(Received for publication, November 6, 1995; and in revised form, February 9, 1996)
From the
Vitronectin (Vn) is not only a major adhesive glycoprotein present in platelets but also regulates proteolytic enzyme cascades, including the blood coagulation, fibrinolytic, and complement systems. In human platelet lysates prepared by freeze-thawing or by the addition of nonionic detergent, the Vn antigen content was drastically reduced in comparison with lysates prepared in the presence of SDS, suggesting that Vn is hydrolyzed by platelet-associated enzymes. Exogenously added purified human Vn and Vn present in plasma were also cleaved by these enzyme systems. Degradation was mediated by a nonsecreted or membrane-associated protease system that was inhibited by E-64, EDTA, and leupeptin but not inhibitors of serine and aspartic proteases, suggesting an involvement of calcium-dependent cysteine proteases. Consistently, calpastatin inhibited the hydrolysis of Vn, suggesting that Vn is a substrate for calpain. This was confirmed in a purified system. Vn was cleaved by calpains I and II in a dose- and time-dependent manner, resulting in defined Vn fragments with similar electrophoretic mobility in comparison with those detected in platelet lysates. Functional characterization of the calpain-hydrolyzed Vn revealed that while the type 1 plasminogen activator inhibitor binding activity was unchanged, the heparin and cell binding functions were destroyed. These results suggest that calpains released upon platelet membrane damage or upon tissue injury and necrosis differentially regulate functional domains of the Vn molecule.
Vitronectin (Vn), ()an adhesive glycoprotein present
in the circulation and in a variety of tissues, appears to have a
number of biological functions. It belongs to a group of adhesion
molecules that mediate attachment of cells by binding to specific cell
surface receptors of the integrin family(1, 2) . Vn
also exerts several regulatory functions in cell-associated proteolytic
enzyme cascades, including the complement, coagulation, and
fibrinolytic systems. For example, Vn binds to and stabilizes the
biological activity of type 1 plasminogen activator inhibitor (PAI-1),
the physiological inhibitor of both tissue-type and urinary-type
plasminogen activators(3) . Binding sites for PAI-1 in Vn have
been mapped to both the N-terminal somatomedin B domain of Vn (4, 5, 6) and the C-terminal heparin binding
domain(7, 8, 9, 10) . The relative
importance of each single site for the binding of PAI-1 has not been
established.
Plasma contains the majority of Vn present in
vivo, and plasma Vn concentrations have been reported to be
200-400 µg/ml(2) . A second circulatory pool of Vn is
contained within platelets(11, 12, 13) , and
the concentration of Vn in platelets has been estimated to be 2-8
µg/10 platelets(12, 13) . Thus,
platelets contain approximately 0.8% of the circulating pool of Vn, and
platelet Vn accounts for 1% of total platelet proteins. Platelet Vn is
compartmentalized in the
-granules and released upon stimulation
with physiological agonists(13) . In contrast to plasma Vn, the
released platelet Vn is present in a high molecular weight form and, at
least in part, in complex with PAI-1(13, 14) .
Moreover, Vn released from stimulated platelets expresses epitopes for
a conformationally sensitive monoclonal antibody (mAb) that is not
exposed in the native plasma form(14) . Taken together, these
observations have led to the concept that the circulation contains two
structurally and functionally distinct pools of Vn.
In studying structure-function relationships of platelet Vn, it became apparent that platelet Vn is rapidly degraded in lysates prepared under nondenaturing conditions. Evidence is provided that Vn is susceptible to proteolytic modification by platelet calpain, resulting in the dissociation of PAI-1, heparin, and cell binding functions of the adhesive glycoprotein, suggesting that the principal PAI-1 binding site in Vn is located in N-terminal Vn fragments. These results suggest that calpains released upon platelet membrane damage or upon tissue injury and necrosis will differentially modify Vn functions in vivo.
Figure 1:
Degradation of Vn in lysates of
nonstimulated platelets. Human platelets were isolated by differential
centrifugation, washed, and lysed either by treatment with SDS or by
freeze-thawing. The resulting polypeptides were fractionated by
SDS-PAGE followed by immunoblotting using antibodies to Vn, or they
were analyzed by silver staining. Panel A, lanes 1-4,
immunoblot of a 9% SDS-PAGE using mAb 1244. Lane 1, 1 µl
of plasma; lane 2, supernatant of nonstimulated platelets; lane 3, pellet nonstimulated platelets lysed in SDS; lane
4, pellet nonstimulated platelets lysed by freeze-thawing. Lanes 5 and 6, immunoblot of a 15% SDS-PAGE using mAb
1244. Lane 5, pellet nonstimulated platelets lysed in SDS; lane 6, pellet nonstimulated platelets lysed by
freeze-thawing. Panel B, immunoblot of a 9% SDS-PAGE using mAb
8E6 (lanes 1 and 2) or rabbit anti-human Vn IgG (lanes 3 and 4); lanes 5 and 6,
silver stain of a 9% SDS-PAGE. Lanes 1, 3, and 5,
pellet nonstimulated platelets lysed in SDS; lanes 2, 4, and 6, pellet nonstimulated platelets lysed by freeze-thawing. The
migration of M standards is
indicated.
Figure 7:
Limited proteolysis of Vn in a purified
protein system and ex vivo results in similarly sized initial
cleavage fragments. Purified denatured Vn (4 µg, lane 1)
was added to washed platelets in platelet wash buffer containing 1
mM Ca (lane 2, 5
10
platelets; lane 3, 1
10
platelets; lane 4, 5
10
platelets; lane 5, 1
10
platelets; lane 6, 5
10
platelets), and lysates were prepared by the addition of Triton
X-100 (0.5% v/v). In lane 7, purified Vn was cleaved with
calpain II at a calpain to Vn molar ratio of 1:10. The samples were
fractionated by SDS-PAGE and analyzed by immunoblotting using mAb 1244.
The mobility of M
standards is
indicated.
The apparent lack of Vn immunoreactivity upon freeze-thawing of platelets raised the possibility that the mAb 1244 epitope was destroyed by limited proteolysis. To explore this possibility, platelet lysates were analyzed by a second mAb with an epitope distinct from that of mAb 1244 (Fig. 1B, lanes 1 and 2) and by a polyclonal antibody (Fig. 1B, lanes 3 and 4). Using these antibodies, Vn immunoreactivity was again drastically reduced by freeze-thawing in comparison with the SDS lysates. These results raised the possibility that the degradation of Vn went to apparent completion.
To exclude the possibility that the reduction of Vn immunoreactivity was due to a general protein degradation induced by freeze-thawing, platelet samples were fractionated by SDS-PAGE followed by staining with silver nitrate. The overall protein profiles were very similar when comparing SDS lysates (Fig. 1B, lane 5) with lysates prepared by freeze-thawing (Fig. 1B, lane 6). These results suggest that the reduction of Vn immunoreactivity was relatively specific.
Figure 2:
Evidence that Vn released upon platelet
stimulation is not susceptible to degradation. Washed platelets were
stimulated with PMA for 10 min. The platelet releasates (lanes 1 and 2) and platelet pellets (lanes 3 and 4) were separated by centrifugation and harvested in SDS (lanes 1 and 3) or by freeze-thawing (lanes 2 and 4). Alternatively, the stimulated platelets were
directly harvested in SDS (lane 5) or by freeze-thawing (lane 6). The proteins were fractionated by SDS-PAGE (9%) and
analyzed by immunoblotting using mAb 1244. The migration of M standards is
indicated.
Figure 3:
Susceptibility of plasma and purified Vn
to cleavage by platelet proteases. Purified denatured Vn (lanes
1-3 and 10-12), native Vn (lanes
4-6), or human plasma (lanes 7-9) were added
to washed platelets or Hep 3B cells. The platelets/cells were lysed by
treatment with SDS (lanes 2, 5, 8, and 11) or by
freeze-thawing (lanes 3, 6, 9, and 12), and the
resulting polypeptides were fractionated by SDS-PAGE (9%) followed by
immunoblotting using mAb 1244. The migration of M standards is indicated to the right.
Figure 4:
Identification of the groups of proteases
involved in the degradation of Vn. Panel A, washed platelets
were lysed either in SDS (lane 1) or by freeze-thawing in the
absence of protease inhibitor (lane 2) and in the presence of
-macroglobulin (2.5 units/ml, lane 3), E-64
(10 µg/ml, lane 4), leupeptin (1 µg/ml, lane
5), phenylmethylsulfonyl fluoride (1 µM, lane
6), pepstatin (1 µg/ml, lane 7), or EDTA (10
mM, lane 8). The samples were fractionated by
SDS-PAGE (9%) and analyzed by immunoblotting using mAb 1244. Panel
B, platelets were lysed in SDS (lane 1) or in the
presence of calpastatin (lane 2, 100 µg/ml; lane
3, 10 µg/ml; lane 4, 1 µg/ml) and characterized
as in panel A. The mobility of M
standards is indicated.
Platelets are an abundant source of both calpains
I and II, Ca-dependent cysteine proteases that differ
in their Ca
requirements to hydrolyze substrate
proteins (22, 23) . While calpain I is active in the
presence of micromolar levels of calcium, calpain II requires
millimolar concentrations of calcium(22) . To more directly
test the involvement of these protease systems, the effects of
calpastatin, a relatively specific inhibitor of calpains I and
II(23) , on the degradation of Vn were tested. Calpastatin
prevented the hydrolysis of Vn in a dose-dependent manner (Fig. 4B), suggesting that platelet Vn is cleaved by
calpain.
Figure 6:
Cleavage of vitronectin by calpain I.
Purified denatured Vn (1 µg) was dissolved in 40 µl of platelet
wash buffer containing the indicated concentrations of calpain I and
Ca. The samples were incubated for 1 h at 37 °C,
fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and
analyzed by immunoblotting using mAb 1244. Panel A, dose
response of Ca
, Vn to calpain molar ratio of 1:1; lane 1, Vn standard, uncleaved; lane 2, 1 µM Ca
; lane 3, 10 µM Ca
; lane 4, 100 µM Ca
; lane 5, 1 mM Ca
. Panel B, dose response of calpain
I, 100 µM Ca
; lane 1, Vn to
calpain molar ratio of 1:5; lane 2, ratio of 1:1; lane
3, ratio of 2:1; lane 4, ratio of 10:1; lane 5,
ratio of 20:1. The mobility of M
standards is
indicated.
Figure 5:
Degradation of vitronectin by calpain II.
Purified denatured Vn (1 µg) was dissolved in 40 µl of platelet
wash buffer containing the indicated concentrations of calpain II and
Ca. The samples were incubated for 1 h at 37 °C,
fractionated by SDS-PAGE, and analyzed by immunoblotting (panels A and C) or by staining with silver nitrate (panel
B). Panel A, lane 1, uncleaved Vn; lanes 2 and 5, calpain to Vn molar ratio of 5:1; lanes 3 and 6, molar ratio of 1:2; lanes 4 and 7, molar ratio of 1:10. Lanes 1-4 were developed with
rabbit anti-human Vn, whereas lanes 5-7 were developed
with mAb 1244. 1 mM Ca
was used in all lanes. Panel B, lane 1, uncleaved Vn; lane 2, calpain to Vn molar ratio of 1:10; lane 3,
100 ng of purified calpain incubated in parallel. 1 mM Ca
was used in all lanes. Panel C, lane 1, 5 mM Ca
; lane 2, 1
mM Ca
; lane 3, 500 µM Ca
; lane 4, 100 µM Ca
. Calpain to Vn molar ratio of 1:10 in all lanes. The mobility of M
standards is
indicated.
In related
studies, the susceptibility of Vn to hydrolysis by calpain I was tested (Fig. 6). Calpain I hydrolyzed Vn in a
Ca-dependent manner (Fig. 6A).
Limited cleavage was observed in the presence of 10 µM Ca
(Fig. 6A, lane 3), and
maximal cleavage was observed at approximately 100 µM Ca
(Fig. 6A, lane 4). Thus, as
expected from the Ca
requirements of calpain I, a
lower Ca
concentration was needed for Vn cleavage in
comparison with calpain II (compare with Fig. 5). The
electrophoretic mobilities of the resulting initial Vn fragments (i.e. M
60,000, 45,000, and 36,000; not shown)
were similar to those observed using calpain II (compare Fig. 5and Fig. 6). The cleavage of Vn by calpain I was
also dose-dependent with respect to the enzyme concentration, and the
higher concentrations of calpain I resulted in the loss of Vn
immunoreactive fragments (Fig. 6B).
Figure 8:
Functional characterization of
calpain-hydrolyzed Vn. Purified denatured Vn (closed circles)
was cleaved at a Vn to calpain II molar ratio of 1:5 (open
circles), 2:1 (closed squares), or 10:1 (open
squares), and the reaction was stopped by the addition of EDTA (panels A and B) or E-64 (panel C). The
resulting cleavage pattern is presented in Fig. 5A. Panel A, increasing concentrations of the Vn samples were
mixed with a constant amount of active PAI-1 and incubated on Vn-coated
wells. Bound PAI-1 was detected by rabbit anti-PAI-1 IgG, followed by
biotin-labeled anti-rabbit IgG, streptavidin alkaline phosphatase
complex, and p-nitrophenyl phosphate. Results are expressed as
percentage binding in the absence of a soluble competitor. Panel
B, heparin-coated wells were incubated with increasing
concentrations of intact Vn or calpain-hydrolyzed Vn, and bound Vn was
detected with rabbit anti-Vn IgG followed by antibody detection steps
as in panel A. mOD, change of absorbance at 405
nm/min. Panel C, microtiter wells were coated with the
indicated concentration of Vn or Vn digest and incubated with
[S]methionine-labeled HT 1080 cells for 1 h.
After gentle washing, bound cells were quantified by
-counting,
and results are expressed as percentage of cells specifically bound
(see ``Experimental
Procedures'').
The binding of Vn to glycosaminoglycans is believed to be of importance for the localization of Vn in tissues and for the regulation of heparin-dependent proteolytic enzyme cascades(1, 2) . The ability of calpain-hydrolyzed Vn to bind to glycosaminoglycans was tested in solid phase binding assays to immobilized heparin (Fig. 8B). While intact Vn and Vn hydrolyzed with low concentrations of calpain bound to heparin in a dose-dependent manner, more extensive cleavage of Vn by calpain resulted in a successive loss of heparin binding. At the highest concentration of calpain, the heparin binding activity of Vn was practically abolished (Fig. 8B). The solid phase heparin binding assay employed polyclonal antibodies to Vn in the detection system, and results presented in Fig. 5A suggested that the immunoreactivity of Vn was reduced upon cleavage with high calpain concentrations. To exclude the possibility that the reduced Vn binding was not due to loss of immunoreactivity but actual reduced heparin binding, the ability of the digest to compete with biotinylated Vn for binding to immobilized heparin was tested. Again, intact and incomplete hydrolyzed Vn competed with the binding of labeled Vn to heparin in a dose-dependent manner, whereas no competition was evident using Vn hydrolyzed by high calpain concentrations (not shown).
The ability of calpain-hydrolyzed Vn to
promote cell adhesion was tested in adhesion assays using the human
fibrosarcoma cell line HT 1080 (Fig. 8C). Microtiter
wells were coated with the indicated concentrations of Vn or Vn
digests, and after blocking, prelabeled cells were allowed to adhere
for 1 h. After washing, bound cells were solubilized in SDS and
quantified by -counting. While intact Vn and Vn cleaved with low
concentrations of calpain II promoted cell adhesion in a dose-dependent
manner, the adhesion was drastically reduced using Vn cleaved by
intermediate or high concentrations of calpain (Fig. 8C). In control experiments, the coating
efficiency of the intact Vn was compared with that of the Vn digests.
While the binding of the polyclonal antibodies to the Vn
fragment-coated wells was unchanged at the three highest coating
concentrations, the binding was slightly reduced (i.e. 3-fold)
at the lowest concentrations in comparison with intact Vn (not shown).
Thus, the reduced binding of cells to the calpain-digested Vn, at least
at the three highest concentrations of Vn employed, was not due to
reduced binding of the digest to the microtiter wells but rather to
destruction of the cell binding domains of Vn. It should be noted that
the functional characterization of the calpain-derived Vn fragments was
performed in the presence of 10 mM EDTA (PAI-1 and heparin
binding) or that hydrolysis was terminated by the addition of an excess
amount of E-64 to both intact and cleaved Vn (cell adhesion) to prevent
potential interference of residual calpain activity with the assay
systems. Thus, hydrolysis of Vn by calpain dissociates the PAI-1, cell,
and glycosaminoglycan binding functions of Vn and suggests that the
principal PAI-1 binding site in Vn is distinct from its
glycosaminoglycan binding domain.
In studies to further characterize the structure-function relationships of platelet Vn, it was noted that the mode of platelet disruption significantly affected the Vn immunoreactivity in platelets (Fig. 1). While platelets lysed by the addition of SDS contained Vn concentrations similar to those reported(12, 13) , platelet lysates prepared by freeze-thawing or treatment with Triton X-100 lacked detectable Vn immunoreactivity (Fig. 1). The hydrolysis was not specific for platelet Vn but was also observed when purified Vn preparations and Vn present in plasma were added to platelet prior to lysis (Fig. 3). This degradation was significantly reduced under conditions promoting the inactivation of divalent ion-dependent cysteine proteases (Fig. 4). In addition, Vn degradation was prevented by calpastatin in a dose-dependent manner (Fig. 4), suggesting an involvement of platelet calpains in the hydrolysis of platelet Vn.
Platelets are an abundant source of calpains I and II, which have similar substrate specificity but differ in their divalent ion concentration requirements(23) . Platelets are special in comparison with most other cell types in that the amount of calpain inhibitor (i.e. calpastatin) is lower than the amount of calpain (22, 23) . Accordingly, the proteolytic balance is shifted toward degradation of calpain-sensitive substrates in platelets but not in most other cell types(22) . Consistently with these considerations, purified Vn added to Hep 3B cells prior to lysis was not susceptible to cleavage (Fig. 3). It should be noted that the protease inhibitor experiment could only provide circumstantial evidence that calpains are involved in Vn degradation. For example, it appears possible that a limited proteolysis of Vn by calpain may cause destabilization of the structural rigidity, making it more sensitive to proteolytic attack by other cellular proteases. Alternatively, other proteases may be activated by calpain and degrade Vn independent of calpain.
To more
directly test the involvement of calpains in the degradation of
platelet Vn, the susceptibility of Vn to cleavage by calpains was
tested in a purified protein system. Purified calpains I and II
degraded Vn in a dose-dependent manner, and hydrolysis required calcium
ions ( Fig. 5and Fig. 6). At relatively high enzyme
concentrations, Vn was digested to low molecular weight fragments,
resulting in reduction or loss of immunoreactivity for both polyclonal
and monoclonal antibodies (Fig. 6). The concentration of enzyme
used in these in vitro experiments is in the range expected in vivo since calpains are in at least 10-fold molar excess
over Vn present in platelets(23, 24) . In the presence
of Ca concentrations lower than the requirements for
calpains I and II, respectively, no cleavage of Vn was detectable,
indicating that the observed cleavage was not due to contaminating
calcium-independent proteases ( Fig. 5and 6). The extensive
digestion of Vn by calpains was rather surprising, since proteolysis
induced by calpain usually produces larger fragments that are not
further susceptible to calpain cleavage(25) . While the
intermediate sized fragments were not observed in the initial
degradation experiments of Vn in platelet lysates (Fig. 1Fig. 2Fig. 3Fig. 4), dose-response
experiments of platelets revealed that identically sized Vn fragments
in comparison with those observed in the purified protein system could
also be recovered from the platelet lysates (Fig. 7). This
observation does not exclude the possibility that lower molecular
weight Vn fragments, undetectable by immunoblotting, are generated in
platelets that are different from those obtained in a purified protein
system. Thus, calpains apparently exert their initial proteolytic
action on Vn present in platelets, resulting in the generation of
relatively large, immunologically detectable fragments without the
involvement of other proteases.
The functional consequences of the degradation of Vn by calpain were further analyzed. In many instances, calpain actually activates biological systems by limited proteolysis, resulting in the gain of function. While we did not observe an increase in biological activities, the modulation of functional activities was domain-specific. For example, the PAI-1 binding activity of the digested Vn was unchanged (Fig. 8), indicating that a high affinity PAI-1 binding site was intact. The observation that the somatomedin B domain, recently identified as a high affinity PAI-1 binding site(5) , is extensively disulfide-linked and thus relatively resistant to proteolytic attack (26) is consistent with this result.
The cell binding domain is located immediately C-terminal of the PAI-1 binding site (amino acids 45-47) in the connecting region of the Vn molecule(27, 28) . Hydrolysis of Vn by calpain completely abolished the binding of cells to Vn, suggesting that either this domain is destroyed or that the domain is not accessible on the surface of these calpain-derived fragments upon immobilization of microtiter wells. In addition, the heparin binding domain located between amino acid 341 and 379 (1, 2) was destroyed by calpain cleavage. Thus, these results indicate that calpain can dissociate specific Vn functions. The heparin binding domain has been implicated in the binding of Vn to tissues and extracellular matrices but also as a second PAI-1 binding site(2) . The results presented here suggest that the PAI-1 binding activity of the heparin binding domain is of minor importance in comparison with N-terminal Vn fragments. It is intriguing to speculate that calpain cleavage will release PAI-1 and Vn fragment complexes in thrombi or in areas of tissue injury, reducing the amount of thrombus- or tissue-associated PAI-1 and thereby shifting the fibrinolytic balance to promote plasminogen activation.
While the
data presented here provide a better understanding of the
structure-function relationships of the Vn molecule, particularly with
respect to the significance of reported PAI-1 binding domains, the
physiological significance of the observed degradation of Vn by calpain
remains to be elucidated. Within the detection limit of our Vn
degradation assay, a biologically active protease was not released upon
stimulation with physiological agonists. It should be noted that upon
stimulation of platelets with thrombin or platelet-activating factor,
approximately 1% of total calpain II immunoreactivity is expressed on
the external platelet surface(24) . It is unknown whether this
membrane-associated calpain is biologically active. The observation
that Vn was not degraded would rather suggest that this form of calpain
is biologically inactive. A number of considerations could account for
this observation, including complex formation of calpain with specific
protease inhibitors (e.g. calpastatin) on the platelet
surface. Alternatively, other released proteins could be more
susceptible to degradation and serve as competing substrates for the
hydrolysis of Vn by calpain. However, since the majority of calpain is
present in the platelet cytoplasm, whereas Vn is contained within the
-granules, both proteins are physically separated unless platelets
are stimulated or damaged or platelet membrane integrity is
compromised. These considerations suggest that the release of calpains
followed by a subsequent degradation of Vn may be important during
prolonged storage of platelet concentrates for transfusion, in
immunologically induced membrane damage, upon dissolution of aging
thrombi, or in tissue injury and necrosis.