In vivo catabolism of alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine

The clearances of 125I-labeled alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine (CH3NH2) were compared in our previously described mouse model. alpha 1-Proteinase inhibitor-trypsin cleared with a t 1/2 of 20 min, antithrombin III-thrombin of 7 min and 125I-labeled alpha 2-macroglobulin-methylamine of 2 min. Competition studies were performed to determine whether one or several pathways clear these three ligands. The clearance of 125I-labeled alpha 1-proteinase inhibitor-trypsin and 125I-labeled antithrombin III-thrombin was blocked by large molar excesses of either ligand, but not by alpha 2-macroglobulin-methylamine. The clearance of 125I-labeled alpha 2-macroglobulin-methylamine can be blocked by a large molar excesses of unlabeled alpha 2-macroglobulin-methylamine but not by alpha 1-proteinase inhibitor-trypsin. These studies demonstrate that the clearance of alpha 1-proteinase inhibitor-trypsin complexes is independent of alpha 2-macroglobulin-methylamine and utilizes the same pathway which is involved in the clearance of antithrombin III-thrombin complexes.     

Effect of antithrombin III, alpha 1-proteinase inhibitor and heparin on amidolytic activity of nerve growth factor (7S-NGF)

A series of potent inhibitors of angiotensin-converting enzyme (dipeptidyl carboxypeptidase, E.C. 3.4.15.1) derived from benzofused 1-carboxyalkyl-3-(1-carboxy-3-phenyl-propylamino) lactams (III) is described. In the most effective inhibitors (I50 2-4 X 10(-9)M) the lactam is 7 or 8 membered and the N-1 side chain is carboxymethyl or carboxyethyl. Conformational and steric factors pertinent to binding to the enzyme are discussed.     

Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.     

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor

-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human -thrombin shows multiple surface exosites for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). _T ^–-Thrombin and thrombin Quick I (Arg 67 Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. -Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (M_r = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5phosphate modifiedthrombin.     

Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases.

Human neutrophils use the H2O2-myeloperoxidase-chloride system to generate chlorinated oxidants capable of activating metalloproteinase zymogens that hydrolyze not only native and denatured collagens, but also the serine proteinase inhibitor (serpin) alpha 1-proteinase inhibitor (alpha 1 PI). To identify the metalloenzyme that hydrolyzes and inactivates alpha 1 PI, neutrophil releasates were chromatographed over gelatin-Sepharose and divided into fractions containing either progelatinase or procollagenase. The gelatinase-containing fraction cleaved alpha 1 PI in a manner inhibitable by native type V, but not type I, collagen. Conversely, while the collagenase-containing fraction also cleaved alpha 1 PI, this activity was inhibited by type I, but not type V, collagen. Because type I and V collagens are competitive substrates for collagenase and gelatinase, respectively, each of the metalloproteinase zymogens were purified to apparent homogeneity and examined for alpha 1 PI-hydrolytic activities. Both purified gelatinase and collagenase inactivated alpha 1PI by hydrolyzing the serpin within its active-site loop at the Phe352-Leu353 and Pro357-Met358 bonds, albeit with distinct kinetic properties. Furthermore, purified collagenase, but not gelatinase, cleaved a second serpin, alpha 1-antichymotrypsin, by hydrolyzing the Ala362-Leu363 bond within its active-site loop. These data demonstrate that human neutrophils use chlorinated oxidants to activate collagenolytic metalloproteinases whose substrate specificities can be extended to members of the serpin superfamily.     

DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor

This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases alpha(1)-proteinase inhibitor (alpha(1)PI), alpha(1)-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment ((30bp)DNA), used as a model of DNA, tightly binds cat G (K(d), 8.5 nM) but does not react with proteinase 3, alpha(1)PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whose K(i) is close to K(d). ACT and alpha(1)PI are slow binding inhibitors of the cat G-(30bp)DNA complex whose second-order rate constants of inhibition are 2300 M(-1) s(-1) and 21 M(-1) s(-1), respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, (30bp)DNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by alpha(1)PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases.     

Effect of heparin on the inhibition of thrombin by alpha 1-proteinase inhibitor.

Heparin interferes with the inhibition of thrombin by α₁-proteinase inhibitor (αPI). The inhibitory effect of heparin is due to its binding to thrombin. Other glycosaminoglycans and carboxyl-modified heparin do not have the same effect as heparin. The results indicate that there are similarities in the structural requirements in heparin, for anticoagulant activity and for the inhibition of αPI interaction with thrombin.     

Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, antithrombin III, alpha 2-antiplasmin, angiotensinogen, and ovalbumin.

Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the proteinase-complexed form, a stable equimolar complex between the inhibitor and a target proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the proteinase component of the serpin-proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit proteinases in a manner similar to that described for other types of protein inhibitors of serine proteinases.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

Several intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli were detected by stopped-flow spectrophotometry and by rapid-scanning spectrophotometry after conventional mixing. Structures were assigned to intermediates on the basis of kinetic and spectral evidence. In the early stages of the reaction an intermediate with the properties expected of a geminal diamine accumulated significantly. Changes consistent with the conversion of this species into the external aldimine were also observed. The course of product formation was determined and linked with spectral changes taking place in the bound coenzyme. The effect of the minor decarboxylation-dependent transamination that accompanies the major reaction was analysed.     

Kinetics and physiologic relevance of the inactivation of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin).

Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.     

Antithrombin action of phosvitin and other phosphate-containing polyanions is mediated by heparin cofactor II

We have examined the antithrombin effects of various phosphate-containing polyanions (including linear polyphosphates, polynucleotides and the phosphoserine glycoprotein, phosvitin) on the glycosaminoglycan-binding plasma proteinase inhibitors, antithrombin III (ATIII) and heparin cofactor II (HCII). These phosphate-containing polyanions accelerate the HCII-thrombin reaction, as much as 1600-fold in the case of phosvitin. The HCII-thrombin reaction with both phosvitin and polynucleotides appears to follow the ternary complex mechanism. The HCII-thrombin complex is rapidly formed in the presence of these phosphate polyanions (each at 10 micrograms/ml) when 125I-labeled thrombin is incubated with human plasma (ex vivo). None of these phosphate polyanions accelerate the ATIII-thrombin reaction. Our results suggest that the antithrombotic effect of these phosphate-containing polyanions is mediated by HCII activation and not by ATIII.     

Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha1-proteinase inhibitor (alpha1PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.     

Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition

The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.     

Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin

'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, and alpha(2)-macroglobulin. The chemically inactivated alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only anti-integrin (alpha(v)beta(3) and alpha(5)beta(1)) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.