Plasma inhibitors

Plasma Inhibitors, In Vivo Anticoagulants. Fourteen naturally occurring compounds that normally exert an inhibiting effect on the activity of coagulation, platelet function, and fibrinolytic activity and complement systems have been identified within the circulating blood. 

The fibrinolytic reactions are controlled by endogenous blood plasma inhibitors as well [3]. 

Stimulation by thrombin does not lead to the generation of leukotriene C4 or B4, whereas stimulation of the same mast cells by the calcium ionophore, A23187, does. The secretory response elicited by thrombin is prevented by preincubation with AT-III, a plasma inhibitor of thrombin, or by hirudin, a thrombin inhibitor derived from the leech [135],... 

Heeb MJ, Espana F. alpha2-macroglobulin and Cl-inactivator are plasma inhibitors of human glandular kallikrein. Blood Cell Mol Dis 1998 24 412 119. 

If the elimination of the enzyme proceeds via binding to plasma inhibitors, it becomes evident that temporary inactivation of the active center blocks at... 

Tlte acylated enzyme does not react with the plasma inhibitors in the same way. However, it is obviously fixed to fibrin and deacylated there, so that the fibrinolytic effect remains restricted to the thrombus (Hg. 9). 

The acyl derivatives of kallikiein were prepared from porcine pancreatic kallikrein. In our experiments we used benzoyl-kallikiein, By deacylation the enzymatically active kallikrein was generated in plasma from benzoy 1-kallikrein demonstrated by means of its amidolytic activity. From the time course of reactivation of benzoy 1-kallikrein, a half-time of reactivation of 54 minutes was calculated. Benzoyl- kallikrein was protected from being inactivated by plasma inhibitors. Therefore, the kallikrein activity in nlasma was hicher after incubation... 

Elastase has been shown to have a pH optimum between 8.0 and 9.0 with most protein substrates and is strongly inhibited by the plasma inhibitor cti-PI. 

M42. Morton, R. E., and Zilversmit, D. B., A plasma inhibitor of triglyceride and cholesteryl ester transfer activities. J. Biol. Chem. 256, 11992-11995 (1981). 

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. 

Proteolytic enzymes (proteases) are involved in a wide variety of physiological processes including digestion, fertilization, coagulation, and the immune response. Outside of their normal environment, proteases can be extremely destructive and natural human plasma inhibitors inhibit most proteases that escape. Imbalance in protease-protease inhibitor systems can lead to a number of diseases of which pulmonary emphysema is one well-characterized example. This disease results when the protease elastase attacks elas-tin, the major elastic protein in the lung. Considerable effort has been devoted to the synthesis of inhibitors of proteolytic enzymes such as elastase for possible therapeutic use. In the future, specific and selective synthetic protease inhibitors should be useful for treating specific diseases that range from the common cold to chronic disorders such as emphysema. 

Since a major component of all cells is protein, proteases could be very destructive if they were not carefully controlled or compartmentalized. The potential seriousness of uncontrolled proteolysis can be recognized by the fact that ca. 10% of the proteins by weight found in human plasma are protease inhibitors. The currently recognized plasma protease inhibitors are listed in Table I (3,4). In addition to the plasma inhibitors, there are other inhibitors that are more localized and have not been as well characterized. 

Lucore CL, Sobel BE. Interactions of tissue-type plasminogen activator with plasma inhibitors and their pharmacologic implications. Circulation 1988 ... 

The proteolytic load a connective tissue endures during inflammation is a balance between the activity of the proteinases secreted by inflammatory cells and the local specific activity of functional plasma inhibitors. During inflammatory episodes, oxidative inactivation of specific inhibitors may move this delicate balance so that connective tissue macromolecule proteolysis is favoured (see Fig. 2). 

In particular, excessive proteolysis of elastin by HLE has been implicated in pulmonary emphysema [19]. In this case, the imbalance appears to result from reduced levels of active extracellular alpha,-proteinase inhibitor (a,-PI), the primary plasma inhibitor of HLE. This decrease is caused either by a genetic disorder (PiZZ phenotype individuals) or by reduction in the elastase inhibitory capacity (EIC) of ai-PI due to its oxidative inactivation by tobacco smoke [20]. The detailed evidence supporting the potential role of elastase in the development of emphysema has been extensively reviewed [21] and will not be repeated here. The fact that HLE is also a potent secretagogue [22] may play a role in several disease states, including cystic fibrosis [23], chronic bronchitis [24], and acute respiratory distress syndrome (ARDS) [25]. The mechanism of the secretagogue activity is not known, but, since the HLE-induced secretion can be blocked by specific HLE inhibitors, it appears to require catalytic activity by the enzyme [26]. 

The two main plasma inhibitors of HLE are a,-proteinase inhibitor (aj-PI) and a2-macroglobulin (CX2-M). Comparison of the ti,2inact calculated for a,-PI and a2-M Table 2.1) indicates that apPI should be considered the primary plasma inhibitor of HLE. This assumption is corroborated by the finding that when mixtures of human serum and labelled HLE are analyzed they show that 92% of the HLE is complexed to aj-PI and only 8% is complexed to 2-M [53]. 

The major plasma inhibitor of Factor XIa is Uj-antitrypsin while antithrombin III, in the presence or absence of heparin, plays a minor role in controlling this enzyme (S5). Interestingly, plant inhibitors like soybean, com, and lima bean trypsin inhibitors are able to block Factor Xlla and kallikrein but only mildly inhibit Factor XI. Thus, one can use these agents to minimize cross-reactivity in an indirect s)mthetic substrate assay for Factor XIa. 

The amount of thrombin inhibited reflects the amount of AT-III in the sample. The addition of heparin reduces interference by other plasma inhibitors like a2-macroglobulin. It has been reported that the species of thrombin, bovine or human, used in the assay can influence abnormally low AT-III values. Friberger and co-workers in a 1982 report recommended bovine thrombin to insure better agreement with immunological AT-III values (F9). 

The enzymatically inactive acyl-urokinase was reactivated in plasma at 37°C with a reactivation half-time of 8 minutes (benzoyl-urokinase) or 10 hours (p-guanidinobenzoyl-urokinase). Upon administration to rabbits, urokinase was more rapidly eliminated than either acyl enzyme (Fig. 11). The results suggest that urokinase is eliminated via the binding to plasma inhibitors. Thus, it could be shown that the clearance of urokinase is modified significantly on acylation. 

Even though serine proteinases share a common mechanism of peptide bond cleavage, they differ dramatically in their primary substrate specificity, exhibiting a preference for a certain type of amino acid residue. Hydrolysis studies using natural and synthetic substrates have demonstrated that NE preferentially cleaves peptide bonds after small aliphatic amino acid residues such as valine and alanine [21]. Elastase has been shown to have a pH optimum between 8.0 and 9.0 with most protein substrates and is strongly inhibited by the plasma inhibitor ai-PI. 

Inhibitors of transfer proteins may be present in the cytosol or plasma. Morton and Zilversmit (1981) have partially purified a human plasma inhibitor of the triglyceride and cholesteryl ester transfer activities. It is a protein with a molecular weight of approximately 35,000. Crude extracts of potato tuber and the bacteria Rhodopseudomonas sphaeroides appear to be devoid of transfer activity (Kader, 1975 Cohen et al., 1979), but activities could be detected after the initial purification steps. 

Spinas, G. A., Bloesch, D., Kaufmann, M. T., Keller, U., and Dayer, J. M. Induction of plasma inhibitors of interleukin-1 and TNF-alpha activity by endotoxin administration to normal humans. Am. J. Physiol 2S9, R993-R997 (1990). 

Specific serum/plasma inhibitor of muscle adenylate deaminase. IRCS Med. Sci. 9 178-179. 

However, the activity tests used were all of the vitro type with no results reported for vivo systems. The same group has found that these compounds act by inhibiting the naturally occurring plasma inhibitors of fibrinolysis thus permitting the flbrinol lc activity normally present in plasma to evince itself strongly. Hansch has developed a physicochemical approach to studying structure-activity relationships of the above classes of fibrinolytics. 

---