©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium-dependent Stabilization of Type I Plasminogen Activator Inhibitor within Platelet -Granules (*)

(Received for publication, August 9, 1995; and in revised form, November 21, 1995)

Irene M. Lang Raymond R. Schleef (§)

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Type 1 plasminogen activator inhibitor (PAI-1) is known to be synthesized in an active conformation but it is rapidly converted into an inactive conformation (t 1 h) upon incubation at 37 °C. This study was initiated to investigate the mechanism that account for the presence of active PAI-1 in anucleated platelets that have a mean life span of 9-12 days in the circulation. Stabilization experiments with a functional immunoassay indicated that the activity of PAI-1 in both platelets and in isolated alpha-granules was prolonged in comparison to the rapid inactivation of this molecule in their lysates (t 1 h). Although combined ligand blot/immunoblot analysis revealed that vitronectin was the major PAI-1 binding protein in platelets, vitronectin/PAI-1 complexes were not detected in alpha-granules using a two-site immunoassay. Co-incubation of alpha-granules with a number of agents that disrupt pH gradients (e.g. ionophores) had no effect on the stability of PAI-1 activity, whereas incubation of alpha-granules with the calcium ionophore A23187 reduced the half-life of PAI-1 to the levels observed for PAI-1 in solution. Addition of calcium ions to intact alpha-granules was an effective means of neutralizing the ionophore's effect on PAI-1 activity. Fractionation of alpha-granule proteins on molecular sieving columns using conditions known to be present within storage granules (e.g. a high calcium concentration) revealed the presence of PAI-1 in fractions with a molecular mass of >10^6 daltons. Immunoabsorption of PAI-1 from these column fractions followed by negative staining revealed 25-nm diameter complexes of alpha-granule proteins under the electron microscope. PAI-1 activity associated with these complexes was prolonged in the presence of calcium ions and these high M(r) complexes were shown to be composed of a defined set of proteins that can be dissociated from PAI-1 by chelation of calcium ions. These data indicate that PAI-1 is stabilized by its packaging with other alpha-granule proteins in a calcium-dependent manner.


INTRODUCTION

Type 1 plasminogen activator inhibitor (PAI-1) (^1)is the primary physiological inhibitor of vascular tissue-type plasminogen activator with rate constants greater than 10^7 (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(r) 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 alpha-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. alpha-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 alpha-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 alpha-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 alpha-granules but this stabilization is not mediated by the binding of PAI-1 to alpha-granule vitronectin or dependent upon the pH of the alpha-granule. In contrast, our data indicate that active PAI-1 is stabilized within alpha-granules in a calcium-dependent process and involves the association of PAI-1 with a series of alpha-granule proteins that result in the formation of a high M(r) complex or aggregate. These data suggest that the interactions between PAI-1 and other proteins in alpha-granules is considerably more complex than previously envisioned.


MATERIALS AND METHODS

Preparation of Platelets

Human blood (500 ml/donor) was collected from the cubital vein into acid citrate dextrose (0.025 M citric acid, 0.85 M sodium citrate, 2% dextrose; one part acid citrate dextrose, five parts whole blood). Platelet-rich plasma was prepared by centrifugation of anticoagulated whole blood (160 times g, 15 min). The platelet-rich plasma was aspirated without agitating the buffy coat and then centrifuged (680 times g, 20 min). The platelet pellet was washed twice by centrifugation with Tris-buffered saline (0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5) containing 2.5 mM EDTA, 1 mM theophylline, and 5.5 mMD-glucose. The resultant pellet was resuspended in the same buffer, and the cells were counted in a Neubauer hemocytometer. Approximately 10 platelets were routinely recovered per unit of blood.

Platelet Homogenization

Platelets were diluted in homogenization buffer (108 mM NaCl, 38 mM KCl, 1.7 mM NaHCO(3), 21.2 mM sodium citrate, 27.8 mMD-glucose, 1.1 mM MgCl(2), 1 mM theophylline, pH 6.5) to a final concentration of 1 times 10^9/ml and sonicated using an Astrason Ultrasonic processor XL (Heat Systems Incorporated, Farmingdale, NY) for five times (4 °C, 3 s of sonication on setting 2 followed by a 15-s pause between each sonication). Samples were centrifuged (15 min, 2,000 times g) and the membrane/organelle/cytosol-containing supernatants were pooled.

Isolation of alpha-Granules

Two consecutive centrifugation protocols were employed to separate alpha-granules from other platelet organelles. First, a metrizamide gradient (38) was utilized to deplete platelet homogenates of dense granules. For this purpose, supernatants of sonicated platelets were mixed 1:1 with a 40% metrizamide solution (Accurate Chemical and Scientific Co., Westbury, NY), and this mixture was layered on top of a two-step gradient consisting of 1 ml of 35% metrizamide underlayered with 0.5 ml of 38% metrizamide. Following centrifugation (1 h, 4 °C, 100,000 times g, VTi-65 rotor, Beckman L7-65 ultracentrifuge), dense granules were located in a compact pellet at the bottom of the centrifuge tube, whereas the alpha-granules were present in a thick band at the interface above the 35% metrizamide solution. Density gradient fractionation on isotonic Percoll gradients (39) was subsequently utilized as the second step to separate alpha-granules from other platelet organelles. This procedure entailed diluting the alpha-granule-rich fractions from the metrizamide gradient with 0.15 M NaCl and mixing the samples 1:1 with 90% stock isotonic Percoll (1 ml of 1.5 M NaCl + 9 ml of Percoll; Pharmacia Biotech Inc.) and subjecting this mixture to ultracentrifugation (4 °C, 30 min, 20,000 times g). The alpha-granules were recovered as an opaque band at a density of 1.06-1.1 g/liter and were washed twice utilizing an Airfuge (100,000 times g, 15 min, 22 °C). Control experiments utilizing platelets incubated with [^3H]serotonin-binoxalate (DuPont) and subcellular fractionation as described above followed by analysis of the radioactivity in the various fractions utilizing a Beckman LS3801 scintillation counter revealed that this two-step gradient procedure efficiently (>98%) depleted the alpha-granule-containing fractions of [^3H]serotonin.

Time Course Stabilization Studies

Platelets (10^8/aliquot) or isolated platelet alpha-granules (1 mg/aliquot) were distributed at 4 °C into two groups of aliquots. Prior to the start of the experiment, one group was freeze-thawed three times by immersion into liquid nitrogen followed by rapid thawing at 37 °C. Stabilization experiments with platelets were performed by incubation of the intact samples and the freeze-thawed samples at 37 °C for 0-8 h. In experiments with alpha-granules, either ionophores (i.e. nigericin, Sigma; calcium ionophore A23187, Sigma), pH-altering chemicals (i.e. NH(4)Cl), or the appropriate diluent were first added both to aliquots containing intact alpha-granules and to aliquots of frozen-thawed alpha-granules, and the samples were subsequently incubated at 37 °C for 0-8 h. Samples were removed at the indicated times, snap frozen in liquid nitrogen, and stored at -70 °C until analysis. All samples were lysed in 0.5% Triton X-100 prior to analysis for PAI-1 activity and antigen.

Purification of PAI-1

Native PAI-1 was purified from the medium conditioned by a transformed human lung fibroblast cell line as described previously(40, 41) . Antibodies to PAI-1 were raised in New Zealand rabbits and affinity-purified on Sepharose-PAI-1 columns as described previously(40, 41) .

Quantitation of PAI-1 Activity and Antigen

PAI-1 activity was quantitated as described previously (10, 42, 43) by using immobilized tissue-type PA (American Diagnostica) to bind active PAI-1 in a sample, and the bound PAI-1 was immunologically detected by incubation with affinity-purified rabbit anti-PAI-1 (10 µg/ml) (41) followed by I-labeled goat anti-rabbit IgG (50,000 cpm/well; Amersham Corp.).

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

Complexes between PAI-1 and vitronectin were quantitated utilizing a two-site immunoassay protocol based upon the assay described by Preissner et al.(33) . Flat-bottom microtiter plates (Immulon II, Dynatech, Chantilly, VA) were coated with monoclonal anti-PAI-1 (2D2)(10) , and the plates were washed and blocked by incubating with blotto (5% w/v skimmed milk powder in 0.01 M Tris-HCl, pH 7.5, 200 µl/well, 2 h, 37 °C). The plates were subsequently incubated with either samples (100 µl/well) or a PAI-1/vitronectin standard curve prepared by incubating increasing amounts of PAI-1 with 300 ng of purified vitronectin. Detection of bound proteins was performed by incubating (37 °C, 1 h) the wells with alkaline phosphatase-labeled goat anti-rabbit lgG (1:2000 dilution, 100 µl/well) followed by incubation with p-nitrophenylphosphate and measurement of the color change at 405 nm over a 10-min period utilizing a Molecular Devices microplate reader. Experiments utilizing affinity-purified rabbit anti-PAI-1 as the immunoabsorbent and detection of the bound complexes utilizing biotinylated rabbit antibodies to vitronectin followed sequentially by alkaline phosphatase-streptavidin conjugate and finally p-nitrophenylphosphate yielded similar results. Purified vitronectin and antibodies directed against vitronectin were a gift of D. Seiffert (Scripps Research Institute).

SDS-PAGE Immunoblotting and Ligand Blotting

SDS-PAGE was performed according to the procedures described by Laemmli(44) , and gels were processed either for Western blot analysis as described previously(40) , for silver staining as described previously(45) , or for ligand blotting as described by Seiffert et al.(46) .

Electron Microscopy

Thin-section electron microscopic analysis of alpha-granules was performed by fixation in modified Karnovsky's solution (1.0% paraformaldehyde, 1.5% glutaraldehyde, 0.1 M cacodylate buffer) and pelleting by centrifugation (14,000 times g, 5 min). Postfixation was carried out using 1.5% OsO(4) in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature, followed by dehydration in a graded ethanol series. Samples were embedded in Epon 812 and cut after polymerization, mounted on 100-mesh parallel lined grids (Ted Pella, Inc., Tustin, CA), and double stained with uranyl acetate and lead citrate. Samples were examined under a Hitachi 12-UA electron microscope as described previously(41) .

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(2), 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 alpha-Granules over Molecular Sieving Columns

Isolated platelet alpha-granules were resuspended to a final volume of 1 ml in the appropriate buffer (i.e. aggregative milieu: 10 mM CaCl(2), 10 mM acid, MES, pH 6.4(47) ; 10 mM CaCl(2), 10 mM MES, pH 7.4; PBS) and lysed by the addition of Triton X-100 to 1%. The preparation was centrifuged (20,000 times g, 15 min, 4 °C) to remove any residual Triton X-100-insoluble material, and the supernatant was fractionated on a Sepharose CL-6B column (95 times 1.5 cm, 30 ml/h, 2 ml/fraction) utilizing the appropriate column buffer supplemented with 0.025% Triton X-100. To further analyze the high M(r) void volume fractions of Sepharose CL-6B columns, these fractions were pooled, concentrated using Centricon 10 spin tubes (Amicon), and fractionated on a Sepharose CL-2B column (95 times 1.5 cm, 30 ml/h) employing a column buffer of 10 mM CaCl(2), 10 mM MES, pH 7.4. Fractions (2 ml) were collected and assayed for A or immunologically for PAI-1 antigen. Fractions containing single-unit 25-nm structures were identified by immunoabsorption on grids followed by negative staining and electron microscopy and these fractions were pooled and chromatographed either on a mAb 2D2 column (0.5 times 0.5 cm, 10 ml/h) or on a normal mouse IgG column (0.5 times 0.5 cm, 10 ml/h) in 10 mM CaCl(2), 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. Fractions were analyzed by SDS-PAGE/silver staining and immunologically for PAI-1 antigen.


RESULTS

Stabilization of PAI-1 in Platelet alpha-Granules

The PAI-1 activity associated with intact platelets incubated at 37 °C declines to a plateau of approximately two-thirds of the initial value over several hours in comparison to a half-life of 1 h for frozen and thawed platelets (Fig. 1A). Because agonist-induced platelet activation is known to cause the release of active PAI-1, which has led to the concept that PAI-1 is stored within platelet alpha-granules(14, 15, 18, 20, 21, 22) , we decided to separate alpha-granules from other platelet organelles (e.g. dense granules) and investigate the stability of PAI-1 within these structures. Control immunoblotting experiments indicated that the majority of PAI-1 activity and antigen co-distributed in the density gradients with other known markers for alpha-granule proteins (data not shown). PAI-1 activity associated with isolated alpha-granules also exhibited a prolonged half-life at 37 °C that was calculated to be 8.2 h in comparison to a half-life of 1 h observed with frozen and thawed alpha-granules (Fig. 1B). Similar results were obtained in a separate series of experiments that compared intact alpha-granules with samples lysed by treatment with 0.5% Triton X-100 (data not shown). Electron microscopic analysis revealed that no morphological changes could be detected in the isolated alpha-granules over the incubation period (Fig. 1, compare Panels C versus D).


Figure 1: Stabilization of PAI-1 activity in intact platelets and alpha-granules. Platelets (Panel A, 10^8 platelets/aliquot) and isolated alpha-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 alpha-granules harvested from four individuals. Thin section electron microscopic analysis of intact platelet alpha-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 alpha-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 alpha-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 alpha-granules (Fig. 2B).


Figure 2: Functional and immunological analyses of the interaction between platelet PAI-1 and vitronectin. Panel A, platelets (10^8/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(r) of endogenous PAI-1. Panel B, platelets (10^8 platelets containing 125.2 ± 11.3 ng of PAI-1 antigen/well, n = 4) or alpha-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 alpha-granules. Table 1indicates that the co-incubation of alpha-granules with NH(4)Cl, 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 alpha-granules could be reduced to the levels observed for this inhibitor in solution by incubating the isolated alpha-granules with the calcium ionophore A23187 (Table 1). Addition of calcium ions to the isolated alpha-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 alpha-granule are participating in the stabilization of PAI-1 activity.



Evidence for the Interaction of PAI-1 with Other Proteins within Platelet alpha-Granules-To understand the interactions that are involved in the calcium-dependent stabilization of PAI-1 activity within alpha-granules, we investigated the applicability of fractionating alpha-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(2), 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 alpha-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 alpha-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 >10^6 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(r) 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(r) 50,000 (data not shown). To investigate the nature of these PAI-1-containing high M(r) 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 alpha-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 alpha-granule proteins on Sepharose CL-6B. Panels A and B, platelet alpha-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 times 1.5 cm, 30 ml/h) either employing a column buffer of 10 mM CaCl(2), 10 mM MES, pH 6.4, 0.025% Triton X-100 (Panel A) or a column buffer of 10 mM CaCl(2), 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(r) 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 times 1.5 cm, 30 ml/h) employing a column buffer of 10 mM CaCl(2), 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 times 0.5 cm, 10 ml/h; open symbols) or on a normal mouse IgG column (0.5 times 0.5 cm, 10 ml/h; closed symbols) in 10 mM CaCl(2), 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(r) 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(r) PAI-1-containing protein complexes that were isolated from the alpha-granule matrix. First, analysis of the high M(r) 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 alpha-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 alpha-granule proteins play a role in stabilizing PAI-1 at a neutral pH. Therefore, isolated alpha-granules were lysed with Triton X-100 in a buffer containing 10 mM CaCl(2), 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 alpha-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 alpha-granules (Table 1). Third, because fractionation of alpha-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(r) 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(2), 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(r) 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(r) 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 alpha-granule proteins from the Sepharose CL-2B column (Fig. 3D). Taken together, these data indicate that the high M(r) 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 alpha-granules by a unique mechanism. First, although our combined Western blotting and ligand blotting experiments revealed that the major PAI-1 binding protein within alpha-granules is vitronectin, a two-site immunoassay was not able to detect complexes between PAI-1 and vitronectin immediately following the lysis of the alpha-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/alpha-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 alpha-granules with a number of agents that disrupt pH gradients (e.g. NH(4)Cl) had no effect on the stability of PAI-1 activity suggesting that the low pH within alpha-granules was not responsible for the stabilization of this inhibitor's activity. Thirdly, the stability of PAI-1 associated with alpha-granules could be reduced to the levels observed for PAI-1 in solution by incubating the isolated alpha-granules with the calcium ionophore A23187. Furthermore, addition of exogenous calcium ions to the isolated alpha-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 alpha-granule are participating in the stabilization of PAI-1 activity.

This concept is further supported by our ability to obtain a high M(r) form of PAI-1 by the fractionation of alpha-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 alpha-granule proteins in a 25-nm diameter unit, which appears to be involved in the stabilization of PAI-1. Although the alpha-granule matrix is highly electron dense, we have been able to identify at high magnification (i.e. 60,000 times magnification) regions within the alpha-granule matrix that resemble 25-nm single or multiunit PAI-1-containing complexes. (^2)Furthermore, we are also able to detect the release of similar structures from alpha-granules that were resuspended in PBS and disrupted by a single freeze-thawing cycle followed immediately by immersion into fixative.^2 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 alpha-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 alpha-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 alpha-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(r) 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 alpha-granules into a physiological milieu, our data support a concept in which PAI-1 would be released from its association with other alpha-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.

(^1)
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.

(^2)
I. M. Lang and R. R. Schleef, unpublished observations.


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