Non-ionic detergents Nonidet P-40 and Triton X-100 increase enzymatic activity of plasmin.

Plasmin is a potent serin protease involved in a variety of biological functions, such as fibrinolysis and tissue remodeling. On performing an in vitro control assay to measure the activity of endogenous plasmin in cell lysates, a stimulatory effect of non-ionic detergent NP-40 on plasmin activity was discovered. Another non-ionic detergent, TX-100, also enhanced plasmin activity, while ionic detergents sodium deoxycholate and sodiem dodecyl sulfate abolished plasmin enzyme activity. Kinetic analysis of plasmin activity in the presence of NP-40 and TX-100 demonstrated an increase in Vmax; however, there was no change in Km values, suggesting that these detergents stimulate plasmin activity in a non-competitive manner. Fibrin plate assay indicates that NP-40 and TX-100 functionally stimulate plasmin activity by showing a dose-dependent increase in fibrinolysis.

A direct oral anticoagulant edoxaban accelerated fibrinolysis via enhancement of plasmin generation in human plasma: dependent on thrombin-activatable fibrinolysis inhibitor.

A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.

Fli1 deficiency contributes to the downregulation of endothelial protein C receptor in systemic sclerosis: a possible role in prothrombotic conditions.

BACKGROUND: Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc). OBJECTIVES: To investigate the potential contribution of EPCR to the development of SSc. METHODS: EPCR expression was examined in skin samples and cultivated dermal microvascular endothelial cells by immunostaining, immunoblotting and/or quantitative reverse-transcription polymerase chain reaction. Fli1, binding to the PROCR promoter, was assessed by chromatin immunoprecipitation. Serum EPCR levels were determined by enzyme-linked immunosorbent assay in 65 patients with SSc and 20 healthy subjects. RESULTS: EPCR expression was decreased in dermal small vessels of SSc lesional skin compared with those of healthy control skin. Transcription factor Fli1, deficiency of which is implicated in SSc vasculopathy, occupied the PROCR promoter, and EPCR expression was suppressed in Fli1 small interfering RNA-treated endothelial cells and dermal small vessels of Fli1(+/-) mice. In patients with SSc, decreased serum EPCR levels were associated with diffuse skin involvement, interstitial lung disease and digital ulcers. Furthermore, serum EPCR levels inversely correlated with plasma levels of plasmin-α2-plasmin inhibitor complex (PIC). Importantly, bosentan significantly reversed circulating EPCR and PIC levels in patients with SSc, and the expression of Fli1 and EPCR in dermal small vessels was elevated in patients treated with bosentan compared with untreated patients. CONCLUSIONS: Endothelial EPCR downregulation due to Fli1 deficiency may contribute to hypercoagulation status leading to tissue fibrosis and impaired peripheral circulation in SSc.

[Thrombolytic agents].

Thrombolytic agents positively resolve existing thrombi by accelerating the activity of plasmin, a key enzyme of the fibrinolytic pathway. The main drug in use is a plasminogen activator (PA) which degrades plasminogen to plasmin. PA is classified into urokinase-type (u-PA) and tissue-type (t-PA). Because t-PA more selectively activates plasmin onto the surface of thrombi, it induces less in terms of systemic hemorrhagic complications. Beside the main effects, some articles have reported that t-PA causes damage to blood brain barrier structures and has a level of neuron toxicity. With this, delayed administration of t-PA for acute ischemic stroke may accelerate ischemic damage of brain tissue. In circulating blood, plasmin itself is rapidly inactivated by a plasmin-inhibitor, so with this, intra-arterial administration of plasmin directly near thrombi site is currently under investigation.

Cysteine-Derived Chiral Carbon Quantum Dots: A Fibrinolytic Activity Regulator for Plasmin to Target the Human Islet Amyloid Polypeptide for Type 2 Diabetes Mellitus.

The fibrillization and deposition of the human islet amyloid polypeptide (hIAPP) are the pathological hallmark of type 2 diabetes mellitus (T2DM), and these insoluble fibrotic depositions of hIAPP are considered to strongly affect insulin secretion by inducing toxicity toward pancreatic islet ß-cells. The current strategy of preventing amyloid aggregation by nanoparticle-assisted inhibitors can only disassemble fibrotic amyloids into more toxic oligomers and/or protofibrils. Herein, for the first time, we propose a type of cysteine-derived chiral carbon quantum dot (CQD) that targets plasmin, a core natural fibrinolytic protease in humans. These CQDs can serve as fibrinolytic activity regulators for plasmin to cleave hIAPP into nontoxic polypeptides or into even smaller amino acid fragments, thus alleviating hIAPP's fibrotic amyloid-induced cytotoxicity. Our experiments indicate that chiral CQDs have opposing effects on plasmin activity. The l-CQDs promote the cleavage of hIAPP by enhancing plasmin activity at a promotion ratio of 23.2%, thus protecting ß-cells from amyloid-induced toxicity. In contrast, the resultant d-CQDs significantly inhibit proteolysis, decreasing plasmin activity by 31.5% under the same reaction conditions. Second harmonic generation (SHG) microscopic imaging is initially used to dynamically characterize hIAPP before and after proteolysis. The l-CQD promotion of plasmin activity thus provides a promising avenue for the hIAPP-targeted treatment of T2DM to treat low fibrinolytic activity, while the d-CQDs, as inhibitors of plasmin activity, may improve patient survival for hyperfibrinolytic conditions, such as those existing during surgeries and traumas.

Elevated urinary plasmin activity resistant to alpha2-antiplasmin in acute poststreptococcal glomerulonephritis.

BACKGROUND: A pathogenic role of intraglomerular plasmin bound to nephritogenic antigen (nephritis-associated plasmin receptor, NAPlr) and resistant to physiologic inhibitors such as alpha(2)-antiplasmin (alpha(2)-AP) has recently been proposed in acute poststreptococcal glomerulonephritis (APSGN). To confirm this concept, we analysed the urinary profile of plasmin cascade in APSGN patients. METHODS: Urine samples from 10 patients with APSGN, 12 patients with IgA nephropathy (IgAN), 10 patients with streptococcal infection without nephritis (SI) and 10 healthy control subjects were analysed. The alpha(2)-AP-resistant plasmin activity was assessed by a chromogenic assay after alpha(2)-AP was added to each urine sample. Urinary plasminogen activator (PA) and plasmin were further analysed by polyacrylamide gel zymography. Urinary NAPlr was assessed by western blot analysis in selected samples. RESULTS: Urinary alpha(2)-AP-resistant plasmin activity corrected for creatinine concentration (units/g x creatinine) was significantly higher in patients with APSGN (2.99 +/- 0.63) than in patients with IgAN (1.02 +/- 0.20, P < 0.01), SI (0.79 +/- 0.17, P < 0.01), or in healthy control subjects (0.73 +/- 0.18, P < 0.01). This tendency was confirmed by casein gel zymography. However urinary PA activity assessed by plasminogen-casein gel zymography did not differ between groups. NAPlr was detected in the urine of APSGN patients. CONCLUSIONS: We found elevated urinary plasmin activity resistant to alpha(2)-AP, which may be due to urinary excretion of NAPlr in patients with APSGN. This result supports the pathogenic role of the NAPlr-plasmin complex in the development of APSGN. Furthermore, alpha(2)-AP-resistant urinary plasmin activity may be useful as a diagnostic marker for APSGN.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

The plasmin system is induced by and degrades amyloid-beta aggregates.

Amyloid-beta (Abeta) appears critical to Alzheimer's disease. To clarify possible mechanisms of Abeta action, we have quantified Abeta-induced gene expression in vitro by using Abeta-treated primary cortical neuronal cultures and in vivo by using mice transgenic for the Abeta precursor (AbetaP). Here, we report that aggregated, but not nonaggregated, Abeta increases the level of the mRNAs encoding tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Moreover, tPA and uPA were also upregulated in aged AbetaP overexpressing mice. Because others have reported that Abeta aggregates can substitute for fibrin aggregates in activating tPA post-translationally, the result of tPA induction by Abeta would be cleavage of plasminogen to the active protease plasmin. To gain insights into the possible actions of plasmin, we evaluated the hypotheses that tPA and plasmin may mediate Abeta in vitro toxicity or, alternatively, that plasmin activation may lead to Abeta degradation. In evaluating these conflicting hypotheses, we found that purified plasmin degrades Abeta with physiologically relevant efficiency, i.e., approximately 1/10th the rate of plasmin on fibrin. Mass spectral analyses show that plasmin cleaves Abeta at multiple sites. Electron microscopy confirms indirect assays suggesting that plasmin degrades Abeta fibrils. Moreover, exogenously added plasmin blocks Abeta neurotoxicity. In summation, we interpret these results as consistent with the possibility that the plasmin pathway is induced by aggregated Abeta, which can lead to Abeta degradation and inhibition of Abeta actions.

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Functional determination of plasmin in arginine-stabilized plasma.

Reliable data on plasmin activities in blood of patients during fibrinolytic treatment are lacking. This is due to continuing plasminogen activation by plasminogen activators after blood withdrawal. The purpose of this study was to establish a new method for stabilization of blood and to detect plasmin activity in stabilized plasma. For optimization of plasma stabilization by arginine, 50 microL pooled normal citrated plasma was incubated with 50 microL of 0 to 1500 mM arginine, pH 8.7, and 25 microL 100 IU/mL u-PA, 1250 IU/mL t-PA, 10000 U/mL reteplase, 400 U/mL plasminogen-streptokinase-activator complex, 10 microg/mL tenecteplase in 6% BSA-PBS or 25 microL 25 microg/mL plasmin in 20% glycerol. Twenty-five microliters 3 mM HD-Val-Leu-Lys-pNA were added immediately (1 step) or after 90 minutes (room temperature [RT]). The same experiment was performed with pooled normal citrated plasma supplemented with 3.2 mg/mL EDTA, preoxidized with 0 mM or 20 mM chloramine-T for 10 minutes (37 degrees C). For optimization of plasmin activity, the oxidation time of the arginine-stabilized plasma sample containing 0.5 U/mL active plasmin and the chloramine-T amount was varied. Citrated plasma is stabilized against the in vitro action of all six plasminogen activators tested if the final arginine concentration is greater than 500 mM. Neither the addition of EDTA nor the addition of chloramine-T changes this plasma-stabilizing power of arginine. The optimized functional plasmin assay consists of incubation of 10 microL arginine-stabilized plasma with 10 microL 1.5 M arginine, pH 8.7, and 10 microL 100 mM CT in PBS. After 30 minutes (37 degrees C), 75 microL 1.2 M KCl, 1.6 M Arg, 0.75 mM Val-Leu-Lys-pNA (Stop-CS Reagent), and 175 microL 6% BSA-PBS are added and the absorbance increase (DeltaA) at 405 nm is determined. With the present arginine stabilization procedure of plasma and the determination of plasmin activity in arginine-stabilized plasma as described, it is feasible to determine the activity of plasmin in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes in the samples.

Enhancement of catalytic activities of serine proteases by tripeptides compounds.

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.

Like fibrin, (DD)E, the major degradation product of crosslinked fibrin, protects plasmin from inhibition by alpha2-antiplasmin.

Plasmin generation is localized to the fibrin surface because tissue-type plasminogen activator (t-PA) and plasminogen bind to fibrin, an interaction that stimulates plasminogen activation over a hundred-fold. To ensure efficient fibrinolysis, plasmin bound to fibrin is protected from inhibition by alpha2-antiplasmin. (DD)E, a major soluble degradation product of cross-linked fibrin that is a potent stimulator of t-PA, compromises the fibrin-specificity of t-PA by promoting systemic activation of plasminogen. In this study we investigated whether (DD)E also protects plasmin from inhibition by alpha2-antiplasmin, facilitating degradation of this soluble t-PA effector. (DD)E and fibrin reduce the rate of plasmin inhibition by alpha2-antiplasmin by 5- and 10-fold, respectively. Kringle-dependent binding of plasmin to (DD)E and fibrin, with Kd values of 52 and 410 nM, respectively, contributes to the protective effect. When (DD)E is extensively degraded by plasmin, yielding uncomplexed fragment E and (DD), protection of plasmin from inhibition by alpha2-antiplasmin is attenuated. These studies indicate that (DD)E-bound plasmin, whose generation reflects the ability of (DD)E to stimulate plasminogen activation by t-PA, has the capacity to degrade (DD)E by virtue of its resistance to inhibition. This provides a mechanism to limit the concentration of (DD)E and maintain the fibrin-specificity of t-PA.

Heparin coating of extracorporeal circuits inhibits contact activation during cardiac operations.

OBJECTIVE: Heparin coating reduces complement activation on the surface of extracorporeal circuits. In this study we investigated its effect on activation of the contact system in 30 patients undergoing coronary artery bypass grafting with the use of a heparin-coated (Duraflo II, Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.; n = 15) or an uncoated extracorporeal circuit (n = 15). METHODS: Plasma markers that reflect activation of contact (kallikrein-C1-inhibitor complexes), coagulation (prothrombin fragments F1 + 2), or fibrinolytic (plasmin-alpha 2-antiplasmin complexes) systems were determined before and during the operation. The generation of kallikrein-C1-inhibitor complexes was reduced by 62% (p = 0.06) after the onset of cardiopulmonary bypass and by 43% (p = 0.026) after the cessation of bypass in the group in which a heparin-coated circuit was used compared with the group in which the circuit was uncoated. Generation was reduced by 58% (p = 0.06) when the ratio of kallikrein-C1-inhibitor to prekallikrein after onset of bypass was considered. We detected significant increases in F1 + 2 levels in both groups and increases in plasmin-alpha 2-antiplasmin complexes in the heparin-coated group at cessation of bypass, but no intergroup differences were observed. Thus use of heparin-coated extracorporeal circuits during cardiac operations reduces formation of kallikrein-C1-inhibitor complexes when compared with use of uncoated circuits. The heparin coating is not accompanied by similar reductions in coagulation or fibrinolysis, suggesting that thrombin and plasmin formation during cardiopulmonary bypass occurs mainly independently of the contact system activation.

The effect of various contrast media on the activation of plasminogen by streptokinase or recombinant tissue plasminogen activator in vitro.

RATIONALE AND OBJECTIVES: Radiologic contrast media (CM) may influence processes of coagulation and fibrinolysis. In the current study, the effects of various CM on the formation of plasmin were examined in an in vitro buffer system. METHODS: The effects of three clinically relevant concentrations of seven different iodine-containing CM and gadolinium-DTPA on streptokinase (SK) or recombinant tissue plasminogen activator (rt-PA)-induced plasmin formation was monitored using a plasmin-sensitive chromogenic substrate. RESULTS: Contrast media generally had an inhibitory effect at the plasminogen activation step; this effect was particularly noticeable with the ionic CM. CONCLUSIONS: Contrast media influence plasminogen activation by SK and rt-PA in vitro. Ionic CM have a more pronounced inhibitory effect than the nonionic media. The ionic Gd-DTPA shows a less inhibitory effect than the ionic iodine-containing CM. However, they must be regarded separately because of the different chemical composition of the magnetic resonance imaging and x-ray CM.

Tranexamic acid attenuates oleic-acid-induced pulmonary extravasation.

OBJECTIVE: Activation of fibrinolysis is implicated in the development of vascular injury in certain lung injuries. It has yet to be reported that activation of plasmin is involved in extravasation caused by oleic acid (OA). We examined whether or not plasmin is involved in pulmonary extravasation by OA. DESIGN: Prospective trial. SETTING: University laboratory. SUBJECTS: A total of 78 guinea pigs (498.9 +/- 10.6 g). INTERVENTIONS: Evans blue (EB) was administered to anesthetized guinea pigs. Subsequently four protocols were followed: (1) After 1 min, 60 micro l/kg of OA was injected. Perfusion was performed 30, 60 or 90 min after OA injection to wash out intravascular EB. (2) After 1 min, 15, 30 or 60 micro l/kg of OA was injected. (3) Tranexamic acid (TA) (2 g/kg) or saline was administered 30 min before OA (15 micro l/kg) injection. (4) Diphenhydramine hydrochloride (2.9 mg/kg) or saline was administered 7 min before OA (15 micro l/kg) injection. MEASUREMENT AND RESULTS: Except in protocol 1, the chest cavity was opened 90 min after OA injection. Perfusion was then performed. Airway was separated into four parts from trachea to distal bronchus. EB was extracted from the tissues and measured. OA caused an extravasation throughout airways in a time- and dose-dependent manner. Extravasation was more conspicuous in peripheral tissues. TA significantly attenuated extravasation, while diphenhydramine hydrochloride did not. CONCLUSIONS: It is suggested that plasmin, but not histamine, is involved in extravasation by OA. Inhibition of plasmin can be an effective strategy for treatment of this kind of lung injury.

Comparison of the effect of histidine-rich glycoprotein and 6-aminohexanoic acid on plasmin production and fibrinolysis in vitro.

Because histidine-rich glycoprotein binds to the kringle 1-3 domain of plasminogen, it may affect fibrinolysis by reducing fibrin-dependent plasmin production, and in this way it could be mechanistically analogous to 6-aminohexanoic acid. We tested this hypothesis by comparing the effects of histidine-rich glycoprotein and 6-aminohexanoic acid in an in vitro assay of fibrin-dependent plasmin production mediated by tissue plasminogen activator. Whereas 1 mM of 6-aminohexanoic acid increased the K(m) of the reaction from approximately 0.22 microM to approximately 1.7 microM, 2 microM of histidine-rich glycoprotein had no discernible effect. Similar results were obtained in an assay based upon fibrin clot lysis. Therefore, we could not document an effect of histidine-rich glycoprotein on the rate of fibrin-dependent plasmin production.

The thrombolytic paradox.

Thrombolytic drugs do not only stimulate the plasmin system but also induce thrombin activation additionally to the preexisting hypercoagulative state in patients with acute myocardial infarction. Testing the in vitro-derived hypothesis of a plasmin-mediated activation of the contact phase of the coagulation leading to the procoagulant effect, several thrombolytic regimen have been evaluated. Paradoxical thrombin activation (referred to as "thrombolytic paradox") was related to absence of fibrin specificity. Highly fibrin-specific drugs like tenecteplase did not cause additional thrombin activation, while non-fibrin-specific drugs like streptokinase caused a marked additional activation of the contact phase and of thrombin. It could be shown that the thrombolytic paradox was related to the extent of systemic plasmin activation confirming the hypothesis of a plasmin-mediated factor XII/kallikrein system activation as cause of the thrombolytic paradox.

Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin.

Evidence has recently been presented that activated macrophages (M phi) express both urinary (u-PA) and tissue type (t-PA) plasminogen activator. Major cell products of M phi and polymorphonuclear neutrophils (PMN) are reactive oxidants of the HOCl/chloramine type. Since PMN and M phi are involved in inflammatory and fibrinolytic processes, we were interested in the interaction of u-PA, t-PA, and plasmin with oxidants of the leukocyte type. The enzymes were treated with chloramine-T, which at pH 8.5 is a selective oxidant for methionine residues. Oxidation by chloramine-T of t-PA abolishes about 40% of both stimulation susceptibility of t-PA by fibrinogen degradation products (FDP) and affinity of t-PA to FDP. However, the plasminogenolytic and amidolytic activity of unstimulated t-PA as well as the plasminogenolytic activity of u-PA and the amidolytic activity of plasmin are not impaired. Identification of the amino acid residues in the t-PA responsible for the interaction with fibrin might be of great importance in order to understand the mechanism of the clot- selectivity of t-PA. The present study gives evidence that fibrin specificity of t-PA partly depends on chloramine oxidizable amino acids, presumably methionine residues. Hence, experimental data on the interaction between t-PA and fibrin, using oxidized and labelled t-PA should be interpreted with caution. It may be suggested that oxidants of the leukocyte type might regulate t-PA activity and selectivity for fibrin.

Platelets and soluble fibrin promote plasminogen activation causing downregulation of platelet glycoprotein Ib/IX complexes: protection by aprotinin.

Blood loss during and after open-heart surgery with cardiopulmonary bypass (CPB) is largely caused by platelet dysfunction. Previous studies indicate that plasmin can induce platelet dysfunction and affect primary hemostasis by proteolytic degradation and/or redistribution of essential platelet membrane glycoprotein complexes such as the glycoprotein Ib/IX complex. In this study, we present a model for plasmin generation localized on the platelet surface. Platelets treated with soluble fibrin or platelets in a mixture with soluble fibrin, t-PA, and plasminogen caused a significantly increased plasmin generation (p<0.01), dependent on t-PA, soluble fibrin, and platelet concentration. The plasmin generation resulted in a downregulation of platelet membrane glycoprotein Ib/IX glycoprotein complexes. Finally, we demonstrated that inhibitors of fibrinolysis, such as %2-antiplasmin, tranexamic acid, and aprotinin, can inhibit plasmin activity in the fluid phase. The downregulation of platelet glycoprotein Ib/IX complexes, however, was only prevented by aprotinin and not by alpha2-antiplasmin and tranexamic acid. These in vitro observations suggest a platelet localized activation of plasminogen, dependent on t-PA, enhanced by the presence of soluble fibrin. Since high concentrations of soluble fibrin and elevated levels of t-PA during CPB are observed, plasmin activity on the platelet surface during this period is anticipated. This plasmin activity reduces platelet metabolic functions and can be directed towards membrane glycoproteins such as glycoprotein Ib/IX complexes, thereby affecting hemostasis during and after CPB.