Type 1 plasminogen activator inhibitor (PAI-1) ()is the primary physiological inhibitor of vascular tissue-type plasminogen activator with rate constants greater than 10 (mol/liter) s for both single- and two-chain tissue-type PA, as well as urokinase, which results in the formation of high molecular weight, inactive PA-PAI-1 complexes (for reviews, see (1, 2, 3) ). The ability of PAI-1 to neutralize the activity of tissue-type PA and other serine proteases (e.g. urokinase, factor XIa, activated protein C) has led to the classification of this inhibitor in the serine protease inhibitor (serpin) superfamily(1, 2) . The role of PAI-1 as a primary regulator of fibrinolysis was initially raised by the association of elevated blood PAI-1 and a number of physiological and pathological processes that carry an increased risk of thrombotic complications, including pregnancy(4, 5) , severe trauma(6) , major surgery(7) , and a wide spectrum of other disease states(8) . This concept is further supported by the correlation of bleeding disorders in a number of patients with a deficiency in blood PAI-1 activity(9, 10, 11, 12, 13) . This inhibitor is produced as a M 50,000 glycoprotein and is present in blood either at low concentrations in plasma (10-25 ng/ml) or in a large storage pool within platelets (14, 15, 16, 17, 18, 19, 20, 21, 22) . Agonist-induced platelet activation is known to cause the release of active PAI-1 suggesting that this inhibitor is stored in conjunction with other hemostatic proteins within platelet -granules (14, 15, 18, 20, 21, 22) . Platelet-rich thrombi are more resistant to thrombolysis than erythrocyte-rich thrombi and this resistance to lysis is mediated by the release of active PAI-1 from activated platelets (23, 24, 25, 26) . The presence of PAI-1 antigen in megakaryocytes(27, 28) , the hemopoietic precursor of platelets, suggests that PAI-1 may be deposited into storage organelles (i.e. -granules) during the maturation of these cells.

Research from a number of laboratories indicates that PAI-1 is synthesized in an active form, but it is a relatively labile inhibitor that is rapidly converted into an inactive form at 37 °C with a half-life of approximately 1 h(2) . The conformation of PAI-1 resulting from inactivation at 37 °C is commonly referred to as ``latent'' because inhibitory activity can be revealed by treatment with denaturants or negatively charged phospholipids(2) . Although a portion of PAI-1 in platelets is in the latent form(15, 16, 22, 29, 30) , little information exists on the mechanisms that account for the presence of active PAI-1 stored within platelets that have low biosynthetic capabilities and have a mean life span of 9-12 days in the circulation(31) . Because vitronectin is known to be capable of increasing by 2-fold the half-life of PAI-1 activity in solution (37 °C) (for review, see (32) ) and complexes between vitronectin and PAI-1 can be detected in the releasates of activated platelets (33) , it is possible that this inhibitor is stabilized within -granules and that this effect is mediated via its intraorganelle association with vitronectin. However, active PAI-1 can also be stabilized by incubation in a low pH environment(34) , which is known to exist in the regulated secretory pathway(35, 36) , as well as via its interaction with other molecules (e.g. arginine)(37) . Based upon this information, this study was initiated to investigate the mechanisms within the -granule that may contribute to the presence of active PAI-1 in platelets. In this report, we document that the activity of PAI-1 is stabilized within intact platelets and isolated -granules but this stabilization is not mediated by the binding of PAI-1 to -granule vitronectin or dependent upon the pH of the -granule. In contrast, our data indicate that active PAI-1 is stabilized within -granules in a calcium-dependent process and involves the association of PAI-1 with a series of -granule proteins that result in the formation of a high M complex or aggregate. These data suggest that the interactions between PAI-1 and other proteins in -granules is considerably more complex than previously envisioned.

MATERIALS AND METHODS

Preparation of Platelets

Platelet Homogenization

Isolation of -Granules

Time Course Stabilization Studies

Purification of PAI-1

Quantitation of PAI-1 Activity and Antigen

Quantitation of PAI-1 antigen was performed as described previously (10) by using an immobilized monoclonal (2D2) antibody against human PAI-1 to bind PAI-1 antigen in a sample, and bound PAI-1 was detected as described above.

Enzyme Immunosorbent Assay for Vitronectin PAI-1 Complexes

SDS-PAGE Immunoblotting and Ligand Blotting

Electron Microscopy

Parloidion/carbon-coated nickel grids were incubated (5 min, 22 °C) with 10 µg/ml of purified rabbit antibodies (i.e. affinity-purified rabbit anti-PAI-1 or normal rabbit IgG), washed with distilled water, blocked by incubation (1 h, 22 °C) with 5 mg/ml goat IgG in Sepharose column buffer (10 mM CaCl, 10 mM MES, Sigma, pH 6.4), and incubated with 100 µl of a column fraction for 1 h at 22 °C. Samples of Sepharose CL-6B column fractions were diluted in 5 mg of goat IgG in column buffer to reduce nonspecific background binding of proteins to the IgG-coated grids. The grids were subsequently washed with 5 mg/ml goat IgG in Sepharose column buffer, washed quickly with distilled water, negatively stained with uranyl acetate, air dried, and examined under a Hitachi 12-UA electron microscope(41) .

Fractionation of -Granules over Molecular Sieving Columns

RESULTS

Stabilization of PAI-1 in Platelet -Granules

Figure 1: Stabilization of PAI-1 activity in intact platelets and -granules. Platelets (Panel A, 10 platelets/aliquot) and isolated -granules (Panel B, 1 mg of protein/aliquot) were incubated either at 37 °C as intact structures (open symbols) or following lysis by freeze-thawing (closed symbols). At the indicated times, the samples were frozen in liquid nitrogen and subsequently assayed for PAI-1 activity utilizing a functional immunoassay as described under ``Materials and Methods.'' Data represent the means ± 1 S.D. from experiments utilizing platelets and -granules harvested from four individuals. Thin section electron microscopic analysis of intact platelet -granules either prior to incubation at 37 °C (Panel C, scale bar, 250 nm) or following an 8-h incubation at 37 °C (Panel D, scale bar, 250 nm).

The data in Fig. 1suggest that PAI-1 activity is stabilized within platelet -granules. Because multimeric forms of vitronectin have been detected within platelets (48) and vitronectin is capable of binding PAI-1(33) , we investigated if PAI-1 could be associated with vitronectin in -granules. Western blotting of platelets for vitronectin revealed two prominent immunoreactive bands with molecular masses of 72 and 74 kDa (Fig. 2A, lane 1). Ligand blotting, a procedure that utilizes the ability of certain molecules to interact with proteins separated by SDS-PAGE and transferred to nitrocellulose, indicated that the major PAI-1-binding proteins present in platelets co-migrated with vitronectin (Fig. 2A, lane 2). However, analysis of PAI-1-vitronectin complexes utilizing a two-site immunological assay revealed that complexes between these two molecules were not present within either platelets or isolated -granules (Fig. 2B).

Figure 2: Functional and immunological analyses of the interaction between platelet PAI-1 and vitronectin. Panel A, platelets (10/lane) were fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose. After blocking, lane 1 was probed with a monoclonal antibody directed against vitronectin (10 µg/ml; mAb 1244) and lane 2 was incubated with 25 ng of PAI-1 followed by rabbit anti-PAI-1 (10 µg/ml). Detection of the bound antibody was performed using the appropriate I-labeled second antibody. Arrow indicates the M of endogenous PAI-1. Panel B, platelets (10 platelets containing 125.2 ± 11.3 ng of PAI-1 antigen/well, n = 4) or -granules (2 mg/ml protein containing 154.2 ± 17.8 ng of PAI-1 antigen/well, n = 4) were sonicated and either assayed immediately for PAI-1-vitronectin complexes or the sonicated material incubated for 1 h at 37 °C and then assayed for PAI-1-vitronectin complexes as described under ``Materials and Methods.''

Because active PAI-1 can also be stabilized by reducing the pH of the buffer(34) , we investigated the effect of several agents that disrupt pH gradients on the stability of PAI-1 associated with -granules. Table 1indicates that the co-incubation of -granules with NHCl, KCl, and/or the ionophore nigericin had no effect on the half-life of PAI-1 activity. However, the stability of PAI-1 associated with -granules could be reduced to the levels observed for this inhibitor in solution by incubating the isolated -granules with the calcium ionophore A23187 (Table 1). Addition of calcium ions to the isolated -granules either prior to or simultaneous with the addition of the calcium ionophore was observed to be an effective means of neutralizing the ionophore's effect on PAI-1 activity (Table 1). These data suggest that endogenous calcium ions within the -granule are participating in the stabilization of PAI-1 activity.

Evidence for the Interaction of PAI-1 with Other Proteins within Platelet -Granules-To understand the interactions that are involved in the calcium-dependent stabilization of PAI-1 activity within -granules, we investigated the applicability of fractionating -granule proteins utilizing a buffer system that would mimic the conditions (e.g. high calcium concentration) known to be present within storage granules(35, 36) . For example, Chanat and Huttner (47) optimized a buffer system containing 10 mM CaCl, 10 mM MES, pH 6.4, which these investigators refer to as aggregative milieu because these conditions were sufficient to maintain the aggregation of several regulated secretory proteins in isolated vesicles of the trans-Golgi network and in isolated secretory granules of PC12 cells. Based upon this information, isolated -granules were lysed into and fractionated on molecular sieving columns in this buffer system which was supplemented with low concentrations of Triton X-100 in order to prevent reassembly of phospholipid membranes/vesicles. Fig. 3A demonstrates a representative experiment that utilized this buffer system and indicates that the majority of -granule derived PAI-1 antigen is present in the void volume of Sepharose CL-6B columns, which has an exclusion volume with a molecular mass of >10 daltons. The fractionation profile shown in Fig. 3A is representative of a series of experiments that were performed with platelets derived from approximately 20 human donors (i.e. each experiment requiring 1 unit of human blood as described under ``Matrials and Methods'') as a means to characterize this high M form of PAI-1. When the fractionation was performed utilizing PBS, the majority of PAI-1 was distributed within the column volume (data not shown) in agreement with our published data utilizing platelet releasates(30) . Chromatography of purified PAI-1 on Sepharose CL-6B columns in the presence of either PBS or aggregative milieu supplemented with 0.025% Triton X-100 resulted in this molecule fractionating within the column volume at M 50,000 (data not shown). To investigate the nature of these PAI-1-containing high M species, affinity purified rabbit antibodies against PAI-1 were bound to electron microscopic grids and used to immunoabsorb PAI-1 and associated proteins within the fractions excluded from the Sepharose CL-6B column. The grids were subsequently negatively stained, and electron microscopic examination revealed complexes of -granule proteins that were 20-30 nm in diameter (Fig. 4). Control experiments utilizing grids coated with normal rabbit IgG or another protein and subsequently incubated with these column fractions were devoid of these structures when examined in the electron microscope.

Figure 3: Fractionation of -granule proteins on Sepharose CL-6B. Panels A and B, platelet -granules (1 ml containing 55 mg of protein) were lysed by the addition of Triton X-100 to a final concentration of 1% and fractionated on a Sepharose CL-6B column (95 1.5 cm, 30 ml/h) either employing a column buffer of 10 mM CaCl, 10 mM MES, pH 6.4, 0.025% Triton X-100 (Panel A) or a column buffer of 10 mM CaCl, 10 mM MES, pH 7.4, 0.025% Triton X-100 (Panel B). Fractions (2 ml) were collected and assayed for A or immunologically for PAI-1 antigen. Panel C, The high M void volume fractions of a Sepharose CL-6B column shown in Panel A were pooled, concentrated using Centricon 10 spin tubes, and fractionated on a Sepharose CL-2B column (95 1.5 cm, 30 ml/h) employing a column buffer of 10 mM CaCl, 10 mM MES, pH 7.4. Fractions (2 ml) were collected and assayed for A or immunologically for PAI-1 antigen. Panel D, single-unit PAI-1-containing spherical structures isolated on a Sepharose CL-2B column (Panel C, fractions 44-48) were pooled and chromatographed either on a mAb 2D2 column (0.5 0.5 cm, 10 ml/h; open symbols) or on a normal mouse IgG column (0.5 0.5 cm, 10 ml/h; closed symbols) in 10 mM CaCl, 0.025% Triton X-100, 10 mM MES, pH 7.4. The column was washed and eluted with 10 mM EDTA, 0.5 M NaCl, 0.025% Triton X-100, 10 mM MES, pH 7.4, followed by 0.2 M glycine-HCl, pH 2.5. Inset shows reducing SDS-PAGE/silver stained gel of following fractions: lane 1, pool of Sepharose CL-2B fractions 44-48, 200 µl; lane 2, fraction 13 of mAb 2D2 column eluted with EDTA buffer, 200 µl; lane 3, fraction 13 of normal mouse IgG column eluted with EDTA buffer, 200 µl; lane 4, fraction 20 of mAb 2D2 column eluted with acidic pH, 200 µl; lane 5, fraction 20 of normal mouse IgG column eluted with acidic pH, 200 µl.

Figure 4: Electron microscopic analysis of PAI-1-containing high M fractions from Sepharose CL-6B columns. Parloidion/carbon-coated nickel grids were incubated with 10 µg/ml affinity-purified rabbit anti-PAI-1 and blocked with 5 mg/ml goat IgG in Sepharose column buffer. Fraction 26 of Sepharose CL-6B column profile shown in Fig. 3A was diluted with 5 mg/ml goat IgG in Sepharose column buffer and incubated (1 h, 22 °C) with the antibody-coated grids. The grids were washed, negatively stained with uranyl acetate, and examined in a Hitachi (12-UA) microscope. Arrow in Panel A (scale bar, 250 nm) indicates area shown in Panel B at higher magnification (scale bar, 250 nm).

A series of experiments were performed to further understand several features of the high M PAI-1-containing protein complexes that were isolated from the -granule matrix. First, analysis of the high M PAI-1-containing void volume fractions of the Sepharose CL-6B column (Fig. 3A) for PAI-1-vitronectin complexes in our two-site immunoassay continued to reveal an absence of complexes between these two molecules (data not shown). Second, our initial observation that the stability of PAI-1 in -granules was not affected by the incubation of these organelles with pH-altering ionophores suggested that we should be able to increase the pH of the lysing and column buffer and determine if the complexes of -granule proteins play a role in stabilizing PAI-1 at a neutral pH. Therefore, isolated -granules were lysed with Triton X-100 in a buffer containing 10 mM CaCl, 10 mM MES, pH 7.4, and fractionated on a Sepharose CL-6B column utilizing this buffer supplemented with 0.025% Triton X-100. Although the protein concentration in the void volume was reduced by approximately one-third, the majority of the PAI-1 antigen continued to elute in the void volume (Fig. 3B). Furthermore, analysis of these fractions under the electron microscope utilizing rabbit anti-PAI-1-coated grids continued to reveal -granule protein complexes. Analysis of the stability of PAI-1 in the void volume fractions revealed a prolonged half-life of 4 h, comparable to the situation for PAI-1 in intact -granules (Table 1). Third, because fractionation of -granules over a Sepharose CL-6B column results in a void volume that contains a heterogenous population of single-unit and multiunit structures, we investigated our ability to subfractionate these populations. Therefore, the high M void volume of a Sepharose CL-6B column was concentrated using Centricon 10 spin tubes and the concentrate was fractionated on a Sepharose CL-2B column utilizing the aforementioned buffer of 10 mM CaCl, 10 mM MES, pH 7.4. PAI-1 antigen was observed to elute either in the void volume of the Sepharose CL-2B column or within the column volume (Fig. 3C). Analysis of the PAI-1-containing fractions on electron microscopic grids indicated that the Sepharose CL-2B void volume fractions were primarily composed of multiunit complexes, whereas the major peak of PAI-1 antigen present in the column volume corresponded to single-unit 25-nm structures. Stability studies with the PAI-1-containing Sepharose 2B column fractions that contained single units revealed a comparable prolonged half-life (Table 1), thus suggesting that the interactions between PAI-1 and the molecules in these structures play a role in stabilizing PAI-1.

Based upon our ability to immunoabsorb PAI-1-containing high M protein complexes to electron microscopic grids, we investigated the possibility of utilizing Sepharose beads conjugated with one of our current mAbs directed against PAI-1 as an affinity matrix to identify the species of proteins that constitute these PAI-1-containing high M protein complexes. For example, mAb 2D2 reacts strongly with solution-phase PAI-1, and we routinely use this mAb in a two-site immunoassay as it detects both free PAI-1 and PAI-1 complexed to tissue-type PA(10) . Fig. 3D indicates an experiment in which mAb 2D2 was coupled to CNBr-activated Sepharose, and this column was used to absorb the PAI-1-containing column fractions from a Sepharose CL-2B column run. The Sepharose-MAB 2D2 column was washed, eluted with an EDTA-containing buffer, and finally with an acidic buffer. A defined set of proteins was found to bind to this affinity column and could be eluted with the EDTA-containing buffer (Fig. 3D, inset), whereas the majority of PAI-1 antigen remained associated with the affinity matrix and required acidic conditions for elution. A control column of mouse IgG bound neither PAI-1 nor any of the -granule proteins from the Sepharose CL-2B column (Fig. 3D). Taken together, these data indicate that the high M PAI-1-containing complexes are composed of a set of defined proteins and that these complexes of proteins play a role in stabilizing PAI-1.

DISCUSSION

Platelet PAI-1 has been established to play a key role in regulating the fibrinolytic system(13, 23, 24, 25, 26) . One unusual characteristic of this inhibitor is its instability at 37 °C(2) . Although PAI-1 was first detected in platelets in 1984(14, 18) , little information exists concerning the mechanisms that stabilize this relatively labile inhibitor within platelets, which have a life span in the circulation of 9-12 days and are formed over a period of 3-5 days during megakaryocytopoiesis(31) . This study presents biochemical evidence indicating that the active PAI-1 in platelets is stabilized within -granules by a unique mechanism. First, although our combined Western blotting and ligand blotting experiments revealed that the major PAI-1 binding protein within -granules is vitronectin, a two-site immunoassay was not able to detect complexes between PAI-1 and vitronectin immediately following the lysis of the -granules, whereas complexes between these two proteins could be readily detected following a 1-h incubation at 37 °C of the lysed organelles. These results are in agreement with the published data of Preissner et al.(33) in which complexes between PAI-1 and vitronectin could be detected in platelet releasates. Because active PAI-1 has been observed to have a significantly higher affinity to vitronectin than the latent form(49, 50) , our ability to detect between 3.2-4.5% of the total platelet/-granule PAI-1 to be complexed with vitronectin following a 1 h incubation at 37 °C is in agreement with published data (15) indicating that approximately 3.5 ± 1.1% of platelet PAI-1 is in an active form. Second, co-incubation of -granules with a number of agents that disrupt pH gradients (e.g. NHCl) had no effect on the stability of PAI-1 activity suggesting that the low pH within -granules was not responsible for the stabilization of this inhibitor's activity. Thirdly, the stability of PAI-1 associated with -granules could be reduced to the levels observed for PAI-1 in solution by incubating the isolated -granules with the calcium ionophore A23187. Furthermore, addition of exogenous calcium ions to the isolated -granules either prior to or simultaneous with the addition of the calcium ionophore was an effective means of neutralizing the ionophore's effect on PAI-1 activity. These data suggest that endogenous calcium ions within the -granule are participating in the stabilization of PAI-1 activity.

This concept is further supported by our ability to obtain a high M form of PAI-1 by the fractionation of -granules proteins on a series of molecular sieving columns (e.g. Sepharose CL-6B) utilizing a high calcium-containing buffer. Immunoabsorption coupled with negative staining electron microscopy indicate that PAI-1 in these column fractions is associated with a number of other -granule proteins in a 25-nm diameter unit, which appears to be involved in the stabilization of PAI-1. Although the -granule matrix is highly electron dense, we have been able to identify at high magnification (i.e. 60,000 magnification) regions within the -granule matrix that resemble 25-nm single or multiunit PAI-1-containing complexes. ()Furthermore, we are also able to detect the release of similar structures from -granules that were resuspended in PBS and disrupted by a single freeze-thawing cycle followed immediately by immersion into fixative. These latter observations suggest that these structural units were derived from the granules and not a result of the buffer environment that was employed to stabilize these structures.

In addition to providing evidence for a novel mechanism that mediates the stabilization of a physiologically relevant form of PAI-1, our data also provide an insight into the processes that target PAI-1 into -granules. Current information concerning the sorting of proteins into the regulated secretory pathway using a number of model cell systems indicate that two distinct mechanisms may participate in this process, protein aggregation and specific sorting signals(35, 36) . Supportive evidence for a specific sorting signal has been provided by transfection experiments utilizing the cDNA for P-selectin (i.e. a platelet -granule protein) and a mouse pituitary cell line (i.e. AtT-20 cells) that contains both a constitutive and a regulated secretory pathway(51) . These studies have indicated that a domain on the P-selectin cytoplasmic region may play a direct role in sorting by permitting its direct interaction with the underlying submembrane cytoskeleton(36, 51) . However, prevailing theories (35, 52) would require that a soluble protein (e.g. PAI-1) contains a signal that interacts with a membrane-associated receptor dedicated to facilitate sorting. Because a highly conserved sequence of amino acids has not been identified within the diverse group of proteins that are routed into the regulated secretory pathway, the ability of certain secretory products to form molecular aggregates with each other and condense into electron dense material within the Golgi has been proposed as an alternative mechanism to initiate sorting by excluding ``nonaggregating'' molecules from the forming dense core secretory granule(36, 52) . It is known that several factors appear to play a role in the aggregation or condensation of molecules within the trans-Golgi, including an elevated calcium concentration and a low pH (36, 52) . Our observation that high concentrations of calcium ions are able to maintain the association between PAI-1 and several -granule proteins suggest that this latter process may be mediating the packaging of PAI-1. The ability to immunoabsorb the PAI-1-containing high M complexes both to electron microscopic grids and to affinity columns indicate that epitopes for the PAI-1 molecule are available on the surface of these units. Importantly, the absence of vitronectin-PAI-1 complexes in the samples raises the possibility that PAI-1 is oriented in a specific manner as to occupy or mask the vitronectin binding region on the PAI-1 molecule delineated by Lawrence et al.(50) . This situation would be physiologically relevant because platelet-released PAI-1 would not be subsequently restricted to an association with only platelet vitronectin. Thus, following the activation of platelets and the release of -granules into a physiological milieu, our data support a concept in which PAI-1 would be released from its association with other -granules proteins and this free form of active PAI-1 would be then able to bind either to vitronectin in the extracellular matrix, present on the platelet surface, or to another molecule (e.g. fibrin).

Transfection experiments with the cDNA for PAI-1 and AtT-20 cells (53) have indicated that PAI-1 contains a functional region or domain that enables this inhibitor to be directed into the regulated secretory pathway. These studies (53) have also indicated that the activity status of PAI-1 stored in the transfected AtT-20 cells is similar to its activity status within porcine(30) , canine (54) and human platelets(15, 17, 20, 30, 54) . Furthermore, experiments utilizing this system (53) revealed that PAI-1 is also stabilized within AtT-20 dense core secretory granules resulting in a prolonged half-life of 5 h. Based upon our previous (53) and current data, we hypothesize that PAI-1 contains a functional domain(s) that permits this molecule to associate in a calcium-dependent manner with specific granule proteins that result in the formation of a structural unit in which active PAI-1 is stabilized while concomitantly masking the vitronectin binding domain on this inhibitor.

FOOTNOTES

*

This research was supported by fellowships from the Tobacco Related Disease Research Program 3FT-0194 and the American Heart Association 93-83 (to I. M. L.), and in part by National Institutes of Health Grants HL45954 and HL49563 (to R. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Vascular Biology (VB-1), The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7129; Fax: 619-784-7323.

()

The abbreviations used are: PAI-1, type 1 plasminogen activator inhibitor; PA, plasminogen activator; mAb, monoclonal antibody; MES, morpholinoethanesulfonic acid; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

()

I. M. Lang and R. R. Schleef, unpublished observations.

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