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Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis Lauren DC Casa 1 Scott E Gillespie 2 Shannon L Meeks 3 and David N Ku 1 1 George W. Woodruff School of Mechanical Engineering Georgia Institute of Technology Atlanta GA 2 Department of Pediatrics Emory University School of Medicine Atlanta GA 3 Aflac Cancer Center and Blood Disorders Service Children’s Healthcare of Atlanta Department of Pediatrics Emory University Atlanta GA Corresponding author: David N Ku MD PhD GWW School of Mechanical Engineering Georgia Institute of Technology 315 Ferst Dr. Room 2307 Atlanta GA 30332-0405 Tel: 404-894-6827 Fax: 404-894-1395 E-mail: david.kume.gatech.edu Received date: July 31 2016 Accepted date: August 12 2016 Published date: August 19 2016 Copyright: © 2016 Casa LDC et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Abstract Platelets and von Willebrand factor VWF are recognized as important mediators of thrombosis at high shear rates but their relative contributions are unclear. We employ a stenotic microfluidic test system to induce thrombus formation to occlusion on collagen at pathologic shear rates of 3500-6000 s -1 . To obtain blood analogs with reduced platelet and VWF concentrations human whole blood was diluted with saline by 90 or 99 and hematocrit restored by adding washed red blood cells. Platelets and VWF were selectively restored to normal levels. Blood from patients with known von Willebrand disease VWD was also investigated with and without the addition of VWF to investigate the contribution of platelet VWF. Normal whole blood led to channel occlusion in all tests occlusion time t occ 6.1 ± 2.2 min. 90 dilution with restored VWF occluded in 6/7 subjects even without restored platelets t occ 16.6 ± 1.4 min. 90 dilution with restored platelets occluded in 2/5 subjects t occ 27.2 ± 1.8 min showing that added VWF is more effective for restoring occlusion than platelets. Addition of plasma VWF to VWD blood restored occlusion in only 1/4 severe subjects VWF:RCo15 and did not reduce normal occlusion time in the occluding subject suggesting that VWF release from platelets plays an important role in high shear thrombosis. Logistic regression shows that VWF concentration and platelet count are strong predictors of thrombotic occlusion. Efforts to control high shear thrombosis may focus on VWF in addition to platelet function. Keywords: Trombosis von W illebrand factor von W illebrand disease Platelets High fuid shear rate Abbreviations: t occ : Occlusion time s VWD: von W illebrand Disease VWF: von W illebrand Factor VWF:RCo: Ristocetin cofactor assay Introduction Myocardial infarction MI and ischemic stroke result from rapid occlusive thrombus formation at the site of atherosclerotic plaque rupture 1. Trombus formation at atherosclerotic lesions occurs at high shear rates 23 and follows distinct pathways 45. Briefy collagen exposed by plaque cap rupture acts as a thrombogenic surface. At high shear rates plasma von W illebrand factor VWF is adsorbed onto the surface. Platelets then bind to VWF at the A1 domain via platelet receptor GPIb. Bound platelets are activated by shear and/or soluble agonists and irreversibly bind via integrin αIIbβ3. Activated platelets release VWF at high local concentration as well as other prothrombotic and proinfammatory factors initiating a feed-forward amplifcation of thrombus growth. Clinical investigations have indicated that increased VWF levels are associated with increased risk of recurrent MI 6 MI in patients with angina pectoris 7 and stroke 8. On the other hand low levels of VWF are characteristic of von W illebrand Disease VWD a bleeding disorder with symptoms including nose bleeding skin bruises and hematomas prolonged bleeding from trivial wounds oral cavity bleeding and excessive menstrual bleeding 9. It is well-accepted that platelets are a key constituent of arterial thrombi. Some investigators have shown that VWF is critical for platelet adhesion at high shear rates but few studies have investigated the independent role of VWF in large-scale thrombus accumulation particularly at high shear rates 3000 s -1 relevant to MI and ischemic stroke. Te objective of the present study is to determine the relative roles and minimum concentrations of plasma VWF and platelets in forming large-scale occlusive thrombus at high shear rates 3000 s -1 . Previously we have theoretically shown that the number of exposed A1 domains on elongated netlike VWF is likely to be the limiting factor in capturing circulating platelets 10. We thus hypothesize that the contribution of VWF to high shear thrombus formation is equal to or greater than that of platelets. Further we hypothesize that the high concentration of VWF released from platelets alpha-granules is important to the continued capture of platelets beyond the initial attachment to collagen. To achieve high shear rates with reasonable blood volumes we employ a microfuidic device in conjunction with human-derived blood analogs containing controlled concentrations of plasma VWF platelets and red blood cells RBC. We also study thrombus formation in blood from patients with known VWD to study the relative contributions of plasma VWF and platelet VWF. Te results of this study lead to a better understanding of the roles of plasma VWF platelet VWF and platelet count in high shear thrombosis. Journal of Hematology Thromboembolic Diseases Casa et al. J Hematol Thrombo Dis 2016 4:4 DOI: 10.4172/2329-8790.1000249 Research Article Open Access J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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Materials and Methods Blood collection and preparation Blood was collected in either 60 mL syringes or 10 mL evacuated blood collection tubes BD Vacutainer Becton Dickinson and Company Franklin Lakes NJ from normal volunteers and patients with known VWD under protocols approved by Georgia Institute of Technology and Emory University IRBs. Blood was anti-coagulated with 15.8 USP units/mL heparin. Blood analogs were produced by diluting whole blood from normal volunteers with normal saline at 90 and 99 dilutions. Hematocrit was restored to 40 by adding washed red blood cells RBCs. To test the contributions of VWF to high shear thrombosis VWF Humate-P CLS Behring King of Prussia PA gif from Hemophilia of Georgia was added at 50 100 and 200 IU/dL plasma corresponding to the normal range of plasma VWF levels 11. Humate-P has been shown to have a similar VWF multimer distribution as human plasma 12. Isolated platelets were added to the blood analog at 120 000 platelets/μL 1314. Additional experiments were conducted adding recombinant human ADAMTS-13 RD Systems Inc. Minneapolis MN which cleaves VWF 15 at normal concentration 0.75 mg/mL 16 to 90 dilutions with restored VWF. One experiment was conducted with ADAMTS-13 added at six times normal concentration. All blood analogs were fltered under gravity at 50 μm using nylon mesh McMaster-Carr Supply Company Elmhurst IL immediately before experimentation. Table 1 summarizes the blood analogs considered. Additional experiments were performed with human fbrinogen F4883 Sigma-Aldrich Co. St. Louis MO added at normal concentration 300 mg/dL. Dilution VWF Added Platelets Added IU/dL saline 10 3 /μL Normal Whole Blood Control 0 0 0 Negative Control 90 0 0 Normal VWF and Normal Platelet 90 100 120 Normal Platelet Only 90 0 120 Normal VWF Only 90 100 0 Low Normal VWF Only 90 50 0 High Normal VWF Only 90 200 0 Normal VWF and Normal Platelet 99 dilution 99 100 120 Normal Platelet Only 99 dilution 99 0 120 Normal VWF Only 99 dilution 99 100 0 Table 1: Blood analogs produced by hemodilution. Microfuidic test platform To obtain high fuid shear rates with small blood volumes blood was perfused through a stenotic microfuidic test section with a gradual contraction zone similar to an atheroma. Perfusion under a constant 65 mm pressure head yielded wall shear rates across 90 of the width of 3500-6000 s -1 in the stenotic region and 500-1000 s -1 in the non-stenotic region corresponding to normal arterial wall shear rates 4. Te microfuidic channels were flled with 0.1 collagen Type I fbrillar solution Chronopar Chronolog Inc. Havertown PA and incubated at room temperature for 24 hours 17. Data acquisition and analysis Te collagen-coated microfuidic chips were installed in the experimental set up shown in Figure 1. Te discharge mass was measured to determine occlusion time t occ determined as the time from frst blood contact with the stenosis to the time when the balance mass stopped increasing for at least 1 minute. Up to 5 mL of blood or blood analog was allowed to fow under a constant pressure head through the channel. Images of thrombus formation were acquired every 0.5 second using a microscope DM6000 B Leica Microsystems Wetzlar Germany ftted with a high-resolution 1392×1020 pixels CCD camera Pixelfy PCO Kelheim Germany. Incident light was fltered using a bandpass flter in the range of 480 nm BG-42 Edmund Optics Inc. Barrington NJ. Image acquisition was facilitated by the μManager open-source microscopy sofware 18. Blood from normal subjects collected in blood collection tubes was tested three times to characterize the experimental platform. Figure 1: Microfuidic experimental set-up. A diagram overall set- up B components of experimental set up C photograph of experimental set-up D diagram of microfuidic test section width is 480 μm into page E photograph of microfuidic chip. Citation: Casa LDC Gillespie SE Meeks SL Ku DN 2016 Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis. J Hematol Thrombo Dis 4: 249. doi:10.4172/2329-8790.1000249 Page 2 of 8 J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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Microscopy images were analyzed using MATLAB R2013b Te Mathworks Inc. Natick MA. First images were converted from 16-bit black-and-white to 8-bit color using the algorithm provided by the camera manufacturer 19. For analysis the total average intensity grayscale of the three color channels was used. A rectangular region of interest comprising the stenotic region was manually selected and the total intensity arbitrary units AU increase since the initial reference image when blood frst contacted the channel was averaged over the area was computed for each time point. See Data Supplement for more detailed methods. Statistical analysis Data is shown as mean ± standard deviation throughout. Statistical signifcance was evaluated at the 0.05 level and data analyses were performed using SAS 9.3 Cary NC and R Project V ienna Austria. Rates of occlusion and thrombus were calculated using frequencies and percents within control and successive blood dilation samples. Rate comparisons for occlusion and thrombus yes vs. no were made using an exact form of the Chi-square goodness of ft test. Time to occlusion was gauged across control and blood dilution groups using Kaplan- Meier curves and log-rank tests. Log-rank p-values were calculated overall and for each pairwise comparison using the Holm-Bonferroni method 20. Samples that did not occlude were censored afer 35 minutes of follow-up. Univariate and multivariable logistic regression was employed to identity signifcant risk factors associated with occlusion. Potential predictors included platelet counts and VWF at baseline and afer dilution. Multivariable analysis was guided by the univariable results and the fnal model was selected based upon the strongest predictors as indicated by statistical signifcance and model ft. Adjustments were made for within-sample correlated data using Generalized Estimating Equations GEEs. Predictive ability of independent variables was assessed via receiver operating characteristic curve ROC statistics and model fts were evaluated with Quasi-Akaike Information Criterion QIC values 21. Results Trombus formation in normal subjects Characteristic normal thrombus growth in the microfuidic test section is shown in Figure 2. Under grayscale imaging the test section initially appears black as no thrombus has formed Figure 2A. Trombus begins to appear as patchy bright regions Figure 2B and 2C. At occlusion large thrombus deposits are clearly visible throughout the stenotic portion of the test section Figure 2D. Te change in total transmitted intensity across the test section is shown in Figure 2E. Initial slow deposition is followed by rapid thrombus accumulation. Te fnal sharp increase in intensity may be due to clot contraction as the channel nears occlusion. Te timescale of the intensity increase is in good agreement with previous results of the timescale of clot and platelet contraction of 8-12 min 2223. Characterization of occlusion time for normal thrombus formation is show in the Data Supplement. All syringe-collected control cases led to channel occlusion measured by mass fow n12 p0.001 Figure 3A with a mean occlusion time of 6.1 ± 2.2 min. Normal subjects had platelets counts of 245 ± 66×10 3 /μL n12 and VWF:RCo of 75 ± 69 IU/dL n6. Figure 2: Normal thrombus formation in microfuidic test section. Te test section appears black before thrombus formation and thrombus appears white within the test section. Te stenotic region is bound by the white lines. Flow is from lef to right. A at initiation of blood fow B at 50 occlusion time C at 75 occlusion time D at full occlusion. E Change in mean transmitted intensity during experiment. Note initial slow-growth phase followed by rapid thrombus growth beginning at approximately 30 seconds. Occlusion occurs at the end of the plot. Trombus formation in 90 dilutions with selectively restored VWF and Platelets None of the 90 dilution cases led to channel occlusion n7 p0.016 indicating that dilution reduces the necessary components of high shear thrombosis to the point of eliminating thrombotic occlusion. 90 dilutions with restored platelets and VWF 100 IU/dL occluded the channel in all cases t occ 13.7 ± 2.5 min. Restoring only platelets to normal levels in 90 dilutions led to occlusion in 2/5 subjects p1 with the remainder showing visible but non-occlusive thrombus t occ 27.2 ± 1.8 min. Restoring only VWF to 100 IU/dL in 90 dilutions restored occlusion in 6/7 subjects p0.125 exact test t occ 16.6 ± 1.4 min p0.0001 compared to control log-rank test. Tus VWF is capable of forming occlusive thrombus even with very low platelet counts. Restoring VWF alone also restored occlusion in more subjects and yielded a more rapid occlusion time than restoring platelets alone. Te further addition of ADAMTS-13 to restored VWF analogs still resulted in high shear thrombosis that occluded in 4/5 subjects and occlusion time was not reduced. Even adding ADAMTS-13 at six times normal concentration did not prevent occlusion n1. Tus ADAMTS-13 does not appear to cancel the formation of VWF-mediated thrombosis in our high shear environment. Kaplan-Meier analysis Figure 3B showed signifcantly lower probability of occlusion in the cases of 90 dilution compared to control p0.001 adjusted pairwise comparison. Te addition of platelets to 90 dilution with normal VWF cases did not have a signifcant efect on occlusion probability p0.073 log-rank test. However the addition of VWF to 90 dilution with normal platelet cases signifcantly increases the probability of occlusion p0.006 log- rank test. Citation: Casa LDC Gillespie SE Meeks SL Ku DN 2016 Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis. J Hematol Thrombo Dis 4: 249. doi:10.4172/2329-8790.1000249 Page 3 of 8 J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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Figure 3: Trombotic occlusion in microfuidic test section for 90 dilutions of whole human blood with normal additions of VWF and platelets. A Occlusion times “No Occlusion” indicates that test section did not occlude within 5 mL of blood analog but thrombus was visible during test “No Trombus” indicates that no thrombus was detected during the experiment B Occlusion times in microfuidic test section for 90 dilutions of whole human blood with VWF additions ranging from 50 to 200 IU/dL. C Kaplan- Meier analysis of patency probability. Characteristic OR 95 CI P-Value Platelet count after dilution 4.43 2.96 – 6.65 1 0.001 VWF after dilution 10.33 2.51 – 42.50 2 0.001 1 OR are based upon 100000 unit increase in platelet characteristics 2 OR are based upon 100 unit increases in VWF characteristics Table 2: Multivariable logistic regression for occlusion yes vs. no. Kaplan-Meyer analysis suggests a trend towards lower probability of occlusion in conditions with decreased platelet count compared to conditions with decreased VWF concentration p0.054. Multivariable logistic regression for occlusion indicated that the Figure 4: A Occlusion times in microfuidics test sections for 99 dilutions of whole human blood with normal additions of VWF and platelets. “No Occlusion” indicates that test section did not occlude within 5 mL of blood analog but thrombus was visible during test “No Trombus” indicates that no thrombus was detected during the experiment B 99 dilution with 100 IU/dL VWF and 120000 platlets/μL forms large thrombus but does not form stable occlusive thrombus. resultant platelet count and resultant VWF concentration afer dilution are signifcant predictors of occlusion Table 2. Te odds ratio OR for a 100000 platelet/μL increase was 4.43 and the OR for a 100 IU/dL increase in VWF is 10.33. Together platelet count and VWF concentration create a very good model for predicting occlusion ROC0.871. In 90 dilutions added VWF was varied from 50 IU/dL to 200 IU/dL Figure 3B.At low- normal levels of VWF 50 IU/dL 2/6 subjects occluded p0.688 exact test. When 100 IU/dL VWF was added to 90 dilution 6/7 subjects occluded p0.125 exact test. At high-normal levels of VWF 200 IU/dL 6/6 subjects occluded p0.031 exact test. Tough additional VWF resulted in more Citation: Casa LDC Gillespie SE Meeks SL Ku DN 2016 Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis. J Hematol Thrombo Dis 4: 249. doi:10.4172/2329-8790.1000249 Page 4 of 8 J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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subjects occluding at each subsequent experimental condition the occlusion times remained rapid at the same mean times. Trombus formation in 99 dilutions with selectively restored VWF and platelets Surprisingly 99 dilutions with restored platelets and VWF 100 IU/dL did not lead to thrombotic occlusion Figure 4A. Rather large- scale thrombus formed but did not fully occlude the vessel. Figure 4B shows the change in transmitted intensity for one such case and rather than rapidly increasing as in Figure 2E the curve shows a dynamic cyclic pattern with large peaks and valleys over a long experiment time. Note that the time scale is in tens of minutes to compress the time. Te addition of fbrinogen to 99 dilutions with restored platelets and VWF resulted in full channel occlusion in 20.1 ± 1.6 min. Tus fbrinogen did not enhance high shear thrombus growth rates but did stabilize the large thrombus enough to cause fow occlusion. Trombus formation in blood from VWD subjects Studies were also performed on patients with VWD to isolate the efects of VWF released by platelets. Blood from VWD patients showed decreased occlusion compared to normal controls Figure 5A. Te normal blood for this comparison was collected in evacuated blood collection tubes for direct comparison with the VWD patient blood. All normal blood collected in evacuated blood collection tubes led to occlusion n15 with a mean occlusion time of 9.5 ± 3.4 min slightly longer than the syringe blood controls but not statistically diferent. Normal subjects had a mean platelet count of 275 ± 69×10 3 / μL. VWD patients had a mean platelet count of 280 ± 109×10 3 /μL. VWD patients were separated post hoc into two groups for analysis. Mild VWD patients had values between 15 and 50 15VWF:RCo50 24. Mild VWD blood occluded in 4/6 subjects tested mean platelet count of 300 ± 10 4 /μL. When VWF was added in vitro to mild VWD samples occlusion was restored in the two non- occluding subjects but the occlusion times for the occluding subjects were not shortened. Severe VWD patients have Ristocetin Cofactor values of 15 VWF:RCo15. Severe VWD blood did not occlude in any of the fve subjects tested mean platelet count of 256 ± 120/μL. Te addition of VWF to severe VWD subjects restored occlusion in only one subject. Kaplan-Meier analysis shows that mild VWD samples and mild VWD samples treated with in vitro VWF have a lower probability of occlusion compared to normal subjects p0.01 Figure 5B. Te probability of occlusion between the in vitro treated and untreated groups was not statistically diferent p0.815. Restoring plasma VWF in subjects with VWD that is in subjects defcient in platelet VWF was insufcient to lead to occlusion. Tus platelet VWF is important for thrombotic occlusion. Discussion Trombus formation under high fuid shear rate is mediated by platelets and VWF. In the present study we utilized dilutions of whole blood to demonstrate that large-scale occlusive thrombus can form in microchannels with a normal concentration of VWF with very low platelet count. VWF was the primary mediator of high shear thrombosis and VWF concentration and platelet count were strong predictors of occlusion. We also utilized blood from patients diagnosed with VWD to show that platelet VWF is important for occlusive thrombus formation. Trombus formation in 90 dilutions with selectively restored VWF and platelets Human blood diluted to 90 with only normal concentrations of VWF added 100 IU/dL led to thrombotic occlusion in most cases despite having only 10 normal platelet count. Hence VWF is capable of supporting high shear thrombosis even with few platelets. Te occlusion times for 90 dilutions with added VWF were 2.7 times longer than for controls 16.6 ± 1.4 versus 6.1 ± 2.2 min. Te increase in occlusion time may be due to the time required for sufcient platelets to be transported to the growing thrombus or to a decrease in VWF release by platelets. It should be noted however that the increase in occlusion time does not indicate that an equal number of platelets must be incorporated into the growing thrombus as in the whole blood case. Figure 5: Trombotic occlusion in microfuidic test section for normal subjects and VWD patients with and without in vitro added VWF. A Occlusion times B Kaplan-Meier analysis of occlusion probability. If the same number of platelets were required to form occlusive thrombus one would expect the occlusion time with 10 of normal platelet count to be 10 times as long as the whole blood control. Te decreased platelet requirement may be facilitated by VWF self- assembly 25 and net formation 26 both processes that are Citation: Casa LDC Gillespie SE Meeks SL Ku DN 2016 Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis. J Hematol Thrombo Dis 4: 249. doi:10.4172/2329-8790.1000249 Page 5 of 8 J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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enhanced by high shear rates. VWF nets have been proposed as a mechanism for rapid and efcient platelet capture at high shear rates 10 and may create thrombotic occlusion in blood analogs with very low platelet counts. 90 dilutions with only platelets restored occluded the microchannel in 2/5 cases with occlusion times 20 minutes which closely recapitulates results for VWD samples. Logistic regression showed vWF concentration afer dilution was more predictive of occlusion than platelet count afer dilution. Our results are consistent with Ogawa et al. 27 who showed that 40 hemodilution decreased thrombus onset and growth and that adding VWF accelerated thrombosis at shear rates of 1100 s -1 . Note that hemodilution also reduces RBC count which is known to reduce difusive transport 28 and slow thrombus formation 29. Additionally the modest hemodilutions of 40 used in the Ogawa study retain high residual concentrations of plasma proteins and platelet count. Te present study employs higher shear rates more relevant to arterial thrombosis fxed hematocrit 40 to control for changes in shear enhanced difusivity and larger dilutions to isolate and analyze the individual contributions of VWF and platelets. Trombus formation in 99 dilutions with selectively restored VWF and Platelets When all other plasma proteins are diluted to 1 of their normal concentrations and VWF and platelets are restored large thrombus formed but did not occlude. Tese results suggested that an additional plasma proteins was necessary for thrombus stability and occlusion but not growth. Addition of fbrinogen permitted stable occlusion of the channel. Tis fnding is in agreement with Maxwell et al. 30 who noted that stable platelet aggregates do not form at shear rates of 1800 s -1 on a VWF-only matrix but do form on a VWF-fbrinogen matrix indicating that fbrinogen plays a stabilizing role in thrombus formation at high shear rates. Trombus formation in blood from VWD subjects: Te role of VWF release from platelets Blood samples from subjects with known VWD were utilized to investigate the contributions of plasma and platelet VWF. VWD blood will have both low plasma VWF and low platelet VWF whereas diluted samples have platelets with normal VWF content. While the in vitro addition of plasma VWF restored occlusion in both non-occluding samples with mild VWD VWF addition did not restore occlusion in subjects with severe VWD. Tus restoring plasma VWF alone is not sufcient to create occlusion if VWF is absent from the platelets. In contrast restored plasma VWF and platelets with VWF do occlude. Tis indicates that platelet VWF is important for thrombotic occlusion at high shear rates. Te notion of VWF release for platelets acting as an important step in initiating rapid occlusive thrombosis is further supported by lag time and growth time data See Data Supplement. A previous study by Turritto et al. showed that VWD blood perfused over rabbit subendothelium yielded normal platelet adhesion to the surface in some cases but reduced thrombus volume growth in the same cases 31. Tese results support the conclusion of the present study that a moderate concentration of plasma VWF is sufcient for surface- platelet adhesion but VWF release from platelets is required for large- scale thrombus growth. Our results support previous biophysics analysis that concluded that high VWF local concentration released from platelets is needed for platelet capture at high shear rates 10. Our results are also consistent with those of Moake et al. 32 who used a cone-and-plate model to show that platelet aggregation under moderately high shear stress up to 60 dynes/cm 2 or a shear rate of approximately 1700 s -1 is mediated by either plasma VWF or VWF released from platelets. Isolated platelets re-suspended in bufer showed little aggregation but VWF added to the suspension induced aggregation under shear while severe VWD platelets in bufer showed no aggregation. Te present study extends these results to a single-pass system at higher shear rates that better mimic the shear conditions of a stenotic artery. Whereas the Moake et al. study investigated only small platelet aggregates the present study used large occlusive thrombus formation as the endpoint to better model clinically relevant thrombi. Te major fnding of the present study is that normal VWF can lead to thrombotic occlusion even with small platelet counts showing the dominance of VWF in high shear thrombosis. Tis is even more surprising as many would guess that platelet bulk would be critical to creating the mass of the thrombus. Taken together it appears that a threshold of plasma VWF is needed to initiate thrombosis at high shear rate while VWF release from platelets produces large rapid occlusive thrombosis. A previous study in mice showed that mice defcient in platelet VWF could support hemostasis based on tail bleeding time and platelet VWF could reduce bleeding time in some VWF-defcient mice 33. Microfuidic experiments in the same study showed that either plasma or platelet-derived VWF supported platelet adhesion to a collagen surface at shear rates of 2000 s -1 . However hemostasis was the focus of the previous study and large-scale platelet thrombus was not studied. Te results of the present study show that while plasma VWF alone may support surface adhesion of platelets VWF release from platelets may be required to create large-scale occlusive thrombus at high shear rates. Platelet-derived VWF has also been shown to have a higher molecular weight distribution than plasma VWF and high molecular weight VWF is more hemostatically active 34. Te same study showed that VWF released by platelets is resistant to cleavage by ADAMTS-13 which may further contribute to rapid thrombus growth leading to occlusion. Our results concur that ADAMTS13 does not prevent occlusive thrombus formation with either plasma or platelet derived VWF. Clinical implications Te results of the present study have clinical implications for the prevention of arterial thrombosis. Presently anti-platelet agents primarily aspirin and P2Y12 inhibitors e.g. clopidogrel prasugrel and ticagrelor are the treatments-of-choice for long-term therapy for their relatively low cost and oral administration. Tat thrombus may form with only 10 of normal platelet count may explain some of the documented aspirin and clopidogrel non-response 3536. Te efectiveness of anti-platelet agents may be in part the result of decreased platelet activation leading to decreased VWF release from platelets. Non-responsiveness may also be due to platelet activation via alternative pathways such as shear 3738 or GPIb binding 39. Alternative anti-thrombotic agents that target VWF are currently under development 40 and may provide an additional or alternative treatment for patients who are non-responsive to anti-platelet drugs. Te present results may also have important implications for bleeding in surgery or trauma. In case of severe bleeding with saline fuid replacement the blood undergoes hemodilution which could lead to reduced hemostasis. Our results indicate that replacing only platelets may be insufcient for restoring hemostatic function as plasma VWF may be of greater importance at high shear rates. Citation: Casa LDC Gillespie SE Meeks SL Ku DN 2016 Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis. J Hematol Thrombo Dis 4: 249. doi:10.4172/2329-8790.1000249 Page 6 of 8 J Hematol Trombo Dis an open access journal ISSN:2329-8790 Volume 4 • Issue 4 • 1000249

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Limitations Te present study has several limitations. Blood was anticoagulated with heparin for transport which reduces the contribution of fbrin formation in thrombus formation. Sodium citrate with subsequent re- calcifcation instead of heparin could be used to retain fbrin formation. For some studies blood was collected in blood collection tubes which induce higher shear rates and may prematurely activate platelets. Tis was controlled for in the present study but may lead to diferences in thrombus growth and occlusion times compared to in vivo thrombosis. Multiple platelet counts were not investigated. Calcium ion concentration was not controlled or restored in hemodilutions which may impair platelet activation. Te VWF concentrate also contained coagulation Factor VIII. Te addition of Factor VIII is not expected to have a large efect on the results since the blood was anticoagulated with heparin. Conclusions In conclusion VWF can lead to fow occlusion by high shear thrombosis even with signifcant dilution of platelets and other plasma proteins. Plasma VWF can support occlusive thrombus formation even with low platelet counts but is insufcient if platelet VWF is absent in the case of severe VWD. For fow occlusion from a stable thrombus approximately 10 fbrinogen level appears to be needed. Greater understanding of the role of plasma and platelet-derived VWF may lead to more efective prevention of high shear thrombosis. Acknowledgements We gratefully acknowledge J. Rhett Mayor GWW School of Mechanical Engineering Georgia Institute of Technology Atlanta GA for the micro-machining of molds for the fabrication of microfuidic test sections. Tis work was supported by a grant from the Center for Pediatric Innovation Georgia Institute of Technology Children’s Healthcare of Atlanta and Emory University Lawrence P . Huang Chair Funds Georgia Institute of Technology and the John and Mary Brock Discover Fund. L.D.C.C. was supported by an American Heart Association Pre-Doctoral Fellowship 14PRE18080005. References 1. Lilly L 2007 Pathophysiology of Heart Disease 4th edn.. Philadelphia PA: Lippincott W illiams W illiams. 2. Bark DL Para AN Ku DN 2012 Correlation of thrombosis growth rate to pathological wall shear rate during platelet accumulation. Biotechnology and Bioengineering 109: 2642-2650. 3. Bark DL Ku DN 2010 Wall shear over high degree stenoses pertinent to atherothrombosis. J Biomech 43: 2970-2977. 4. Jackson SP 2007 Te growing complexity of platelet aggregation. Blood 109: 5087-5095. 5. Casa LD Deaton DH Ku DN 2015 Role of High Shear Rate in Trombosis. J Vasc Surg 61: 1068-1080. 6. Jansson J Kilsson T Johnson O 1991 von W illebrand factor in plasma: A novel risk factor for recurrent myocardial infarction and death. Br Heart J 66: 351-355. 7. 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