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Keywords:

  • ADAMTS-13;
  • flow shear stress;
  • ultra-large von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

Summary.  von Willebrand factor (VWF) freshly released from endothelial cells is normally cleaved by the ADAMTS-13 metalloprotease to prevent the direct release of these ultra-large (UL) and hyper-reactive multimers into plasma. The balance of ULVWF proteolysis may be regulated by the amount of ULVWF released and the processing capacity of ADAMTS-13. The former associates with the size of ULVWF storage pool, sensitivity of vascular endothelial cells to stimulation, and the type of agonists, whereas the latter associates with the activity of ADAMTS-13. These parameters may vary significantly among individuals. We have determined the variations of ADAMTS-13 activity in 68 normal individuals by a flow-based assay and a static assay using ULVWF strings and recombinant VWF A2 domain as substrates, respectively. We found that the levels of ADAMTS-13 activity required to cleave the platelet-decorated ULVWF strings under flow is significantly higher than that of static assays. Normal plasma diluted to 25% significantly reduced its ability to cleave ULVWF strings under flow, whereas 2% plasma retained 48% enzyme activity in static assay. ADAMTS-13 activity varied from 33 to 100% among individuals and the variations were greater at shorter incubations of plasma with the substrate. Furthermore, the production of ULVWF from endothelial cells also varied among individuals. These results suggest that the commonly used static assays may underestimate the ADAMTS-13 activity required to cleave newly released ULVWF. They also demonstrated that the proteolysis of ULVWF may vary significantly among individuals, potentially contributing to the individual's vulnerability to thrombosis so that measurement of ADAMTS-13 may serve as a marker for TTP and other thrombotic diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

von Willebrand factor (VWF), present primarily in vascular subendothelium, platelets, and plasma, mediates platelet tethering and adhesion to the injured vessel wall. Tethering of platelets to subendothelial VWF initiates a cascade of events that lead to the formation of a platelet plug to stop bleeding. In addition, VWF plays key roles in the development of atherosclerosis and thrombosis [1–4]. For example, plasma VWF, which does not bind to circulating platelets, can aggregate platelets in the area of pathologically high fluid shear stress, as it is found in the area of severe stenosis [5] and deposit onto damaged vascular endothelial cells [3].

Synthesis of VWF occurs in two sites [1]: in megakaryocytes, where it is stored in α-granules that are later differentiated into platelets, and [2] in endothelial cells, where it is either secreted constitutively or stored in granules called Weibel–Palade bodies for secretion upon stimulation. The VWF stored in Weibel–Palade bodies is rich in ultra-large (UL) forms that are hyper-reactive in their capacity to bind the GP Ib-IX-V complex with high strength bonds [6,7]. Upon secretion, ULVWF multimers are rapidly, but partially cleaved by the metalloprotease ADAMTS-13 at the Y842/M843 peptide bond in the VWF A2 domain [8–11]. The proteolysis not only reduces the size of ultra-large VWF multimers to smaller forms in vitro[8,12,13], but also renders cleaved VWF multimers less active, interacting with the GP Ib-IX-V complex only in the presence of modulators or high shear stress. The importance of ADAMTS-13 cleavage of ULVWF is clinically demonstrated by the severe thrombotic microangiopathy called thrombotic thrombocytopenic purpura (TTP) that is attributed to the deficiency of this metalloprotease [14].

Current methods measure ADAMTS-13 activity by incubating the metalloprotease with the VWF substrate for a long period of time (up to 24 h) and are performed under static conditions [8,13,15–17]. Using these methods, the metalloprotease activity in patients with clinically diagnosed TTP varies from 0 to 100%, but most studies define ADAMTS-13 deficiency as 5% or less of normal activity [16–21]. Very recently, we reported two rapid ways to detect cleavage activity of ADAMTS-13 using a recombinant VWF-A2 as substrate [22,23]. Both methods measure the ADAMTS-13 activity under non-denaturing conditions and in a shorter period (< 5 h). The static assays have recently been evaluated extensively for their ability to measure ADAMTS-13 activity and as a diagnostic tool for TTP and potentially other thrombotic diseases.

In addition to these static assays, we have recently demonstrated that the cleavage of newly released ULVWF by ADAMTS-13 may also occur on the surface of endothelial cells [24]. We found that the newly released ULVWF multimers are anchored to the surface of endothelial cells in a P-selectin-dependent manner [25] and form extremely long string-like structures to which platelets adhere [24]. ADAMTS-13 binds and cleaves these ULVWF strings much faster than when under static conditions (less than 2 min as compared to several hours) [24,26]. These results demonstrate the importance of fluid shear stress in the proteolysis of ULVWF, consistent with other studies [27,28]. Upon anchorage to endothelial cells, ULVWF multimers are stretched by fluid shear stress to expose either the site for the metalloprotease to tether or the cleavage site in the VWF-A2 domain, which is likely to be hidden by its two neighboring A1 and A3 domains. Upon cleavage, VWF fragments are released into the plasma and released from the stretching force to allow the encryption of the ADAMTS-13 cleavage site, thereby limiting further proteolysis.

A common finding of the static assays is the wide variations of the metalloprotease activity among patients with clinical diagnosis of TTP and normal donors, but such variations have not been evaluated in the flow-based assay. The individual variations may be important for evaluating ADAMTS-13 activity in patients with TTP and other conditions where the metalloprotease may be affected. They are probably determined by multiple factors such as the rate and amount of ULVWF released from endothelial cells, and the rate and capacity of the metalloprotease to process the newly released ULVWF. Studying these variations could provide us new insights not only into how ULVWF is cleaved in vivo, but also how changes in the rate of ULVWF proteolysis may contribute to the development of thrombotic diseases other than TTP. For example, over-production and release of ULVWF may transiently exhaust the VWF cleaving metalloprotease, leading to consumptive deficiency of ADAMTS-13 [29]. The possibility can be significantly magnified for individuals who inherently have a larger pool of stored ULVWF, hypersensitive endothelial cells in releasing stored ULVWF, and/or an intrinsic low activity of ADAMTS-13.

In the current studies, we have examined the individual variations in the production of ULVWF multimers from human umbilical vein endothelial cells and in ADAMTS-13 activity as determined by its ability to cleave ULVWF strings on endothelial cells under flow and the recombinant VWF-A2 under static conditions. The results of these studies may help us to identify new risk factors for thrombotic diseases.

Platelet and plasma preparations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

Blood was obtained from 68 healthy donors under a protocol approved by the Institutional Review Board of the Baylor College of Medicine for using human subjects in biomedical research. All donors signed consent forms before blood was drawn. There were 29 males and 39 females with an age range of 22–56 years.

Blood was processed to obtain washed platelets and plasma (as the source of ADAMTS-13). For the former, blood was drawn into 10% acid–citrate dextrose buffer (ACD, 85 mmol L−1 sodium citrate, 111 mmol L−1 glucose, and 71 mmol L−1 citric acid) and centrifuged at 150 × g for 15 min at 24 °C to first obtain PRP, which was then centrifuged at 900 × g for 10 min. Platelet pellets were washed once with CGS buffer (13 mmol L−1 sodium citrate, 30 mmol L−1 glucose and 120 mmol L−1 sodium chloride, pH 7.0) and then resuspended in Ca2+ and Mg2+-free Tyrode's buffer (138 mmol L−1 sodium chloride, 5.5 mmol L−1 glucose, 12 mmol L−1 sodium bicarbonate, 2.9 mmol L−1 potassium chloride, and 0.36 mmol L−1 dibasic sodium phosphate, pH 7.4) [24]. For the latter, blood was drawn using PPACK (final concentration of 75 µmol L−1) as anticoagulant and then centrifuged at 150 × g for 15 min at 24 °C to collect PRP, which was then centrifuged at 900 × g for 10 min to obtain platelet-poor plasma.

Endothelial culture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

Endothelial cells were obtained from human umbilical veins (HUVECs) as described previously [24,30]. The use of human umbilical cords has been approved by the Institutional Review Board of the Baylor College of Medicine. Briefly, the umbilical cords were infused and incubated with collagenase (0.02%, Invitrogen Life Technologies, Carlsbad, CA, USA) for 30 min. Endothelial cells were then collected and grown in Medium 199 (Invitrogen Life Technologies) containing 20% heat-inactivated fetal calf serum and 0.2 mmol L−1 of l-glutamine on culture dishes coated with 1% gelatin until confluent.

Two types of HUVECs were obtained. For studying individual variations in the production of ULVWF, individual cords were collected and HUVECs from each cord separately grown. To test the ADAMTS-13 activity, HUVECs pooled from 10 to 20 cords were used. To induce the release of ULVWF, endothelial cells were stimulated with 25 µmol L−1 histamine (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature immediately before the perfusion experiments.

Parallel-plate flow chamber

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

We induced the formation of ULVWF strings on endothelial cells through a previously described method using a parallel-plate flow chamber [24]. Briefly, HUVECs were first stimulated with histamine and then assembled to form the bottom of the parallel-plate flow chamber (Glycotech, Rockville, MD, USA) connected to a syringe pump that draws the washed platelets suspended in Tyrode's buffer through the chamber at defined flow rates to generate specific shear stresses. The flow chamber was mounted onto an inverted-stage microscope (Nikon, Eclipse TE300, Garden City, NY, USA) and entire experiments were conducted at 37 °C with a thermostatic air bath.

The ADAMTS-13 activity was defined in this assay as the ability of plasma to clear ULVWF strings formed on the histamine-stimulated HUVECs. For this, plasma was mixed with washed platelets (1 : 1) and perfused over stimulated HUVECs at 2.5 dyn cm−2 shear stress. After 2 min perfusion, the numbers of ULVWF strings formed in 20 continuous view fields (400 ×) were quantitated.

Measurement of ADAMTS-13 activity under a static condition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

A static assay using the recombinant VWF A2 as the substrate has been described previously [22,23]. The ADAMTS-13 activity measured by this method has been compared to that by a modified assay from Furlan et al. [16] and found to be well correlated [23]. Briefly, complementary DNA encoding the human VWF A2 domain (amino acids 718–905) was generated by PCR using full-length human VWF cDNA as the template and subcloned into pQE9 vector (Qiagen, Chatsworth, CA, USA) for expression in Escherichia coli as a His tag-fusion protein. The transfected bacteria were cultured, induced and lyzed as described earlier [22]. For the purification, the washed pellet was solubilized by the addition of 7.5 mol L−1 urea in 50 mmol L−1 Tris-HCl, pH 7.5 and the solubilized protein passed over a Co2+-chelated Sepharose (TALON Superflow, Clontech, Palo Alto, CA, USA) column equilibrated with 5 mol L−1 urea, 50 mmol L−1 Tris-HCl, 500 mmol L−1 NaCl, pH 7.4 buffer. VWF-A2 protein was eluted from the column with 150 mmol L−1 imidazole. The buffer was rapidly exchanged to 25 mmol L−1 Tris-HCl, 150 mmol L−1 NaCl, 0.05% Tween-20, pH 7.4 (TBS-T) by using a desalting column (Amersham). Protein concentration was determined by the BCA method (Pierce Chemical Co., Rockford, IL, USA).

To evaluate the ADAMTS-13 activity in plasma, 10 µL of normal plasma were incubated with recombinant VWF-A2 (30 µg/mL in TBS) at 37 °C [22]. Two microliters of reaction mixture were taken from each sample at 0, 15 and 30 min, and 1, 3 and 6 h and mixed with non-reduced sample buffer (10 µL). The proteins were separated by 12% SDS-PAGE and transferred onto a polyvinylidine difluoride (PVDF, Waters, Milford, MA, USA) membrane. To detect the A2 cleavage, the transferred membrane was first incubated with 5% non-fat dry milk in TBS-T for 30 min and then with a monoclonal antihistidine antibody (Sigma-Aldrich) diluted in TBS-T (1 : 5000) for 30 min at room temperature. The cleaved products were visualized with enhanced chemiluminescence (Pierce).

To measure the ADAMTS-13 activity in plasma the ELISA based method, described previously, was used [23]. Briefly, either citrated (0.38% sodium citrate) or PPACK (75 µmol L−1) normal plasma were diluted in 25 mmol L−1 Tris-HCl, 150 mmol L−1 NaCl, pH-7.4 (TBS) and mixed with recombinant VWF-A2 peptide (5 µg mL−1 in TBS). Two different dilutions of plasma [1: 1 and 1: 50]: were tested. The mixture was added into microtiter wells coated with Ni2+ (Ni-NTA HisSorb Strips, Qiagen) and incubated for 2 h at 37 °C. After incubation for 2 h, the wells were washed with TBS, and monoclonal antibody Tag-100 (against Tag-100) (Qiagen) (1 : 2000 in TBS) was added and incubated for one additional hour at 37 °C. The wells were then washed with TBS and incubated with a 1 : 3000 dilution of goat peroxidase-conjugated monoclonal antimouse IgG antibody (Sigma, St. Louis, MO, USA) for 45 min at 37 °C. The wells were washed, and the substrate o-phenylenediamine (Sigma) was added. After 10 min of substrate conversion, reactions were stopped with 0.025 mL of 2 mol L−1 H2SO4, and the plates were read at 490 nm.

ADAMTS-13 activity required to cleave ULVWF strings under flow

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

We have previously shown that normal plasma at 50% dilution cleaved more than 95% of ULVWF-platelet strings formed on the histamine-stimulated endothelial cells within 2 min of perfusion at 2.5 dyn cm−2 in most normal individuals [24]. We therefore set this condition as the maximal activity of ADAMTS-13 (100%) and then measured the cleavage of ULVWF strings using different amounts of plasma under flow. Diluted normal plasma mixed with washed platelets was perfused over the histamine-stimulated HUVECs and the numbers of ULVWF strings formed counted after 2 min perfusion. We found that the ADAMTS-13 activity for cleaving ULVWF strings under flow was reduced to 50% with 25% plasma (49.4 ± 16.1% vs.100% for 25 and 50% plasma, respectively, Fig. 1A). In comparison, plasma diluted to 20% retained more than 70% of ADAMTS-13 activity in the static assay (72.6 ± 12.3% vs.100% for 20 and 50% plasma, respectively, Fig. 1B). ADAMTS-13 activity in 5% plasma was close to 50% in the same static assay. These results suggest that the activity of ADAMTS-13 required to cleave endothelial cell-bound ULVWF strings under flow was significantly higher than that of static condition. Although the static assay used the VWF-A2 instead of ULVWF as substrate, we have previously demonstrated that ADAMTS-13 activity measured by this assay is closely correlated to the static assay using ULVWF as the substrates [22].

image

Figure 1. ADAMTS-13 activity required to cleave ULVWF strings under flow. The rates of VWF cleavage were measured under flow and static conditions. The former measured the cleavage of ULVWF strings formed on histamine-stimulated HUVECs (A), whereas the latter measured the cleavage of the recombinant VWF-A2 by ELISA (B). In both cases, cleavage with 50% plasma was used as baseline. A higher ADAMTS-13 activity (less diluted plasma) was required for cleaving the ULVWF strings under flow as compared to cleavage of the VWF-A2 under static condition. The figures are mean ± SEM, n = 68.

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In addition, the rate of ULVWF cleavage under flow monitored for longer periods was proportional to plasma concentration (Fig. 2). At 50% dilution, more than 95% of ULVWF strings were cleaved after a 2 min perfusion, whereas 51, 68, and 69% of strings remained on endothelial cells after the 2-min perfusion at 25, 12.5, and 6.25% dilutions, respectively. Even after 8 min perfusion, 24, 39, and 54% strings remained. Due to the technical difficulties, we were unable to determine the initial rate of cleavage, which may be more helpful for understanding the kinetics of ULVWF cleavage under flow. Such a limitation may not affect our observations for the individual variations in ADAMTS-13 activity, but may fail to account for difference in the initiation phase of the cleavage kinetics.

image

Figure 2. The time course of cleaving ULVWF strings under flow condition. Washed platelets were mixed with various amounts of normal plasma and perfused over histamine-stimulated HUVECs under 2.5 dyn cm−2 shear stress for 2 min. The number of ULVWF strings was determined at the end of 2 min perfusion. The mean number of ULVWF strings was less than one with 50% plasma, whereas significantly higher numbers of ULVWF strings were detected with normal plasma diluted to 25, 12.5 and 6.25%. The continuous cleavage was observed in all plasma dilutions except 6.25, where cleavage reached plateau at 6 min perfusion. The figures are mean ± SEM, n = 34.

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Individual variations in ADAMTS-13 activity under flow and static conditions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

In addition to significantly higher ADAMTS-13 activity needed for cleaving ULVWF strings under flow, we have also observed significant variations in ADAMTS-13 activity among 68 individuals that we have tested. The ADAMTS-13 activity of 50% plasma in the flow assay ranged from 34 to 99% (coefficient variation = 147, n = 68, Fig. 3A).

image

Figure 3. Individual variations for the plasma cleavage of VWF substrate. ADAMTS-13 activity in plasma was determined by either the cleavage of ULVWF strings under flow (50% dilution of plasma) or the recombinant VWF-A2 under static conditions (50 and 2% dilution of plasma (A). Results were from 68 (for flow assay) or 40 donors (for static assay). ADAMTS-13 activity was measured over different incubation times under a static condition. Cleavage occurred after a 15-min incubation of plasma with the VWF A2 substrate and reached maximum after 3 h. The metalloprotease activity of donor A was significantly higher than that of donor B with shorter incubation times (15 and 30 min), whereas it was similar between two samples after 3 h or longer incubation (B).

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Similar variations were also found in the cleavage of the recombinant VWF-A2 under a static condition. Using the ELISA based assay, the ADAMTS-13 activity with 50% and 2% plasma ranged from 20 to 100% and 0–100%, respectively (Fig. 3A). The greatest variation was observed within the first hour of incubation as demonstrated by Western blot (Table 1). For example, ADAMTS-13 activity from two normal donors was similar after 1-h incubation of plasma with the VWF-A2 substrate, but the metalloprotease activity of donor A was nearly five- and twofold of donor B at 15- and 30-min incubations, respectively (Fig. 3B).

Table 1.  Variations of individual capacity to cleave VWF-A2 under static conditions
Activity (%)Incubation time between plasma and VWF-A2 (min)
30601803001440
  1. CV, coefficient of variation.

Mean17.735.054.769.171.3
SD21.721.617.917.513.7
CV122.761.632.725.319.2

Individual variations in ULVWF release from HUVECs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

We have previously showed that ULVWF newly released from HUVECs upon histamine stimulation produce extremely long string-like structures on which platelets tether. These results were obtained using endothelial cells pooled from multiple cords and therefore failed to take the potential individual variations in ULVWF release into consideration. To address this concern, we grew HUVECs from 35 individual umbilical cord veins and then measured the formation of ULVWF strings on the histamine-stimulated HUVECs. The cords were collected 1–48 h after placenta was removed and HUVECs used after 5–7 days of culture. The numbers of ULVWF strings produced in 20 continuous view fields varied significantly, independent of the lag time between the delivery and culture of endothelial cells (correlation analysis, r2 = 0.2644, n = 35, Fig. 4). Finally, we have also determined the correlation between the perfusion time and the amount of ULVWF strings formed on HUVECs using the pooled HUVECs (data not shown). The number of ULVWF strings increased proportionately with the perfusion time in histamine-stimulated HUVECs. Shear stress alone also produced ULVWF strings, but at a significantly lower numbers (72.7 ± 12.0 vs. 4.7 ± 2.0 strings/20 continuous view fields).

image

Figure 4. Individual variations in the production of ULVWF strings on endothelial cells under flow. Endothelial cells were collected from individual human umbilicus and tested for their ability to produce ULVWF strings under a 2.5 dyn cm−2 shear stress in response to 25 µmol L−1 of histamine. The numbers of ULVWF strings counted over 20 continuous 400 × viewfields ranged from 26 to 197 and the variation was not affected by the time interval between placenta removal and HUVEC culture. The figure was from 35 human umbilici.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References

In this study we have demonstrated that the activity of ADAMTS-13 required to cleave the platelet-adhering ULVWF strings formed on HUVECs under flowing condition is much higher than to cleave VWF (ULVWF or recombinant VWF-A2) substrates under static condition. We found that the proteolytic activity of ADAMTS-13 was markedly reduced with 25% of normal plasma under flow conditions (Fig. 1), whereas the cleavage of the recombinant VWF A2 was observed with 2% plasma for most donors. The mean activity of ADAMTS-13 using 2% plasma in the static assay was significantly greater than 12.5% plasma used in the flow assay (48.2 ± 21.4% for the static vs. 38.5 ± 11.7% for the flow assay, Student's t-test, n = 52, P < 0.01). Although it is difficult to quantitate the VWF concentration in the form of ULVWF strings, the amount of the VWF-A2 substrate used in our ELISA assay (5 µg mL−1) is significantly more than the amount of ULVWF strings produced from HUVECs of 0.6 cm2 area (the surface area of the flow chamber).

The results reported here suggest that the ADAMTS-13 activity measured by static assays may significantly underestimate the activity required to cleave the newly released ULVWF under flow. Although both are in vitro results, the flow-based assay is likely to be more physiologically relevant because its takes into consideration the vascular endothelial cells and fluid shear stress, two parameters that may play critical roles in the proteolysis of VWF in vivo. Furthermore, the assay was conducted at physiological pH without denaturing the VWF substrate. This high activity requirement in the flow assay may explain why ADAMTS-13 activity in some TTP patients was found to be much higher than 5% that has been defined in most studies as the threshold for ADAMTS-13 deficiency [31–33]. More importantly, our results suggest that conditions where ADAMTS-13 activity is found to be moderately reduced, including pregnancy [34,35], autoimmune diseases [36], infection [37], trauma and surgery [38], and tumors [39,40], may have more profound thrombotic implications.

Another key finding of our studies is the significant variations in the rate and amount of ULVWF production and cleavage. The variation of plasma ADAMTS-13 activity among normal individuals is nearly three-fold ranging from 34 to 99% (Fig. 3). The variations are also observed in the static assays, using either ULVWF or the VWF-A2 as substrates [23], suggesting that the variation is intrinsic, independent of the type of assays used. We found that the rate to cleave VWF-A2 protein markedly varies within the first hour of incubation under static conditions. For example, after 15- and 30-min incubation, the ADAMTS-13 activity measured for one donor is five- and twofold higher than the other, even though both measure similar activity when plasma was incubated with the A2 substrate for longer than 3 h. These results suggest that most static assays, which measure the enzyme activity after a long incubation of up to 24 h, may have greatly underestimated the individual variations for ADAMTS-13 activity.

Similar variations are also observed for the production of ULVWF from endothelial cells upon stimulation. We have observed a five- to sevenfold difference in the number of ULVWF strings formed on HUVECs in response to the same stimulation (Fig. 4), suggesting that some individuals may have inherently larger storage pools of ULVWF or that their endothelial cells are more sensitive to stimulations.

Such marked variations could make the VWF proteolysis in some individuals more efficient than others, due either to smaller pools of stored ULVWF in endothelial cells or to higher ADAMTS-13 activity. Similarly, some may be prone to thrombosis because of larger pools of ULVWF storage or inherently low ADAMTS-13 activity. The latter has recently been reported in nearly 10% of the Japanese population due to a gene polymorphism [41]. For these individuals, conditions such as inflammation and pregnancy, which induce vascular endothelial cells to release ULVWF, may result in transient deficiency of ADAMTS-13 and subsequent accumulation of ULVWF in plasma. In a recent report, Reiter et al. [29] demonstrated that infusion of desmopressin, which stimulates the release of ULVWF from endothelial cells, resulted in moderate deficiency of ADAMTS-13 and detection of ULVWF in plasma. Unlike the absolute deficiency of ADAMTS-13 as it is found in TTP patients, consumptive deficiency of ADAMTS-13 may be transient and its clinical relevance remains largely unknown. Our studies suggest the needs for such investigations and the population-based studies to determine whether variations in the proteolysis of ULVWF could serve as a risk factor for thrombosis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Platelet and plasma preparations
  6. Endothelial culture
  7. Parallel-plate flow chamber
  8. Measurement of ADAMTS-13 activity under a static condition
  9. Statistical analysis
  10. Results
  11. ADAMTS-13 activity required to cleave ULVWF strings under flow
  12. Individual variations in ADAMTS-13 activity under flow and static conditions
  13. Individual variations in ULVWF release from HUVECs
  14. Discussion
  15. Acknowledgements
  16. References
  • 1
    Ruggeri ZM. Von Willebrand factor, platelets and endothelial cell interactions. J Thromb Haemost 2003; 1: 133542.
  • 2
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