Human plasma alpha 2-macroglobulin : an inhibitor of plasma kallikrein
01 Aug 1970-Journal of Experimental Medicine (The Rockefeller University Press)-Vol. 132, Iss: 2, pp 329-352
TL;DR: The studies suggest that the α2-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kall Kikrein enzyme systems.
Abstract: Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified alpha(2)-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the alpha(2)-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the alpha(2)-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, alpha(2)-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the alpha(2)-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The alpha(2)-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the alpha(2)-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The alpha(2)-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the alpha(2)-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.
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TL;DR: In this paper, it was found that α2-macroglobulin is comprised of subunit chains of 185,000 molecular weight as analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate.
Abstract: Human plasma α2-macroglobulin is an inhibitor of circulating proteases that function in hemostatic and inflammatory reactions but the biochemical nature of its interaction with these enzymes is not well defined. This investigation has found that α2-macroglobulin is comprised of subunit chains of 185,000 molecular weight as analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Trypsin, thrombin, plasmin, and plasma kallikrein in amounts completely bound to α2-macroglobulin attacked one region in the subunit chain producing a single derivative with a molecular weight of 85,000 indicating that hydrolysis occurred at or near the center of the parent chain. The proteolytic derivative was also identified in an α2-macroglobulin preparation from plasma incubated with the plasminogen activator, urokinase. α2-macroglobulin functionally capable of binding enzyme appeared to be required both for limiting tryptic hydrolysis and for confining the concentration dependent increase in the derivative chain to the 1st min of incubation since acid-denatured α2-macroglobulin that failed to bind trypsin was extensively degraded. Three derivative chains resulted from the interaction of α2-macroglobulin with chymotrypsin demonstrating the presence of at least two chymotrypsin susceptible regions in the precursor chain. Reduction of the α2-macroglobulin-enzyme mixture was required for the identification of the derivative subunit chains establishing that these cleavage products were covalently linked to the parent molecule by disulfide bridges. Thus, α2-inacroglobulin acts as a substrate for circulating proteases, a finding which may also pertain to the mechanism of action of other plasma enzyme inhibitors.
337 citations
Cites background or methods from "Human plasma alpha 2-macroglobulin ..."
...Prior studies have indicated that plasma proteases may be activated and bound to ~z2-macroglobulin during the procedures involved in blood collection and protein purification unless appropriate precautions are employed (4, 8)....
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...The substrates used were BAPNA for trypsin (20), AGLMe for plasmin (4), and TAMe for ka]likrein (8)....
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...The activity of plasmin preparations was assayed by a standard caseinolytic assay (21); and that of kallikrein by its TAMe esterase activity (l U of activity equaling that volume of enzyme which hydrolyzed 1 #mol TAMe per min) (8)....
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...--Partially purified human plasma kallikrein was prepared as described (8)....
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...ce2-macroglobulin was prepared from human plasma by a modification of previously described methods (8)....
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TL;DR: New information is provided concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits to provide new information concerning the enzyme-inhibitor reaction.
Abstract: This study has explored the nature of the molecular events which occur when C1 inactivator, a human plasma inhibitor of the complement, kinin-forming, coagulation, and fibrinolytic enzyme systems, interacts with C1s, plasmin, and trypsin. Purified inhibitor preparations demonstrated two bands, when examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The molecular weights of the major and minor bands were 105,000 and 96,000 daltons, respectively. The minor component appeared to be immunologically and functionally identical to the main C1 inactivator component. Loss of C1s and plasmin functional activity was associated with the formation of a 1:1 molar complex between the inhibitor and each enzyme. These complexes were stable in the presence of SDS and urea. The light chain of both these enzymes provided the binding site for C1 inactivator. Complex formation and enzyme inhibition occurred only with native and not with an inhibitor preparation denatured by acid treatment, thereby demonstrating the importance of conformational factors in the enzyme-inhibitor reaction. Although peptide bond cleavage of the C1 inactivator molecule by C1s was not documented, plasmin was found to degrade the inhibitor with the production of several characteristic derivatives. At least one of these products retained the ability to complex with C1s and plasmin. Trypsin, which failed to form a complex with C1 inactivator, degraded the inhibitor in a limited and sequential manner with the production of nonfunctional derivatives one of which appeared structurally similar to a plasmin-induced product. These studies therefore, provide new information concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits.
199 citations
185 citations
TL;DR: The results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma, and that high-Mr kininogen-deficient plasma reconstituted with high- Mr kin inogen does not protect kall Kikrein from inactivation in the plasma milieu.
Abstract: Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.
164 citations
TL;DR: Results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, alpha(2)-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors.
Abstract: This study demonstrates that human plasma α2-macroglobulin preparations possess an enzymic activity that degrades fibrinogen, resulting in the formation of products whose structure resembles that of circulating fibrinogen catabolites. The sequence of degradation is similar to that observed in plasmin-catalyzed digests, in that Aα-chain fragmentation precedes that of Bβ-chain. The addition of plasminogen activators to plasma induced an increase in the N-α-tosyl-l-arginine methyl ester HCl esterase and fibrinogenolytic activity associated with α2-macroglobulin purified from this plasma, indicating that the enzymic activity of the complex was preserved and could be increased in the presence of other plasma enzyme inhibitors. Immunochemical studies demonstrated that an α2-macroglobulin-plasmin complex had formed in urokinase-treated plasma. This α2-macroglobulin preparation manifested an esterolytic profile like that of a complex prepared from plasmin and purified α2-macroglobulin. After complex formation with α2-macroglobulin in plasma, plasmin retained less than 0.1% of its fibrinogenolytic activity. That plasmin expressed its activity while bound to α2-macroglobulin was suggested by immunoprecipitation of this activity with α2-macroglobulin antibody and by the demonstration that pancreatic trypsin inhibitor did not effectively inhibit its fibrinogenolytic or esterolytic activity. These results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, α2-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors.
157 citations
Cites background or methods or result from "Human plasma alpha 2-macroglobulin ..."
...pared from platelet-poor human plasma, processed entirely in plastic containers in the presence of soybean trypsin inhibitor, was associated with a low level of TAMe esterolytic activity, a finding previously reported (7) and extended in this study to include those processed in the presence of Polybrene or e-aminocaproic acid....
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...a2-Macroglobulin was prepared from these plasma samples by the following procedures, described previously in detail (7)....
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...One such inhibitor, plasma a2-macroglobulin, apparently participates in the regulation of several different plasma enzyme systems, since it has been shown in vitro to form a complex with plasmin (1-3), thrombin (4-6), and kallikrein (7-9), resulting in inhibition of proteolytic activity....
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...kallikrein (7), or cationic aspartate aminotransferase (44)....
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References
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TL;DR: Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin, which results in a proportional gain in the inhibiting activity.
Abstract: A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mmicro.
2,335 citations
1,208 citations
"Human plasma alpha 2-macroglobulin ..." refers methods in this paper
...Immunodectrophoresis (22), double diffusion analysis (23) and quantitative radial immunodiffusion (24) were performed by established techniques....
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TL;DR: Waaler has demonstrated that long times with normal plasma may be caused by inadequate contact activation of the test plasma when, and it is shown that the partial thromboplastin time technic is not widely used in clinical laboratories.
Abstract: The partial thromboplastin time (PPT) test consists of recalcifying plasma in the presence of a lipid reagent that supplies optimal platelet thromboplastic factor-like activity. Thus, the test measures the overall adequacy of the intrinsic plasma-clotting factors. It has proven particularly valuable in the recognition of hemophilia and the hemophilioid states, i.e., deficiencies of antihemophilic globulin (AHG, Factor VIII), of plasma thromboplastin component (PTC, Factor IX), of plasma thromboplastin antecedent (PTA), and of Hageman factor (HF). Despite its simplicity, the PTT is not widely used in clinical laboratories. Instead, when faced with the task of screening patients for possible plasma thromboplastic factor deficiencies, most laboratories either rely upon insensitive or erratic technics, such as clotting times or prothrombin consumption tests, or resort to the complicated thromboplastin generation test. This neglect of the partial thromboplastin time technic has stemmed partly from the lack of a commercially available partial thromboplastin and partly from a failure to obtain short clotting times consistently with normal plasma in some laboratories. Waaler has demonstrated that long times with normal plasma may be caused by inadequate contact activation of the test plasma when
994 citations
TL;DR: Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin, which results in a proportional gain in the inhibiting activity.
Abstract: A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound.
The inhibitor has no effect on pepsin.
The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity.
Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin.
Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ.
815 citations
"Human plasma alpha 2-macroglobulin ..." refers background in this paper
...1 to convert it to the concentration of SBTI expressed in milligrams per milliliter (16)....
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TL;DR: In this paper, the control of Dicumarol therapy and the Quantitative Determination of Prothrombin and Proconvertin were discussed, and the authors proposed a method for the quantification of ProConvertin and Thrombin.
Abstract: (1951). The Control of Dicumarol Therapy and the Quantitative Determination of Prothrombin and Proconvertin. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 3, No. 3, pp. 201-208.
653 citations