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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From INSERM U489 and Association Claude Bernard, Tenon
Hospital, and Unité de Pharmacologie Cellulaire, Unité
Associée, Pasteur Institute INSERM U485, Paris, France.
Matrix metalloproteinase 2 (MMP2) has been reported to be
secreted by collagen-stimulated platelets, and active MMP2 has been shown to play a role in platelet aggregation. It has been demonstrated that MMP2 activation is dependent on the complex (membrane type 1 [MT1]-MMP/tissue inhibitor of MMP2 [TIMP2]) receptor and MMP2. We
have investigated human platelets as a possible source of MT1-MMP, and
we have studied its role in MMP2 activation and in platelet aggregation. Gelatin zymograms showed the existence of MMP2 at proforms
(68 kd) and activated-enzyme forms (62-59 kd) in supernatants of
resting and activated platelets, respectively. No gelatinolytic activity was associated with the platelet pellet after aggregation, suggesting a total release of MMP2 during cell activation. By Western
blot analysis in nonreduced conditions, MT1-MMP was found on resting
platelet membranes in 2 forms-the inactive 45-kd form and an apparent
89-kd form, which totally disappeared under reduced conditions. After
platelet degranulation, only the 45-kd form was detected. Reverse
transcription-polymerase chain reaction experiments showed the
expression in platelets of messenger RNA encoding for MMP2, MT1-MMP,
and TIMP2. Flow cytometry analysis showed that MT1-MMP, MMP2, and TIMP2
expressions were enhanced at the activated platelet surface. MMP
inhibitors, recombinant TIMP2, and synthetic BB94 inhibited
collagen-induced platelet aggregation in a concentration-dependent
manner, indicating the role of activated MT1-MMP in the modulation of
platelet function. In conclusion, our results demonstrate the
expression of the trimolecular complex components (MT1-MMP/TIMP2/MMP2)
by blood platelets as well as the ability of MMP inhibitors to modulate
the aggregating response.
(Blood. 2000;96:3064-3069) Remodeling of the extracellular matrix plays a
critical role in the reorganization of connective tissue under both
normal and pathophysiologic conditions. Extracellular matrix turnover is initiated by proteolytic enzymes, mainly by serine proteinases of
the plasminogen system and matrix metalloproteinases
(MMPs).1 MMPs are a family of related proteinases whose
principal function appears to be the breakdown of extracellular matrix
proteins during processes of tissue remodeling associated with growth,
development, and repair. Several MMPs are expressed in cancers at
levels that are higher than those found in normal tissue or benign
adenomas. It has been suggested that MMPs and tissue inhibitors of
metalloproteinase (TIMPs) in circulating body fluids may contribute to
the regulation of tumor metastasis, invasion, and
angiogenesis.2,3 Recent studies have shown that the
MMP2:TIMP2 ratio could be a predictor of invasion, metastasis, and
recurrence in urothelial4 and in prostate cancer
patients.5 In addition, an increasing number of studies of
MMPs in soluble form Blood platelets, principal cells of primary hemostasis, play major
roles in thrombosis, clot lysis, atherosclerosis, metastasis, and
inflammation. At the site of vessel injury, platelets become activated
and release several mediators that modify tissue integrity. They may be
considered secreting cells, and their ability to release proteinases
during activation was recently emphasized.23 It is well
known that interactions between soluble molecules and platelets result
in a clot formation, but platelets have also been shown to facilitate
metastasis by interacting with tumor cells. Interestingly, it was shown
that tumor cells, able to cause metastasis in vivo, had the ability in
vitro to induce platelet aggregation.24 To date, only 2 papers have reported a relationship between gelatinase secretion and
human platelets. One paper described tumor cells' ability to induce in
vitro platelet aggregation and showed that platelets could stimulate
tumor cells to secrete MMPs, thereby facilitating
metastasis.25 More recently, Sawicki et al26
reported the release of MMP2 by human platelets and claimed that MMP2
was involved in platelet-aggregating response. They showed that active
but not latent MMP2 enhanced platelet aggregation triggered by collagen
and that the inhibition of MMP2 activation inhibited platelet
aggregation. However, the mechanism of MMP2 activation was not elucidated.
In the present work, we show the expression of MT1-MMP in blood
platelets and its role in platelet aggregation within the trimolecular
complex MT1-MMP/TIMP2/MMP2. We also demonstrate the modulatory effect
of natural (TIMP2) and synthetic (BB94) MMP inhibitors on the platelet response.
Materials
Preparation of human platelets
Platelet aggregation Aggregation was monitored at 37°C by measuring the variation of light transmission according to method of Born29 with a Chronolog aggregometer. Briefly, 400-µL samples of washed platelets supplemented with fibrinogen (0.28 mg/mL) and stirred at 1100 rpm were preincubated with MMPs inhibitors, TIMP1, TIMP2, and BB94 at different concentrations. After 2 minutes of incubation, pretreated platelets were then challenged with different agonists (AA, collagen, ADP, thrombin). The percentage of aggregation was determined 3 minutes after addition of the stimulating agent.Flow cytometry analysis Membrane surface expression of MMP2, MT1-MMP, TIMP2, and MMP9 by resting and activated platelets was studied by flow cytometry. Nonstirred washed platelets (5 × 108/mL) were incubated in an aggregometer cuve at 37°C for 5 minutes in the presence or in the absence of 0.5-µg/mL collagen. The platelets were then immediately fixed in 1% formaldehyde for 30 minutes, adjusted to 106 cells/mL, and distributed in a 96-well culture plate. Platelets were incubated 30 minutes in the dark at 4°C with MMP2, MT1-MMP, TIMP2, or MMP9 monoclonal antibody or with a mouse IgG (control isotype). The samples were then washed twice in Tyrode's buffer and incubated for 30 minutes at 4°C with fluorescein isothiocyanate-conjugated goat antimouse IgG (20 µg/mL). Finally, samples were washed, centrifuged, and resuspended in 0.6 mL of Tyrode's buffer. Analysis was performed using FACScan instrument (Becton Dickinson, San Jose, CA).Membrane preparation Plasma membranes were prepared from human mesangial cells and from human washed platelets as previously described30 and stored at 80°C.
Gelatin zymography and reverse zymography The gelatinolytic activity in pellets and supernatant of resting and activated platelets was visualized on zymograms performed under nonreduced conditions, using SDS-polyacrylamide gels containing 1-mg/mL gelatin (7.5% PAGE for gelatin zymography and 14% for reverse zymography). After electrophoresis the gels were soaked in 2.5% Triton X-100 for 1 hour to remove SDS and incubated in Tris-HCl (50 mmol/L, pH 7.5), CaCl2 (5 mmol/L), and ZnCl2 (1 µmol/L) overnight at 37°C and stained with Coomassie blue dye. Reverse gelatin zymography was performed by incubating SDS-PAGE gels in the same buffer containing 1 volume of HT1080 serum-free conditioned medium.Immunoblot analysis After SDS-PAGE, in nonreduced or reduced conditions (1-mmol/L -mercaptoethanol), the proteins were transferred onto polyvinylidene difluoride membrane (Millipore Corp, Bedford, MA) with a Trans-Blot semidry transfer cell (Bio-Rad). After blocking 1 hour at 37°C in
Tris-HCl (50 mmol/L, pH 7.5), NaCl (100 mmol/L),
triethanolamine-buffered saline (TBS) containing 2% bovine serum
albumin, and 0.1% Tween 20, the membrane was incubated with
monoclonal antibodies anti-TIMP2 (1 µg/mL) or anti-MT1-MMP (10 µg/mL) for 18 hours at 4°C. After extensive washing in TBS-0.1%
Tween 20, the membrane was incubated with goat antimouse IgG for 1 hour
at room temperature. After extensive washing, the blots were revealed
by chemoluminescence with the ECL kit.
Amplification of platelet MT1-MMP, MMP2, and TIMP2 Total RNA from human platelets was isolated by the method of Chomczynski and Sacchi.31 The mRNA was reverse transcribed using random hexamers. A total of 25 ng of complementary DNA was used as a template in each polymerase chain reaction (PCR). The sense primer 5'-CCCTATGCCTACATCCGTGA-3' and the antisense primer 5'-TCCATCCATCACTTGGTTAT-3', complementary to nucleotides 598 to 617 and 1148 to 1167, respectively, of human MT1-MMP were used as described.15,18 For MMP2, the sense primer 5'-TTTTCTCGAATCCATGATGG-3' and the antisense primer 5'-CTGGTGCAGCTCTCATATTT-3', complementary to nucleotides 428 to 447 and 1028 to 1047, respectively, were used according to Martin et al.32 For TIMP2, the sense primer 5'-GTTTTGCAATGCAGATGTAG-3' and the antisense primer 5'-ATGTGGAGAAACTCCTGCTT-3', complementary to nucleotides 381 to 400 and 901 to 920, respectively, were used as described.32 PCR was performed by standard techniques using Taq polymerase, repeating 35 cycles of a 60-second denaturation step at 94°C, a 60-second annealing step at 60°C, and a 60-second extension step at 72°C for MT1-MMP; denaturation was 60 seconds at 95°C and annealing 90 seconds at 52°C for MMP2 and TIMP2. The respective amplified PCR products were analyzed on a 1.5% agarose gel and scanned.
Expression and release of MMP2, MT1-MMP, and TIMP2 by aggregated platelets Control and collagen- (1 µg/mL) aggregated platelets were centrifuged, and the supernatants and the pellets were collected and analyzed by gelatin zymography under nonreduced conditions. In the supernatants of resting platelets, gelatin zymograms showed the presence of a unique gelatinolytic band of 68 kd corresponding to latent MMP2 (Figure 1, lane1). After stimulation, 2 activated forms of 62 and 59 kd appeared in the supernatants (Figure 1, lane 2). No gelatinolytic activity associated with the platelet pellet could be detected (Figure 1, lane 3).
Western blot analysis of cell membranes showed that resting platelets
(Figure 2A, lane 3) expressed MT1-MMP in
2 forms: the inactive 45-kd MT1-MMP similar to that in membranes of
urokinase-treated mesangial cells (Figure 2A, lane 2) and another
unusual 89-kd form. After platelet aggregation, only the 45-kd band
remained associated with the platelet membranes (Figure 2A, lane 4). To study whether the 89-kd band was a new molecular form of MT1-MMP or a
complex containing MT1-MMP, we performed SDS-PAGE under reduced conditions (1-mmol/L
By reverse zymography, both TIMP1 and TIMP2 were detected in platelet
supernatants of resting (Figure 3, lane
3) and activated (Figure 3, lane 4) platelets as well as in purified
platelet membranes (Figure 3, lane 6).
Expression of mRNAs encoding for MMP2, MT1-MMP, and TIMP2 in platelets RT-PCR was performed on total RNA extracted from washed human platelets. The resulting products were compared with those obtained from human mesangial cells as control. We found mRNAs encoding for MT1-MMP, MMP2, and TIMP2 as single bands of 550, 620, and 540 base pairs, respectively, as predicted by the selected primers. RT-PCR products from platelets were of the same size as in control mesangial cells, but mRNA transcript levels were in lower amounts in platelets than in mesangial cells (Figure 4).
Enhanced expression of trimolecular complex components during platelet activation To study platelet surface expression of MT1-MMP, MMP2, and TIMP2, resting and activated platelets were used. Flow cytometry analysis showed the expression at the cell surface of MMPs and of TIMP2 before activation. As shown by an increase of fluorescence intensity when platelets were incubated with specific monoclonal antibody to MMP2 and MT1-MMP, compared with appropriate isotype, platelet activation enhanced the expression of MMP2 and MT1-MMP (Figure 5C-D). In addition, a large amount of TIMP2 was released and remained associated with platelet membranes during platelet activation (Figure 5E). A very small amount of MMP9 was detected on resting platelets and did not increase after platelet activation (Figure 5B).
Inhibition of platelet aggregation and of MMP2 activation by MMP inhibitors The role of MMP inhibitors in platelet aggregation was studied using various agonists. Washed platelets (108 cells/mL) were preincubated with MMP-specific tissue inhibitors TIMP1 (1.5 µg/mL), TIMP2 (0.5-1.5 µg/mL) or a synthetic compound BB94 (2.5-4.0 µmol/L). After 2 minutes, platelets were exposed to collagen (1 µg/mL), AA (10 µmol/L), ADP (10 µmol/L), or thrombin (0.075 IU/mL). Both TIMP2 and BB94 inhibited collagen-induced platelet aggregation in a concentration-dependent manner (Figure 6A, upper panel). The inhibition of platelet aggregation was associated with the absence of activation of MMP2 as shown by zymography analysis of the same supernatants (Figure 6B). In contrast, TIMP1 had no significant inhibitory effect (Figure 6A, upper panel). Similar inhibition of platelet aggregation was observed with TIMP2 and BB94 when platelets were triggered by AA or by ADP (Figure 6A, lower panel). When platelets were stimulated by thrombin, only a partial inhibition of platelet response and MMP2 activation was observed in the presence of TIMP2 (Figure 7).
Recently, Sawicki et al26 have reported the release of
pro-MMP2 by platelets and have demonstrated a proaggregatory effect of
active human recombinant MMP2, but the mechanism of pro-MMP2 activation
was not elucidated and, in particular, the expression of MT1-MMP by
platelets has never been addressed. MT1-MMP has been described in at
least 3 membrane-associated forms: a native 63-kd, a 45-kD
inactive-processed, and the 60-kd furin-processed form.33,34 We have previously shown the existence of a
soluble MT1-MMP of 55 kd in human mesangial cell
supernatants,30 and recently we found it in human plasma
and serum (I.K. et al, unpublished data, 1998). This finding lead us to
study MT1-MMP secretion by human platelets. The results of Western blot
experiments on human resting platelet membranes showed 2 major
immunoreactive bands of 45 and 89 kd. The 89-kd band, recognized by the
MT1-MMP monoclonal antibody, was never described before. To study
whether this 89-kd protein could represent a new molecular form of
MT1-MMP or a complex containing MT1-MMP, the platelet membranes were
also analyzed in reduced conditions. In the presence of
The apparent discrepancy between the increase of MT1-MMP expression observed by flow cytometry during platelet activation and the decrease of total MT1-MMP protein on membranes after collagen-induced aggregation, visualized by Western blot, may be explained by differences in the experimental conditions. In the flow cytometry experiments, platelets were activated without stirring and were not aggregated; MT1-MMP was expressed at the cell surface but not consumed. Then platelets were fixed and stained with anti-MT1-MMP antibody. In contrast, in Western blot experiments, platelets were recovered by centrifugation at the end of the aggregation and lysed in the sample buffer. During platelet aggregation, MT1-MMP had been totally activated and processed into the 45-kd inactive form. Pro-MMP2 activation on the cell surface is a complex process involving a trimolecular complex composed of TIMP2 interacting simultaneously with the catalytic site of MT1-MMP and with the C-terminal domain of pro-MMP2.21,36 Then, a second "free" MT1-MMP molecule cleaves and activates pro-MMP2, generating an intermediate 64-kd form.37-39 The autocatalytic cleavage of MMP2 generates the active 62- and 59-kd forms. MT1-MMP activation sequentially generates the 60-kd active and a 45-kd inactive species, the latter being a product of MMP2 activation.40,41 Indeed, we observed in membranes of activated platelets the total disappearance of the 89-kd MT1-MMP concomitantly to the generation of the 62- to 59-kd active forms of MMP2 in platelet supernatant. Therefore, our results indicate that MT1-MMP is not only a major determinant of pro-MMP2 activation on platelets but also that this activation has taken place within the trimolecular complex MT1-MMP/TIMP2/MMP2. This finding is further supported by the presence of TIMP2 in platelets. The platelet collagenolytic activity has been studied in guinea pigs42 and in human beings,26 and the presence of TIMP1 and TIMP2 in human megakaryocytes and platelets has been established recently.43 The fact that TIMP1 was also expressed by platelets could explain the existence of latent MMP2 in both resting and activated platelets. MMP2 activity is sensitive to inhibition by both TIMP1 and TIMP2,21 but recent studies have reported contrary effects of TIMP1 and TIMP2 on MMP2 and MT1-MMP activity. TIMP1 appears to be an effective inhibitor of MMP244 but is not able to form a complex with pro-MMP2.45 TIMP2 has dual effects on MMP2. At stochiometric concentrations, TIMP2 allows pro-MMP2 activation in the trimolecular complex and, in excess, TIMP2 binds free MT1-MMP, therefore preventing pro-MMP2 cleavage and activation by free MT1-MMP. MT1-MMP is also sensitive to the inhibition by TIMPs; however, unlike TIMP2, TIMP1 even at high concentration is unable to inhibit the processing of MT1-MMP.40 The fact that platelet aggregation could be inhibited by BB94 indicates that this inhibition is rather due to the inhibition of MMP2 and of MT1-MMP than to a direct effect of TIMP2 on platelets. In addition, the inhibition of platelet aggregation by TIMP2, but not by TIMP1, indicates an inhibitory profile characteristic of MT1-MMP.35,36 The partial inhibition of thrombin-induced platelet aggregation could be explained by the direct proteolysis and activation of MMP2 by thrombin,46 bypassing the inhibitory effect of TIMP2 on the activation of MMP2 within the trimolecular complex. In conclusion, our results have demonstrated the presence of MT1-MMP in human platelets, the release of the components of the trimolecular complex MT1-MMP/TIMP2/MMP2 during platelet activation, and MT1-MMP's role in the modulation of platelet aggregation.
We thank Dr Agnes Noel for helpful suggestions.
Submitted January 13, 2000; accepted June 28, 2000.
Supported by a grant from the French Academy of Medicine.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Isabelle Kazes, INSERM U489, Hôpital Tenon, 4 rue de la Chine, 75020, Paris, France; e-mail: kazes{at}b3e.jussieu.fr.
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 F. Mannello, G.A.M. Tonti, and F. Canestrari The `never-ending story' of the influence of blood specimen collection methods affecting the concentration, the zymographic profile and the usefulness of matrix metalloproteinases and their tissue inhibitors in multiple sclerosis diagnosis/prognosis: a landmark for limiting the misuse of serum samples Multiple Sclerosis, June 1, 2007; 13(5): 687 - 690. [PDF]
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 H.-J. Cho, J. H. Kang, J.-Y. Kwak, T.-S. Lee, I.-S. Lee, N. G. Park, H. Nakajima, J. Magae, and Y.-C. Chang Ascofuranone suppresses PMA-mediated matrix metalloproteinase-9 gene activation through the Ras/Raf/MEK/ERK- and Ap1-dependent mechanisms Carcinogenesis, May 1, 2007; 28(5): 1104 - 1110. [Abstract] [Full Text] [PDF]
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 D. Alonso-Escolano, C. Medina, K. Cieslik, A. Radomski, P. Jurasz, M. J. Santos-Martinez, T. Jiffar, P. Ruvolo, and M. W. Radomski Protein Kinase C{delta} Mediates Platelet-Induced Breast Cancer Cell Invasion J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 373 - 380. [Abstract] [Full Text] [PDF]
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S. Hong, K.-K. Park, J. Magae, K. Ando, T.-S. Lee, T. K. Kwon, J.-Y. Kwak, C.-H. Kim, and Y.-C. Chang Ascochlorin Inhibits Matrix Metalloproteinase-9 Expression by Suppressing Activator Protein-1-mediated Gene Expression through the ERK1/2 Signaling Pathway: INHIBITORY EFFECTS OF ASCOCHLORIN ON THE INVASION OF RENAL CARCINOMA CELLS J. Biol. Chem., July 1, 2005; 280(26): 25202 - 25209. [Abstract] [Full Text] [PDF]
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 N. P. Kadoglou, S. S. Daskalopoulou, D. Perrea, and C. D. Liapis Matrix Metalloproteinases and Diabetic Vascular Complications Angiology, March 1, 2005; 56(2): 173 - 189. [Abstract] [PDF]
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J. A. Coppinger, G. Cagney, S. Toomey, T. Kislinger, O. Belton, J. P. McRedmond, D. J. Cahill, A. Emili, D. J. Fitzgerald, and P. B. Maguire Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions Blood, March 15, 2004; 103(6): 2096 - 2104. [Abstract] [Full Text] [PDF]
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 M. Jayachandran, W. G. Owen, and V. M. Miller Effects of ovariectomy on aggregation, secretion, and metalloproteinases in porcine platelets Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1679 - H1685. [Abstract] [Full Text] [PDF]
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Y. Wu, N. Asazuma, K. Satoh, Y. Yatomi, T. Takafuta, M. C. Berndt, and Y. Ozaki Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase Blood, May 1, 2003; 101(9): 3469 - 3476. [Abstract] [Full Text] [PDF]
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 V. Fontaine, M.-P. Jacob, X. Houard, P. Rossignol, D. Plissonnier, E. Angles-Cano, and J.-B. Michel Involvement of the Mural Thrombus as a Site of Protease Release and Activation in Human Aortic Aneurysms Am. J. Pathol., November 1, 2002; 161(5): 1701 - 1710. [Abstract] [Full Text] [PDF]
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 S. W. Galt, S. Lindemann, L. Allen, D. J. Medd, J. M. Falk, T. M. McIntyre, S. M. Prescott, L. W. Kraiss, G. A. Zimmerman, and A. S. Weyrich Outside-In Signals Delivered by Matrix Metalloproteinase-1 Regulate Platelet Function Circ. Res., May 31, 2002; 90(10): 1093 - 1099. [Abstract] [Full Text] [PDF]
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K. Lehti, J. Lohi, M. M. Juntunen, D. Pei, and J. Keski-Oja Oligomerization through Hemopexin and Cytoplasmic Domains Regulates the Activity and Turnover of Membrane-type 1 Matrix Metalloproteinase J. Biol. Chem., March 1, 2002; 277(10): 8440 - 8448. [Abstract] [Full Text] [PDF]
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 P. AUKRUST, T. WAeHRE, J. K. DAMAS, L. GULLESTAD, and N. O. SOLUM Inflammatory role of platelets in acute coronary syndromes Heart, December 1, 2001; 86(6): 605 - 606. [Full Text] [PDF]
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D. V. Rozanov, E. I. Deryugina, B. I. Ratnikov, E. Z. Monosov, G. N. Marchenko, J. P. Quigley, and A. Y. Strongin Mutation Analysis of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP). THE ROLE OF THE CYTOPLASMIC TAIL CYS574, THE ACTIVE SITE GLU240, AND FURIN CLEAVAGE MOTIFS IN OLIGOMERIZATION, PROCESSING, AND SELF-PROTEOLYSIS OF MT1-MMP EXPRESSED IN BREAST CARCINOMA CELLS J. Biol. Chem., July 6, 2001; 276(28): 25705 - 25714. [Abstract] [Full Text] [PDF]
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B. G. Galvez, S. Matias-Roman, J. P. Albar, F. Sanchez-Madrid, and A. G. Arroyo Membrane Type 1-Matrix Metalloproteinase Is Activated during Migration of Human Endothelial Cells and Modulates Endothelial Motility and Matrix Remodeling J. Biol. Chem., September 28, 2001; 276(40): 37491 - 37500. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
detected and measured in plasma and serum
-32P) dCTP were from Amersham Life Science
(Buckinghamshire, United Kingdom). The MMP synthetic inhibitor
BB94 (Batimastat; P. D. Brown, L. Bawden, K. Miller, Patent, WO
93/21942, 1993
