Cross-linking of alpha 2-plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor

During blood coagulation alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked with fibrin by an activated fibrin-stabilizing factor (FSFa) plasma transglutaminase, activated coagulation factor XIII). When alpha 2PI was treated with FSFa in the absence of acceptor amino groups, the inhibitor lost more than 90% of its capacity to be cross-linked to fibrin because of hydrolysis of the gamma-carboxamides of FSFa-susceptible glutamine residues. Chemical modifications of the inhibitor's lysine epsilon-amino groups did not affect the cross-linking capacity of the inhibitor with fibrin, whereas the same chemical modifications in fibrinogen resulted in a remarkable loss of cross-linking capacity. These observations suggest that alpha 2PI plays a role as an acyl donor with its FSFa-susceptible glutamine residues in the cross-linking reaction with fibrin, and fibrin serves as an acyl acceptor with its lysine residues. The number of FSFa-susceptible glutamine residues/molecule of the inhibitor was estimated by measuring the maximum incorporation of [3H]histamine into the inhibitor and by analyzing the distribution of radioactivity in a tryptic digest of [14C]histamine-incorporated alpha 2PI.l It was found that each inhibitor molecule has one glutamine residue that is most susceptible to FSFa. When the radioactive histamine-incorporated inhibitor was reacted with excess amounts of plasmin, a small fragment carrying all the released radioactivity was rapidly released from the NH2-terminal part of the inhibitor moiety of the complex. The NH2-terminal amino acid sequence of the inhibitor was analyzed before and after treatment with FSFa or before and after incorporation of radioactive histamine. The glutamine residue at the second position from the NH2-terminal end was converted to a glutamic acid residue when the inhibitor was treated with FSFa. When the radioactive histamine-incorporated inhibitor ws analyzed, the radioactivity was found predominantly at the second position from the NH2-terminal end. These results indicate that the glutamine residue susceptible to FSFa in alpha 2PI is located next to the NH2-terminal residue.     

Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor

A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI.     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.     

Purification and characterization of alpha 1-thiol proteinase inhibitor and its identity with kinin- and fragment 1.2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

The primary inhibitor of plasmin in human plasma.

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.     

Application of antibody to synthetic peptides for characterization of the intact and truncated alpha 22 protein specified by herpes simplex virus 1 and the R325 alpha 22- deletion mutant.

The alpha 22 protein is one of five proteins synthesized immediately after infection of permissive cells with herpes simplex virus 1 and 2 (HSV-1 and HSV-2). On the basis of the reported nucleotide sequence of the HSV-1 gene, we synthesized two peptides containing the predicted amino acids 12 through 23 (12 residues) and 21 through 36 (16 residues) in two hydrophilic domains near the N terminus of the protein. Rabbit antisera made against these peptides were then used to characterize the alpha 22 protein made by wild-type HSV-1(F) strain and by an HSV-1 mutant, R325, carrying a 500-base-pair deletion within the coding domain of the gene. The results were as follows. (i) Both antisera reacted with HSV-1(F) alpha 22 protein in lysates electrophoretically separated in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet; neither antiserum reacted with the corresponding HSV-2 protein. The protein accumulated at 34 and 39 degrees C in the nucleus of infected permissive HEp-2 and baby hamster kidney (BHK) cells. The protein formed at least five spots differing in charge, mobility, and extent of phosphorylation on two-dimensional electrophoretic separation. (ii) The antisera reacted with a truncated nuclear protein (33,700 apparent molecular weight) in permissive HEp-2 and restrictive BHK cells infected with R325 and incubated at 39 degrees C but not at 34 degrees C. The truncated protein represents, therefore, the product of the undeleted 5' domain of the alpha 22 gene in R325. (iii) The presence of identical as well as slower migrating, reactive proteins in infected BHK cell lysates indicated that wild-type and truncated alpha 22 proteins are processed differently in BHK and HEp-2 cells.     

Expression, Purification, and Characterization of Recombinant Human Factor X

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the −2 residue (Thr → Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure–function relationships by mutational analysis.     

Alpha 2-antiplasmin Enschede is not an inhibitor, but a substrate, of plasmin.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.     

Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Location of four plasmin attack points and a covalent cross-linking site.

The amino acid sequence of a 38-residue midsection piece of the alpha chain of human fibrinogen has been determined using a combination of plasmin-derived peptides and cyanogen bromide fragments. The segment contains several important features, including four early plasmin attack points, one of the two alpha-chain cross-linking acceptor sites, and a peptide homologous to one isolated from plasmin digests of bovine fibrinogen, and reported to have anticoagulant activity. The segment is sequentially adjacent to and overlapping with a large molecular weight (20000-25000) fragment released during plasminolysis. This latter material is very rich in glycine and serine and deficient in nonpolar amino acids. It also contains the other alpha-chain cross-linking acceptor site.     

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.     

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.     

High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system

The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells. About 25% of the active enzyme was a novel, partially processed product of pOTC containing four extra amino acids at the amino terminus of OTC. The most abundant protein found in mitochondria from infected insect cells was the normal processing intermediate iOTC, which contains 8 extra amino acids at the amino terminus of OTC. Whereas this species, present at 20 micrograms/10(6) cells, was not active and did not bind the transition-state analog inhibitor of OTC, delta-PALO, the novel processing product did bind and was affinity-purified, along with mature OTC, on a PALO-affinity column. The OTC expressed in insect cells was located in the same compartment of the mitochondrion as in rat liver. The incomplete processing occurred in vitro in both noninfected and infected insect cells. The high level of expression of iOTC using the baculoviral expression system provides a means of overproducing an obligatory intermediate in the mitochondrial import process.     

Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets by (NH4)2SO4 fractionation and affinity chromatrography on Sepharose-linked purified plasminogen. The material contained none of the known plasma proteinase inhibitors when studied by crossed-immunoelectrophoresis and electroimmunoassay, but inhibited a clot-lysis-time assay and an esterolytic assay that used the synthetic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide). The inhibitory activity had the same mobility as the alpha 2-plasma proteins on preparative agarose-gel electrophoresis. Titration of the inhibitor preparation by active-site-titrated plasmin demonstrated a dissociation constant of approx. 0.1 nM. The inhibition was complete within 1 min. The inhibitor increased the mobility in agarose-gel electrophoresis of purified activator-free plasmin or 125I-labelled plasmin, as demonstrated by crossed-immunoelectrophoresis against specific immunoglobulins against plasminogen or by radioautography. The results strongly suggest the presence in platelets of a plasmin inhibitor different from the known plasma proteinase inhibitors.     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Expression of human alpha 2-macroglobulin cDNA in baby hamster kidney fibroblasts: secretion of high levels of active alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase, alpha 2M entraps the proteinase molecule in a reaction that involves large conformational changes in alpha 2M. We describe the molecular cloning of alpha 2M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human alpha 2M with a specific enzyme-linked immunosorbent assay. Human recombinant alpha 2M (r alpha 2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to alpha 2M purified from human serum. The results show that r alpha 2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r alpha 2M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference. Moreover, r alpha 2M-trypsin complex bound to purified human placental alpha 2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived alpha 2M and trypsin.