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Keywords:

  • ADAMTS-13;
  • platelets;
  • ULVWF

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

Summary.  The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

Von Willebrand factor (VWF) is a multimeric glycoprotein that mediates the initial tethering of platelets to vascular subendothelium. It is also critical for platelet aggregation under high fluid shear stress as found in arterial stenosis [1,2]. VWF is synthesized in endothelial cells and megakaryocytes/platelets [3,4]. In endothelial cells, newly synthesized VWF multimers are either directly released into plasma through a constitutive pathway or targeted for storage in Weibel-Palade bodies, where they are released upon stimulation [5]. VWF released from Weibel-Palade bodies is enriched in ultra-large (UL) forms that are hyperactive in their interaction with the GP Ib-IX-V complex on platelets [6,7]. Upon release, these highly prothrombotic ULVWF multimers are rapidly cleaved by ADAMTS-13, a member of the ADAMTS metalloprotease family [8,9], to smaller and less active forms. The cleavage appears to occur on the surface of endothelial cells under flow conditions [10]. Deficiency in ULVWF proteolysis, caused by lacking or inhibition of ADAMTS-13 activity, is associated with thrombotic thrombocytopenic purpura (TTP) [11,12].

Platelets contain up to 25% of total circulating VWF [4,13]. Platelet-derived VWF is stored in α-granules that are structurally similar to the Weibel-Palade bodies of endothelial cells and also contains ULVWF [14–16]. Release of platelet ULVWF can be induced by agonists that also stimulate endothelial cells to release ULVWF [14,17,18]. Like the endothelial cell-derived form, platelet-derived ULVWF can also spontaneously aggregate platelets or render them much more adhesive to vascular endothelial cells. However, platelet-derived ULVWF is hardly detected in the supernatant of thrombin-stimulated platelets, due to its re-association with platelets. Even in the platelet lysates, it is only detected in the presence of a divalent cation-dependent protease [2,6,19,20–22], indicating that platelet ULVWF may also undergo proteolysis at the time of release. This notion is further supported by the report of Kunicki et al. [23] showing that a calcium-activated sulfhydryl-dependent neutral protease cleaves platelet VWF. In a more indicative study, Fernandez et al. [24] found that thrombin stimulates platelets to release ULVWF in the presence of EDTA, but much smaller VWF multimers if EDTA was absent. The loss of ULVWF in the supernatant of thrombin-stimulated platelets was attributed to the re-association of these hyperactive ULVWF with platelets. However, considering the similarities in VWF synthesis and release between endothelial cells and megakaryocytes/platelets as well as the proteolysis of endothelial cell-derived ULVWF by ADAMTS-13, one may hypothesize a similar proteolytic process for platelet-derived ULVWF. Such a possibility is supported by the recent finding that platelets contain an intracellular pool of ADAMTS-13 [25], but its activity in resting and activated platelets has not been characterized. In this report, we show that ADAMTS-13 can be detected on the surface of platelets and the expression increases upon the platelet activation by the thrombin receptor-activating peptide (TRAP), but not by ADP. The platelet-derived ADAMTS-13 is fully functional in cleaving endothelial cell-derived ULVWF under flow and static conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

Preparation of washed human platelets

Blood was obtained from 18 healthy donors (11 males and seven females of 22–47 years of age) under a protocol approved by the Institutional Review Board of Baylor College of Medicine for use of human subjects in biomedical research. All donors signed consent forms before blood was drawn. To obtain washed platelets, blood was collected in ACD (85 mm of sodium citrate, 111 mm of Glucose, 71 mm of citric acid, and 50 ng mL−1 of PGI2). Whole blood was centrifuged at 150 g for 15 min at 24 °C to obtain platelet-rich plasma (PRP), which was then centrifuged at 750 g for 10 min at 24 °C. After removal of platelet-poor plasma, platelet pellets were washed once with CGS buffer (13 mm of sodium citrate, 30 mm of glucose, 120 mm of sodium chloride, pH 7.0) and then resuspended in Tyrode's buffer (138 mm of sodium chloride, 5.5 mm of glucose, 12 mm of sodium bicarbonate, 2.9 mm of potassium chloride, and 0.36 mm of dibasic sodium phosphate, pH 7.4). For assays on whole blood and PRP, blood was collected in 0.38% of sodium citrate instead of ACD.

Analysis of VWF multimers by agarose gels

SDS-1% VWF agarose gel electrophoresis was used to analyze multimeric composition of platelet-derived VWF [11,26]. Briefly, washed platelets (1 × 108) were lyzed with a Tris-buffered saline (TBS) containing 1% of sodium dodecylsulfate (SDS) in the presence and absence of 5 mm of EDTA, vortexed, and heated at 60 °C for 20 min. The platelet lysates were separated by SDS-1% of agarose gel electrophoresis under non-reducing conditions and transferred to PVDF membranes. For immunoblotting, the membrane was first blocked with 5% non-fat dry milk in TBS containing 0.2% of Tween 20 (TBS-T) for 1 h at room temperature. It was then incubated with a goat anti-VWF antibody (200 ng mL−1, Bethyl Laboratories, Houston, TX, USA) for 2 h and horseradish peroxidase-labeled rabbit antigoat IgG (Bethyl Laboratories) for an additional 2 h at room temperature. After washing to remove unbound antibody, the membrane was incubated with the chemiluminescent substrate (Amersham Bioscience, Piscataway, NJ, USA) for 10 min and exposed to X-ray film (CL-X posureTM film; Pierce Biotechnology Inc., Rockford, IL, USA). VWF purified from normal human plasma and ULVWF released from cultured human umbilical vein endothelial cells (HUVECs) stimulated with 100 μm of histamine (30 min at 37 °C) were used as controls.

ADAMTS-13 activity under static conditions

ADAMTS-13 activity was measured under static conditions as previously described [26]. Platelet lysates were obtained by lyzing washed platelets (1 × 108) in a TBS containing 1% of SDS for 20 min at 4 °C. They were then mixed with ULVWF from the conditioned medium of the histamine-stimulated HUVECs in the presence of 1.5 m of urea, and 10 mm of BaCl2 (pH 8) for 24 h at 37 °C. Plasma and recombinant ADAMTS-13 were used as positive controls and lysis buffer for negative control. The sample mixtures were then separated by SDS-1% of agarose gel electrophoresis and transferred to PVDF membranes. VWF multimers were recognized by immunoblotting. ADAMTS-13 activity was measured by the disappearance of ULVWF and defined as 100% for normal plasma.

ADAMTS-13 activity under flow conditions

Platelet-derived ADAMTS-13 activity was measured under flow conditions as previously described [10,26]. For the flow assay, washed platelets from 10 ml of PRP were sonicated and lyzed in a Triton buffer (20 μm of Tris–HCl, 50 μm of NaCl, and 0.5% of Triton X-100, pH 7.0) for 20 min at room temperature. The Triton X-100 in the platelet lysates was then removed by extracti-gel D gel (Pierce Biotechnology Inc.) as previously described [27]. The Triton-free platelet lysates were then concentrated 15-fold using a spin column (Amicon Ultra 15–50 000 MWCO, Millipore, Bedford, MA, USA). The concentrated platelet lysates were mixed with homologous washed platelets (1:1 ratio, v/v) and perfused over HUVECs that were stimulated with 25 μm of histamine under a shear stress of 2.5 dyn cm2. To calculate ADAMTS-13 activity, the numbers of ULVWF strings after 2-minute perfusion of the Triton-free lysis buffer were counted and defined as 0% activity. The numbers of strings formed during the same period when the platelet lysates were perfused as compared with controls were defined as the percent cleavage of ULVWF strings. In some experiments, the platelet lysates were first incubated with ADAMTS-13 autoantibody (IgG fraction and used at 20 μg mL−1) purified from plasma of a patient with adult acquired TTP (AffinityPak protein A columns; Pierce Biotechnology Inc.).

ADAMTS-13 Immunoblotting of platelet lysates

Washed platelets were resuspended in the Triton lysis buffer containing a cocktail of protease inhibitors (10 mm of benzamidine, 1 mm of PMSF, 20 μg mL−1 aprotinin, 20 μg mL−1 of leupeptin, 100 μg mL−1 of soybean trypsin inhibitor, and 5 mm of EDTA) for 20 min at room temperature. Platelet-derived ADAMTS-13 was first immunoprecipitated from platelet lysates by incubating with a mouse monoclonal ADAMTS-13 antibody that binds to the catalytic domain of the metalloprotease (kindly proved by Drs F. Scheiflinger and B. Plaimauer of Baxter BioScience, Austria) for 3 h at 4 °C, followed by sheep anti-mouse IgG linked to Dynabeads M280 (Dynal ASA, Oslo, Norway) for an additional hour at 4 °C. The beads were collected at 1000 g at 4 °C and washed with a SDS buffer. The immunoprecipitated samples were boiled for 5 min in a SDS reducing sample buffer and then separated by 10% of SDS polyacrylamide gel electrophoresis (PAGE). The separated samples were transferred to PVDF membranes. For immunoblotting, the membranes were first blocked with 5% non-fat dry milk in TBS-T for 1 h at room temperature. They were then incubated overnight at 4 °C with one of two affinity-purified goat anti-ADAMTS-13 antibodies made against two synthetic peptides from the disintegrin (clone 154) and the fourth TSP-1 repeat (Clone 156, Bethyl Laboratories), respectively. After washing to remove unbound antibody, the membrane was incubated with HRP-conjugated rabbit antigoat IgG for 1 h at room temperature. The bound antibody was detected using the chemiluminescent substrate (Super signalR west pico, Pierce Technology Inc.).

For measuring the antigen levels of platelet-derived ADAMTS-13 by an ELISA assay, microtiter plates were first coated with a goat ADAMTS-13 antibody (clone 156 at 400 ng/well, Bethyl laboratories) overnight at 4 °C and then washed with PBS. The non-specific binding was blocked by incubating the wells with 3% of BSA for 1 h at 37 °C. After washing thoroughly, 200 μL of platelet lysates (from 1 × 108 platelets) or pooled plasma from five healthy donors was incubated with the coated antibody for 3 h at 37 °C. The bound ADAMTS-13 was detected by the monoclonal ADAMTS-13 antibody (37 °C for 45 min) and a HRP-conjugated goat antimouse IgG (room temperature for 30 min). The platelet ADAMTS-13 antigen levels for five healthy donors were then calculated as the percentage of ADAMTS-13 antigen in normal plasma.

Detection of ADAMTS-13 on platelet surfaces by immunostaining

Surface-bound ADAMTS-13 was detected by double immunostaining with a goat ADAMTS-13 antibody (clone 156) and an R-Phycoerythrin (PE)-conjugated monoclonal GP Ibα antibody (BD, Palo Alto, CA, USA). For these experiments, washed platelets were spotted onto fibrinogen immobilized onto glass coverslips (coating density was 30 μg/coverslip). After 30 min incubation, the coverslips were washed with Tyrode's buffer to remove unbound platelets. Attached platelets were fixed with 3.7% of paraformaldehyde in PBS containing 2% of sucrose at 4 °C for 15 min. The fixed platelets were incubated with the goat anti-ADAMTS-13 (clone 156, 10 μg mL−1) for 1 h at room temperature. After washing three times to remove unbound antibody, platelets were incubated with a FITC conjugated rabbit antigoat antibody (20 μg mL−1, Pierce Technology Inc.) for 20 min and then a PE-conjugated GP Ibα antibody for 1 h at room temperature. The coverslips were then washed and mounted for reviewing under a NIKON Eclipse E800 upright microscope with a 100 × superfluo objective. Single staining for each antibody was used to compensate for cross-detection and goat antiserum as a negative control. In a subset of experiments, the adherent platelets were stimulated with the thrombin-receptor activating peptide (TRAP at a final concentration of 5 μm) for 10 min before immunostaining.

Detection of ADAMTS-13 on platelets by flow cytometry

Expression of ADAMTS-13 on platelet surfaces was also measured by flow cytometry. Washed platelets, whole blood, and PRP (10 μL) were incubated with an ADAMTS-13 antibody and an FITC-conjugated secondary antibody for 10 min at room temperature. The stained samples were then mixed with 1 mL of PBS containing 1% of paraformaldehyde and the surface-bound antibody was detected on a Coulter Epics XL.MCL flow cytometer (Beckman Coulter, Miami, FL, USA). Three ADAMTS-13 antibodies were used for the assay: the monoclonal antibody SZ112 [28] and two affinity-purified goat ADAMTS-13 antibodies (Bethyl Laboratories). Isotype IgG was used as controls. The measurements were made on unstimulated platelets and platelets stimulated with either 2 μm of TRAP or 10 μm of ADP. To measure the absorption of recombinant ADAMTS-13 to platelets, recombinant ADAMTS-13 (20 μg mL−1) was incubated with washed platelets for 30 min at room temperature and detected by a monoclonal antimyc (the C-terminal tag, Sigma-Aldrich, St Louis, MO, USA) on flow cytometry.

Statistical analysis

The data were analyzed by the standard Student's t-test. All values were reported as mean ± SEM. A P-value of 0.05 or less was considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

ULVWF was present in platelet lysates

Washed platelets were lyzed in the presence and absence of EDTA, and VWF multimers analyzed on SDS-1% of agarose electrophoresis followed by immunoblotting. When lyzed in the presence of 5 mm of EDTA, platelets contained ULVWF multimers that were similar in pattern to those released from HUVECs (Fig. 1). In comparison, ULVWF was not detectable when platelets were lyzed in the absence of EDTA and VWF multimer distribution was similar to that of plasma VWF (Fig. 1). The ULVWF multimer that was freshly released from endothelial cells was bigger than that found in the plasma of patients with TTP [11], possibly because it was mechanically broken by higher fluid shear stress due to systemic thrombosis.

image

Figure 1. Platelets contained ULVWF. Washed platelets were lyzed in a lysis buffer in the presence and absence of 5 mm of EDTA, and separated by SDS-1% of agarose gel electrophoresis. VWF multimers were recognized by a polyclonal VWF antibody. ULVWF was detected in platelet lysates in the presence (lane 2), but not in the absence (lane 1) of EDTA. For comparison, lanes 3 and 4 showed VWF multimers obtained from plasma and supernatant of cultured HUVECs stimulated with histamine. The figure is representative of four separate experiments.

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Platelets contained VWF-cleaving activity under static and flow conditions

The loss of ULVWF in platelet lysates in the absence of EDTA suggests that platelets contain a divalent cation-dependent VWF-cleaving activity. To further evaluate this activity, we tested the ability of platelet lysates to cleave ULVWF released from HUVECs under static and flow conditions. VWF-cleaving activity under static conditions was measured by the disappearance of ULVWF in the supernatant of histamine-stimulated HUVECs [26]. As shown in Fig. 2, platelet lysates cleaved EC-derived ULVWF as did normal plasma. This activity was inhibited by 5 mm of EDTA. Measurements of VWF-cleaving activity under static conditions required prolonged incubation in the presence of urea and barium [26,29,30]. These conditions cannot be found in vivo, indicating that the cleavage may require other factors such as shear stress. To address this concern, we also measured the VWF-cleaving activity under more physiologically relevant flow conditions [10,31]. This method measures the cleavage of ULVWF strings formed on the surface of histamine-stimulated endothelial cells under fluid shear stress. Under these conditions, there were 68.4 ± 8.2 strings when Tyrode's buffer was perfused (0% activity), whereas there were only 5.2 ± 0.1 strings after 2-minute perfusion of platelet lysate (Student's t-test, n = 6, P < 0.001, Fig. 3), similar to that of recombinant ADAMTS-13 (3.1 ± 0.1). The cleavage of ULVWF strings was inhibited by preincubation of platelet lysates with an ADAMTS-13 antibody purified from a patient with acquired idiopathic TTP (Fig. 3). Releasates from washed platelets stimulated with TRAP, but not ADP, also cleaved ULVWF strings under flow conditions (albeit at a lower level, Fig. 3), indicating that TRAP induced platelet release of ADAMTS-13.

image

Figure 2. Platelet lysates cleaved endothelial cell-derived ULVWF under static conditions. Washed platelets were lyzed and incubated with ULVWF from the supernatants of histamine-stimulated HUVECs (100 μm) in the presence of 1.5 m of urea and 10 mm of BaCl2. The cleavage of ULVWF was indicated by the disappearance of UL forms of VWF on immunoblots. Platelet lysates cleaved ULVWF into smaller VWF multimers similar to those of plasma and the cleavage was inhibited by 5 mm of EDTA. The figure is representative of four separate experiments.

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image

Figure 3. Platelet lysates cleaved ULVWF strings formed on endothelial cells under flow conditions. Lysates of washed platelets were perfused over a monolayer of cultured HUVECs stimulated with 25 μm of histamine under 2.5 dyn cm2. Perfusion of Tyrode's buffer for 2 min produced significantly more ULVWF strings (A) than perfusion of platelet lysates (B). Perfusion of platelet lysate reduced the numbers of strings as did recombinant ADAMTS-13 (C). The cleavage of ULVWF strings by platelet lysate was inhibited by an antibody purified from a patient with acquired idiopathic TTP. Perfusion of supernatants from TRAP-stimulated platelets yielded significantly less ULVWF strings compared with that of unstimulated platelets (C). Figures in panel C were mean ± SEM, Student's t-tests, n = 6, *P < 0.001 compared with the buffer control.

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Platelets contained ADAMTS-13-like metalloprotease

To further determine that the platelet-derived VWF-cleaving activity is due to ADAMTS-13, we immunoblotted platelet lysates for ADAMTS-13 using two antibodies that were generated against synthetic peptides derived from the fourth TSP-1 repeat (clone 156) and disintegrin domain (clone 154), respectively. As shown in Fig. 4, both antibodies reacted with a polypeptide of approximately 170 kDa that was slightly larger than recombinant ADAMTS-13 expressed in Chinese Hamster Ovary fibroblasts (CHO cells). In addition, antibody 154 also detected two additional bands of approximately 160 and 140 kDa, and the antibody 156 detected 160 and 42 kDa bands. These additional bands may represent the different isoforms and the degradation product of the metalloprotease. The 170 kDa band was also detected in the supernatant of TRAP-stimulated washed platelets, along with a degradation product of 50 kDa (Fig. 4).

image

Figure 4. Detection of ADAMTS-13 in platelet lysates. Washed platelets were lyzed in a Triton lysis buffer. ADAMTS-13 was first immunoprecipitated with a monoclonal ADAMTS-13 antibody, subjected to SDS-PAGE, and then immunoblotted by each of two goat ADAMTS-13 antibodies that were made against synthetic peptides from the fourth TSP-1 repeat (lane 2, clone 156) or disintegrin domain (lane 3, clone 154). ADAMTS-13 was also detectable in the supernatant of TRAP-stimulated platelets (lane 4, clone 154 antibody). Recombinant ADAMTS-13 expressed in CHO cells (lane 1) was used as control. The figure is representative of four independent experiments.

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Using an ELISA assay, the amount of ADAMTS-13 from 1 × 108 platelets was equivalent to 16.4% ± 5.2% of that in 1 mL of plasma. The ADAMTS-13 antigen level was therefore calculated to be 164 ± 5.2 ng/108 platelets based on the calculated plasma ADAMTS-13 of 1 μg mL−1 [32].

ADAMTS-13 expressed on the surface of platelets

We utilized two technical means to determine whether ADAMTS-13 is detected on the surface of platelets: immunofluorescence staining and flow cytometry. For the former, non-permeablized platelets were spotted onto immobilized fibrinogen and stained with ADAMTS-13 antibody. They were also counter-stained with GP Ibα, the platelet receptor for VWF, to identify platelets. As shown in Fig. 5, platelets were positively stained for ADAMTS-13 (Fig. 5). ADAMTS-13 staining delineated platelet spreading induced by TRAP, showing a typical pattern of surface labeling.

image

Figure 5. ADAMTS-13 was detected on the surface of platelets by immunofluorescence. Washed platelets were spotted onto immobilized fibrinogen and double stained with a goat ADAMTS-13 antibody (followed by FITC-conjugated rabbit antigoat IgG) and a PE-conjugated GP Ibα antibody. The non-permeabilized platelets stained positively for ADAMTS-13 (A), but not an isotype control IgG (B). The ADAMTS-13-positive platelets were also stained for GP Ibα (C). ADAMTS-13 was distributed uniformly on the surface of resting platelets (E), whereas it was in the center and edges of platelets stimulated with TRAP (F). The figure is representative of three separate experiments (bar = 400× for A–D and 1000× for E and F).

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As platelets are often partially activated upon immobilization onto fibrinogen, we also measured the surface-bound ADAMTS-13 by flow cytometry. Washed platelets, PRP, and whole blood were incubated with the monoclonal ADAMTS-13 antibody SZ112, followed by FITC-conjugated rabbit antimouse IgG. We found that SZ112 bound to washed platelets and platelets in PRP and whole blood (Fig. 6). Two goat antibodies 154 and 156 also bound platelets in a pattern similar to that of SZ112 (data not shown). Interestingly, expression of ADAMTS-13 on the platelet surface was significantly increased when washed platelets were stimulated with TRAP and to much smaller extend, by ADP (Fig. 6, lower panel). As negative control, we used CHO cells instead of platelets from congenital TTP patients for a technical reason. ADAMTS-13 deficiency found in these patients can be caused by defects in synthesis or function of the metalloprotease. As the result of the latter, non-functional ADAMTS-13 may be detected in some of the patients.

image

Figure 6. ADAMTS-13 was detected on platelets by flow cytometry. (A) Washed platelets, PRP, and whole blood were incubated with a mouse monoclonal ADAMTS-13 antibody followed by a FITC-conjugated rabbit antimouse IgG. ADAMTS-13 antibody, but not isotype control-bound washed platelets and platelets in PRP and whole blood. (B) Washed platelets were treated with either 2 μm of TRAP or 10 μm of ADP for 5 min at room temperature and then stained for ADAMTS-13. ADAMTS-13 on TRAP-stimulated platelets was significantly increased compared with the unstimulated or ADP-stimulated platelets. Figures were mean ± SEM of five (A) and six (B) separate experiments (Student's t-tests, n = 9, *P < 0.001 compared with isotype control).

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The increase in surface-bound ADAMTS-13 may be attributed to the release of intracellular pool or absorbance from plasma. To distinguish the two possibilities, we added recombinant ADAMTS-13 to washed platelets before stimulation. The recombinant ADAMTS-13 is expressed with a C-terminal myc tag so that, if the surface-bound ADAMTS-13 is primarily from the absorption, it should be detected by myc antibody and SZ112, whereas the native ADAMTS-13 should only be stained with SZ112. Using this strategy, we found that resting platelets predominantly presented the native ADAMTS-13, whereas the binding of both SZ112 and myc antibodies increased significantly upon TRAP, but not ADP, stimulation (Fig. 7), indicating that the increase in the surface-bound ADAMTS-13 may derive from intracellular pool as well as plasma.

image

Figure 7. Increase in surface-bound ADAMTS-13 was due to endogenous release and absorbance. Washed platelets were stimulated with either 2 μm of TRAP or 10 μm of ADP in the presence of recombinant ADAMTS-13 and then stained for surface-bound native ADAMTS-13 by SZ112 or recombinant ADAMTS-13 by a monoclonal myc antibody. The data were expressed as mean ± SEM and analyzed by Student's t-tests (n = 3).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

We have demonstrated that ADAMTS-13 can be detected on platelet surface by four antibodies that bind to different domains of the metalloprotease. We further show that platelet-derived ADAMTS-13 is responsible for the observed VWF-cleaving activity of platelet lysate under static and flow conditions because the activity is inhibited by EDTA or an ADAMTS-13 antibody from a patient with acquired TTP. Finally, we show that the surface expression of ADAMTS-13 is significantly increased on platelets stimulated by TRAP, due to endogenous secretion as well as absorbance from plasma. TRAP also induces the release of soluble ADAMTS-13 from platelets.

These results are consistent with several early observations. Firstly, ULVWF multimers are detected in platelets lyzed in the presence, but not absence of EDTA, a calcium chelator that inhibits ADAMTS-13 activity [29,30]. Secondly, ADAMTS-13 mRNA and antigen are intracellularly detected in platelets [25]. Thirdly, a platelet-derived VWF-cleaving activity has previously been reported [23]. Finally, Fernanda et al. [24] showed that ULVWF can be detected in the releasates of washed platelets stimulated with thrombin in the presence of EDTA, whereas only smaller multimer is detected in the absence of the calcium chelator. The loss of ULVWF in platelet releasates has been attributed to the binding of newly secreted ULVWF to platelets [24], primarily through integrin αIIbβ3 [19,20,25]. It has indeed been demonstrated that platelet-bound VWF increases upon thrombin activation of platelets [24,33,34]. However, the multimer composition of the platelet-bound VWF has not been examined.

Platelets contain up to 25% of circulating VWF that can be released by various agonists [13]. Without proper cleavage, platelet ULVWF may be directly released into plasma or re-associate with platelets, leading to platelet aggregation in ways indistinguishable from that of endothelial cell-derived ULVWF. In addition, platelets with the surface-bound ULVWF are more adhesive to endothelial cells [35]. In this regard, platelet-derived ADAMTS-13 is likely to serve as a critical safeguard to prevent the direct release of prothrombotic ULVWF into plasma in the event of platelet activation. One question is why platelet-bound ADAMTS-13 fails to cleave ULVWF strings, to which platelets tether under flow. We have previously shown that ADAMTS-13 immobilized onto polystyrene beads cleaved ULVWF strings under flow conditions, but at an extremely slow rate [36]. The slow kinetic is likely due to either the small amount of the metalloprotease available on each bead or immobilization reduces the activity, or both. As the results, cleavage of ULVWF strings by platelet-bound ADAMTS-13 is not detectable during the period of 2-minute perfusion.

One interesting observation is that the surface-bound ADAMTS-13 increases upon platelet activation through intracellular release and plasma absorbance (Fig. 7). On one hand, the increase at a time of ULVWF release (platelet activation) may efficiently prevent the direct release of ULVWF into plasma. On the other hand, it may reduce available ADAMTS-13 in plasma to cleave the endothelial cell-derived ULVWF, which is often released by the stimulation that activates platelets. Which of the two mechanisms is dominant may vary among individuals and different condition. Further investigations in this regard are critical because they may provide clue as how ULVWF proteolysis is regulated in vivo. The observation also raises a question as how ADAMTS-13, which is a secret enzyme, is captured on platelet surface. A candidate molecule is platelet-bound VWF [19,37]. We have previously shown that ADAMTS-13 binds to the A1 and A3 domains of VWF under both static and flow conditions [36]. This interaction may allow ADAMTS-13 to form a complex with ULVWF on platelets to initiate proteolysis, through a mechanism similar to that of endothelial cells [10,31]. Finally, it is also not clear where ADAMTS-13 is stored within platelets. TRAP, but not ADP, increases the expression of ADAMTS-13 on platelet surfaces, event though both agonists induce ULVWF release from platelet α-granules [38], suggesting that it may be stored in different granules. Lysosomes are likely candidates because they hold most platelet enzymes and are induced to release by strong agonists such as thrombin, but not weak ones such as ADP.

In summary, we have shown that platelets express ADAMTS-13 that is active in cleaving ULVWF under static and flow conditions. ADAMTS-13 can be detected on the surface of unstimulated platelets and the expression increases significantly upon stimulation by TRAP, but not by ADP. The platelet-derived ADAMTS-13 in vivo may provide a safeguard to prevent the direct release of hyperactive ULVWF into plasma upon platelet activation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

This work was supported by NIH grants P50-HL65967 and HL71895, and the Mary R. Gibson Foundation. J.F.D is an established investigator of the American Heart Association.

Reference

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference
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