Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor

Blood can become hypercoagulable by shear‐induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood‐shearing device. Platelet activation (P‐selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear‐induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM‐vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.     

The CentriMag Centrifugal Blood Pump as a Benchmark for In Vitro Testing of Hemocompatibility in Implantable Ventricular Assist Devices

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge‐to‐transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10^?5), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10^?5) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.     

High clotting factor activities and enhanced platelet-related haemostasis in a patient with recurrent episodes of thrombotic-thrombocytopenic purpura: additional risk factors for onset and recurrence?

Sir, Thrombotic–thrombocytopenic purpura (TTP) is a life-threateningdisorder characterized by microvascular thrombi, mainly in arteriolesand capillaries, low platelet count, haemolysis and neurologicaldeficits [1]. Impaired activity of the von Willebrand factor(vWF) cleaving protease ADAMTS 13, leading to the accumulationof ultralarge vWF multimers, was found to play a major rolein the pathogenesis of TTP [2]. Consequently, plasma exchangebecame the therapy of choice supplying     

Review article: unexpected bleeding in the operating room: the role of acquired von Willebrand disease

Acquired von Willebrand disease (AvWD) is a rare bleeding disorder that occurs in association with a variety of underlying disorders and can lead to unforeseen bleeding in surgical patients. Cardiovascular as well as malignant and immunological diseases may be associated with AvWD, and several pathophysiological mechanisms have been proposed. von Willebrand factor (vWF) is a plasma glycoprotein that mediates platelet adhesion to subendothelial collagen and causes platelet aggregation under high shear stress. Additionally, vWF acts as a specific carrier for coagulation factor VIII (FVIII) in the plasma. AvWD results from a reduced rate of vWF synthesis, an increased rate of vWF removal, or a final generation of lower-molecular-weight, less active subunits or multimers. In contrast to inherited von Willebrand disease patients, who are characterized by lifelong bleeding episodes, AvWD patients present with a sudden onset of bleeding symptoms, which can induce acute bleeding episodes during critical surgical procedures. Typically, no family history of bleeding is found. The clinical visualization of AvWD is similar to that of the hereditary form with mucocutaneous bleeding and increased perioperative bleeding, ranging from mild to severe bleeding. Laboratory evaluation of AvWD is mainly based on the measurement of vWF activity and antigens as well as on the multimeric analysis of vWF. A variety of therapeutic approaches have been used depending on the underlying disease and pathophysiological mechanisms. Treatment options to control acute hemorrhages or to prevent bleeding complications during surgery include desmopressin, FVIII/vWF concentrates, high-dose IV immunoglobulins, and plasma exchange. Because the half-life of vWF is reduced in AvWD, high doses of FVIII/vWF concentrates administered at frequent intervals may be necessary during bleeding episodes. In cases of unresponsiveness to standard therapy, recombinant activated factor VIIa may be an alternative option. However, the most effective therapy is the resolution of the underlying disease. In the present review, we focus on the current understanding of AvWD, outlining the associated disorders, underlying pathophysiological mechanisms, and possible treatment options.     

Stress and Exposure Time on von Willebrand Factor Degradation

Despite the prevailing use of the continuous flow left ventricular assist devices (cf‐LVAD), acquired von Willebrand syndrome (AvWS) associated with cf‐LVAD still remains a major complication. As AvWS is known to be dependent on shear stress (τ) and exposure time (t_exp), this study examined the degradation of high molecular weight multimers (HMWM) of von Willebrand factor (vWF) in terms of τ and t_exp. Two custom apparatus, i.e., capillary‐tubing‐type degrader (CTD) and Taylor‐Couette‐type degrader (TCD) were developed for short‐term (0.033 sec ≤ t_exp ≤ 1.05 s) and long‐term (10 s ≤ t_exp ≤ 10 min) shear exposures of vWF, respectively. Flow conditions indexed by Reynolds number (Re) for CTD were 14 ≤ Re ≤ 288 with corresponding laminar stress level of 52 ≤  ≤ 1042 dyne/cm². Flow conditions for TCD were 100 ≤ Re ≤ 2500 with corresponding rotor speed of 180 ≤ _o ≤ 4000 RPM and laminar stress level of 50 ≤  ≤ 1114 dyne/cm². Due to transitional and turbulent flows in TCD at Re > 1117, total stress (i.e., = laminar + turbulent) was also calculated using a computational fluid dynamics (CFD) solver, Converge CFD (Converge Science Inc., Madison, WI, USA). Inhibition of ADAMTS13 with different concentration of EDTA (5 mM and 10 mM) was also performed to investigate the mechanism of cleavage in terms of mechanical and enzymatic aspects. Degradation of HMWM with CTD was negligible at all given testing conditions. Although no degradation of HMWM was observed with TCD at Re < 1117 ( = 1012 dyne/cm²), increase in degradation of HMWM was observed beyond Re of 1117 for all given exposure times. At Re ~ 2500 ( = 3070 dyne/cm²) with t_exp = 60 s, a severe degradation of HMWM (90.7 ± 3.8%, abnormal) was observed, and almost complete degradation of HMWM (96.1 ± 1.9%, abnormal) was observed with t_exp = 600 s. The inhibition studies with 5 mM EDTA at Re ~ 2500 showed that loss of HMWM was negligible (<10%, normal) for all given exposure times except for t_exp = 10 min (39.5 ± 22.3%, borderline‐abnormal). With 10 mM EDTA, no degradation of HMWM was observed (11.1 ± 4.4%, normal) even for t_exp = 10 min. This study investigated the effect of shear stress and exposure time on the HMWM of vWF in laminar and turbulent flows. The inhibition study by EDTA confirms that degradation of HMWM is initiated by shear‐induced unfolding followed by enzymatic cleavage at given conditions. Determination of magnitude of each mechanism needs further investigation. It is also important to note that the degradation of vWF is highly dependent on turbulence regardless of the time exposed within our testing conditions.     

Development of a Hyperactive Primary Hemostatic System During Off‐Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13

An unbalance between the platelet‐adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 is a risk factor for thrombosis. Here, we assessed levels and functionality of VWF and ADAMTS13 in patients undergoing off‐pump lung transplantation. We analyzed plasma of 10 patients and distinguished lung transplantation‐specific effects from those generally accompanying open‐chest surgeries by comparing results with 11 patients undergoing off‐pump coronary bypass graft (CABG) surgery. Forty healthy volunteers were included for reference values. VWF antigen levels as well as the VWF ristocetin cofactor activity/VWF antigen ratio increased during lung transplantation and after CABG surgery. An increase in VWF propeptide levels was paralleled by a decrease in ADAMTS13 activity. This was more pronounced during lung transplantation. Similarly, the capacity of plasma to support platelet aggregation under shear flow conditions in vitro was more increased during lung transplantation. The proportion of high molecular weight VWF multimers was elevated in both groups without evidence for ultra‐large VWF. VWF's collagen binding activity remained unchanged. In conclusion, a hyperactive primary hemostatic system develops during lung transplantation resulting both from a pronounced (functional) increase of the VWF molecule and decrease of ADAMTS13. This may increase the risk of platelet thrombosis within the allograft.     

Acquired Von Willebrand syndrome is an early-onset problem in ventricular assist device patients

Objective: Acquired Von Willebrand syndrome (AVWS) can contribute to bleeding complications in patients with ventricular assist devices (VADs). AVWS results from shear stress, which causes unfolding of the high-molecular-weight (HMW) multimers of Von Willebrand factor (VWF) with subsequent cleavage. Loss of the HMW multimers of VWF is the leading finding in AVWS. In consequence, binding of VWF to collagen and to platelets is impaired. The onset of AVWS after VAD implantation is not yet determined. We examined VAD patients for presence of an AVWS in the early, intermediate, and late phase after VAD implantation. Methods: Patients with a biventricular Thoratec-PVAD^® (BVAD, n = 6) or a left-ventricular HeartMateII^® (HMII, n = 11) were analyzed prior to VAD implantation and after 1, 3, 14, 30, and 60 days. Diagnosis of AVWS based on VWF:ristocetin cofactor activity/VWF:VWF antigen (VWF:RCo/VWF:Ag), collagen-binding capacity:VWF antigen (VWF:CB/VWF:Ag), and multimeric analysis. In addition, we analyzed the number of bleeding episodes, which required surgical intervention. Results: No patient had an AVWS prior to VAD implantation. An AVWS was identified already in the very early postoperative period, that is, in almost all patients on the first day and in all patients on the third day. The AVWS was also detected in the majority of patients in the further course. Nine of all 17 patients suffered bleeding complications and required a total of 25 interventions due to hemorrhages. Forty percent of re-interventions were carried out within the first 10 days after implantation; five of these were necessary within the first 24 h. Conclusion: The AVWS is present already in the early postoperative phase after VAD implantation. Therefore, reduced shear stress has to be an important feature of newly developed assist devices in the future.     

手术室中不可预测的出血：获得性血管性血友病

获得性血管性血友病（acquired von willebrand disease,AvWD）是一种-7见的、与多种基础疾病相关的出血性疾病,可以导致不可预测的手术患者出血。心血管疾病、恶性肿瘤和免疫疾病可能导致AvWD的发生,关于其病理生理机制有几种提法。ron Willebrand因子（vorl Willebrand factor,vWF）是一种血浆糖蛋白。可介导血小板黏附内皮下胶原蛋白,引起高剪切应力下血小板聚集。此外,vWF还是血浆中凝血因子Ⅷ（FⅧ）的特定载体。vWF合成量减少、清除率增加、低分子量终产物生成、不太活跃的亚基或者多聚体的生成,可导致AvWD。相对于遗传性血管性血友病患者终身出血的特点,AvWD患者表现为突然发作的出血,可诱发外科手术过程中的急性出血。典型病例没有发现出血家族史。与遗传性者相似,AvWD临床表现为皮肤黏膜出血及围术期出血增加,可以是轻度也可能为重度。AvWD的实验室诊断主要是基于对vWF活性和抗原的测定,以及对vWF的多重分析。依据基础疾病和病理生理机制,AvWD有多种治疗方法。控制急性出血或者预防手术过程中出血并发症的治疗方案包括去氨加压素、凝血因子Ⅷ／岍浓缩物、高剂量免疫球蛋白和血浆置换。由于AvWD中vWF的半衰期缩短,在出血时高剂量凝血因子Ⅷ／VWF多次重复使用是有必要的。标准治疗无反应的情况下,重组活化因子Ⅶa可以作为一治疗选择。然而,最有效的疗法是基础疾病的治疗。本综述中,我们着重于阐述目前对AvWD的研究理解、相关疾病的概述及其病理生理机制以及可能的治疗方案。     

Rituximab therapy in two children with autoimmune thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare disease among pediatric patients, in whom it may be mistaken for hemolytic uremic syndrome (HUS) and idiopathic thrombocytopenic purpura (ITP). Familial forms are caused by mutations in the ADAMTS13 gene, whereas acquired forms may result from an inhibitory antibody directed against ADAMTS13, a metalloprotease that cleaves very large multimers of Von Willebrand factor (VWF), thereby preventing platelet aggregation in blood vessels. We report two cases of TTP. The first was a 15-year-old girl with her first episode of TTP that failed to respond after 10 days of plasmapheresis and was treated with rituximab; she has remained in remission at 12 months of follow-up. The second was a 6-year-old boy with acquired relapsing TTP previously managed with plasmapheresis and prednisolone, who presented with a third relapse that was treated with plasmapheresis and rituximab; he remains in remission 17 months after treatment. Rituximab has been used by pediatricians for treating B cell malignancy, autoimmune diseases and antibody-mediated diseases, such as the Factor VIII inhibitors in hemophilia A, and may also have a promising role in children with acute refractory or chronic relapsing TTP.     

Differential thrombogenicity of artery and vein: The role of von Willebrand factor

There exists clinical evidence for a difference in the relative thrombogenicity of arterial and venous surfaces. We studied this phenomenon in an in vitro model where the effects of hemodynamic differences could be controlled. Nonanticoagulated human blood was perfused across injured (air-insufflated) arterial and venous surfaces in a recirculating perfusion system at shear rates of 500 and 1500/sec. Thrombus formation was assessed by measuring radiolabeled platelet (¹¹¹ In) and fibrin (¹²⁵I)deposition on the surfaces. The role of von Willebrand factor (vWF) in arterial and venous thrombosis was evaluated by blocking its effect with polyclonal anti-vWF antibody (vWF Ab). Raw data were converted to log ₁₀ for statistical analysis. The increased thrombogenicity of injured venous vs. arterial segments was confirmed by these studies (p<0.001).The addition of vWF Ab decreased platelet and fibrin deposition (p<0.001)and these effects were greater in veins than in arteries. The difference in platelet deposition between arteries and veins was more pronounced at lower shear rates (p<0.05),an effect not observed with fibrin deposition. To determine whether the increased thrombogenicity of veins could be explained by an increased content of subendothelial vWF, the amount of vWF was assessed by incubating injured vessels with ¹²⁵ I vWF Ab and then measuring the radioactivity of the vessels. Veins had a higher content of vWF than arteries, as implied by the higher amount of radiolabeled vWF Ab (813±90 in veins vs. 2173±317 in arteries,p<0.001).These observations suggest that increased thrombogenicity of veins may in part be explained by intrinsic differences in subendothelial vWF and subsequent platelet attachment.Supported by grant HL 40889 from the National Institutes of Health, Bethesda, Md.Presented at the Nineteenth Annual Meeting of the Peripheral Vascular Surgery Society, Seattle, Wash., June 5, 1994.     

Platelet dysfunction in acute type A aortic dissection evaluated by the laser light-scattering method

Objectives

Platelet dysfunction contributes to bleeding tendency in acute type A aortic dissection. Particle counting by new laser light-scattering methods more accurately quantifies changes in the number of different-sized platelet aggregates than do conventional optical density methods. We studied platelet aggregation kinetics and patterns of aggregation deficiency in acute-phase aortic dissection with laser light scattering.

Methods

Blood from 20 acute type A aortic dissection patients undergoing surgery was sampled during acute (9.1 ± 6.8 hours from onset) and chronic (postoperative day 20, control) phases of aortic dissection. Platelet count and aggregability were assessed by optical density and laser light-scattering methods after aggregation was induced (addition of 2.0 μg/mL collagen to samples).

Results

Optical density showed significant reduction in acute-phase platelet aggregation (acute vs chronic: 65 ± 27% vs 77 ± 17%, P < .03). Laser light scattering showed significant reduction in medium (25-50 μm) and large (50-70 μm) but not small aggregate (9-25 μm) generation (acute vs chronic: small, 1.2 ± 0.6 × 10⁷ vs 1.5 ± 1.0 × 10⁷, NS; medium, 0.6 ± 0.3 × 10⁷ vs 1.1 ± 0.5 × 10⁷, P < .001; large, 1.4 ± 1.2 × 10⁷ vs 2.6 ± 1.7 × 10⁷, P < .001). Acute- versus chronic-phase platelet counts were significantly decreased (1.7 ± 0.1 × 10⁵/μL vs 3.6 ± 0.3 × 10⁵/μL, P < .001).

Conclusions

Platelet aggregation is suppressed in acute-phase aortic dissection. This suppression does not occur in the initial phase of small aggregate formation; rather, it occurs during the conglomeration of small aggregates into larger aggregates.     

Von Willebrand Factor Deposition and ADAMTS-13 Consumption in Allograft Tissue of Thrombotic Microangiopathy-like Disorder After Living Donor Liver Transplantation: A Case Report

Background

Thrombotic microangiopathy (TMA) pathogenesis after living donor liver transplantation (LDLT) is thought to be caused by release of unusually large von Willebrand factor multimers (UL-vWFMs) resulting from sinusoidal endothelial cell damage and induction of platelet adhesion and aggregation. A decrease in a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13 (ADAMTS-13) that cleave UL-vWFMs might cause excessive UL-vWFMs activity and result in platelet thrombus formation. However, this phenomenon has not undergone a full pathologic assessment.

The role of PI3K/Akt signaling pathway in non‐physiological shear stress‐induced platelet activation

The PI3K/Akt signaling pathway has been implicated in playing an important role in platelet activation during hemostasis and thrombosis involving platelet‐matrix interaction and platelet aggregation. Its role in non‐physiological shear stress (NPSS)‐induced platelet activation relevant to high‐shear blood contacting medical devices (BCMDs) is unclear. In the context of blood cells flowing in BCMDs, platelets are subjected to NPSS (>100 Pa) with very short exposure time (<1 s). In this study, we investigated whether NPSS with short exposure time induces platelet activation through the PI3K/Akt signaling pathway. Healthy donor blood treated with or without PI3K inhibitor was subjected to NPSS (150 Pa) with short exposure time (0.5 s). Platelet activation indicated by the surface P‐selectin expression and activated glycoprotein (GP) IIb/IIIa was quantified using flow cytometry. The phosphorylation of Akt, activation of the PI3K signaling, was characterized by western blotting. Changes in adhesion behavior of NPSS‐sheared platelets on fibrinogen, collagen, and von Willebrand factor (vWF) were quantified with fluorescent microscopy by perfusing the NPSS‐sheared and PI3K inhibitor‐treated blood through fibrinogen, collagen, and vWF‐coated microcapillary tubes. The results showed that the PI3K/Akt signaling was involved with both NPSS‐induced platelet activation and platelet‐matrix interaction. NPSS‐sheared platelets exhibited exacerbated platelet adhesion on fibrinogen, but had diminished platelet adhesion on collagen and vWF. The inhibition of PI3K signaling reduced P‐selectin expression and GPIIb/IIIa activation with suppressed Akt phosphorylation and abolished NPSS‐enhanced platelet adhesion on fibrinogen in NPSS‐sheared blood. The inhibition of PI3K signaling can attenuate the adhesion of unsheared platelets (baseline) on collagen and vWF, while had no impact on adhesion of NPSS‐sheared platelets on collagen and vWF. This study confirmed the important role of PI3K/Akt signaling pathway in NPSS‐induced platelet activation. The finding of this study suggests that blocking PI3K/Akt signaling pathway could be a potential method to treat thrombosis in patients implanted with BCMDs.     

Spleen size improvement in advanced heart failure patients using a left ventricular assist device

The spleen has been recognized as an important organ that holds a reserve of 20% to 30% of the total blood volume. Spleen contraction and splenic volume reduction occur in patients with hypovolemic shock. However, the change in the spleen volume and the association between spleen size and hemodynamic parameters remain unclear in patients with advanced heart failure (HF) who need left ventricular assist device (LVAD) support. This study was performed to investigate the change in spleen size and the relationship between spleen size and hemodynamic parameters before and after LVAD implantation in patients with advanced HF. We enrolled 20 patients with advanced HF on LVAD support. All patients underwent right heart catheterization and computed tomography before and after LVAD implantation. The spleen size was measured by computed tomography volumetry. We excluded patients with a mean right atrial pressure (RAP) of <5 mm Hg because of the possibility of hypovolemia and those with a cardiac index of >2.2 L/min/m² before LVAD implantation. The splenic volume significantly increased from 160.6 ± 46.9 mL before LVAD implantation to 224.6 ± 73.5 mL after LVAD implantation (P < .001). Before LVAD implantation, there was a significant negative correlation between spleen volume and systemic vascular resistance (SVR). After LVAD implantation, however, there were significant correlations between spleen volume and the cardiac index, RAP, and pulmonary capillary wedge pressure despite the absence of a significant correlation between spleen volume and SVR. Furthermore, one patient developed reworsening HF because of LVAD failure due to pump thrombosis. In this case, the splenic volume was 212 mL before LVAD implantation and increased to 418 mL after LVAD implantation, although it decreased to 227 mL after LVAD failure. The spleen size may change depending on hemodynamics in patients with advanced HF with LVAD support, reflecting sympathetic nerve activity and the systemic volume status.     

Validation of mitral regurgitation reversibility in patients with HeartMate 3 LVAD implantation

The resolution of functional mitral valve regurgitation (MR) in patients awaiting left ventricular assist device (LVAD) implantation is discussed controversially. The present study analyzed MR and echocardiographic parameters of the third-generation LVAD HeartMate 3 (HM3) over 3 years. Of 135 LVAD patients (with severe MR, n = 33; with none, mild, or moderate MR, n = 102), data of transthoracic echocardiography were included preoperatively to LVAD implantation, up to 1 month postoperatively, and at 1, 2, and 3 years after LVAD implantation. Demographic data and clinical characteristics were collected. Severe MR was reduced immediately after LVAD implantation in all patients. The echocardiographic parameters left ventricular end-diastolic diameter (P < .001), right ventricular end-diastolic diameter (P < .001), tricuspid annular plane systolic excursion (P < .001), and estimated pulmonary artery pressure (P < .001) decreased after HM3 implantation independently from the grade of MR prior to implantation and remained low during the 2 years follow-up period. Following LVAD implantation, right heart failure, ventricular arrhythmias, ischemic stroke as well as pump thrombosis and bleeding events were comparable between the groups. The incidences of death and cardiac death did not differ between the patient groups. Furthermore, the Kaplan-Meier analysis showed that survival was comparable between the groups (P = .073). HM3 implantation decreases preoperative severe MR immediately after LVAD implantation. This effect is long-lasting in most patients and reinforces the LVAD implantation without MR surgery. The complication rates and survival were comparable between patients with and without severe MR.     

The Effect of Shear Stress on the Size,Structure, and Function of Human von Willebrand Factor

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.     

Preclinical Models for Translational Investigations of Left Ventricular Assist Device‐Associated von Willebrand Factor Degradation

Evidence suggests a major role for von Willebrand factor (vWF) in left ventricular assist device (LVAD)‐associated bleeding. However, the mechanisms of vWF degradation during LVAD support are not well understood. We developed: (i) a simple and inexpensive vortexer model; and (ii) a translational LVAD mock circulatory loop to perform preclinical investigations of LVAD‐associated vWF degradation. Whole blood was obtained from LVAD patients (n = 8) and normal humans (n = 15). Experimental groups included: (i) blood from continuous‐flow LVAD patients (baseline vs. post‐LVAD, n = 8); (ii) blood from normal humans (baseline vs. 4 h in vitro laboratory vortexer, ～ 2400 rpm, shear stress ～175 dyne/cm², n = 8); and (iii) blood from normal humans (baseline vs. 12 h HeartMate II mock circulatory loop, 10 000 rpm, n = 7). vWF multimers and degradation fragments were characterized with electrophoresis and immunoblotting. Blood from LVAD patients, blood exposed to in vitro supraphysiologic shear stress, and blood circulated through an LVAD mock circulatory loop demonstrated a similar profile of decreased large vWF multimers and increased vWF degradation fragments. A laboratory vortexer and an LVAD mock circulatory loop reproduced the pathologic degradation of vWF that occurs during LVAD support. Both models are appropriate for preclinical studies of LVAD‐associated vWF degradation.     

Biology of von Willebrand factor

Von Willebrand factor (VWF) is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells. It is stored in platelets and endothelial cells and secreted towards subendothelium and plasma. VWF multimers consist of linear arrangements of identical subunits with a molecular weight of 270 kDa. The longest multimers reach more than 20 x 10(6) Da in storage granules. In the circulation, the multimer size is limited by the specific protease ADAMTS13. In primary hemostasis, VWF plays a key role as a molecular bridge in adhesion between platelets and subendothelium and between platelets during their aggregation. These functions, which involve the interaction with platelet glycoprotein lb, are mainly enhanced by VWF immobilization onto hydrophobic surfaces (collagen, cell membrane) and by high shear rates found in microcirculation and stenosed arteries. In these functions, the higher molecular weight forms are the most efficient. Under such hemodynamic conditions, proteolytic activity of ADAMTS13 is also optimal and limits the multimer size to about 15 x 10(6) Da as soon as their secretion. Thus ADAMTS13 appears as a key physiologic regulator of the VWF platelet functions. In the microcirculation, the lack of ADAMTS13 activity can result in the formation of VWF-rich platelet aggregates responsible for thrombotic thrombocytopenic purpura.     

Computational fluid dynamics analysis to determine shear stresses and rates in a centrifugal left ventricular assist device

Axial flow left ventricular assist devices (LVADs) are a significant improvement in mechanical circulatory support. However, patients with these devices experience degradation of large von Willebrand factor (vWF) multimers, which is associated with bleeding and may be caused by high shear stresses within the LVAD. In this study, we used computational fluid mechanics to determine the wall shear stresses, shear rates, and residence times in a centrifugal LVAD and assess the impact on these variables caused by changing impeller speed and changing from a shrouded to a semi-open impeller. In both LVAD types, shear rates were well over 10,000/s in several regions. This is high enough to degrade vWF, but it is unclear if residence times, which were below 5ms in high-shear regions, are long enough to allow vWF cleavage. Additionally, wall shear stresses were below the threshold stress of 10Pa only in the outlet tube so it is feasible to endothelialize this region to enhance its biocompatibility.