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Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
The Effect of Shear Stress on the Size, Structure, and Function of Human von Willebrand Factor *Chris Hoi Houng Chan, *Ina Laura Pieper, *Scott Fleming, *Yasmin Friedmann, †Graham Foster, *Karl Hawkins, *Catherine A. Thornton, and *Venkateswarlu Kanamarlapudi *Institute of Life Science, College of Medicine, Swansea University; and †Institute of Life Science, Calon Cardio-Technology Ltd, Swansea, Wales, UK
Abstract: Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for
vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices. Key Words: von Willebrand factor—Shear stress—Platelet aggregation—Multimer analysis.
During the past few decades, ventricular assist devices (VADs) have emerged as an increasingly
popular therapy in patients with advanced heart failure who do not respond to medical or resynchronization therapy. VADs are used either as a bridge-to-transplant or as destination therapy in patients deemed ineligible for cardiac transplantation, or those who cannot receive a transplant due to the limited supply of donors (1). While VADs have already benefitted many patients, VAD-related blood damage remains a major issue. This includes hemolysis (2,3), platelet activation (4), alteration of the coagulation cascade and thrombosis (5), reduced functionality of leukocytes (6), release of
doi:10.1111/aor.12382 Received April 2014; revised June 2014. Address correspondence and reprint requests to Dr. Chris Hoi Houng Chan, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales SA2 8PP, UK. E-mail: [email protected]; [email protected] Presented in part at the 21st Congress of the International Society for Rotary Blood Pumps held September 26–28, 2013 in Yokohama, Japan.
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microparticles (7), and degradation of von Willebrand factor (vWF) (8,9). Gastrointestinal (GI) bleeding is one complication associated with the placement of a VAD, with rates as high as 65% within the first year after VAD placement (10,11). The mechanism underlying this problem in patients on long-term continuous flow mechanical support is not well understood (1,9,11– 22). This cannot be explained by the anticoagulation regimen alone, but may be symptomatic of acquired von Willebrand syndrome (AvWs) which could be the result of shear stress caused by the VAD. In a literature review of clinical studies describing patient populations suffering from GI bleeding, two populations in particular were found to stand out: those suffering from aortic valve stenosis and those implanted with axial flow VADs (Table 1). In aortic stenosis, there is a high level of shear stress and loss of HMW vWF multimers (23,24). In contrast to axial flow pumps, centrifugal flow VADs and total artificial hearts (TAHs) have fewer reported events of GI bleeding. The centrifugal pump with the most reports of GI bleeding is the novel HeartWare device (20,25– 28). In patients implanted with VentrAssist and EVAHEART, there have been few reports of bleeding, although they have been diagnosed with AvWs, showing a loss of high molecular weight (HMW) vWF multimers and decreased ratios of collagen binding capacity and ristocetin cofactor activity to vWF antigen (17). Comparing the shear stress in these devices provides a partial explanation as the characteristic hydraulic performance of axial flow usually involves high rotational pump speed compared with centrifugal flow resulting in higher shear stress (29). Furthermore, TAHs typically have lower
shear stress than centrifugal pumps due to their pulsatile operation (11). Thus, the hypothesis is that the higher the shear stress, the more damage caused to vWF, resulting in decreased platelet aggregation and thus bleeding. In light of this, it is important to understand the point at which shear stress becomes damaging, and why centrifugal VAD patients do not suffer GI bleeding to the same extent, although obviously suffering impairment of vWF. It is difficult to calculate shear stress in VADs both in vitro and in vivo. Therefore, it is important to establish an in vitro laboratory model with greater sensitivity and the ability to better quantify HMW vWF multimer loss while furthering our understanding of the relationship between HMW vWF abundance and activity. Research focusing on the impact of wall shear rate caused by perfusing normal plasma through long capillary tubing, and that of pumping plasma in a mock circulatory loop driven by a VAD has shown a relationship between the shear rate and the loss of HMW vWF multimers (8,30,31). However, neither of these models provide a way to subject the plasma to accurate and controlled levels of shear rate environment. A rheometer can be used to apply controlled levels of shear rate during timed intervals. Therefore, the purpose of this study was to: (i) quantify HMW vWF breakdown at specified levels of shear rate using a rheometer; (ii) correlate the loss of HMW multimers to vWF functionality; and (iii) investigate the effect of mechanical shear on platelet aggregates. This information would be of value to VAD developers who could modify device design to avoid damaging shear stress levels.
TABLE 1. Literature review describing patient populations suffering from GI bleeding and vWF diagnostic methods vWF diagnostic methods
Device or medical condition CF-VAD
Axial flow
Centrifugal flow
Pulsatile TAH No device
HeartMate II Jarvik 2000 MicroMed DeBakey Thoratec BVAD VentrAssist CentriMag BiVAD HeartWare EVAHEART Duraheart CardioWest HeartMate XVE Aortic valve stenosis
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GI bleeding
HMW vWF loss (multimer analysis)
Yes Yes Yes Yes No N/A Yes No N/A No No Yes
Yes N/A N/A Yes Yes Yes N/A No No No No Yes
Decrease in vWF : RCo / vWF : Ag activity Yes N/A N/A Yes Yes N/A N/A Yes N/A No N/A N/A
Decrease in vWF : CB / vWF : Ag activity Yes N/A N/A Yes Yes N/A N/A N/A N/A No N/A Yes
Reference 9,11–13,16–19 15 11 18,19 11,18 16 20,25–28 29 16 18 11,16 23,24
VON WILLEBRAND FACTOR AND SHEAR STRESS MATERIALS AND METHODS Blood preparation Peripheral blood (72 mL) was collected from six healthy volunteers in vacutainers containing 9NC coagulation sodium citrate 3.2% (455322, Greiner Bio-one, Wemmel, Belgium). Platelet-rich plasma (PRP) was prepared by centrifuging whole blood for 7 min at 500 × g at room temperature. Platelet-poor plasma (PPP) was prepared by centrifuging PRP for 5 min at 13 000 × g at room temperature. The PPP was extracted and analyzed using the automated hematology analyzer CELL-DYN Ruby (Abbott Diagnostics, Abbott Park, IL, USA) to ensure that the plasma was void of blood cells. PPP was used instead of whole blood to ensure that any potential effect of shear on vWF was not a result of cellular interactions. This study was approved by the South Wales Research Ethics Committee and all donors gave informed written consent. Rheometry PPP was subjected to shear stress rates of 4000 and 8000/s in an AR-G2 rheometer (TA Instruments, New Castle, DE, USA) at 37°C. The rheometer was equipped with double concentric geometry in which a rotating inner cylinder cup allows generation of uniformly established shear flow at a well-defined shear rate. The PPP was sampled at hourly intervals (6 h in total). Static PPP at 37°C was used as a control. The sheared PPP was analyzed by flow cytometry immediately after the shear tests and the remaining plasma samples were stored at −80°C for ≤5 days prior to analysis for the vWF multimer by immunoblotting. Plasma vWF multimer analysis by immunoblotting Sheared and nonsheared PPP was subjected to electrophoresis on high gelling temperature agarose (0.6% agarose, w/v, Sea Kem, FMC Bioproducts, Rockland, ME, USA) in a horizontal gel apparatus (81–2325, Galileo Bioscience, Cambridge, MA, USA) at 4°C. Electrophoresis was performed at 30 mA for 30 min and then at 50 mA until the dye front had migrated 10–12 cm from the origin (total gel running time was 6 h). vWF multimers separated on agarose gel were transferred to polyvinylidene difluoride (PVDF) 0.45 μm membrane (IPVH304F0, Immobilon-P, Millipore Corporation, Billerica, MA, USA) for 15–17 h at 70 mA in the electroblotting tank (91–2020-TB, Galileo Bioscience). vWF detection using anti-human vWF primary (ab6994, Abcam, Cambridge, UK) and horseradish peroxidise (HRP)-conjugated secondary (ab6721, Abcam) antibodies, and chemiluminescence substrate (170–5060,
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Bio-Rad, Hercules, CA, USA) was performed as described by Krizek et al. (32). The shear-induced changes in the size distribution of plasma vWF multimers were studied by densitometric scanning initiated from the origin (Quantity One software v4.6.8, BioRad, Hertfordshire, UK). The loss of HMW vWF multimers was quantified by the rate of change in gradient of HMW vWF. vWF : Ristocetin (vWF : RCo) assay vWF functionality was assessed using the vWF : Ristocetin (vWF : RCo) assay developed by Chen et al. (33). The assay measures the ability of vWF (in PPP) to bind to platelets in the presence of ristocetin resulting in aggregation of the platelets. Human platelets (50 000 platelets per μL) were stained either green (CellTracker Green CMFDA, Life Technologies, Glasgow, UK) or red (MitoTracker Red FM, Life Technologies), and incubated with 2 μL PPP and 1 mg/mL ristocetin solution for 45 min at room temperature in the dark rotating at 30 rpm (SB3 Rotator, Stuart, Bibby Scientific, Staffordshire, UK). Nonaggregated platelets (green or red events) and aggregated platelets (doublelabeled events) were quantified by flow cytometry (FACSAria I, BD Biosciences, Oxford, UK) and the percentage of double-labeled events was measured (FACS Diva 6.1.3, BD Biosciences). The greater the percentage of double-labeled events, the higher the platelet aggregation and hence the vWF functionality. Confocal microscopy of platelet aggregates vWF : RCo assays were performed as described above. Post 24 h incubation at room temperature, 70 μL reaction mixture samples containing vWF and red/green platelets were stained with anti-human vWF antibody (ab6994, Abcam) labeled with Pacific Blue (Z25041, Zenon Pacific Blue mouse IgG1 labelling kit, Life Technologies) in an 8-well borosilicate chamber slide (155411, Thermo Scientific, New York, NY, USA). Individual and aggregated fluorescent platelets adhered by vWF were fixed by Vectashield hardset mounting medium (H-1400, Vector Laboratories, Peterborough, UK) and examined with a confocal microscope (LSM 710, Zeiss, Jena, Germany). Images of green and red fluorescent platelets and blue fluorescent-stained vWF were captured and analyzed using ZEN imaging software 2012. vWF ELISA The concentration of vWF : antigen (vWF : Ag) in the sheared PPP was quantified by a vWF specific enzyme-linked immunosorbent assay (ELISA) kit (ab108918, Abcam) according to manufacturer’s Artif Organs, Vol. 38, No. 9, 2014
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FIG. 1. Measurement of vWF multimers by immunoblotting. HMW and LMW vWF multimer immunoblot images (left panel) and densitometric analysis (right panel) for human PPP under different shear rates: (A) static controls 0/s, (B) 4000/s, and (C) 8000/s at hourly intervals during 6 h at 37°C.
instructions and absorbance at 450 nm measured (POLARstar Omega, BMG LABTECH, Ortenberg, Germany). Statistical analysis Due to multiple measurements made per PPP sample at different time points and at different shear rates, a repeated measures analysis of variance on the data sets was conducted. The shear rates and the time points were treated as fixed effects and the sample as a random effect. Once significant effects were found, pairwise comparison tests were conducted, adjusted for the multiple comparisons using Bonferroni’s method. Analysis was performed using the RStudio v 0.97.551 (RStudio, Boston, MA, USA) and R statistical environment, v3.0.2 (R Core Team, Vienna, Austria). Artif Organs, Vol. 38, No. 9, 2014
RESULTS Quantification of the rate of change of HMW and LMW vWF using densitometry The rate of change of HMW and low molecular weight (LMW) multimers was quantified using densitometry (Fig. 1A–C, right panel) and the results are summarized in Fig. 2A (HMW vWF) and 2B (LMW vWF). HMW vWF Repeated measures analysis of variance shows a very significant dependence of the densitometry values on the shear rate (P < 10−6) and on time (P < 10−6). The interaction between time and shear rate is also significant (P < 0.002). Pairwise comparisons (with Bonferroni correction) confirm the
VON WILLEBRAND FACTOR AND SHEAR STRESS
1.2 1.0 0.8 0.6 0.4 0.2 0.0
Mean normalized HMW vWF densitometry values
A
Shear rate: static 4000 1/sec 8000 1/sec
0
1
2
3
4
5
6
time (h)
2.0 1.8
LWM vWF Repeated measures analysis of variance showed a significant dependence of the densitometry values on the shear rates (P < 0.002) and on time (P < 10−5). There was no significant interaction between shear rate and time in this case. Pairwise comparisons with Bonferroni correction show that the significant differences are between the static and high shear values, and that measurements need to be at least 5 h apart to show significant changes. This is summarized in Fig. 2B, which shows three distinct curves describing mean densitometry values of the three shear rates. The curves representing sheared samples exhibit an upward trend with time of the densitometry values. To summarize, the results reflect loss of HMW vWF at 4000 and 8000/s, compared with the static control at hourly intervals during 6 h at 37°C in Fig. 1 (left panel). There is a positive correlation between shear rate and degradation of vWF, with 8000/s causing the greatest loss of HMW bands compared with the static control. Similarly, there is a correlation between shear rate and an increase in LMW bands, suggesting that the HMW bands may be cleaved into smaller fragments. This conclusion is strengthened by the quantification of vWF antigen below.
0.8
1.0
1.2
1.4
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Shear rate: static 4000 1/sec 8000 1/sec
0.6
Mean normalized LMW vWF densitometry value
B
745
0
1
2
3 time (h)
4
5
6
Quantification of vWF antigen by ELISA The total vWF antigen decreases during the 6-h test in the static control and the sheared samples (Fig. 3). However, as there is no difference between the groups, we can conclude that the HMW band loss demonstrated by immunoblotting (Fig. 1) in the sheared samples is due to a cleavage of HMW multimers into LMW multimers.
FIG. 2. (A) Means (over the different samples) ± SE of the normalized results of densitometric analysis for HMW vWF in human PPP under different shear rates (static control 0, 4000, and 8000/s at hourly intervals during 6 h at 37°C). (B) Means (over the different samples) ± SE of the normalized results of densitometric analysis for LMW vWF in human PPP under different shear rates (static control 0, 4000, and 8000/s at hourly intervals during 6 h at 37°C).
significant difference between any two levels of shear rate and show that significant differences are achieved between measurements that are at least 4 h apart. This is summarized in Fig. 2A which shows three distinct curves. The mean densitometry values in the control sample remain high while there is a decrease with time in the sheared samples, with a stronger decrease in the sample subjected to the higher shear.
FIG. 3. Total vWF antigen quantified by ELISA in postsheared human PPP at different shear rates for 6 h. Human PPP subjected to 0/s (static control), 4000 or 8000/s, and then analyzed by ELISA. Artif Organs, Vol. 38, No. 9, 2014
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vWF functionality assessed using flow cytometry and visualized using confocal microscopy The assessment of the vWF functionality was performed using a ristocetin-dependent flow cytometry assay of platelet aggregation. When equal concentrations of green and red platelets, resolved by the FITC and APC channels, respectively (Fig. 4A, left panel), were mixed in the absence of PPP, single green and red platelets dominated and double positive events (platelet aggregates) were negligible (20 000/s (8). Another limitation was the residence time (6 h) of the PPP which is continuously sheared in the rheometer, whereas the blood passes through the VAD in a period of milliseconds. However, this work provides a simple and accurate way to expose the blood plasma samples to controlled shear rates ranging from physiological to pathological levels which allow evaluation of the impact of shear rate on the vWF. This is of value to provide further insight into bleeding complications in patients with VAD. Our future work will focus on
FIG. 8. Confocal microscopy of platelet aggregates stained with red or green in the presence of human PPP form the static control (A) and sheared at 8000/s (B) after 6 h. The nonsheared PPP sample results in less dispersed and larger platelet aggregates compared with the sheared vWF sample.
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VON WILLEBRAND FACTOR AND SHEAR STRESS further exploration of the shear stress and time domains of vWF mechanoenzymatic stability, especially at higher ranges of shear stress and shorter exposure times. In addition, the use of whole blood instead of PPP is the next step to mimic physiological conditions, although shear stress might also damage or activate platelets causing release of ADP and vWF multimers into the plasma. Furthermore, the role of ADAMTS13, the protease known to break down vWF (31), in HMW vWF destruction during high shear stress will be considered. CONCLUSIONS The results shown here confirmed that the longer human platelet-poor plasma is exposed to high shear rate, the greater the loss of the high molecular weight von Willebrand factor multimers and decline in the functional activity of vWF will be. There is a strong linear correlation between HMW vWF multimer loss and the lower functional activity of vWF. Therefore, this study indicates that a simple, fast, accurate flow cytometric evaluation of the vWF activity is sensitive enough for a comparative acquired von Willebrand syndrome study for in vitro testing of different shear conditions. This work could improve in vitro ventricular assist device evaluation and bring it a step closer to assessment of total blood trauma. It is recommended that the effort of blood pump design should not only focus on mechanical stability and hemolysis, but also on the other blood components that could be damaged such as leukocytes, platelets, and soluble factors such as vWF and factor VIII. Future in vitro device evaluation could benefit from inclusion of assays such as this to provide a more complete picture of the overall effect on blood function.
7.
8.
9.
10. 11. 12. 13.
14. 15.
16.
17. 18.
19.
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in vitro testing of ventricular assist devices. Artif Organs 2013;37:793–801. Diehl P, Aleker M, Helbing T, et al. Enhanced microparticles in ventricular assist device patients predict platelet, leukocyte and endothelial cell activation. Interact Cardiovasc Thorac Surg 2010;11:133–7. Egger C, Maas J, Hufen T, Schmitz-Rode T, Steinseifer U. Establishing a method for in vitro investigation of mechanical parameters causing acquired von Willebrand syndrome in ventricular assist devices. Artif Organs 2013;37:833–9. Malehsa D, Meyer AL, Bara C, Struber M. Acquired von Willebrand syndrome after exchange of the HeartMate XVE to the HeartMate II ventricular assist device. Eur J Cardiothorac Surg 2009;35:1091–3. Slaughter MS, Rogers JG, Milano CA, et al. Advanced heart failure treated with continuous-flow left ventricular assist device. NEJM 2009;361:2241–51. Crow S, John R, Boyle A, et al. Gastrointestinal bleeding rates in recipients of nonpulsatile and pulsatile left ventricular assist devices. J Thorac Cardiovasc Surg 2009;137:208–15. Meyer AL, Malehsa D, Bara C, et al. Acquired von Willebrand syndrome in patients with an axial flow left ventricular assist device. Circ Heart Fail 2010;3:675–81. Klovaite J, Gustafsson F, Mortensen SA, Sander K, Nielsen LB. Severely impaired von Willebrand factor-dependent platelet aggregation in patients with a continuous-flow left ventricular assist device (HeartMate II). J Am Coll Cardiol 2009;53:2162–7. Eckman PM, John R. Bleeding and thrombosis in patients with continuous-flow ventricular assist devices. Circulation 2012;125:3038–47. Letsou GV, Shah N, Gregoric ID, Myers TJ, Delgado R, Frazier OH. Gastrointestinal bleeding from arteriovenous malformations in patients supported by the Jarvik 2000 axialflow left ventricular assist device. J Heart Lung Transplant 2005;24:105–9. Uriel N, Pak S-W, Jorde UP, et al. Acquired von Willebrand syndrome after continuous-flow mechanical device support contributes to a high prevalence of bleeding during long-term support and at the time of transplantation. J Am Coll Cardiol 2010;56:1207–13. Crow S, Chen D, Milano C, et al. Acquired von Willebrand syndrome in continuous-flow ventricular assist device recipients. Ann Thorac Surg 2010;90:1263–9. Heilmann C, Geisen U, Beyersdorf F, et al. Acquired von Willebrand syndrome in patients with ventricular assist device or total artificial heart. Thromb Haemost 2010;103:962–7. Geisen U, Heilmann C, Beyersdorf F, et al. Non-surgical bleeding in patients with ventricular assist devices could be explained by acquired von Willebrand disease. Eur J Cardiothorac Surg 2008;33:679–84. Hayes HM, Dembo LG, Larbalestier R, O’Driscoll G. Management options to treat gastrointestinal bleeding in patients supported on rotary left ventricular assist devices: a single-centre experience. Artif Organs 2010;34:703– 6. Stern DR, Kazam J, Edwards P, et al. Increased incidence of gastrointestinal bleeding following implantation of the HeartMate II LVAD. J Card Surg 2010;25:352–6. Islam S, Cevik C, Madonna R, et al. Left ventricular assist devices and gastrointestinal bleeding: a narrative review of case reports and case series. Clin Cardiol 2013;36:190– 200. Vincentelli A, Susen S, Le Tourneau T, et al. Acquired von Willebrand syndrome in aortic stenosis. N Engl J Med 2003;349:343–9. Pareti FI, Lattuada A, Bressi C, et al. Proteolysis of von Willebrand factor and shear stress-induced platelet aggregation in patients with aortic valve stenosis. Circulation 2000;102:1290–5.
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25. Lalonde SD, Alba AC, Rigobon A, et al. Clinical differences between continuous flow ventricular assist devices: a comparison between HeartMate II and HeartWare HVAD. J Card Surg 2013;28:604–10. 26. Slaughter MS, Pagani FD, McGee EC, et al. HeartWare ventricular assist system for bridge to transplant: combined results of the bridge to transplant and continued access protocol trial. J Heart Lung Transplant 2013;32:675–83. 27. Saeed D, Albert A, Kamiya H, Maxhera B, Westenfeld R, Lichtenberg A. Five days of no anticoagulation or antiplatelet therapy and NovoSeven administration in a HeartWare HVAD patient. Artif Organs 2012;36:751–3. 28. Popov AF, Hosseini MT, Zych B, et al. Clinical experience with HeartWare left ventricular assist device in patients with end-stage heart failure. Ann Thorac Surg 2012;93:810–5. 29. Ichihara Y, Nishinaka T, Komagamine M, et al. Gastrointestinal bleeding was rare with centrifugal type continuous flow left ventricular assist device EVAHEART. J Heart Lung Transplant 2013;32:S233–4.
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30. Tsai HM, Sussman II, Nagel RL. Shear stress enhances the proteolysis of von Willebrand factor in normal plasma. Blood 1994;83:2171–9. 31. Dassanayaka S, Slaughter MS, Bartoli CR. Mechanistic pathway(s) of acquired von Willebrand syndrome with a continuous-flow ventricular assist device: in vitro findings. ASAIO J 2013;59:123–9. 32. Krizek DR, Rick ME. A rapid method to visualize von Willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection. Thromb Res 2000;97:457–62. 33. Chen D, Daigh CA, Hendricksen JI, et al. A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF : RCo) activity assay by flow cytometry. J Thromb Haemost 2008;6:323–30. 34. Sobieski MA, Giridharan GA, Ising M, Koenig SC, Slaughter MS. Blood trauma testing of CentriMag and RotaFlow centrifugal flow devices: a pilot study. Artif Organs 2012;36: 677–82.
Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
The Effect of Shear Stress on the Size, Structure, and Function of Human von Willebrand Factor *Chris Hoi Houng Chan, *Ina Laura Pieper, *Scott Fleming, *Yasmin Friedmann, †Graham Foster, *Karl Hawkins, *Catherine A. Thornton, and *Venkateswarlu Kanamarlapudi *Institute of Life Science, College of Medicine, Swansea University; and †Institute of Life Science, Calon Cardio-Technology Ltd, Swansea, Wales, UK
Abstract: Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for
vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices. Key Words: von Willebrand factor—Shear stress—Platelet aggregation—Multimer analysis.
During the past few decades, ventricular assist devices (VADs) have emerged as an increasingly
popular therapy in patients with advanced heart failure who do not respond to medical or resynchronization therapy. VADs are used either as a bridge-to-transplant or as destination therapy in patients deemed ineligible for cardiac transplantation, or those who cannot receive a transplant due to the limited supply of donors (1). While VADs have already benefitted many patients, VAD-related blood damage remains a major issue. This includes hemolysis (2,3), platelet activation (4), alteration of the coagulation cascade and thrombosis (5), reduced functionality of leukocytes (6), release of
doi:10.1111/aor.12382 Received April 2014; revised June 2014. Address correspondence and reprint requests to Dr. Chris Hoi Houng Chan, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales SA2 8PP, UK. E-mail: [email protected]; [email protected] Presented in part at the 21st Congress of the International Society for Rotary Blood Pumps held September 26–28, 2013 in Yokohama, Japan.
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microparticles (7), and degradation of von Willebrand factor (vWF) (8,9). Gastrointestinal (GI) bleeding is one complication associated with the placement of a VAD, with rates as high as 65% within the first year after VAD placement (10,11). The mechanism underlying this problem in patients on long-term continuous flow mechanical support is not well understood (1,9,11– 22). This cannot be explained by the anticoagulation regimen alone, but may be symptomatic of acquired von Willebrand syndrome (AvWs) which could be the result of shear stress caused by the VAD. In a literature review of clinical studies describing patient populations suffering from GI bleeding, two populations in particular were found to stand out: those suffering from aortic valve stenosis and those implanted with axial flow VADs (Table 1). In aortic stenosis, there is a high level of shear stress and loss of HMW vWF multimers (23,24). In contrast to axial flow pumps, centrifugal flow VADs and total artificial hearts (TAHs) have fewer reported events of GI bleeding. The centrifugal pump with the most reports of GI bleeding is the novel HeartWare device (20,25– 28). In patients implanted with VentrAssist and EVAHEART, there have been few reports of bleeding, although they have been diagnosed with AvWs, showing a loss of high molecular weight (HMW) vWF multimers and decreased ratios of collagen binding capacity and ristocetin cofactor activity to vWF antigen (17). Comparing the shear stress in these devices provides a partial explanation as the characteristic hydraulic performance of axial flow usually involves high rotational pump speed compared with centrifugal flow resulting in higher shear stress (29). Furthermore, TAHs typically have lower
shear stress than centrifugal pumps due to their pulsatile operation (11). Thus, the hypothesis is that the higher the shear stress, the more damage caused to vWF, resulting in decreased platelet aggregation and thus bleeding. In light of this, it is important to understand the point at which shear stress becomes damaging, and why centrifugal VAD patients do not suffer GI bleeding to the same extent, although obviously suffering impairment of vWF. It is difficult to calculate shear stress in VADs both in vitro and in vivo. Therefore, it is important to establish an in vitro laboratory model with greater sensitivity and the ability to better quantify HMW vWF multimer loss while furthering our understanding of the relationship between HMW vWF abundance and activity. Research focusing on the impact of wall shear rate caused by perfusing normal plasma through long capillary tubing, and that of pumping plasma in a mock circulatory loop driven by a VAD has shown a relationship between the shear rate and the loss of HMW vWF multimers (8,30,31). However, neither of these models provide a way to subject the plasma to accurate and controlled levels of shear rate environment. A rheometer can be used to apply controlled levels of shear rate during timed intervals. Therefore, the purpose of this study was to: (i) quantify HMW vWF breakdown at specified levels of shear rate using a rheometer; (ii) correlate the loss of HMW multimers to vWF functionality; and (iii) investigate the effect of mechanical shear on platelet aggregates. This information would be of value to VAD developers who could modify device design to avoid damaging shear stress levels.
TABLE 1. Literature review describing patient populations suffering from GI bleeding and vWF diagnostic methods vWF diagnostic methods
Device or medical condition CF-VAD
Axial flow
Centrifugal flow
Pulsatile TAH No device
HeartMate II Jarvik 2000 MicroMed DeBakey Thoratec BVAD VentrAssist CentriMag BiVAD HeartWare EVAHEART Duraheart CardioWest HeartMate XVE Aortic valve stenosis
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GI bleeding
HMW vWF loss (multimer analysis)
Yes Yes Yes Yes No N/A Yes No N/A No No Yes
Yes N/A N/A Yes Yes Yes N/A No No No No Yes
Decrease in vWF : RCo / vWF : Ag activity Yes N/A N/A Yes Yes N/A N/A Yes N/A No N/A N/A
Decrease in vWF : CB / vWF : Ag activity Yes N/A N/A Yes Yes N/A N/A N/A N/A No N/A Yes
Reference 9,11–13,16–19 15 11 18,19 11,18 16 20,25–28 29 16 18 11,16 23,24
VON WILLEBRAND FACTOR AND SHEAR STRESS MATERIALS AND METHODS Blood preparation Peripheral blood (72 mL) was collected from six healthy volunteers in vacutainers containing 9NC coagulation sodium citrate 3.2% (455322, Greiner Bio-one, Wemmel, Belgium). Platelet-rich plasma (PRP) was prepared by centrifuging whole blood for 7 min at 500 × g at room temperature. Platelet-poor plasma (PPP) was prepared by centrifuging PRP for 5 min at 13 000 × g at room temperature. The PPP was extracted and analyzed using the automated hematology analyzer CELL-DYN Ruby (Abbott Diagnostics, Abbott Park, IL, USA) to ensure that the plasma was void of blood cells. PPP was used instead of whole blood to ensure that any potential effect of shear on vWF was not a result of cellular interactions. This study was approved by the South Wales Research Ethics Committee and all donors gave informed written consent. Rheometry PPP was subjected to shear stress rates of 4000 and 8000/s in an AR-G2 rheometer (TA Instruments, New Castle, DE, USA) at 37°C. The rheometer was equipped with double concentric geometry in which a rotating inner cylinder cup allows generation of uniformly established shear flow at a well-defined shear rate. The PPP was sampled at hourly intervals (6 h in total). Static PPP at 37°C was used as a control. The sheared PPP was analyzed by flow cytometry immediately after the shear tests and the remaining plasma samples were stored at −80°C for ≤5 days prior to analysis for the vWF multimer by immunoblotting. Plasma vWF multimer analysis by immunoblotting Sheared and nonsheared PPP was subjected to electrophoresis on high gelling temperature agarose (0.6% agarose, w/v, Sea Kem, FMC Bioproducts, Rockland, ME, USA) in a horizontal gel apparatus (81–2325, Galileo Bioscience, Cambridge, MA, USA) at 4°C. Electrophoresis was performed at 30 mA for 30 min and then at 50 mA until the dye front had migrated 10–12 cm from the origin (total gel running time was 6 h). vWF multimers separated on agarose gel were transferred to polyvinylidene difluoride (PVDF) 0.45 μm membrane (IPVH304F0, Immobilon-P, Millipore Corporation, Billerica, MA, USA) for 15–17 h at 70 mA in the electroblotting tank (91–2020-TB, Galileo Bioscience). vWF detection using anti-human vWF primary (ab6994, Abcam, Cambridge, UK) and horseradish peroxidise (HRP)-conjugated secondary (ab6721, Abcam) antibodies, and chemiluminescence substrate (170–5060,
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Bio-Rad, Hercules, CA, USA) was performed as described by Krizek et al. (32). The shear-induced changes in the size distribution of plasma vWF multimers were studied by densitometric scanning initiated from the origin (Quantity One software v4.6.8, BioRad, Hertfordshire, UK). The loss of HMW vWF multimers was quantified by the rate of change in gradient of HMW vWF. vWF : Ristocetin (vWF : RCo) assay vWF functionality was assessed using the vWF : Ristocetin (vWF : RCo) assay developed by Chen et al. (33). The assay measures the ability of vWF (in PPP) to bind to platelets in the presence of ristocetin resulting in aggregation of the platelets. Human platelets (50 000 platelets per μL) were stained either green (CellTracker Green CMFDA, Life Technologies, Glasgow, UK) or red (MitoTracker Red FM, Life Technologies), and incubated with 2 μL PPP and 1 mg/mL ristocetin solution for 45 min at room temperature in the dark rotating at 30 rpm (SB3 Rotator, Stuart, Bibby Scientific, Staffordshire, UK). Nonaggregated platelets (green or red events) and aggregated platelets (doublelabeled events) were quantified by flow cytometry (FACSAria I, BD Biosciences, Oxford, UK) and the percentage of double-labeled events was measured (FACS Diva 6.1.3, BD Biosciences). The greater the percentage of double-labeled events, the higher the platelet aggregation and hence the vWF functionality. Confocal microscopy of platelet aggregates vWF : RCo assays were performed as described above. Post 24 h incubation at room temperature, 70 μL reaction mixture samples containing vWF and red/green platelets were stained with anti-human vWF antibody (ab6994, Abcam) labeled with Pacific Blue (Z25041, Zenon Pacific Blue mouse IgG1 labelling kit, Life Technologies) in an 8-well borosilicate chamber slide (155411, Thermo Scientific, New York, NY, USA). Individual and aggregated fluorescent platelets adhered by vWF were fixed by Vectashield hardset mounting medium (H-1400, Vector Laboratories, Peterborough, UK) and examined with a confocal microscope (LSM 710, Zeiss, Jena, Germany). Images of green and red fluorescent platelets and blue fluorescent-stained vWF were captured and analyzed using ZEN imaging software 2012. vWF ELISA The concentration of vWF : antigen (vWF : Ag) in the sheared PPP was quantified by a vWF specific enzyme-linked immunosorbent assay (ELISA) kit (ab108918, Abcam) according to manufacturer’s Artif Organs, Vol. 38, No. 9, 2014
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FIG. 1. Measurement of vWF multimers by immunoblotting. HMW and LMW vWF multimer immunoblot images (left panel) and densitometric analysis (right panel) for human PPP under different shear rates: (A) static controls 0/s, (B) 4000/s, and (C) 8000/s at hourly intervals during 6 h at 37°C.
instructions and absorbance at 450 nm measured (POLARstar Omega, BMG LABTECH, Ortenberg, Germany). Statistical analysis Due to multiple measurements made per PPP sample at different time points and at different shear rates, a repeated measures analysis of variance on the data sets was conducted. The shear rates and the time points were treated as fixed effects and the sample as a random effect. Once significant effects were found, pairwise comparison tests were conducted, adjusted for the multiple comparisons using Bonferroni’s method. Analysis was performed using the RStudio v 0.97.551 (RStudio, Boston, MA, USA) and R statistical environment, v3.0.2 (R Core Team, Vienna, Austria). Artif Organs, Vol. 38, No. 9, 2014
RESULTS Quantification of the rate of change of HMW and LMW vWF using densitometry The rate of change of HMW and low molecular weight (LMW) multimers was quantified using densitometry (Fig. 1A–C, right panel) and the results are summarized in Fig. 2A (HMW vWF) and 2B (LMW vWF). HMW vWF Repeated measures analysis of variance shows a very significant dependence of the densitometry values on the shear rate (P < 10−6) and on time (P < 10−6). The interaction between time and shear rate is also significant (P < 0.002). Pairwise comparisons (with Bonferroni correction) confirm the
VON WILLEBRAND FACTOR AND SHEAR STRESS
1.2 1.0 0.8 0.6 0.4 0.2 0.0
Mean normalized HMW vWF densitometry values
A
Shear rate: static 4000 1/sec 8000 1/sec
0
1
2
3
4
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time (h)
2.0 1.8
LWM vWF Repeated measures analysis of variance showed a significant dependence of the densitometry values on the shear rates (P < 0.002) and on time (P < 10−5). There was no significant interaction between shear rate and time in this case. Pairwise comparisons with Bonferroni correction show that the significant differences are between the static and high shear values, and that measurements need to be at least 5 h apart to show significant changes. This is summarized in Fig. 2B, which shows three distinct curves describing mean densitometry values of the three shear rates. The curves representing sheared samples exhibit an upward trend with time of the densitometry values. To summarize, the results reflect loss of HMW vWF at 4000 and 8000/s, compared with the static control at hourly intervals during 6 h at 37°C in Fig. 1 (left panel). There is a positive correlation between shear rate and degradation of vWF, with 8000/s causing the greatest loss of HMW bands compared with the static control. Similarly, there is a correlation between shear rate and an increase in LMW bands, suggesting that the HMW bands may be cleaved into smaller fragments. This conclusion is strengthened by the quantification of vWF antigen below.
0.8
1.0
1.2
1.4
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Shear rate: static 4000 1/sec 8000 1/sec
0.6
Mean normalized LMW vWF densitometry value
B
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0
1
2
3 time (h)
4
5
6
Quantification of vWF antigen by ELISA The total vWF antigen decreases during the 6-h test in the static control and the sheared samples (Fig. 3). However, as there is no difference between the groups, we can conclude that the HMW band loss demonstrated by immunoblotting (Fig. 1) in the sheared samples is due to a cleavage of HMW multimers into LMW multimers.
FIG. 2. (A) Means (over the different samples) ± SE of the normalized results of densitometric analysis for HMW vWF in human PPP under different shear rates (static control 0, 4000, and 8000/s at hourly intervals during 6 h at 37°C). (B) Means (over the different samples) ± SE of the normalized results of densitometric analysis for LMW vWF in human PPP under different shear rates (static control 0, 4000, and 8000/s at hourly intervals during 6 h at 37°C).
significant difference between any two levels of shear rate and show that significant differences are achieved between measurements that are at least 4 h apart. This is summarized in Fig. 2A which shows three distinct curves. The mean densitometry values in the control sample remain high while there is a decrease with time in the sheared samples, with a stronger decrease in the sample subjected to the higher shear.
FIG. 3. Total vWF antigen quantified by ELISA in postsheared human PPP at different shear rates for 6 h. Human PPP subjected to 0/s (static control), 4000 or 8000/s, and then analyzed by ELISA. Artif Organs, Vol. 38, No. 9, 2014
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vWF functionality assessed using flow cytometry and visualized using confocal microscopy The assessment of the vWF functionality was performed using a ristocetin-dependent flow cytometry assay of platelet aggregation. When equal concentrations of green and red platelets, resolved by the FITC and APC channels, respectively (Fig. 4A, left panel), were mixed in the absence of PPP, single green and red platelets dominated and double positive events (platelet aggregates) were negligible (20 000/s (8). Another limitation was the residence time (6 h) of the PPP which is continuously sheared in the rheometer, whereas the blood passes through the VAD in a period of milliseconds. However, this work provides a simple and accurate way to expose the blood plasma samples to controlled shear rates ranging from physiological to pathological levels which allow evaluation of the impact of shear rate on the vWF. This is of value to provide further insight into bleeding complications in patients with VAD. Our future work will focus on
FIG. 8. Confocal microscopy of platelet aggregates stained with red or green in the presence of human PPP form the static control (A) and sheared at 8000/s (B) after 6 h. The nonsheared PPP sample results in less dispersed and larger platelet aggregates compared with the sheared vWF sample.
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VON WILLEBRAND FACTOR AND SHEAR STRESS further exploration of the shear stress and time domains of vWF mechanoenzymatic stability, especially at higher ranges of shear stress and shorter exposure times. In addition, the use of whole blood instead of PPP is the next step to mimic physiological conditions, although shear stress might also damage or activate platelets causing release of ADP and vWF multimers into the plasma. Furthermore, the role of ADAMTS13, the protease known to break down vWF (31), in HMW vWF destruction during high shear stress will be considered. CONCLUSIONS The results shown here confirmed that the longer human platelet-poor plasma is exposed to high shear rate, the greater the loss of the high molecular weight von Willebrand factor multimers and decline in the functional activity of vWF will be. There is a strong linear correlation between HMW vWF multimer loss and the lower functional activity of vWF. Therefore, this study indicates that a simple, fast, accurate flow cytometric evaluation of the vWF activity is sensitive enough for a comparative acquired von Willebrand syndrome study for in vitro testing of different shear conditions. This work could improve in vitro ventricular assist device evaluation and bring it a step closer to assessment of total blood trauma. It is recommended that the effort of blood pump design should not only focus on mechanical stability and hemolysis, but also on the other blood components that could be damaged such as leukocytes, platelets, and soluble factors such as vWF and factor VIII. Future in vitro device evaluation could benefit from inclusion of assays such as this to provide a more complete picture of the overall effect on blood function.
7.
8.
9.
10. 11. 12. 13.
14. 15.
16.
17. 18.
19.
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