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Sulfated motifs on heparan sulfate (HS) are involved in various extracellular processes from cell signaling to enzymatic regulation, but the structures of these motifs are obscure. We have developed a strategy to determine the structure of sulfotransferase recognition sites which constitute these motifs. Stable isotope is first introduced into specific sites on HS with HS sulfotransferases and the modified HS is then digested into oligosaccharides of differing sizes. The overlapping oligosaccharides containing the introduced stable isotope are identified by changes in the m/z profiles by mass spectrometry, and their relationships are elucidated. In this way, the HS structures in the vicinity of the sulfotransferase recognition site are quickly determined and groups on precursor structures of HS that direct the action of HS sulfotransferases are pinpointed.

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-(35)S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of DeltaUA2S-GlcNS-IdoUA2S-[(35)S]GlcNH(2)3S and DeltaUA2S-GlcNS-IdoUA2S-[3-(35)S]GlcNH(2)3S6S . The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N-unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3-OST-3A-modified heparan sulfate.

To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction.

3-O-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellular production of the critical active structure is controlled by the rate-limiting enzyme, heparan sulfate d-glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base with the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to isolate full-length cDNAs. Clones corresponding to predominant transcripts were obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3A, and 3-OST-3B, respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous carboxyl-terminal sulfotransferase domain of ∼260 residues. Also recovered were partial length clones for 3-OST-4. Expression of the full-length enzymes confirms the 3-O-sulfation of specific glucosaminyl residues within heparan sulfate (Liu, J., Shworak, N. W., Sinay, P., Schwartz, J. J. Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185–5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 genes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy each in mice. Intriguingly, the entire sulfotransferase domain sequence of the 3-OST-3B cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3A. Together, these data argue that the structure of this functionally important region is actively maintained by gene conversion between 3OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified the loci for mouse3Ost genes and syntenic assignments of corresponding human isologs were confirmed by the identification of mapped sequence-tagged site markers. Northern blot analyses indicate brain exclusive and brain predominant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas, 3-OST-3A and 3-OST-3Bisoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferase domain suggest that this structure is a self-contained functional unit. Moreover, the extensive number of 3OST genes with diverse expression patterns of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST-1, regulate important biologic properties of heparan sulfate proteoglycans.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999)J. Biol. Chem. 274, 5170–5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate d-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3′-phosphophate 5′-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide.

Retinoic acid (RA) and dibutyryl cAMP plus theophilline (CT) trigger F9 cells to differentiate into parietal endoderm. The differentiation induces a 9-fold increase in total heparan sulfate (HStotal) biosynthesis and a 170-fold increase in anticoagulantly active HS (HSact) biosynthesis. Measurement of 3-O-sulfotransferase-1 mRNA and enzymatic activity demonstrated an increase of over 100-fold whereas determination of N-, 2-O, and 6-O-sulfotransferase enzymatic activities showed elevations of 2-, 3. 5-, and 3.7-fold, respectively. HSact precursor pool measurements reveal that 30% of control F9 HStotal can be converted into HSact while only an additional 10% of RACT F9 HStotal can be transformed into HSact. Disaccharide analysis of metabolic labeled HS indicated that 32% 3-O-sulfate containing disaccharides, i.e. GlcA-anManR3S and GlcA-anManR3S6S, are present in HSact and 68% GlcA-anManR3S and GlcA-anManR3S6S are found in anticoagulantly inactive HS (HSinact). By using adenosine 3'-phosphate 5'-phosphosulfate and purified 3-O-sulfotransferase-1, 30% of 3-O-sulfation occurs in HSact and 70% of 3-O-sulfation occurs in HSinact. The similar ratio of 3-O-sulfate distribution in HSact versus HSinact suggests that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. Extensively 3-O-sulfated HSinact may play an important functional role under in vivo conditions within the murine placenta.

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitro transcription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5'-untranslated region of variable length, which results from the insertion of 0-1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are approximately 85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (approximately 34 kDa) and the previously purified enzyme (approximately 46 kDa). The two 3-OST species also exhibit approximately 50% similarity with all previously identified forms of the heparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain.

LTA cells synthesize a minor population of heparan sulfate proteoglycans (HSPGact) bearing anticoagulant heparan sulfate (HSact) with a specific monosaccharide sequence that accelerates the action of antithrombin (AT). LTA cells also synthesize a major population of heparan sulfate proteoglycans endowed with nonanticoagulant heparan sulfate (HSinact) lacking the AT-binding site. To investigate the pathway-specific features of HSPGact generation, we established a novel detergent-containing cell-free system with unlabeled and labeled microsomes from wild-type and variant LTA cells, respectively. The unlabeled microsomes provide “HSact conversion activity” that requires 3′-phosphoadenosine 5′-phosphosulfate to convert [35S]HSPGinact into [35S] HSPGact, presumably by sulfation. The reaction kinetics demonstrate that the rate of HSact synthesis is constant over the first 4 h of incubation. During this time, the rate of HSact production is linearly dependent on the amount of unlabeled LTA microsomal protein over a range of 10 to 50 μg as well as on the level of [35S]HS substrate over a range of 0.4 to 4.0 μg, microsomal protein. Compared with labeled microsomes, equivalent or slightly greater levels of HSact were generated from 35S-labeled HSPG, microsomal HS, or cell surface HS, which demonstrates that HSinact is the minimal substrate and that large amounts of HSact precursor exit the Golgi apparatus. Indeed, extensive modification of wild-type LTA cell surface [35S]HS elevated HSact content from 9 to 35%. The hypothesis that microsomal HSact conversion activity predicts the cellular rate of HSact generation was tested with wild-type or variant LTA cells in which production of HSact has been significantly altered by mutagenesis or overexpression of core protein or growth conditions. The data demonstrate that microsomal HSact conversion activity accurately reflects the cellular rate of HSact synthesis over a very wide range of conditions. The possibility that the reduced HSact generation is due to an inhibitor was excluded by mixing experiments. The possibility that reduced HSact generation is caused by decreased levels of HSact precursor was excluded as equivalent levels of HSact were formed from wild-type and variant [35S]HS. Based upon the above data, the LTA cell microsomal HSact conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HSact generation and the levels of HSact precursor in HS greatly exceed HSact production.

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 micronol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.

We have investigated the characteristics of clonal L cell mutant VI-7 that has previously been demonstrated to exhibit reduced levels of cell surface proteoglycans containing heparan sulfate (HS) with regions of defined monosaccharide sequence that interact with antithrombin (HSact) (de Agostini, A. L., Lau, H. K., Leone, C., Youssoufian, H., and Rosenberg, R. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9784-9788). Pulse labeling revealed that the synthesis of HSact in mutant cells, as compared to wild-type cells, was reduced by 5-7-fold, which is identical to the previously observed reduction in cell surface-bound antithrombin. This alteration is independent of growth state since production of HSact by mutant cells, as compared to wild-type cells, is decreased to a similar extent in exponentially growing and post-confluent cultures. The synthetic defect was specific for HSact since production of total HS and chondroitin sulfate by mutant cells, as compared to wild-type cells, was identical in magnitude. The synthetic abnormality is not due to an alteration in core protein since complementation was not observed when mutant cells were stably transfected with the epitope-tagged ryudocan cDNA which can initiate HSact production. Structural analyses revealed that HS from mutant cells, as compared to wild-type cells, exhibited normal molecular weight, extent and distribution of sulfate, and disaccharide composition, which indicate that the mutation did not affect HS biosynthetic enzymes. Together, the data suggest that mutant VI-7 is defective in a regulatory component which directly or indirectly coordinates HS biosynthetic enzymes to specifically generate the defined monosaccharide sequence of HSact.

L cells and endothelial cells synthesize a heparan sulfate (HS) subpopulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lacks this region of defined structure and is anticoagulantly inactive. HSact biosynthesis was examined in these two cell types by stably expressing epitope-tagged rat ryudocan (ryudocan12CA5), which possesses three glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact were present on ryudocan12CA5 isolated from L cells and endothelial cells; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan12CA5 variants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of both types of GAGs to a similar extent. Most importantly, in both cell types total HSact generation declined as a function of ryudocan12CA5 overexpression even though HSinact production increased linearly as a function of this variable. This discordant relationship is a general property of the biosynthetic machinery since in both cell types HSact production was reduced to an equal extent on protein cores of either exogenous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembrane and cytoplasmic domains or by a GAG acceptor site mutated form of core protein incapable of augmenting GAG synthesis. These results suggest that elevated intracellular levels of core protein saturate the capacity of a critical component of the HSact biosynthetic machinery. This critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no structural changes were observed in either GAG during overexpression of core protein. Based upon the above data, we conclude that increased intracellular levels of ryudocan probably act by saturating the capacity of components which regulate HSact production by coordinating the function of biosynthetic enzymes.

The specificity of the glycosaminoglycan (GAG) acceptor sites of ryudocan was examined by stably expressing epitope-tagged ryudocan cDNA constructs in mouse L cells, which normally produce this proteoglycan. Immunopurified ryudocan was glycanated with both heparan sulfate (HS) and chondroitin sulfate (CS). The attachment of GAGs to ryudocan was prevented by creating Ser-->Thr mutations in all possible combinations at positions 44, 65, and 67. The resulting ryudocan of exogenous origin was immunopurified and evaluated with regard to attached GAG chains. The data reveal that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of GAG lyase digests of intact ryudocan reveal the production of the following multiple isoforms: pure HS-ryudocan, various HS/CS-hybrids, and pure CS-ryudocan. The data suggest that the occupancy bias of each site for HS or CS is slight and that each site functions in a relatively independent fashion. The GAG lyase analysis of partially purified L cell proteoglycans shows two pure CS-homoglycans with core proteins of M(r) = 130,000 and 52,000, respectively. A similar analysis of immunopurified L cell glypican demonstrates that this species only exists as a pure HS-homoglycan. The production of pure homoglycans by this clonal cell line strongly suggests that the functional promiscuity of GAG attachment sites of ryudocan must be encoded in the core protein structure. This property of ryudocan is not peculiar to L cells, as ryudocan synthesized by early passage human endothelial cells also bears both HS and CS. The production of multiple isoforms of ryudocan may serve to expand the functional versatility of this cell surface component and allow it to participate in many different biologic processes.

The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.

The intracellular concentration of ionized calcium is involved in regulating mitosis. However, little is known about intracellular levels of calcium during G1. We have demonstrated in vascular smooth muscle cells a mid-G1 decrease in ionized calcium concentration followed by a 2-fold rise at the G1/S interface (44 nM +/- 0.6 nM versus 98 nM +/- 1.1 nM, p < 0.01). The elevation of intracellular calcium is preceded by an increase in c-myb mRNA levels and is abolished with antisense but not missense c-myb oligonucleotides. Furthermore, cells stably transfected with c-myb show a similar 2-fold augmentation in intracellular calcium concentrations, as compared with untransfected cells, which is also abolished by antisense c-myb oligonucleotides. The c-myb-induced rise in intracellular calcium is dependent upon the presence of extracellular calcium and is not suppressed by L type calcium channel blockers. We conclude that c-myb induces an elevation in intracellular calcium levels of vascular smooth muscle cells at the G1/S interface which provides a novel role for this proto-oncogene as well as a potentially important control point for cell cycle regulation.

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.

Phagocyte-mediated oxidant damage to vascular endothelium is likely involved in various vasculopathies including atherosclerosis and pulmonary leak syndromes such as adult respiratory distress syndrome. We have shown that heme, a hydrophobic iron chelate, is rapidly incorporated into endothelial cells where, after as little as 1 h, it markedly aggravates cytotoxicity engendered by polymorphonuclear leukocyte oxidants or hydrogen peroxide (H2O2). In contrast, however, if cultured endothelial cells are briefly pulsed with heme and then allowed to incubate for a prolonged period (16 h), the cells become highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products. This protection is associated with the induction within 4 h of mRNAs for both heme oxygenase and ferritin. After 16 h heme oxygenase and ferritin have increased approximately 50-fold and 10-fold, respectively. Differential induction of these proteins determined that ferritin is probably the ultimate cytoprotectant. Ferritin inhibits oxidant-mediated cytolysis in direct relation to its intracellular concentration. Apoferritin, when added to cultured endothelial cells, is taken up in a dose-responsive manner and appears as cytoplasmic granules by immunofluorescence; in a similar dose-responsive manner, added apoferritin protects endothelial cells from oxidant-mediated cytolysis. Conversely, a site-directed mutant of ferritin (heavy chain Glu62—-Lys; His65—-Gly) which lacks ferroxidase activity and is deficient in iron sequestering capacity, is completely ineffectual as a cytoprotectant. We conclude that endothelium and perhaps other cell types may be protected from oxidant damage through the iron sequestrant, ferritin.

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.

The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.

Proteoglycans are dominant glycoconjugates located on the cell surface and in extracellular spaces and consist of a core protein with one or more glycosaminoglycan side chains linked covalently. Heparan sulfate (HS) belongs to the family of glycosaminoglycans. HS has been assigned a variety of physiological and pathological functions, such as cell-cell adhesion, cell-matrix adhesion, cell proliferation, motility and differentiation, lipoprotein metabolism, blood coagulation, inflammation, tissue regeneration, tumor progression and invasion, pathogenic infection by bacteria, protozoa, and viruses, through specific interaction with a wide array of proteins, ligands, receptors, and pathogens (Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729-777). We have shown here for the first time that light induces changes in pineal HS fine structure and that occurrence of the rare 3-O sulfation catalyzed by HS 3-O-sulfotransferase (3-OST2) is predominantly restricted to daytime pineal glands.

An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.

The activation of Factor XII occurs via fragmentation of this zymogen into a diverse spectrum of enzymatically potent molecular species. To study the interaction of antithrombin-heparin cofactor and heparin with activated Factor XII, we have employed two forms of this enzyme with widely differing physical characteristics and biologic potencies. Antithrombin-heparin cofactor was found to be a progressive, time-dependent inhibitor of both forms. The addition of heparin dramatically accelerated the rates of these interactions. Furthermore, sodium dodecyl sulfate gel electrophoresis of reduced proteins has indicated that antithrombin-heparin cofactor functions by forming an undissociable complex with either species of the enzyme. This complex represents a 1:1 stoichiometric combination of activated Factor XII and inhibitor. In the presence of heparin, both species undergo virtually instantaneous complex formation with antithrombin-heparin cofactor without exhibiting alterations in dissociability or stoichiometry.

Bovine aortic endothelial cells release a heparin-like substance in the presence of 0.4% fetal calf serum. This substance inhibited the growth of smooth muscle cells in vitro by about 70%. Substitution of platelet-poor plasma for serum resulted in minimal liberation of inhibitory activity from the cells unless at least 10-fold higher concentrations of platelet-poor plasma were utilized. This suggested that a platelet product was involved in the release process. Therefore, we examined the ability of the platelet heparitinase described in the preceding communication to release heparin-like species from cultured endothelial cells. Our results show that when endothelial cells were exposed to serum-free medium containing 1 ng/ml of the purified platelet endoglycosidase, at least as much inhibitory activity was released as was obtained with 0.4% serum. Dose response experiments indicated that only 10 pg/ml of the enzyme were necessary to liberate 50% of the inhibitory activity from endothelial cells. The heparin-like nature of the inhibitory substance was demonstrated by its sensitivity to Flavobacterium heparinase. Utilizing appropriate controls, the release of heparin-like material by the endoglycosidase was shown to be enzyme-specific and was not due to artifacts of experimental manipulations. In addition, this enzyme did not convert prereleased material to an active component, but directly liberated the active heparin-like species from endothelial cells. A simple model describing the possible role of heparin-like components and the endoglycosidase in the normal and injured wall is presented.

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12–16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.

The interaction of purified human antithrombin-heparin cofactor and human plasmin was studied in the presence and absence of heparin. Antithrombin is a progressive, timedependent inhibitor of the proteolytic and esterolytic activities of plasmin. Incubation of plasmin with antithrombin for 15 to 30 min results in 90 to 100% inhibition of both activities of the enzyme. The presence of heparin dramatically accelerates the rate of interaction of antithrombin and plasmin, with nearly complete inhibition within 30 s of incubation. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin functions as a potent antiplasmin by forming an undissociable complex which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.

Equilibrium-binding data of highly purified avian lipoprotein lipase to cultured bovine endothelial cells demonstrate the presence of a class of high affinity sites. Analysis of the binding function by weighted least squares technique yielded an association constant of K = 0.7 X 10(7) M-1 and a maximum binding capacity of 1.6 micrograms/1.9 X 10(6) cells. Lipoprotein lipase was monitored both by its catalytic activity and a sensitive radioimmunoassay which permitted the accurate measurement of nanogram quantities of enzyme protein. Specific activity of the bound enzyme was similar to that of the initial purified enzyme. Lipoprotein lipase binding to endothelial cells was inhibited 80% by preincubating cells in 0.1% trypsin for 3 min at 37 degrees C, 92% by 0.01% pronase, and 91% by 0.008% proteinase K. Heparin was most efficient in releasing lipoprotein lipase from endothelial cells. Fifty per cent of the enzyme appeared in the medium at a concentration of 3 micrograms/ml of heparin. At the same concentration of heparan sulfate, 20% of the enzyme was released. Hyaluronic acid and chondroitin sulfate were not effective in stimulating enzyme release. Preincubating endothelial cells with purified human platelet endoglucuronidase for 1 h at 37 degrees C led to a 90% reduction in lipoprotein lipase binding. Endoglucuronidase was purified 20,000-fold as compared to the initial platelet lysate by a 5-step purification method. The extent of inhibition of binding was shown to be dependent on concentration of endoglucuronidase in the preincubation medium. The specificity of platelet endoglucuronidase and the demonstration that the preparation utilized contained no detectable protease activity is further evidence that lipoprotein lipase is bound to endothelial cell heparan sulfate or heparan sulfate-like molecules.

We have raised antisera against human prothrombin activation fragment F2 in rabbits and have chromatographed the respective immunoglobulin G fractions on prothrombin-Sepharose, Pr1-Sepharose, and F2-Sepharose immunoadsorbents. The specific antibody population obtained was utilized to construct a double antibody radioimmunoassay capable of measuring as little as 0.8 ng/ml of this component. Our studies suggest that the immunoreactive site defined by this antibody population is most probably located within the negatively charged COOH-terminal region of F2. The immunologic expression of this area is unaffected by denaturation or reduction-alkylation of F2 as well as by attachment of polypeptide to the NH2-terminal of this component. However, the presence of covalently bound polypeptide at the COOH-terminal of F2 reduces its immunologic reactivity by 300- to 400-fold. Prothrombin, Pr1, and Pr*1, which contain the F2 region as part of their covalent structure, are at least 4000 to 7000 times less immunoreactive than F2 on a molar basis. Conversion of these components to thrombin as well as activation fragments generates the theoretically predicted level of immunoreactivity. Masking of the immunoreactive site within these zymogens is due to two phenomena. Firstly, covalent attachment of polypeptides on the COOH-terminal of the F2 segment significantly depresses the reactivity of this region. Secondly, a critical S--S bridge aids in the sequenstration of the immunoreactive site. This cross-link may facilitate interactions between the COOH-terminal of the F2 segment and other regions of the zymogen.

The plasma esterase inhibitors alpha2-macroglobulin, alpha1-antitrypsin, C1-inhibitor, antithrombin-heparin cofactor, and, as previously described, soybean trypsin inhibitor (Kunitz) and diisopropylphosphorofluoridate (9) enhance the response of guinea pig macrophages to migration inhibitory factor. To obtain this effect, macrophages are incubated with inhibitors prior to assay. The data suggest that (a) the enhancement of migration inhibitory factor response is due to the inhibition of esterases associated with the macrophage through a distinct active site on the inhibitors, and (b) that the active sites of antithrombin-heparin cofactor and soybean trypsin inhibitor, which interact with the macrophage enzymes, are different from the active sites of these inhibitors which interact with thrombin and trypsin respectively. Chemical modification of the active site of antithrombin-heparin cofactor for thrombin and of soybean trypsin inhibitor for trypsin does not affect their capacity to enhance the migration inhibitory factor response. From studies with thrombin, it was known that antithrombin-heparin cofactor has a heparin binding site. Addition of heparin was found to prevent the migration inhibitory factor-enhancing effect of antithrombin-heparin cofactor. The present results suggest that plasma esterase inhibitors may play a regulatory role in the response of macrophages to mediators of cellular immunity.

In this communication we describe the first method for isolating human Factor V. The final product contains no other coagulation components as judged by functional assays and is physically homogeneous as shown by isofocusing gel electrophoresis. In addition, we present a means for obtaining intrinsically activated human Factor X-a. This preparation is usually homogeneous as judged by isofocusing gel electrophoresis. However, on occasion, an additional minor electrophoretic species with Factor X-a activity is observed. Furthermore, we describe the use of isoelectric focusing in sucrose density gradients to free human prothrombin from contamination by coagulation factors and other components. These homogeneous human proteins are employed to examine the conversion of prothrombin to thrombin in the presence and absence of human antithrombin. The latter component is responsible for virtually all of the plasm's capacity to neutralize Factor X-a and thrombin. In the absence of antithrombin, prothrombin (67,800) is converted to the precursor P-2 (51,600) and the fragment F-a (19,500). Subsquently, P-2 is cleaved to form the precursor P-3 (37,000), and the fragment F-b (11,500). Finally, P3 IS proteolyzed to form the heavy chain T-h (29,500) and the light chain T-L (6,500) of active thrombin. In the presence of antithrombin, an additional prothrombin conversion pathway is observed in which the zymogen is directly cleaved to form P-3 and F-A + B (30,000) prior to thrombin generation. Trace amounts of free thrombin remain uninhibited by antithrombin and could bias the zymogen activation pathway. Hirudin is known to neutralized thrombin instantaneoulsly. We demonstrate that the purified leech protein also binds to P-3 and prevents thrombin formation. When hirudin is added to activation mixtures at concentrations sufficient to virtually suppress P-3 conversion to thrombin, molecular species from both activation pathways are observed. Thus two human prothrombin conversion sequences appear to be initiated by Factor X-3 and may be of physiological significance.

Preparations of low molecular weight porcine heparin with an average specific anticoagulant activity of 94 units/mg were fractionated into "active" and "relatively inactive" forms of the mucopolysaccharide of approximately 6000 daltons each. The active fraction was further subdivided into various species with descending but significant affinities for the protease inhibitor as well as decreasing but substantial anticoagulatn potencies. "Highly active" heparin (approximately 8% of the low molecular weight pool) possesses a specific anticoagulant activity of 350 +/- 10 units/mg. The relatively inactive fraction (67% of the low molecular weight pool) exhibits a specific anticoagulant activity of 4 +/- 1 units/mg. The binding of highly active heparin to antithrombin is accurately described by a single-site binding model with a KHep-ATDISS of approximately 1 X 10(-7) M. Variations in this binding parameter secondary to changes in environmental variables indicate that charge-charge interactions as well as an increase in entropy are critical to the formation of the highly active heparin-antithrombin complex. The interaction of relatively inactive heparin with the protease inhibitor is characterized by an apparent KHep-ATDISS of 1 X 10(-4) M. In large measure, this is due to small amounts of residual active mucopolysaccharide (0.5%). The ability of the highly active heparin to accelerate the thrombin-antithrombin interaction was also examined. We were able to demonstrate that the mucopolysaccharide acts as a catalyst in this process and is able to initiate multiple rounds of enzyme-inhibitor complex formation. The rate of enzyme neutralization is increased to a maximum of 2300-fold as the concentration of heparin is raised until the inhibitor is saturated with mucopolysaccharide. Further increases in heparin concentration result in a reduction in the speed of enzyme neutralization. This appears to be due to the formation of thrombin-heparin complexes. A mathematical model is given which provides a relationship between the initial velocity of enzyme neutralization and reactant concentrations.

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.

Quantitative measurements have been made of the interaction of human complement subcomponent C1q with mucopolysaccharides. The binding of C1q to heparin was quantitatively examined by utilizing an assay that employs a 125I-labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of binding sites were detected. The first class of sites bound 2.02 mol of LMW-Hep/mol of C1q with a Kd of 76.6 nM. The second class of sites complexes with 12 mol of LMW-Hep with a Kd of 1.01 microM. The higher affinity-binding site for LMW-Hep could be assigned to the collagenous region of C1q (C1q-c); 2.2 mol of 125I-LMW-Hep were bound/mol of purified isolated C1q-c with a Kd = 381 nM. In contrast, the isolated C1q globular region did not bind to 125I-LMW-Hep. The binding of LMW-Hep to C1q and the C1q-c region was confirmed by fluorescence polarization experiments; C1q and C1q-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/mol of protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction of C1q with 125I-LMW-Hep, the most effective being heparan sulfate and dermatan sulfate. LMW-Hep (2.5 nM) inhibited the ability of C1q (0.5 nM) to recombine with C1r (1.4 nM) and C1s (1.6 nM) to form hemolytically active C1. At 250 mM, LMW-Hep inhibited the hemolytic activity of reconstituted C1. The ability of mucopolysaccharides to interact with purified C1q suggests a role for such molecules in the regulation of the first component of complement.

A new, highly discriminating affinity chromatographic technique has been developed which employs antithrombin and concanavalin A-Sepharose to fractionate heparin species of all molecular sizes. This methodology is able to subdivide the active mucopolysaccharide pools of molecular weight 6,000 to 8,000 (LMW) or 18,000 to 22,000 (HMW) into various species with descending affinities for antithrombin as well as decreasing anticoagulant potencies. The upper 10% of these two pools, either LMW or HMW highly active heparin, appears to be relatively homogeneous with respect to interactions with antithrombin and possessed anticoagulant potencies of 350 units/mg and 731 units/mg, respectively. The HMW highly active heparin has been examined by analytic ultracentrifugation. It exhibited a charge-connected weight-average molecular weight of 22,000 +/- 2,000 with minimal size heterogeneity. The stoichiometries of interaction of antithrombin and platelet factor 4 with HMW highly active heparin as determined by fluorescence spectroscopy indicated that 2 molecules of either protein are able to bind to 1 molecule of the mucopolysaccharide. These studies also reveal that the binding of antithrombin to HMW highly active heparin is characterized by KDISSHAT = 5.0 X 10(-8) M and KDISSHAT2 = 1.0 x 10(-7) M, respectively. The avidity of platelet factor 4 for HMW highly active heparin could not be quantitated but appears to be at least 10 to 100 times greater than that of antithrombin for mucopolysaccharide.

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.

Megakaryocyte stimulatory factor (MSF) has been purified to homogeneity (7.5 X 10(5)-fold) from serum-free conditioned medium obtained from cultured human embryonic kidney cells and to near homogeneity (1.44 X 10(7)-fold) from thrombocytopenic rabbit plasma. MSF activity from either source was assayed by its ability to enhance the rate of synthesis of platelet factor 4-like proteins in a rat promegakaryoblast cell line. The 125I-labeled factor prepared from human embryonic kidney cell conditioned medium is homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing in the presence of 9.2 M urea. MSF obtained from the above source is an acidic protein (pI = 5.1) with an Mr = 15,000 which stimulates platelet factor 4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF was also purified from thrombocytopenic rabbit plasma by a nearly identical isolation procedure, and 125I-labeled factor prepared from this source also possessed an Mr = 15,000. MSF exhibited no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage.

Purified thrombins are isolated from Parke-Davis thrombin topical and from bioactivated crude prothrombins obtained from single animal plasmas. Six thrombin components are present in the former by gel electrophoresis at pH 8.9, ionic strength 0.16, and 30°, but interband protein is high. Ultracentrifugation and gel filtration studies show that, as either ionic strength or temperature is decreased, reversible thrombin self-association and interaction with the gel matrix increase. Electrophoretic mobility and resolution decrease, the latter to give a single diffuse band. Partial fractionation of the six components, numbered according to increasing anodal mobility, was accomplished by differential elution from cellulose phosphate. T2 and T6 were isolated essentially in pure form. Specific activities and component abundances of fractions revealed that T1, T2, and T3 have significantly higher activities than T4, T5, and T6. Thrombins from 60 single animal plasmas contained only T1, T2, and T3, in two distribution types: I, having T1, T2, and T3 in a ratio of 1:2:1, or II, having T1 and T2 as a 1:1 ratio, with T3 absent or present at about 5% of total thrombin. Distribution type for pedigreed animals correlates (a) with age: below 8 months, only I was observed (5 animals); between 8 and 30 months, either I or II was observed (16 animals); and above 30 months, only II was observed (11 animals); and (b) with the two-stage clotting time of the original plasma, with the use of the corresponding serum to supply activators. Other correlations were not found. Component distribution is not a simple, genetically determined characteristic. During activation to produce Distribution I, T1 and T2 may first appear at a ratio of 1:2. An attractive model is as follows. Two prothrombins, P1 and P2, are present in equal amounts. P2 yields T2, and P1 yields T1 and T3, but not sequentially. As the animal ages, the pathway to T3 is lost. Reduction and alkylation of thrombin from thrombin topical results in a dispersion of five fragments between molecular weight of ∼32,000 and ∼5,000. Thrombins from single animal plasmas yield only these two. Probably T4, T5, and T6 are derived from T1, T2, and T3 as a result of peptide cleavages in the larger fragment.

A procedure is presented for purifying antithrombin-heparin cofactor from human plasma. The final product is homogeneous as judged by disc gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, and immunoelectrophoresis. The final yield averages 12%. A specific antibody directed against pure inhibitor preparations precipitates virtually all of both antithrombin and heparin cofactor activity from defibrinated plasma. The purification, the immunoprecipitation, and other data, indicate that both activities are properties of a single molecular species. The inhibitor and thrombin form a 1:1 stoichiometric complex which cannot be dissociated with denaturing and reducing agents. Addition of heparin, a widely used anticoagulant which specifically accelerates the action of our inhibitor, increases the rate of formation of this complex without altering its stoichiometry or its dissociability. Interaction of thrombin with antithrombin-heparin cofactor requires the presence of the active center serine of the enzyme and arginine residue(s) on the inhibitor, since chemical modification of either of these critical residues inhibits complex formation both in the presence and absence of heparin. We suggest that, in analogous fashion to trypsin-trypsin inhibitor systems, a specific interaction occurs between the active center serine of thrombin and a unique arginine-x reactive site on the antithrombin-heparin cofactor. Furthermore, lysyl residues of the inhibitor probably serve as a binding site for heparin, since chemical modification of these residues virtually eliminates heparin cofactor activity with only minimal reduction of antithrombin activity. We postulate that heparin binds to the inhibitor and causes a conformational change which results in a more favorable exposure of the arginine reactive site, allowing a rapid interaction with thrombin.