined tissue plasminogen activator (t-PA) catalyzed plasminogen activation on the liposome surface. Kinetic analyses showed a marked increase in the affinity of t-PA for Lys-plasminogen in the presence of heparin-inserted phosphatidylcholine (PC) liposomes. The catalytic efficiency (kcat/Km) of t-PA for the plasminogen activation on the surface of heparin-inserted PC liposomes was 5.4 times that on the surface of heparin-free PC liposomes. This stimulatory action of immobilized heparin was apparently affected by changing the phospholipid component of liposomes. Phosphatidylethanolamine or stearylamine, having a positively charged group, reduced the catalytic efficiency of t-PA by raising its Km value (10-fold), whereas negatively charged phospholipids, phosphatidylserine and phosphatidylinositol, did not affect the efficiency. t-PA and generated plasmin bound to the liposome surface heparin were protected from inhibition by plasminogen activator inhibitor type 1 and alpha 2-plasmin inhibitor, respectively. t-PA-induced clot lysis of euglobulin or whole plasma, which contained native (Glu-) plasminogen and the above inhibitors, was also accelerated by addition of heparin-inserted PC liposomes. These results suggest that the vascular surface heparin-like molecules may play an important role in modulating fibrinolytic events. The principles of conjugation of t-PA with a biologically active liposome will be applied to the construction of better thrombolytic agents.     

Affinity-chromatographic purification of human alpha 2-antiplasmin.

A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.     

Increased tissue-plasminogen activator (t-PA) levels in patients under oral anticoagulant therapy

The fibrinolytic system was investigated in 30 patients under oral anticoagulant therapy, and in 23 control patients not receiving oral anticoagulants. Patients under oral anticoagulant therapy had significantly higher tissue-plasminogen activator (t-PA) antigen levels than patients in the control group. Mean t-PA levels before venous occlusion were 18.4 ng/ml in the anticoagulated patients vs. 7.9 ng/ml in the control patients (p less than 0.001). After venous occlusion for 10 minutes, t-PA levels were 45.0 ng/ml in the anticoagulated patients and 24.2 ng/ml in the control patients (p less than 0.01). Plasminogen activator inhibitor (PAI) capacity was not significantly different in the two groups before venous occlusion (VO) but differed slightly (p less than 0.05) after VO. The net decrease in euglobulin lysis time (ELT) after venous occlusion (= ELT before VO - ELT after VO), indicating the relative potency of the fibrinolytic activity in blood, was also significantly higher in the anticoagulated patients (median 240 min vs. 125 min, p less than 0.001). These data indicate that oral anticoagulant therapy increases the fibrinolytic activity in blood, and thus may have an additional therapeutic effect in addition to anticoagulation.     

On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen.

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and alpha2-antiplasmin by competing with plasmin for the complementary site(s) in alpha2-antiplasmin. The dissocwation constant Kd for the interaction between intact plasminogen (Glu-plasminogen) and alpha2-antiplasmin is 4.0 microM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1--3 in the plasmin A-chain is mainly responsible for the interaction with alpha2-antiplasmin. The interaction between Glu-plasminogen and alpha2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and alpha2-antiplasmin by competing with alpha2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The Kd obtained for these interactions varied between 0.2 microM and 1.4 microM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH2-terminal and COOH-terminal regions; these sites are to a large extent.     

Tissue-type plasminogen activator inhibits aggregation of platelets in vitro

The effects of tissue-type plasminogen activator (t-PA) on the platelet aggregation were studied using citrated whole blood and platelet-rich plasma (PRP) obtained from human donors. t-PA suppressed adenosine 5'-diphosphate (ADP)- or collagen-induced platelet aggregation in a dose-dependent manner. The 50% inhibitory concentration (IC50) for t-PA was lower by one order of magnitude than that for urokinase (UK) in whole blood and PRP. The suppression of platelet aggregation was not completely inhibited by alpha-2-antiplasmin. t-PA did not cause the degradation of fibrinogen or fibrin in PRP, whereas UK caused the reduction of fibrinogen and fibrin, and the increase of fibrinogen- and fibrin-degradation products (FDP). These results suggest that the mode of action of t-PA in inhibiting platelet aggregation may be different from that of UK.     

Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase.

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy for patients with acute myocardial infarction. Due to its rapid rate of clearance from the circulation, continuous intravenous administration of approximately 100 mg over 3 h is recommended. We have previously characterized novel thrombolytic variant forms of t-PA which offer the potential of administration by bolus injection and reduced dosage due to their slower rates of clearance, relative to t-PA. This study was undertaken to quantitatively compare the pharmacokinetics, thrombolytic activity, and hemostatic effects of two of these variant forms, called delta FE1X and delta FE3X plasminogen activator (PA), with commercially available recombinant t-PA (Activase). These evaluations were performed in rabbits after bolus intravenous injection of the proteins. Following injection of 0.25 mg of protein/kg of body weight, the rates of clearance for delta FE3X and delta FE1X PA antigen were decreased approximately 9- and 18-fold, respectively, relative to Activase. Plasma plasminogen activator activity was also measured and the rates of clearance of delta FE3X and delta FE1X PA activity were similarly decreased by approximately 9- and 22-fold, respectively, relative to Activase. To quantitate thrombolytic activity we used the rabbit jugular vein thrombosis model and demonstrated that approximately 50% thrombolysis was achieved with delta FE1X and delta FE3X PA at approximately an 8.6- and 3-fold lower dose than Activase, respectively. No major differences in fibrinogen and alpha 2-antiplasmin depletion were observed among the agents at doses required to produce 50% thrombolysis, indicating similarities in fibrin specificities among these agents. These results demonstrate a reciprocal relationship between thrombolysis and rate of clearance for these thrombolytic proteins. The 8.6-fold increase in potency of delta FE1X PA relative to Activase supports the future clinical testing of this novel engineered protein as a thrombolytic agent.     

Fibrinogen lysine residue A alpha 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator

In previous studies, we have shown that the stretch 148-197 of the fibrinogen A alpha chain plays a crucial role in the acceleration of the tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation. In this study we have synthesized parts of A alpha 148-197 and analogues thereof. We found that the peptides with sequences identical with A alpha 148-161 and A alpha 149-161 of human fibrinogen accelerate the plasminogen activation by t-PA, whereas the corresponding peptides in which lysine residues A alpha 157 had been replaced by valine or arginine had no accelerating capacity. Furthermore, succinylation of the lysine residue(s) in the synthesized peptides A alpha 148-161 and A alpha 149-161 leads to loss of accelerating action. These findings show that lysine residue A alpha 157 is crucial for the accelerating action of fibrin on the t-PA-catalyzed plasminogen activation.     

A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.     

Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator

The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.     

Effect of prolonged physical exercise on the fibrinolytic system

The effect of a test marathon race on plasma fibrinolytic activity (FA) was studied in 16 endurance athletes before, immediately after, 3 h, and 31 h after the run. Tissue plasminogen activator (t-PA) activity increased about 31-fold immediately after the run. Similar increases were found in t-PA antigen concentration. Plasminogen activator inhibitor (PAI) was not detectable immediately after the race and was significantly decreased 3 h (P less than 0.05) and 31 h (P less than 0.01) later. B beta 15-42 peptide increased by 0.63 pmol.ml-1 (P less than 0.001), D-dimer by 68.3 ng.ml-1 (P less than 0.05). Euglobulin lysis time (ELT) was reduced from 109 to 18 min (P less than 0.001). The increased t-PA activity and t-PA antigen concentration disappeared in the course of the first 3 h after exertion. ELT also reached its pre-exercise levels at this time. Thirty-one hours after the race ELT and t-PA antigen levels were slightly but significantly reduced (P less than 0.05), whereas B beta 15-42 peptide remained increased (P less than 0.05). t-PA activity was unchanged compared with pre-exercise values. It seems that the exercise-induced FA is mainly caused by the marked increase of t-PA antigen and t-PA activity.     

Activation of plasminogen by pro-urokinase. I. Mechanism

The mechanism of the activation of plasminogen by recombinant pro-urokinase (Rec-pro-UK), obtained by expression of the human pro-urokinase gene in Escherichia coli, was investigated in purified systems. In mixtures of Rec-pro-UK and plasminogen, both active urokinase and plasmin are quickly generated. Addition of plasmin inhibitors (aprotinin or alpha 2-antiplasmin) abolishes the conversion of Rec-pro-UK to urokinase but not the activation of plasminogen to plasmin, suggesting that Rec-pro-UK activates plasminogen directly. Human plasma competitively inhibits the activation of plasminogen by pro-urokinase with a Ki of 0.2% (v/v). This explains the relative stability of Rec-pro-UK in plasma and the lack of activation of the plasma fibrinolytic system in the absence of fibrin. The competitive inhibition by plasma is abolished by the addition of CNBr-digested fibrinogen although Rec-pro-UK has no specific affinity for fibrin. These findings suggest that the fibrin specificity of the activation of plasminogen by pro-urokinase is due to neutralization by fibrin of the competitive inhibition exerted by plasma and not to fibrin-enhanced activation of plasminogen.     

A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo

Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 ("Lys-plasminogen"), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.     

Alpha 2-antiplasmin in childhood

In 119 children, predominantly newborns and babies with sepsis, alpha 2-Antiplasmin was determined by the use of the chromogenic substrate S-2251. In healthy newborns, the inhibitor level averaged 65 per cent of the adult level. Already in the initial phase of sepsis, enhanced alpha 2-antiplasmin values were observed. During the further course, they increased markedly. Thus, alpha 2-antiplasmin proved to be an acute phase reactant together with fibrinogen, factors II and X, and alpha 1-antitrypsin measured as trypsin inhibitor capacity. The correlation analysis in all subgroups showed moderately tight binding to fibrin. In patients with shock or in those who decreased, lower levels were measured. The overproduction is assumed to be caused by disseminated intravascular coagulation processes. In other diseases such as respiratory distress, alpha 2-antiplasmin was reduced. In case of disseminated intravascular coagulation that was not caused by sepsis consumption of components dominated. In the probability paper, distribution of the values of the subgroups was found to differ markedly. Thus, the inhibitor proved to be of diagnostic value.