The molecular interaction of human salivary histatins with polyphenolic compounds.

Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst-polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mM dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.

Proteolytic processing of a human salivary proline-rich protein precursor by proprotein convertases.

Salivary proline-rich proteins (PRPs) are synthesized as precursors that are cleaved before secretion giving rise to glycosylated PRPs which have lubricating function and basic PRPs which are potent precipitators of dietary tannins. The putative cleavage sites in the precursors for basic and glycosylated PRPs all conform to the sequence RSXR downward arrowS (X can be A, S or P) in agreement with the recognition sequence (RXXR downward arrow) for various proprotein convertases. PRB4S, a proprotein giving rise to a basic PRP (IB-5) as well as a glycosylated PRP (II-1) was synthesized by in vitro transcription-translation. It was cleaved by furin at RSAR downward arrowS(173-178) giving rise to two proteins II-1 and IB-5. Similarly another precursor with the sequence RSAR downward arrowS(173-178) was also cleaved by furin. This together with previous results show that in vitro furin can cleave all RSXR downward arrowS sequences in the proproteins that give rise to glycosylated and basic PRPs. To demonstrate cellular cleavage, a human submandibular cell line (HSG) was transfected with a vector encoding PRB4S. This resulted in secretion of II-1 and IB-5. The degree of cleavage was enhanced by coexpressing furin and PRB4S. No cleavage occurred if the cells expressed a mutant PRB4S, R177Q, where the furin cleavage site had been destroyed. Cleavage was also inhibited if a furin inhibitor was coexpressed with PRB4S. Incubating the cells at 20 degrees C which blocks exit of proteins from the trans-Golgi network demonstrated that cleavage occurs before exit of the proteins from this network. These results show that furin may be responsible for in vivo cleavage of PRP precursors. Transfecting furin-deficient RPE.40 cells with a vector encoding PRB4S also led to secretion of II-1 and IB-5 showing that convertases other than furin can also cleave PRB4S in tissue culture.

Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein.

Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.

Interaction of tannin with human salivary histatins.

The ability of all major human salivary histatins to precipitate condensed tannin was demonstrated, and it was found that histatins 3 and 5 share the same condensed tannin-binding region but less tannin bound to histatin 1. The condensed tannin-binding region of histatin 5 includes both the N- and the C-terminal parts, although more tannin binding occurs in the C-terminal region. Epigallocatechin gallate (EGCG) showed similar binding characteristics as condensed tannin, but much less EGCG was precipitated. Pentagalloyl glucose (PGG) was precipitated equally well by histatins 1, 3, and 5 and bound equally well to the N- and C-terminal regions of histatin 5. In contrast to condensed tannin, cleaving histatin 5 into N- and C-terminal fragments increased their ability to precipitate PGG. Together, these results show a number of differences in the nature of interaction of histatins with condensed tannin, EGCG, and PGG. Most of the condensed tannin-protein complexes remained insoluble under conditions similar to those in the stomach and the small intestine, suggesting that histatins may act as a defense against dietary tannin in humans.

Encoding of human basic and glycosylated proline-rich proteins by the PRB gene complex and proteolytic processing of their precursor proteins.

Proline-rich proteins (PRPs) constitute a family of about 20 members in human saliva that are encoded by six genes. Assignment of genomic DNA coding regions is complicated because of the occurrence of many alleles and the great similarity of amino acid sequences of PRPs. To overcome these problems, the nucleotide sequences of the genes encoding basic and glycosylated PRPs from one person were determined and then aligned with her previously determined protein sequences. This, together with additional protein data, has also resolved various discrepancies between corresponding protein and DNA sequences. For the first time in one person it is now possible to account for all the regions in the PRB genes encoding basic and glycosylated PRPs, and the primary structures of all secreted basic and glycosylated PRPs have been determined. Each gene encodes a precursor protein that subsequently undergoes proteolytic cleavage, thereby giving rise to the secreted proteins. The results have allowed identification of all the proteolytic cleavage sites in the precursor proteins, which all conform to a consensus cleavage site for furin. To evaluate if furin is responsible for the precursor protein cleavages, a recombinant precursor protein was synthesized by in vitro transcription translation of a PRB1 allele. The protein was shown to be correctly cleaved by furin, giving rise to the expected secreted proteins.

Interaction of tannin with human salivary proline-rich proteins.

Tannins are polyphenolic compounds, widely distributed in plant-based foods, which have harmful effects on animals including humans. Salivary proline-rich proteins (PRPs) may act as a defence against tannins by forming complexes with them and thereby preventing their interaction with other biological compounds and absorption from the intestinal canal. The aim here was to compare the ability of members of the family of human PRPs to form insoluble complexes with tannin and to assess the stability of such complexes under conditions similar to those in the alimentary tract. Basic PRPs (BPRPs), which have no other known biological functions, were very effective in forming insoluble complexes with both condensed tannin and tannic acid. Practically no tannin bound to acidic PRPs (APRPs) and glycosylated PRPs (GPRPs), suggesting that tannin in the diet would not affect their biological activities. There were only small differences in the tannin-precipitating ability of various BPRPs of different sizes or sequences, indicating that, although there is considerable phenotypic variation of PRPs, it is not likely to cause marked individual variation in tannin-binding ability. Tryptic digestion of an APRP led to a marked increase in tannin binding to the resulting proline-rich peptides, supporting observations in other studies that there may be an interaction between the proline-poor N-terminal and the proline-rich C-terminal regions in native APRPs, which inhibits the biological activities of the proteins. Deglycosylation of a GPRP also led to a dramatic increase in tannin-binding ability, showing that the carbohydrate side-chains prevent binding of tannin. Most of the condensed tannin-PRP complexes remained insoluble under conditions similar to those in the stomach and small intestine, supporting the proposal that PRPs act as a defence against tannin.

Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins.

Phosphoproteins in human saliva include proline-rich proteins, statherins, histatin 1 and cystatin SA-III. The presence of phosphate in these proteins is necessary for various functions in the mouth including calcium binding, inhibition of precipitation of calcium phosphate, inhibition of growth of hydroxyapatite crystals and adherence to hydroxyapatite. To elucidate the process of phosphorylation of these proteins, the phosphorylation of a peptide (APRP8) with an amino acid sequence identical to one of the phosphorylated sites in acidic proline-rich proteins by a kinase from the human sublingual gland was investigated. The kinase, which was highly labile, was purified 58-fold by fractionation of sublingual gland homogenate and gel filtration, but the enzyme was inactivated when further purification by chromatographic techniques commonly used for protein kinases was attempted. To compare the enzyme with other kinases, and to obtain information that could be used in its further purification, a characterization was undertaken. The enzyme required 10 mM Mg2+ for optimum activity, it had a KM of 0.09 mM for ATP and the KM for the peptide substrate APRP8 was 0.42 mM. It was not activated by cAMP or calmodulin, characteristics that are shared with casein kinases and mammary gland kinase. The sublingual kinase as well as casein kinase 2 were inhibited by heparin, but in other respects the two kinases had different properties. While casein kinase 2 is activated by polylysine and has optimal activity in 150 mM KCl, sublingual kinase was inhibited by polylysine and the addition of KCl. Moreover, casein kinase 2 can utilize both ATP and GTP as phosphoryl donors, but GTP was not a substrate for sublingual kinase. The sublingual kinase shared a substrate recognition sequence with mammary gland kinase, but, unlike that kinase, it could not utilize Ca2+ instead of Mg2+. While the sublingual kinase thus shared some properties with both casein kinase 2 and mammary gland kinase, distinct differences were also seen and the relationship to these enzymes remains to be determined. The characterization of the sublingual kinase will be useful in its further purification.

D-dimer specific monoclonal antibodies react with fibrinogen aggregates.

Human fibrinogen exposed to 46.5 degrees C was subjected to gel permeation chromatography. The protein eluted in two distinct peaks. The first peak appeared in the void volume containing soluble fibrinogen aggregates, while the other peak represented monomeric fibrinogen. In contrast to the monomeric peak material, the aggregate fraction reacted with a panel of monoclonal antibodies specific for fragment D-dimer using an ELISA system. Edman degradation showed that both the aggregate and the monomeric fractions were devoid of soluble fibrin, and immunoblots of SDS-PAG electrophoretic profiles disclosed no sign of stabilized high molecular weight derivatives. We have previously shown that the aggregate fraction of similarly treated fibrinogen, in contrast to the monomeric fraction, stimulates the t-PA catalyzed conversion of plasminogen to plasmin and concomitantly exposes the sequences Aalpha-(148-160) and gamma-(312-324) involved in t-PA stimulation. Our present and previous findings suggest that soluble fibrinogen aggregates possess a fibrin-like structure, and that fibrin or fibrinogen polymer formation is a prerequisite for the enhancing effect on t-PA-mediated plasminogen to plasmin conversion which is seen even with the polymers in the soluble state.

Aggregated, conformationally changed fibrinogen exposes the stimulating sites for t-PA-catalysed plasminogen activation.

The present paper shows that conformationally changed fibrinogen can expose the sites A alpha-(148-160) and gamma-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5 degrees C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, has exposed the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is not longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during self-association.

Identification of histatins as tannin-binding proteins in human saliva.

Tannins have a number of detrimental biological effects and these include interference with normal growth and metabolism if they are present in the feed of various animals. Proline-rich proteins (PRPs) in saliva have been shown to provide protection against tannin, but little is known about the mechanism of protection and interaction of other salivary proteins with tannin. To identify tannin-binding human salivary proteins, parotid and submandibular/sublingual saliva samples were adsorbed with tannin. PRPs, and in particular a group of low-M(r) proteins, were readily precipitated by tannin. The low-M(r) proteins were purified from parotid saliva and demonstrated to be histatins, a family of well-characterized histidine-rich salivary proteins. The ability of synthetic histatin 5, as well as an acidic PRP (PRP-1) and gelatin to precipitate quebracho condensed tannin and tannic acid was determined. At pH 7.4 histatin 5 was the most effective precipitant of both condensed tannin and tannic acid and it also precipitated the largest amount of condensed tannin at pH 3.0, but the smallest amount of tannic acid at that pH. In contrast PRP-1 showed a greater ability to precipitate both condensed tannin and tannic acid at pH 3.0 than at pH 7.4. Under most circumstances histatin 5 was therefore more effective in precipitating tannins than proteins with high proline content which generally have been recognized as strong precipitants of tannin. Pre-incubation of tannic acid with alpha-amylase inhibited the enzyme, but addition of histatin 5 or the acidic PRP PIF-s protected amylase from inhibition by tannin. Similarly salivary proteins may protect other biological activities in the digestive tract from inhibition by dietary tannin.

Stimulating effect on tissue-type plasminogen activator--a new and sensitive indicator of denatured fibrinogen.

A high clottability and a short thrombin clotting time have routinely been considered as evidence of genuineness of the fibrinogen molecule. Since denatured fibrinogen stimulates the t-PA-catalysed conversion of plasminogen to plasmin, it was of interest to study the sensitivity of t-PA-stimulation as evidence of fibrinogen denaturation. Therefore, fibrinogen was intentionally exposed to various denaturating conditions (freeze-drying, heating, EDTA, alkali), and the clottability, the thrombin clotting time and the t-PA-stimulating effect were recorded. We found that the clottability was a poor indicator of fibrinogen denaturation, whereas the t-PA-stimulating effect could detect even mild fibrinogen denaturation. The thrombin clotting time was shortened after freeze-drying or heating at 47 degrees C, in spite of what might have been expected. Thus, denaturation is not necessarily accompanied by a prolonged clotting time. In some instances therefore, the t-PA-stimulation is an even more sensitive and reliable indicator of fibrinogen denaturation than is the thrombin clotting time. Consequently, this parameter should be combined with the thrombin clotting time to characterise preparations of fibrinogen.

Fibrinogen present in EDTA--anticoagulated plasma stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

The presence of soluble fibrin in plasma is an early and sensitive indicator of activation of the coagulation system. Quantitative spectrophotometric assays for soluble fibrin can be based on the principle that soluble fibrin stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. It was previously shown that treatment of purified fibrinogen by EDTA, which removes the three tightly bound Ca2+ ions, results in exposure of tissue-type plasminogen activator-catalytic sites similar to those unveiled by thrombin. Since EDTA is a common anticoagulant, it was of interest to study the effect of EDTA on a test based on plasminogen activation. It is concluded that the determination of soluble fibrin in EDTA-anticoagulated plasma from healthy individuals gives a false positive indication of the presence of soluble fibrin. This was true irrespective of whether the test was performed at pH 7.4, 7.8 or 8.5. The most probable explanation is that tissue-type plasminogen activator-stimulating sites are exposed in fibrinogen by EDTA. Therefore, EDTA-plasma is unsuitable for assaying soluble fibrin with tests based on the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin.

Freeze-dried fibrinogen or fibrinogen in EDTA stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

Both soluble and insoluble fibrin stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. Whether fibrinogen can exert a similar effect has been a controversial issue. The present investigation shows that while fibrinogen purified by beta-alanine precipitation does not stimulate the tissue-type plasminogen activator-catalysed plasminogen activation, fibrinogen which has been either lyophilized or stripped of bound Ca2+ ions by EDTA chelation, stimulates this reaction. The data indicate that such procedures alter the molecular conformation of fibrinogen, and expose stimulatory sites which are hidden in the native fibrinogen molecule. These results may explain previous findings concerning the capacity of fibrinogen as a stimulator of the tissue-type plasminogen activator-catalysed plasminogen activation. Since even slight alteration of the molecular structure of fibrinogen leads to an increase in the tissue-type plasminogen activator stimulation, the authors suggest that this can be used to test if the fibrinogen is in a native state.

A rapid method for selecting specific hybridoma clones using paramagnetic Dynabeads.

This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X-63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti-mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD. Mixing such beads with the fused cells allowed selective harvesting of cells with membrane-expressed anti-DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96-well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40-79% of the tested clones. It is concluded that it is possible to use antigen-coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest.

Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer.

Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B). The results obtained show that paramagnetic particles coated with mAb S4 catch intact D-dimer as well as a variety of cross-linked fibrin molecules of high-molecular-weight (HMW) in plasma. The existence of HMW derivatives in fibrinaemic serum indicates that some of the HMW fibrin related material in such plasma is not clottable. Fibrinogen/fibrin monomers and some of the fibrinogen/fibrin related derivatives found in the eluates were probably non-covalently bound to the complexes caught by mAb S4 coated particles. The present technique combines the selective concentrating power of immunoparticles and the sensitivity of immunovisualization and allows rapid and direct identification of minute amounts of circulating immunoreactive fibrinogen/fibrin derivatives.

Alignment of amino acid and DNA sequences of human proline-rich proteins.

Human proline-rich proteins (PRPs) constitute a complex family of salivary proteins that are encoded by a small number of genes. The primary gene product is cleaved by proteases, thereby giving rise to about 20 secreted proteins. To determine the genes for the secreted PRPs, therefore, it is necessary to obtain sequences of both the secreted proteins and the DNA encoding these proteins. We have sequenced most PRPs from one donor (D.K.) and aligned the protein sequences with available DNA sequences from unrelated individuals. Partial sequence data have now been obtained for an additional PRP from D.K. named II-1. This protein was purified from parotid saliva by gel filtration and ion-exchange chromatography. Peptides were obtained by cleavage with trypsin, clostripain, and N-bromosuccinimide, followed by column chromatography. The peptides were sequenced on a gas-phase protein sequenator. Overlapping peptide sequences were obtained for most of II-1 and aligned with translated DNA sequences. The best fit was obtained with clones containing sequences for the allele PRB4M (Lyons et al., 1988). However, there was not complete identity of the protein amino acid sequence and the DNA-derived sequences, indicating that II-1 is not encoded by PRB4M. Other PRPs isolated from D.K. also fail to conform to any DNA structure so far reported. This shows the need to obtain amino acid sequences and corresponding DNA sequences from the same person to assign genes for the PRPs and to determine the location of the postribosomal cleavage points in the primary translation product.

Localization of salivary proteins in granules of human parotid and submandibular acinar cells.

Electron microscopic immunocytochemistry was used to investigate the distribution of four secretory proteins within the granules of human parotid and submandibular gland acinar cells. The results demonstrate that the distribution of the individual proteins differs significantly. Amylase is concentrated within the electron-dense regions of the granules, whereas agglutinin is present mainly in the regions of lower density. In contrast, the proline-rich proteins and histatins are distributed throughout the content of the granules and do not appear to be concentrated in either the high- or low-density regions. The results also suggest that different mechanisms, such as self-aggregation or ionic complexes with metal ions and charged macromolecules, may be involved in the packaging of individual proteins within the same granule. Further, they also show that among the salivary glands of various species, the intragranular distribution of similar proteins (e.g., amylase and proline-rich proteins) may differ.

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.

Soluble, cross-linked fibrin(ogen) hybrid oligomers do not stimulate t-PA conversion of plasminogen.

Cross-linked hybrid oligomers of fibrinogen and fibrin are found in plasma from fibrinaemic patients and normal individuals as well as in preparations of purified human fibrinogen. The present study was undertaken to see if such hybrid oligomers have the same stimulatory effect on tissue plasminogen activator (t-PA) conversion of plasminogen as do polymeric and monomeric fibrin. Hybrid oligomeric fibrin(ogen) material was provided by subjecting purified human fibrinogen to gel filtration in urea-containing buffer at pH 5.6. Well separated fractions of hybrid oligomeric material and monomeric fibrinogen were thus obtained. Some of this material was converted to soluble polymeric or monomeric fibrin using insolubilized thrombin. Hybrid polymeric fibrin, polymeric fibrin or monomeric fibrin were then added to citrated, normal plasma to 2.5 or 5 per cent of the plasma fibrinogen concentration. The added material was kept in solution by plasma fibrinogen. The "COA-SET Fibrin Monomer Test" (Kabi,Stocholm,Sweden), based on the ability of fibrin monomers to enhance t-PA mediated plasminogen-plasmin conversion, was used to compare the potential stimulatory effect of the preparations above. The results led to the following conclusions: 1) Cross-linked, soluble fibrin(ogen) hybrid polymers in a concentration of 5 per cent of plasma fibrinogen concentration (w/w) do not stimulate t-PA. 2) Thrombin conversion of the fibrin-fibrinogen hybrid material resulted in an increase in the rate of t-PA mediated plasminogen conversion, corresponding to the one observed with equivalent (w/w) amounts of fibrin monomers. Compared on a mole to mole basis, fibrin oligomers are more powerful than fibrin monomers as stimulators of t-PA activity.