Anticoagulant heparan sulfate proteoglycans expression in the rat ovary peaks in preovulatory granulosa cells.

Ovarian granulosa cells synthesize anticoagulant heparan sulfate proteoglycans (aHSPGs), which bind and activate antithrombin III. To determine if aHSPGs could contribute to the control of proteolytic activities involved in follicular development and ovulation, we studied the pattern of expression of these proteoglycans during the ovarian cycle. aHSPGs were localized on cells and tissues by (125)I-labeled antithrombin III binding followed by microscopic autoradiography. Localization of aHSPGs has shown that cultured granulosa cells, hormonally stimulated by gonadotropins to differentiate in vitro, up-regulate their synthesis and release of aHSPGS: In vivo, during gonadotropin-stimulated cycle, aHSPGs are present on granulosa cells of antral follicles and are strongly labeled in preovulatory follicles. These data demonstrate that aHSPG expression in the ovarian follicle is hormonally induced to culminate in preovulatory follicles. Moreover, we have shown that five heparan sulfate core proteins mRNA (perlecan; syndecan-1, -2, and -4; and glypican-1) are synthesized by granulosa cells, providing attachment for anticoagulant heparan sulfate chains on the cell surface and in the extracellular matrix. These core proteins are constantly expressed during the cycle, indicating that modulations of aHSPG levels observed in the ovary are likely controlled at the level of the biosynthesis of anticoagulant heparan sulfate glycosaminoglycan chains. This expression pattern enables aHSPGs to focus serine protease inhibitors in the developing follicle to control proteolysis and fibrin formation at ovulation.

Characterization and hormonal modulation of anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells.

Anticoagulant heparan sulfate proteoglycans endow the vascular endothelium with antithrombotic properties, but their role outside the vascular bed is unknown. Granulosa cells form an avascular compartment in the ovarian follicle, in which a heparin-like activity has been described. At ovulation extravascular coagulation occurs around ovulatory follicles, and after expulsion of the oocyte, a fibrin clot forms in the antral cavity. Granulosa cells synthesize two major heparan sulfate proteoglycans, whose anticoagulant nature has not been investigated. The purpose of this study was to characterize anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells. Affinity purified 35S-labeled anticoagulant heparan sulfate glycosaminoglycans represent 6.5% of the total heparan sulfate synthesized, and they contain 13% 3-O-sulfated disaccharides that are markers of the antithrombin-binding site of heparin. The biological activity of granulosa cell heparan sulfate proteoglycans was demonstrated by their ability to bind antithrombin and to accelerate the formation of thrombin-antithrombin complexes. The impact of hormonal stimulation on granulosa cell anticoagulant heparan sulfate proteoglycans was studied using 125I-antithrombin binding assays. Follicle-stimulating hormone induced a redistribution of anticoagulant heparan sulfate proteoglycans from the granulosa cell layer to the culture medium, indicating that their distribution could be modulated according to the stage of follicular development. These results suggest that anticoagulant heparan sulfate might be critically located in the follicle to maintain fluidity around the oocyte until its expulsion at ovulation.

Characterization of a cell mutant specifically defective in the synthesis of anticoagulantly active heparan sulfate.

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.

Differential partition of anticoagulant heparan sulfate proteoglycans synthesized by endothelial and fibroblastic cell lines.

The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface-associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface-associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface-associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface-associated and soluble aHSPGs. These observations indicate that different cell types can preferentially accumulate aHSPGs as cell surface-associated or soluble forms which could reflect alternate biological functions.

New approaches for defining sequence specific synthesis of heparan sulfate chains.

Mammalian cells synthesize heparan sulfate proteoglycans (HSPG) which consist of core proteins with covalently linked glycosaminoglycans (GAGs) of 50-150 disaccharide units. The GAGs exhibit great structural diversity which arise from differing arrangements of alternate disaccharide units. It has been hypothesized that HSPG may be involved in regulating the most basic aspects of cell biologic systems such as adhesion, proliferation and differentiation. However, considerable doubt exists about the specific nature of the above interactions because of a failure to isolate GAGs of unique monosaccharide sequence with appropriate biologic activities. We have demonstrated that mouse LTA cells synthesize cell surface heparan sulfate proteoglycans with regions of defined monosaccharide sequence that specifically interact with antithrombin (HSPGact). However, it remains unclear how HSPGact can be generated by a biosynthetic pathway with no simple template for directing the ordered assembly of monosaccharide units. To examine this issue, we treated LTA cells with ethylmethane sulfonate and then identified mutants that exhibit decreased antithrombin binding to heparan sulfate chains but possess no gross defects in glycosaminoglycan biosynthesis. After screening 40,000 colonies, we isolated 7 stable mutants which synthesize 8-27% of the wild type HSPGact but produce normal amounts of other HSPG. These mutants are recessive in nature, and fall into at least two different complementation groups. The delineation of the molecular basis of these defects should greatly improve our understanding of how cells synthesize HSPG with regions of defined monosaccharide sequence.

New approaches for defining the molecular basis of anticoagulantly active heparan sulfate production.

Mammalian cells synthesize heparan sulfate proteoglycans, which consist of core proteins with covalently linked glycosaminoglycans of 50-150 disaccharide units. The GAGs exhibit great structural diversity, which arises from differing arrangements of alternate disaccharide units. It has been hypothesized that HSPG may be involved in regulating the most basic aspects of cell biologic systems, such as adhesion, proliferation, and differentiation. However, considerable doubt exists about the specific nature of the above interactions because of a failure to isolate GAGs of unique monosaccharide sequence with appropriate biologic activities. We have demonstrated that mouse LTA cells synthesize cell surface heparan sulfate proteoglycans with regions of defined monosaccharide sequence that specifically interact with antithrombin (HSPGact). However, it remains unclear how HSPGact can be generated by a biosynthetic pathway with no simple template for directing the ordered assembly of monosaccharide units. To examine this issue, we treated LTA cells with ethylmethane sulfonate and then identified mutants that exhibit decreased antithrombin binding to heparan sulfate chains but possess no gross defects in glycosaminoglycan biosynthesis. After screening 40,000 colonies, we isolated seven stable mutants that synthesize 8-27% of the wild type HSPGact but produce normal amounts of other HSPG. These mutants are recessive in nature and fall into at least two different complementation groups. The delineation of the molecular basis of these defects should greatly improve our understanding of how cells synthesize HSPG with regions of defined monosaccharide sequence.

Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta.

We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.