Plasmin substrate

Fibrinolytics. Figure 2 Various fibrin structures for plasmin. Fibrinogen (Fg) is converted to fibrin (F) by thrombin (T), and thrombin can also convert factor XIII (XIII) to activated factor XIII (Xllla). The latter produces crosslinks between fibrins (FxxF) and also may crosslink fibrin with a2-plasmin inhibitor (FxxFxxPI). The efficiency of digestion of these plasmin substrates by plasmin, resulting in the soluble fibrin degradation products (FDP), is different. The amount of FDP formed in time is expressed in arbitrary units. 

Relative Sensitivity and Selectivity of Various Plasmin Substrates"... 

The kinetic data for the action of plasmin on different substrates under various conditions have been summarized. The MichaeHs-Menten constant (iC ) varies between 10 and 1000 mAf and the catalytic constant between 1 and 75. ... 

One of the most efficient plasmin inhibitor is a2-PI (70 kDa), which is synthesized by the liver, secreted into the blood circulation, where its concentration is 1 pM. It rapidly forms equimolar complex with plasmin, and in this complex, the active site of the enzyme is irreversibly blocked. The complex, thereafter, is removed by the liver. It is remarkable that when plasmin is bound to its substrate (fibrin), it is protected against its primarily inhibitor, a2-PI the rate of inactivation decreases by 400-fold (Fig. 4) [3]. 

Fibrinolytics. Figure 3 Plasminogen activation (a) Kinetics of plasminogen activation by uPA (urokinase-type) and tPA (tissue-type) plasminogen activators. Effect of fibrin (b) Ternary complex formation between enzyme (tPA), substrate (Pg) and cofactor (F) Abbreviations plasmin (P), fibrin (F), plasminogen (Pg). Plasmin, formed in time, is expressed in arbitrary units. 

The carboxy-terminal region in apolipoprotein (a) closely resembles the protease domain in plasminogen [eight amino acid substitutions, nine amino acid deletions, and one insertion in apo(a) relative to plasminogen, with 94% overall nucleotide sequence identity] (G28). The most important difference is the substitution of arginine by serine in the site responsible for proteolytic activity (position 4308) (G28). As a result, Lp(a) has no protease activity towards substrates for plasmin (J3). Salonen (SI) reported a serine-protease activity of Lp(a) towards fibronectin, a glycoprotein present in connective tissue matrices. 

It is generally believed that proteases enhance cancer spread primarily by catalyzing degradation of the extracellular matrix. Since multiple substrates are encountered in this matrix, a number of different proteases are likely to be required to complete the metastatic process. Multiple proteases may also be required to activate different inactive precursor forms. Thus, in vitro, plasmin (D7), cathepsin B (D7), and a trypsin-like protease (K12) can all activate pro-uPA, while plasmin, which results from the action of uPA on plasminogen, can activate certain metalloproteases (M4). As mentioned earlier, completion of the metastatic process may require a cascade of different proteases operating, as shown in Fig. 2. 

Interestingly, a closely related prodrug of doxorubicin that has the same spacer (i.e., 6.33) was found to be a good substrate for plasmin while being stable in buffer and blood serum [55], A marked selectivity was seen against a plasmin-generating MCF-7 breast cancer cell line. 

Backes BJ, Harris JL, Leonetti F, et al. Synthesis of positional-scanning hbraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol 2000 18 187-93. 

Overall, therefore, activation of the thrombolytic cascade occurs exactly where it is needed— on the surface of the clot. This is important as the substrate specificity of plasmin is poor, and circulating plasmin displays the catalytic potential to proteolyse fibrinogen, factor V and factor VIII. Although soluble serum tPA displays a much reduced activity towards plasminogen, some free circulating plasmin is produced by this reaction. If uncontrolled, this could increase the risk of subsequent haemorrhage. This scenario is usually averted, as circulating plasmin is rapidly... 

Tincture of the dried seed, on agar plate at a concentration of 30 p,L/disc, was inactive on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Extract of 10 g plant material in 100 mL ethanol was used b Anticoagulation activity. Serpin BSZx (an inhibitor of trypsin and chemotrypsin) inhibited thrombin, plasma kallikrein, factor Vlla/tissue factor, and factor Xa at heparin-independent association rates. Only factor Xa turned a significant fraction of BSZx over as substrate. Activated protein C and leukocyte elastase were slowly inhibited by BSZx, whereas factor Xlla, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein, and elastase were not or only weakly affected. Trypsin from Fusarium was not inhibited, while interaction with subtilisin Carlsberg and Novo was rapid, but most BSZx was cleaved as a substrate L... 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

A fibrin clot containing adsorbed plasmin inhibitors is difficult to define in a chemical or physical sense. Generally, when enzyme reactions occur at surfaces, the porosities and adsorption properties erf which are variable, the reproducibility of enzyme assay methods is questionable. The proteinoses, to which belong the most important pharmaceutical enzymes, may present some difficulties when natural substrates (protein ) are prescribed. Here, the application of a parallel run with a reference standard is recommended. 

Fig. 3. uPA and plasmin cleavage sites in the linker region and in domain I of uPAR. The uPA cleavage sites in uPAR have been experimentally determined [17]. The plasmin (pli) cleavage sites have been deduced from its substrate specificity hydrolyzing peptide bonds with R or K residues on their carboxyl side. The amino acids of the functional epitope of mAb R3 are indicated [8],... 

It is well known that the specificity of an enzyme such as thrombin and plasmin is very close to that of trypsin. In this respect, inverse substrates for trypsin also are expected to be susceptible to the catalysis by these enzymes. In the kinetic analysis of trypsin-like enzymes toward p-amidinophenyl esters, it was found that the inverse concept is also applicable to thrombin, plasmin, urokinase, kallikrein, and trypsins from various origins 74 - 75). These enzymes are not distinctively different from bovine-... 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

A new approach to thrombosis therapy using acyl plasmins has been reported by Smith et al.78). Acyl plasmin is catalytically inert and unable to react with plasma inhibitors but still can bind to a fibrin clot. Thus, after the administration, acyl plasmin can circulate without being trapped by the inhibitors and can come into contact with fibrin. Deacylation may then occur to give a fibrin-plasmin complex and this active enzyme is expected to lead to fibrinolysis. The preparation of acyl plasmin of appropriate stability was realized by using the general procedure for the specific synthesis of an acyl enzyme — the inverse substrate method. 

C-Methylated -Casein as a Substrate for Plasmin and Its Application to the Study of Milk Protein Transformations... 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

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