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Background: The clinical success of liver transplantation has led to increased demand, requiring further expansion of the donor pool. Therapeutic interventions to optimize organs from donation after circulatory death (DCD) have significant potential to mitigate the organ shortage. Dysfunction in DCD liver grafts is mediated by microvascular thrombosis during the warm ischemic period, and strategies that reduce this thrombotic burden may improve graft function. We hypothesized that the administration of the fibrinolytic enzyme plasmin to the donor organ during the cold storage period would reduce the thrombotic burden and improve DCD liver graft function., Methods: In 2 separate cohorts, 32 syngeneic orthotopic rat liver transplants were performed in Lewis rats. Livers were procured from donors with 45 min of warm ischemic injury. Liver grafts were flushed with histidine-tryptophan-ketoglutarate preservation solution mixed with either plasmin (experimental group) or albumin (control group). All investigators were blinded to treatment group. After preparing the liver for implant using a modified cuff technique, the liver was stored for 1 h by static cold storage at 4 °C. Immediately before implantation, the liver graft was flushed, and this effluent was analyzed for fibrin degradation products to determine graft clot burden. Twenty-four hours following transplantation, animals were euthanized, and samples were collected., Results: Recipient survival was significantly higher for DCD liver grafts treated with plasmin compared with control. Moreover, histology of liver graft tissue immediately before implant reflected significantly reduced congestion in plasmin-treated livers (score, mean ± SD: 0.73 ± 0.59 versus 1.12 ± 0.48; P  = 0.0456). The concentration of fibrin degradation products in the final flush before implantation was significantly reduced in plasmin-treated livers (743 ± 136 versus 10 919 ± 4642 pg/mL; P  = 0.0001), reflecting decreased clot burden in the graft., Conclusions: The present study demonstrates that plasmin improves survival and may reduce thrombotic burden in DCD liver grafts with prolonged warm ischemic injury, meriting further study., Competing Interests: M.G.H. and A.S.B. are members of the Medical Advisory Board of BMI OrganBank. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Introduction: Plasmin is a direct-acting thrombolytic agent with a favorable safety profile upon intra-arterial delivery in pre-clinical and phase I studies. However, the thrombolytic efficacy of plasmin, relative to that of rt-PA, remains to be established. We have compared the dynamics of clot lysis with plasmin or rt-PA in an in vitro perfusion system, in which thrombolytic agent is administered locally, allowed to induce lysis for short intervals, then washed with plasma in a re-circulation circuit., Materials and Methods: Whole blood human clots were prepared in observation chambers, exposed to plasmin or rt-PA at equimolar concentrations (1.2/1.0, 1.8/1.5 and 2.4/2.0 mg/ml) for measured intervals of time, followed by perfusion with human plasma. Clot size was monitored by digital analysis of sequential photographs obtained through an optical microscope., Results: Plasma perfusion after incubation with thrombolytic agent rapidly removed superficial clot fragments. This initial decrease in clot size was greater with plasmin than with rt-PA when tested at the highest concentrations of agent (0.63 ± 0.11 vs. 0.30 ± 0.11, p=0.001 for clots with non-cross-linked fibrin and 0.53 ± 0.15 vs. 0.14 ± 0.15, p=0.02, for clots with cross-linked-fibrin). Subsequent clot lysis during plasma flow was greater after prior incubation with rt-PA. Longer incubation times of plasmin resulted in larger portions of the clot being washed free. Repeated plasmin incubations and plasma perfusions of a clot successfully induced stepwise reductions in clot size., Conclusions: Initial clot lysis is greater with direct exposure using plasmin than rt-PA. During washout and circulation with plasma, rt-PA induced continued clot lysis, while plasmin lysis was curtailed, presumably because of plasmin inhibition., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plasmin, a directly acting thrombolytic agent, demonstrated a very favorable safety profile upon intra-arterial delivery to the clot site; however, its thrombolytic efficacy remains to be further assessed. In this study, differences in thrombolysis between clots exposed to equimolar concentrations of plasmin and recombinant tissue-type plasminogen activator (rt-PA) after partial vessel recanalization were tested in a model system. Model blood clots were prepared in glass chambers enabling direct observation by dynamic optical microscopy. The incubation of clots with plasmin (2.4 mg/ml) or rt-PA (2.63 mg/ml), allowing for the initial biochemical clot degradation, was followed by 'flushing' the clots with tangentially directed plasma flow devoid of a thrombolytic agent, mimicking blood flow after partial vessel recanalization. The acquired images were analyzed for nondissolved blood clot area as a function of time. With both thrombolytic agents, the relative clot area decreased rapidly in the first 30 s after initiation of perfusion due to 'flushing' the degraded clot fragments (after plasmin by 0.26 ± 0.22 and after rt-PA by 0.34 ± 0.21, P = 0.60). In the following minutes, the clot size showed a linear time dependence: after incubation with plasmin the clot size did not change substantially any more, whereas after incubation with rt-PA the clot size continually decreased. The slopes of the regression lines differed significantly (r(pl) = -8.9 10 vs. r(rtPA) = -44.1 10/min, P < 0.01). In conclusion, the thrombolytic action of plasmin was terminated rapidly by contact with flowing blood plasma, whereas the thrombolytic action of rt-PA was prolonged.

Background: Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat., Methods: Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm., Results: The main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2-3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats., Conclusions: The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

We previously demonstrated a significant margin of haemostatic safety for full-length plasmin in comparison with tissue plasminogen activator (t-PA). We now report studies that compare haemostatic safety of full-length plasmin with a novel recombinant plasmin derivative, (Δ K2-5) plasmin, consisting of kringle 1 linked to the serine protease domain of plasmin. Agent was administered intravenously in a randomised, blinded manner in a rabbit model of fibrinolytic haemorrhage. A dose-related decrease in α2-antiplasmin, factor VIII, and fibrinogen followed administration of 1.8, 2.7, 3.7 and 4.6 mg/kg of (Δ K2-5) plasmin, with nadir fibrinogen concentrations of 65%, 40%, 30%, and 0% of initial levels, respectively. Mean primary bleeding time was undisturbed at 1.8 mg/kg (2.2 ± 0.7 minutes), minimally prolonged at 2.7 or 3.7 mg/kg (5 ± 2.9 and 4.4 ± 2.2 minutes), and prolonged at the purposefully toxic 4.6 mg/kg dose (12.8 ± 18.8 minutes). Equimolar amounts of (Δ K2-5) plasmin and full-length plasmin had equal in vitro clot lysis efficacy, but in the bleeding model, (Δ K2-5) plasmin showed better haemostatic competency than full-length plasmin. This safety advantage may be explained by higher residual amounts of plasma fibrinogen in animals given (Δ K2-5) plasmin rather than full-length plasmin. We demonstrate that a unique recombinant plasmin mutant, (Δ K2-5) plasmin, possesses an advantage in hemostatic safety over an equimolar amount of full-length plasmin.

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d)=11.3+/-2.3 microM) and fibrin (C(50) approximately 0.3 microM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1+/-0.1 x 10(7) M(-1) s(-1)) and alpha(2)-macroglobulin (7.6+/-0.6 x 10(5) M(-1) s(-1)). In an in-vitro model, Delta(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by alpha(2)-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Delta[K2-K5]Pm) are warranted.

The physiological or pharmacological dissolution of thrombi is ultimately accomplished by the serine protease plasmin. Plasmin is derived from its precursor plasminogen in a reaction catalyzed by plasminogen activators (PAs) such as tissue-type plasminogen activator (tPA). In the middle of the last century, plasmin was investigated as a potential thrombolytic agent. However, technical obstacles led to the abandonment of this agent, and by the 1970s PAs had become the standard of care for pharmacological management of various thrombotic conditions. Talecris Biotherapeutics has developed a methodology to prepare the plasmin product (Human) TAL-05-00018 that is rendered inactive by low pH (pH 3.0-4.0) until it is delivered directly to the neutral environment of a thrombus by catheter-assisted administration. TAL-05-00018 undergoes a rigorous manufacturing process to ensure a final product free from unactivated plasminogen, streptokinase, enveloped and non-enveloped viruses and prion proteins; generating an extremely high-purity product with a shelf life of three years at ambient temperature. TAL-05-00018 has shown promise in in vitro and pre-clinical studies, and in early clinical trials, demonstrating a dose-dependant reduction in clot weight that compares favorably to that seen with tPA. Several other direct thrombolytics have also been developed, including the recombinant, modified deletion mutant of plasmin TAL6003 (Talecris Biotherapeutics), which retains all the major functional attributes of full-length plasmin.

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.

The direct fibrinolytic enzyme, plasmin, was compared with tissue plasminogen activator (TPA) in rabbit models of local thrombolysis and fibrinolytic hemorrhage. Plasmin was produced by solid-phase urokinase activation of plasminogen and purified on benzamidine Sepharose. Applied as an intra-arterial infusion into the thrombosed abdominal aorta under conditions of unimpeded blood flow, plasmin (4 mg/kg) and TPA (2 mg/kg) achieved equivalent clot dissolution and flow restoration. Using the model of restricted blood flow into the thrombosed aorta, which limits local plasminogen supply, plasmin was superior to TPA in clot lysis and vascular reperfusion. Using similar dosages of plasmin (2 or 4 mg/kg) and TPA (1 or 2 mg/kg) in the earpuncture rebleed model. TPA induced rebleeding in a dose-dependent manner from prior puncture sites in 9 of 10 animals, while none of the 10 animals exposed to plasmin rebled from these sites. These results suggest that plasmin is an effective, unique thrombolytic agent, distinguished from the plasminogen activators in current usage by its striking safety profile.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.

The gelatin-binding region of fibronectin contains three Asn-linked carbohydrate moieties, one on the second type II module and two on the eighth type I "finger" module. Carbohydrate groups were enzymatically removed from two nonoverlapping gelatin-binding fragments (GBFs), 21-kDa GBF (modular composition I8-I9) and, with much greater difficulty, 30-kDa GBF (modular composition I6-II1-II2-I7). The gelatin-binding properties of these fragments were affected only slightly or not at all. Fluorescence and calorimetric analyses indicated that module I8 was strongly destabilized by deglycosylation such that the apo form melts near physiological temperature. A similar effect was caused by decreasing the pH of the holo form to 6.0, suggesting that one or more histidines are important for stability of module I8. The 21-kDa fragment exhibited an acid-induced change in fluorescence that occurred at higher pH in the deglycosylated derivative, providing further evidence of a stabilizing role for one or both carbohydrate moieties. By contrast, the stability of module II2 was unaffected by removal of its single carbohydrate. The effects of carbohydrate on the stability of module I8 may be important in efforts to express it or fragments containing it in bacteria.

The stability of the Fib-1 (29 kDa) and Fib-2 (19 kDa) fragments of human fibronectin as well as several different subfragments and isolated type I "finger" modules were studied under various solvent conditions by differential scanning calorimetry and fluorescence spectroscopy. It was established that all fibronectin fingers constitute independently folded domains whose melting temperatures range from 54 to 108 degrees C. The difference between heat capacities of the native and denatured states (delta Cp) is low, about 0.03 cal/K-g, which is consistent with the relatively low percentage of hydrophobic amino acids and the consequent small change in non-polar surface area exposed to the solvent upon denaturation. The free energy of unfolding at 25 degrees, as calculated from the calorimetric data or measured directly by titration with GdmSCN is also small, in the range of 2.4 to 6.7 kcal/mol. The small delta G value and its flat dependence on temperature (determined by delta Cp) translates the small variations in delta G between fingers into large variations in tm. The small value of delta G also indicates that finger modules are structurally rather fragile which may account for their sensitivity to proteolysis; almost any cleavage within either of the two disulfide loops destroys the cooperative structure and abolishes the corresponding melting transition. The fact that some fingers exhibit large decreases in tm upon separation from more stable neighbors with which they interact can also be viewed as a consequence of the low values of delta G and delta Cp.

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

The domain structures and stabilities of fragments isolated from the so-called 'hep 2' region of plasma fibronectin have been investigated by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The 30 kDa hep-2A fragment contains three type III modules (III12 to III14), whereas the 40 kDa hep-2B fragment contains four such modules (III12 to III15). Melting of these fragments at neutral pH was irreversible and accompanied by rapid aggregation. In contrast, melting was completely reversible in 50 mM-glycine at pH 2.7, where DSC measurements revealed the presence of three independently folded domains in 30kDa hep-2A and four in 40 kDa hep-2B. That each domain represented a single module was confirmed by measurements with four single-module subfragments, all of which melted reversibly, even at neutral pH. At neutral pH in the presence of 6 M-urea, 30 kDa hep-2A melted reversibly in a sharp peak from which only two transitions could be resolved by deconvolution. Only the larger of these was stabilized by heparin and was assigned to modules III13 and III14. Upon isolation, module III13 melted at lower temperature than in the parent fragment where it is stabilized through an interaction with module III14. We conclude that all type III modules in the hep-2 region of fibronectin constitute independently folded domains. Modules III13 and III14 form a highly co-operative structure through functionally significant interactions that can be disrupted with acid or sufficient concentrations of urea or guanidinium chloride.

Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.

The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties.

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.

The interaction of the isolated kringles 4 and 5 from human plasminogen with 6-aminohexanoic acid, pentylamine, pentanoic acid and arginine has been quantitatively characterized by scanning calorimetry and fluorescent spectroscopy. It has been found that the ligands with the positively charged group have a good binding ability while pentanoic acid in comparison with 6-aminohexanoic acid being devoid of amino group does not interact with the kringles under study. The positively charged group of the ligand is suggested to play a crucial role in ligand binding with the lysine-binding site.

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.