Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3

Purified plasma and urinary protein C inhibitors (PCI) formed heparin-dependent complexes with activated protein C (APC) which were detected by immunoblotting after nondenaturing gel electrophoresis. Bands representing APC.PCI complexes were also seen on immunoblots after incubation of plasma with APC and heparin. The same immunoblot pattern of complexes was detected by three different methods: method A, monoclonal antibody to plasminogen activator inhibitor-3 (PAI-3, urinary urokinase inhibitor) + 125I-labeled anti-mouse IgG; method B, polyclonal antibodies to PCI + 125I-labeled purified plasma PCI; and method C, monoclonal antibody to protein C + 125I-protein C. Plasma depleted of PAI-3 by immunoadsorption with insolubilized monoclonal antibody to PAI-3 showed no detectable antigen or complexes with APC as visualized by methods A or B. This PAI-3-depleted plasma had less than 10% of the heparin-dependent inhibitory activity of normal plasma toward APC. Purified plasma PCI was fully reactive in an enzyme-linked immunoabsorbent assay for PAI-3, and plasma and urinary PCI inhibited urokinase activity in a heparin-dependent manner. These data indicate that heparin-dependent plasma and urinary PCI and PAI-3 are immunologically and functionally very similar if not identical. This observation identifies a new interrelation between the protein C anticoagulant and the fibrinolytic systems. In addition, plasma contains a heparin-independent inhibitor of APC which is not immunologically related to plasma PCI or to PAI-3.     

Assignment of the structural gene coding for albumin to human chromosome 4

Summary Albumin is a developmentally regulated serum protein synthesized in the liver mainly during adulthood. Family studies using variant forms of albumin established autosomal linkage between albumin and group-specific component protein (GS). Since GC has been assigned to human chromosome 4, albumin can be indirectly assigned to the same chromosome; however no direct assignment has been made. Recently, the human albumin cDNA probe has been isolated and characterized. It thus permits a direct chromosomal assignment of the albumin gene in the human genome. When the cDNA probe was hybridized to the HindIII digested total human DNA, an intense band at 6.8 kb was present. When the probe was hybridized to the HindIII digested Chinese hamster CHO-K1 DNA, a less intense band at 3.5 kb was found, plus three other faint bands. When the probe was hybridized to a series of human/CHO-K1 cell hybrids retaining a complete hamster genome and various combinations of human chromosomes, it was evident that hybrids containing human albumin gene sequences could be readily distinguished from hybrids containing no human albumin gene. Analysis of 22 primary cell hybrids for the presence or absence of human albumin sequences has assigned the albumin gene to human chromosome 4. Similar results were obtained using another restriction endonuclease EcoR1. Thus, by direct assay of the genomic albumin gene sequences in the cell hybrids, we provide evidence for a direct assignment of the structural gene for human albumin to chromosome 4.     

Assignment of a gene for tryptophanyl-transfer ribonucleic acid synthetase (E.C. 6.1.1.2) to human chromosome 14

We describe a simple method for locating tryptophanyl-tRNA synthetase (E.C. 6.1.1.2) on cellulose acetate gels (Cellogel) following electrophoresis. Employing electrophoretic conditions which result in the separation of mouse and human tryptophanyl-tRNA synthetases, we have analyzed extracts of a number of independently derived mouse-human somatic cell hybrids and subclones derived from these hybrids for the presence of human tryptophanyl-tRNA synthetase. Electrophoretic patterns of hybrid extracts which contain human tryptophanyl-tRNA synthetase exhibit three bands. This is consistent with published evidence that the enzyme from mammalian cells is a homologous dimer. The electrophoretic patterns derived from some hybrids are unusual in that the human and hybrid bands of activity are more intense than the mouse band from the same hybrid. An analysis of hybrid cells and extracts indicates that human tryptophanyl-tRNA synthetase segregates with human chromosome 14 and with the only enzyme marker which has previously been assigned to this chromosome, nucleoside phosphorylase.R. M. D. was supported by a postdoctoral fellowship from the Damon Runyon Fund for Cancer Research. The work described was supported in part by grants from Cancer Research Campaign, the Medical Research Council, and NATO.     

Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2.

The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.     

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C.

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).     

Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP-1, another activating protein, or with extracts of CHO-K1 cells. A series of 22 human/Chinese hamster ovary cell hybrids containing different human chromosomes were examined by this method. All eight hybrid clones containing human chromosome 10 were found to have crossreacting protein in this region. Other chromosomes could be excluded by this method. From these results, we conclude that the gene coding for human SAP-2 is located on chromosome 10.     

Assignment of the gene coding for hepatocyte growth factor-like protein to mouse chromosome 9

The gene coding for hepatocyte growth factor-like protein has been localized to mouse chromosome 9 at a locus (Hgfl) distal to the Trf locus. The likely gene order in this region is centromere-Trf-Gnai-2-Hgfl-Cck. The region surrounding the Hgfl locus shows homology of syntenty to band p21 on human chromosome 3.     

Assignment of the human gene for histidine-rich glycoprotein to chromosome 3

Histidine-rich glycoprotein (HRG) is a monomeric plasma glycoprotein involved in the modulation of coagulation and fibrinolysis. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HRG-specific cDNA probe, the HRG gene was assigned to chromosome 3. One hybrid that was known to contain only a segment of chromosome 3 also reacted positively with the HRG probe. Hybridization analysis with a set of chromosome 3-specific probes showed that the segment of chromosome 3 present in this hybrid is missing the region pter-p14, which indicates that HRG is not located in this region. No restriction fragment length polymorphisms were detected for HRG with 10 commonly used restriction enzymes.     

Assignment of a structural gene for beta-glucuronidase to human chromosome C7.

An electrophoretic technique was developed which allows the separation of human beta-glucuronidase (GUS EC 3.2.1.3.1) from the enzyme present in cultured murine. Chinese and Syrian hamster cells in one buffer system on Cellogel. Using this technique a number of independent human-mouse somatic cell hybrids have been analyzed for the segregation of GUS, other enzyme markers, and all human chromosomes. The results indicate that a structural gene for human beta-glucuronidase is located on chromosome C7.     

Assignment of the human protein C gene (PROC) to chromosome region 2q14----q21 by in situ hybridization

The protein C gene (PROC) was mapped by in situ hybridization. A genomic DNA probe containing the first three exons was 3H-labeled by nick translation, and this was then hybridized in situ to human chromosome preparations. The results localize the gene to 2q14----q21.     

Assignment of the gene coding for human peroxisomal 3-oxoacyl-CoA thiolase (ACAA) to chromosome region 3p22----p23

The chromosomal location of the human gene coding for peroxisomal 3-oxoacyl-CoA thiolase (ACAA) was determined with the aid of cDNA and genomic probes by screening of rodent x human somatic cell hybrids and in situ hybridization. The results localize the gene to chromosome region 3p22----p23.     

Assignment of human pepsinogen C (PGC) gene to chromosome 6

cDNA of rat pepsinogen C (PGC) hybridizes to, among others, a 3.2-kb band in Southern blot analysis of BamHI-cleaved human genomic DNA. This property was employed to localize the human PGC gene. Use of flow-sorted human chromosomes and 12 human x mouse somatic cell hybrid lines demonstrated that the gene is located on chromosome 6.     

Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.     

Structure of the gene for human plasminogen activator inhibitor-2. The nearest mammalian homologue of chicken ovalbumin

Plasminogen activator inhibitor-2 (PAI-2) can regulate the formation of plasmin by inhibiting urokinase and tissue plasminogen activator. PAI-2 is induced in monocytes and endothelium by inflammatory mediators, and it is made in the placenta during pregnancy. PAI-2 is a member of the serine protease inhibitor gene family, and it is particularly similar to chicken ovalbumin. Like ovalbumin, PAI-2 is secreted without cleavage of a signal peptide. To determine the structure of the PAI-2 gene, two bacteriophage lambda human genomic DNA libraries were screened with PAI-2 cDNA probes. Characterization of three positive clones shows that the human PAI-2 gene spans 16.5 kilobases and has eight exons. The 5'-untranslated sequence of the PAI-2 mRNA is 77 base pairs in length as suggested by primer extension and S1 nuclease mapping. The eukaryotic consensus sequence TATAAAA is found 22 base pairs 5' of the proposed cap site. The PAI-2 gene is on chromosome 18q21-23 as determined by hybridization to flow-sorted chromosomes and by in situ hybridization. There appear to be two common PAI-2 alleles that differ by six nucleotides in exons 1, 4, and 8. The structure of the PAI-2 gene is quite different from that of PAI-1 although these two inhibitors have common target protease specificity. In contrast, the structure of the PAI-2 gene is very similar to that of the chicken ovalbumin gene. When protein sequences are aligned to obtain maximal identity, six of the seven intron positions in the PAI-2 gene are identical to those in the chicken ovalbumin gene. We conclude that PAI-2 is the closest mammalian homologue of avian ovalbumin.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.