(Received for publication, August 9, 1995; and in revised form, November 21, 1995)
From the
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
-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
-granules using a two-site immunoassay.
Co-incubation of
-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
-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
-granules was an effective means of neutralizing the
ionophore's effect on PAI-1 activity. Fractionation of
-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
daltons. Immunoabsorption of
PAI-1 from these column fractions followed by negative staining
revealed 25-nm diameter complexes of
-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
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
-granule proteins in a calcium-dependent
manner.
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.
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.
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) .
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
NH
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
-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.
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. NH
Cl) 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.