Reduced Von Willebrand Factor Research Articles

Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice

BackgroundFree labile hemin acts in a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. ObjectivesTo investigate the antithrombotic potential of hydroxychloroquine in hemolysis-induced arterial thrombosis ex vivo, hemin-induced platelet activation, ferric chloride–induced arterial thrombosis, and lung perfusion following hemin injection in mice. ResultsErythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride–induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. ConclusionHydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.

ELABELA/APJ Axis Prevents Diabetic Glomerular Endothelial Injury by Regulating AMPK/NLRP3 Pathway

ELABELA (ELA), a recently discovered peptide, is highly expressed in adult kidneys and the endothelium system. It has been identified as a novel endogenous ligand for the apelin receptor (APJ). This study aims to investigate the role of ELA in diabetic glomerular endothelial pyroptosis and its underlying mechanism. Initially, a significant decrease in ELA mRNA levels was observed in the renal cortex of db/db mice and high glucose–treated glomerular endothelial cells (GECs). It was also found that ELA deficiency in ELA+/− mice significantly accelerated diabetic glomerular injury, as shown by exacerbated glomerular morphological damage, increased serum creatine and blood urea nitrogen, and elevated 24-h urinary albumin excretion. In addition, in vivo overexpression of ELA prevented diabetic glomerular injury, reduced von Willebrand factor expression, restored endothelial marker CD31 expression, and attenuated the production of adhesive molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Furthermore, in vitro studies confirmed that treatment with ELA inhibited GEC injury by regulating the NOD-like receptor protein 3 (NLRP3) inflammasome, as indicated by blocking NLRP3 inflammasome formation, decreasing cleaved Caspase-1 production, and inhibiting interleukin-1β and interleukin-18 production. Moreover, in vitro experiments demonstrated that the protective effects of ELA in GECs during hyperglycemia were diminished by inhibiting adenosine monophosphate–activated protein kinase (AMPK) using Compound C or by APJ deficiency. Taken together, this study provides the first evidence that ELA treatment could prevent diabetic glomerular endothelial injury, which is partly mediated by the regulation of the AMPK/NLRP3 signaling pathway. Therefore, pharmacologically targeting ELA may serve as a novel therapeutic strategy for diabetic kidney disease.

Successful Chemical Synovectomy in a Patient with Acquired von Willebrand Syndrome with Chronic Synovitis Due to Recurrent Knee Hemarthrosis: A Case Report.

Acquired von Willebrand syndrome (AVWS) is a rare, non-hereditary bleeding disorder related to heterogeneous medical conditions such as hematological malignancies and cardiovascular and autoimmune diseases. We describe the clinical course of a 62-year-old man with polycythemia vera who experienced post-traumatic knee and leg swelling due to hemarthrosis. He was treated at another center with low molecular weight heparin due to misdiagnosed deep vein thrombosis further exacerbating the ongoing bleeding. At our center, he was diagnosed with AVWS with reduced von Willebrand factor (VWF):GPIbR plasma activity and loss of high molecular weight multimers (HMWM). He was treated with compressive bandages with resolution. Five months later, on clinical recurrence of knee and leg swelling, knee ultrasound scan showed the presence of chronic synovitis and a hemorrhagic Baker’s cyst with signs of rupture. The treatment consisted of chemical synovectomy with rifampicin and steroids preceded by systemic replacement therapy using plasma-derived factor VIII-VWF concentrate. At the end of the treatment cycle, our patient reported complete resolution of knee pain and restoration of joint range of motion and function. Ultrasound evaluation confirmed complete resolution of knee capsule distension and Baker’s cyst. Hemarthrosis is an anecdotal presentation of AVWS and chemical synovectomy was successful in treating this complication. A multidisciplinary approach allowed an effective management of this rare complication.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40744-022-00477-2.

Individual Variability in von Willebrand Factor Fragility in Response to Shear Stress: A Possible Clue for Predicting Bleeding Risk.

Acquired von Willebrand syndrome (AVWS), characterized by reduced von Willebrand factor (VWF) large multimers, has recently been implicated as the principal mechanism underlying bleeding in patients implanted with left ventricular assist devices (LVADs). Hematological severity of AVWS varies among patients, even if an identical device is implanted. We investigated whether this diversity in hematological severity is due to individual variability in VWF fragility, according to responses to incremental shear stress. Whole-blood samples were sheared at 20,000-40,000 s -1 shear rate, an index of shear stress, using a custom-made shear stressor that could generate shear stress compatible with that produced by an LVAD. The degree of VWF large multimers degradation was evaluated using the VWF large multimer index. A significant inverse correlation was observed between the VWF large multimer index and LVAD-compatible magnitudes of shear stress: the VWF large multimer indices were 68.5 ± 18.3, 48.0 ± 13.9, 33.9 ± 12.1, 23.7 ± 7.9, and 18.7% ± 8.7% at 20,000, 25,000, 30,000, 35,000, and 40,000 s -1 of shear rates, respectively ( P < 0.0001). Furthermore, experimental VWF large multimer index values were compatible with those derived from patients with implanted LVADs (median; 28.9%). Finally, reduction in the VWF large multimer index corresponding to shear stress showed individual variation. We demonstrated that the combined use of a novel high shear stress loading device and quantitative evaluation of VWF large multimers may predict risk of bleeding before LVAD implantation.

Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival.

Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

Criteria for low von Willebrand factor diagnosis and risk score to predict future bleeding

BackgroundImportant diagnostic and clinical aspects of moderately reduced von Willebrand factor (VWF) levels are still unknown. There is no clear evidence which cutoff value (0.50 vs 0.60 IU/ml) should be used to diagnose “low VWF.” Also, the incidence of bleeding after the diagnosis has been made, and risk factors for bleeding are unknown yet. ObjectivesTo investigate the incidence of postsurgical bleeding, postpartum hemorrhage (PPH), and traumatic and spontaneous bleeding after low VWF diagnosis, and to develop a risk score to predict future bleeding. MethodsWe performed a cohort study in patients with historically lowest VWF levels of 0.31 to 0.60 IU/ml. Clinical data of patients were retrospectively collected. ResultsWe included 439 patients with low VWF. During a follow‐up of 6.3 ± 3.7 years, 259 surgical procedures, 81 deliveries, and 109 spontaneous and traumatic bleeding episodes were reported. The incidence of postsurgical bleeding was 2.7%, whereas 10% of deliveries was complicated by PPH. Overall, 65 patients (14.8%) had bleeding requiring treatment, which was not different between patients with historically lowest VWF levels of 0.31–0.50 and 0.51–0.60 IU/ml (p = .154). Age <18 years, abnormal bleeding score at diagnosis, and being referred for bleeding symptoms at the time of diagnosis were independent risk factors for bleeding during follow‐up, and therefore included in the risk score. ConclusionsThe cutoff value of low VWF diagnosis should be set at 0.60 IU/ml. Furthermore, a risk score is developed to identify individuals with a high risk for bleeding after low VWF diagnosis.

Anti-platelet aggregation of Panax notoginseng triol saponins by regulating GP1BA for ischemic stroke therapy

BackgroundPanax notoginseng triol saponins (PTS) has been used clinically for ischemic stroke therapy (IST) in China for more than 17 years due to its anti-platelet aggregation and neuro-protective effects, but its mechanism of action is not fully understand. In this study, anti-platelet aggregation-related protein analysis and computer simulations of drug-protein binding interactions were performed to explore the mechanism of the effects of PTS against ischemic stroke in an ischemia reperfusion model.MethodsThree oral doses of PTS were administered in a model of middle cerebral artery occlusion (MCAO) in rats. Panax notoginseng total saponins (PNS) and a combination of PTS and aspirin were chosen for comparison. To evaluate therapeutic effects and explore possible mechanisms of anti-platelet aggregation, we measured cerebral infarct size and water content in brain tissue, histomorphological changes, expression of related factors (such as arachidonic acid metabolites) and platelet receptors in serum, as well as the binding affinity of PTS for platelet adhesion receptors.ResultsCompared with PNS, PTS showed a stronger and more potent anti-platelet aggregation effect in MCAO model rats. The combination of PTS and aspirin could reduce adverse gastrointestinal effects by regulating the TXA2/PGI2 ratio. We demonstrated for the first time that PTS was able to regulate Glycoprotein Ib-α (GP1BA) in a model animal. The binding of ginsenoside Rg1 and GP1BA could form a stable structure. Moreover, PTS could reduce von Willebrand factor (VWF)-mediated platelet adhesion to damaged vascular endothelium, and thus enhance the probability of anti-platelet aggregation and anti-thrombosis under pathological conditions.ConclusionsOur results showed that GP1BA was closely related to the anti-platelet aggregation action of PTS, which provided new scientific and molecular evidence for its clinical application.

Vascular factor dynamics in therapy for microangiopathy with underlying type 1 diabetes mellitus

Background. The adverse impact of chronic hyperglycaemia on vascular wall in diabetes mellitus includes endothelial dysfunction with subsequent development of diabetic microangiopathy. Microangiopathy can be corrected via adequate glycaemic control for establishing a target level of glycated haemoglobin. Considering a multiplex nature of metabolic and vascular regulation, a comprehensive approach is required for simultaneous correction of rheological disorders, hypercoagulation and endothelial dysfunction.Objectives. Estimation of vascular factors (von Willebrand factor, desquamated endothelium, antithrombin III, protein C, VEGF) and capillaroscopic patterns in therapy for type 1 diabetes with methylethylpyridinol in comparison with sulodexide.Methods. A total of 89 patients with type 1 diabetes were examined and separated by two cohorts: 42 patients receiving sulodexide (cohort 1) and 47 patients receiving methylethylpyridinol (cohort 2). Therapy duration was 14 days. Both cohorts were estimated pre- and post-treatment endothelial conditions (activity of von Willebrand factor, VEGF, desquamated endothelial cell count), anticoagulant indicators (activity of antithrombin III, protein C) and had capillaroscopy with functional test and oximetry.Results. Diabetes patients in pre-treatment exhibited signs of endothelial dysfunction, reduced blood anticoagulant protection and capillary constriction. Both cohorts in post-treatment showed the significantly reduced von Willebrand factor, VEGF activity and desquamated endothelial cell count. The anticoagulant system revealed positive dynamics; capillaroscopy reported limiting of the capillary transition zonal diameter and a certain improvement in functional performance.Conclusion. Patients with type 1 diabetes were revealed with endothelial dysfunction and an increased blood procoagulant activity. Both sulodexide and methylethylpyridinol treatments improved endothelial dysfunction and anticoagulant blood protection. Both preparations can be used for complex microangiopathy correction in patients with &lt;10-years history of type 1 diabetes mellitus.

SUN-552 Follistatin-Like 3 (FSTL3), a Transforming Growth Factor β (TGFβ) Ligand Inhibitor, Regulates Placental Development in Mice

Follistatin-like 3 (FSTL3), a glycoprotein that inhibits transforming growth factor-β (TGFβ) ligands such as activin, is expressed highly in the placenta and other vascular tissues. In addition, FSTL3 is strongly induced in pre-eclamptic placenta. To test the hypothesis that FSTL3 function is required for capillary bed structure and function we studied the placenta in FSTL3 gene deleted mice (FSTL3 KO). We have previously shown that FSTL3 deletion produces striking defects in the placenta when compared to WT. Placental size increases significantly in comparison to WT, at 16.5 and 18.5 dpc, with concurrent reduction in placental efficiency at 18.5 dpc. Histological analyses reveal structural differences in placental junctional zones in FSTL3 KO placenta compared to WT. Morphometric analyses show that the labyrinth area compared to the placenta area is significantly reduced in FSTL3 KO mice. We also found that activin-responsive FSTL3-synexpression genes are upregulated in FSTL3 KO placenta. Of these, EPHB4 protein is induced in the placenta along with its ligand EphrinB2. Here we show that FSTL3 deletion leads to endothelial cell expansion but reduction in blood vessel density along with increased extracellular matrix deposition. Further investigation of the placental phenotype revealed differential expression patterns of desmin and cytokeratin protein, reduced von Willebrand factor (VWF) and increased CD31 and VEGFR2 labelling within FSTL3 KO mice placental labyrinths. To identify mechanisms that might lead to the altered placental development in FSTL3 KO mice qPCR analyses were performed. Our results identified differences in the expression of crucial transcripts, such as Cdh5, Pgf, Fra1and Cited1, that are associated with the regulation of vascular biology. Additionally, we find increased Histone3 and SMAD2 phosphorylation in FSTL3 KO placenta indicating increased proliferation and activin signalling, respectively. These findings suggest that the balance between cellular proliferation and differentiation might be altered in the absence of FSTL3. Thus, we conclude that FSTL3 function, at least partly through the inhibition of activin action, is necessary for normal placental circulation and development.

Clinical significance of slightly reduced von Willebrand factor activity.

Von Willebrand disease (VWD) is the most common congenital bleeding disorder, with a clinical presentation of mucocutaneous and surgical bleeding varying from mild to severe. It is inherited in an autosomal dominant or autosomal recessive manner. The disease is caused by quantitative or qualitative deficiency of the von Willebrand factor (VWF) and is classified as type 1, 2 (2A, 2B, 2M, 2N), and 3. Although type 1 VWD is the most common form of VWD, the f ormal cutoff for diagnosis remains a subject of debate. In our paper, we present results of studies regarding the clinical and laboratory importance of a new type of bleeding disorder called low VWF. The new guidelines for VWD diagnosis and management suggested that patients with historically type 1 VWD should be divided into 2 subsets: type 1 VWD with a VWF antigen level (VWF:Ag) of less than 30 IU/dl or less than 40 IU/dl, in which about 80% of patients exhibited VWF gene mutations, and low VWF with a VWF:Ag level of 30 to 50 IU/dl or 40 to 50 IU/dl, in which the causative mutation is detected in merely 40% of patients and in most families, inheritance is not dependent on the locus of VWF on chromosome 12. Previously, moderately reduced VWF levels (30-50 IU/dl) were considered a risk factor for bleeding, but not a true bleeding disorder, and this condition was named low VWF. Recently, it was documented in a large group of patients with type 1 VWD and low VWF that bleeding score does not correlate with VWF:Ag and bleeding symptoms in type 1 VWD (<30 IU/dl) and low VWF can change from infrequent and moderate to severe bleeds. Because the plasma concentration of VWF depends on many physiological and pathological factors that may mask the diagnosis of VWD, separation of the group of patients with low VWF (30-50 IU/dl) from those with type 1 VWD may delay or prevent them from receiving appropriate treatment. Diagnosis of VWD in each case, particularly those with a slight decrease in VWF (30-50 IU/dl), should be based primarily on the clinical manifestations and family history of hemorrhagic diathesis.

Reduced Activity of von Willebrand Factor after Flow-Diverting Stent Implantation for Intracranial Aneurysms: A Link to Acquired von Willebrand Disease?

Vascular devices generating high shear stress can cause type 2A acquired von Willebrand disease, which is characterized by low von Willebrand factor activity accompanied by hemorrhagic complications. The braided mesh structure of flow-diverting stents with a relatively small strut size can create abnormally high shear stress while arterial blood flows through the stent struts into the aneurysm, and flow-diverting stent may be associated with reduced von Willebrand factor activity. Aneurysmal morphologic parameters and patient data were examined retrospectively among patients who had an unruptured intracranial aneurysm treated with a flow-diverting stent. The RISTOtest (test for whole blood ristocetin-induced platelet aggregation) for von Willebrand factor activity, as well as tests for aspirin and clopidogrel/prasugrel effectiveness, were performed immediately before the endovascular procedure and 24 hours later by multiple electrode aggregometry. A total of 39 patients with 56 aneurysms were recruited, and statistical analyses were performed in 32 patents with 49 aneurysms. Compared with the baseline values, von Willebrand factor activity was reduced in 16 patients but increased in 23 patients. Aneurysmal variables (eg, neck area, volume, volume-to-neck area ratio, size ratio, and morphologic index) clearly distinguished patients with reduced von Willebrand factor activity from those with nonreduced von Willebrand factor activity. The receiver operating characteristic curve showed that the morphologic index and volume had the highest discriminative power, with an area under the curve of 0.99. In high-volume/large-neck aneurysms, flow-diverting stent implantation can cause reduced von Willebrand factor activity, which may be linked causally to acquired von Willebrand disease.

Impact of lipoprotein apheresis on thrombotic parameters in patients with refractory angina and raised lipoprotein(a): Findings from a randomized controlled cross-over trial

Raised lipoprotein(a) [Lp(a)] is a cardiovascular risk factor common in patients with refractory angina. The apolipoprotein(a) component of Lp(a) exhibits structural homology with plasminogen and can enhance thrombosis and impair fibrinolysis. The objective of the study was to assess the effect of lipoprotein apheresis on markers of thrombosis and fibrinolysis in patients with high Lp(a). In a prospective, single-blind, crossover trial, 20 patients with refractory angina and raised Lp(a)>50mg/dL were randomized to three months of weekly lipoprotein apheresis or sham. Blood taken before and after apheresis/sham was assessed using the Global Thrombosis Test, to assess time taken for invitro thrombus formation (occlusion time) and endogenous fibrinolysis (lysis time), as well as von Willebrand Factor, fibrinogen, D-dimer, thrombin/anti-thrombin III complex, prothrombin fragments 1+2, and thrombin generation assays. Lp(a) was significantly reduced by apheresis (100.2 [interquartile range {IQR}, 69.6143.0] vs 24.8 [17.2,34.0] mg/dL, P=.0001) but not by sham (P=.0001 between treatment arms). Apheresis prolonged occlusion time (576±116 s vs 723±142 s, P<.0001) reflecting reduced platelet reactivity and reduced lysis time (1340 [1128, 1682] s vs 847 [685,1302] s, P=.0006) reflecting enhanced fibrinolysis, without corresponding changes with sham. Apheresis, but not sham, reduced von Willebrand Factor (149 [89.0, 164] vs 64.2 [48.5, 89.8] IU/dL, P=.0001), and fibrinogen (3.12±0.68 vs 2.20±0.53g/L, P<.0001), and increased prothrombin fragments 1+2 (158.16 [128.77, 232.09] vs 795.12 [272.55, 1201.00] pmol/L, P=.0006). There was no change in D-dimer, thrombin/anti-thrombin III complex, or thrombin generation assay with apheresis or sham. Lipoprotein apheresis reduces Lp(a) and improves some thrombotic and fibrinolytic parameters in patients with refractory angina.

A Mechanistic Model to Quantify von Willebrand Factor Release, Survival and Proteolysis in Patients with von Willebrand Disease.

A reduced von Willebrand factor (VWF) synthesis or survival, or its increased proteolysis, alone or in combination, contributes to the development of von Willebrand disease (VWD).We describe a new, simple mechanistic model for exploring how VWF behaves in well-defined forms of VWD after its 1-desamino-8-D-arginine vasopressin (DDAVP)-induced release from endothelial cells. We aimed to ascertain whether the model can consistently predict VWF kinetic changes. The study involved 9 patients with VWD types Vicenza (a paradigmatic form with a reduced VWF survival), 8 type 2B, 2 type 2A-I, 1 type 2A-II (associated with an increased VWF proteolysis), and 42 normal controls, whose VWF levels were measured after a 24-hour-long DDAVP test. The rate constants considered were: k0, associated with the VWF release phase; k1, illustrating the phase of conversion from high- to low-molecular-weight VWF multimers; and ke, associated with the VWF elimination phase. The amount of VWF released (D) was also measured. ke and D were significantly higher in O than in non-O blood group controls; k1 was also higher, but less markedly so. All the parameters were accelerated in type Vicenza, especially ke (p < 0.0001), which explains the significant reduction in VWF half-life. In types 2B and 2A-II, k1 was one order of magnitude higher than in controls, which explains their loss of large VWF multimers. All parameters except ke were lower in type 2A-I.The proposed mechanistic model clearly describes the altered biochemical pathways in well-characterized VWD, prompting us to suggest that it might help clarify elusive forms of VWD too.

The role of von Willebrand factor in primary haemostasis under conditions of haemodilution

IntroductionSevere blood loss and related haemodilution during cardiac surgery result in a reduced platelet count which may lead to impaired primary haemostasis. Additionally, the reduced haematocrit lowers rheological forces in circulation and may account for lowered platelet adhesiveness and potentially reduced von Willebrand factor (VWF) activity. These mechanisms may lead to postoperative bleeding. Aim of this study was the examination of VWF activity and VWF-mediated platelet adhesion to collagen under conditions of haemodilution. Materials and methodsAn in vitro flow chamber was utilized to examine the primary haemostasis under a high arterial shear rate of 1500s−1 at variable VWF concentrations, platelet counts and haematocrit levels. ResultsUnder a high arterial shear rate, VWF activity is highly dependent on blood viscosity. Both VWF-collagen binding and VWF-mediated platelet adhesion to collagen were significantly increased with increasing haematocrit. Interestingly, we found slight differences in the VWF multimer sizes able to bind collagen under different shear stress conditions. Under conditions of haemodilution, platelet adhesion was strongly dependent on VWF concentration. Increasing VWF concentration improved platelet adhesiveness under low haematocrit conditions (30%) and variable platelet counts (80, 150 and 250×109/L). This effect was nearly abolished at very low platelet count levels of 50×109/L. ConclusionsVWF improves platelet function under conditions of haemodilution. Therefore, increasing VWF concentration may represent a complementary strategy to administration of platelet concentrates for the management of bleeding in thrombocytopenia.

Endothelial Function, Inflammation, Thrombosis, and Basal Ganglia Perivascular Spaces in Patients with Stroke

Background and ObjectiveRecent studies suggest perivascular spaces are a marker of small vessel disease, blood–brain barrier permeability, and inflammation, but little is known about their risk factors and associations with peripheral blood markers.Materials and MethodsIn prospectively recruited patients with recent minor ischemic stroke, we investigated the influence of age, sex, hypertension, diabetes, and smoking on the severity of perivascular spaces in the basal ganglia seen on T2-weighted magnetic resonance imaging. We assessed plasma markers of endothelial function (von Willebrand factor, intracellular adhesion molecule-1), inflammation (interleukin-6, tumor necrosis factor-alpha, C-reactive protein), and thrombosis (fibrinogen, prothrombin fragments 1 + 2, thrombin–antithrombin complex, tissue plasminogen activator, D-dimer). We used a validated semi-automated method to measure basal ganglia perivascular spaces count and volume. We tested uni- and multivariable associations between blood markers and basal ganglia perivascular spaces count and volume.FindingsIn 100 patients (median age: 67 years, range: 37-92), on adjusted analysis, basal ganglia perivascular spaces count was associated with age (r = .117, P = .003) and hypertension (r = 2.225, P = .013). On multivariable linear regression, adjusted for age, sex, hypertension, smoking and diabetes, reduced von Willebrand factor was associated with increased basal ganglia perivascular spaces count (r = −.025, P = .032).ConclusionThe association of increased basal ganglia perivascular spaces count with reduced von Willebrand factor is novel. As von Willebrand factor may promote cerebral endothelial integrity, insufficient von Willebrand factor is consistent with dysfunctional cerebral endothelium and increased basal ganglia perivascular spaces in cerebral small vessel disease. Quantitative perivascular spaces measurement may increase sensitivity to detect cerebral endothelial dysfunction.

Lenalidomide as a novel treatment for refractory acquired von Willebrand syndrome associated with monoclonal gammopathy

Essentials Treatment options are limited for refractory bleeding in acquired von Willebrand Syndrome (AVWS). Lenalidomide therapy was studied in two patients with AVWS due to monoclonal gammopathy (MG). Lenalidomide increased von Willebrand factor (VWF), lowered VWF clearance and resolved bleeding. Lenalidomide is a potential treatment option for refractory bleeding in AVWS secondary to MG. Background Acquired von Willebrand syndrome (AVWS) is associated with lymphoproliferative disorders, including monoclonal gammopathy (MG) of undetermined significance (MGUS) and multiple myeloma. Patients commonly present with significant bleeding complications that are difficult to manage, owing to a markedly reduced von Willebrand factor (VWF) half-life. Objectives To investigate the use of the immunomodulatory drug lenalidomide in two patients with severe refractory bleeding caused by AVWS associated with MGs. Results In both patients, lenalidomide treatment resulted in significant clinical improvement, and marked increases in plasma VWF antigen (VWF:Ag) and VWF ristocetin cofactor levels. This normalization in plasma VWF levels was sustained for > 2 years in both patients. Furthermore, in one patient, plasma VWF levels remain normal for at least 14 months following discontinuation of lenalidomide treatment. To investigate the molecular mechanisms underlying these observations, VWF propeptide (VWFpp)/VWF:Ag ratios were analyzed to assess VWF clearance. At enrolment, plasma VWFpp/VWF:Ag ratios were significantly elevated in both patients. Importantly, lenalidomide treatment resulted in normalization of VWFpp/VWF:Ag ratios in both patients. These novel data suggest that lenalidomide functions to attenuate enhanced VWF clearance in AVWS. Interestingly, in a patient with MGUS, lenalidomide treatment was associated with a significant increase in plasma VWF levels, despite no major change in paraprotein level. Conclusions Collectively, our findings suggest that lenalidomide constitutes a novel therapeutic option for the management of AVWS associated with MG. The biological mechanism(s) through which lenalidomide causes a sustained increase in plasma VWF levels in AVWS independently of paraprotein level requires further study, but is in part modulated through inhibition of enhanced VWF clearance.

Pathologic von Willebrand factor degradation with a left ventricular assist device occurs via two distinct mechanisms: Mechanical demolition and enzymatic cleavage

Bleeding is an important source of morbidity in patients with a left ventricular assist device. Evidence suggests a major role for von Willebrand factor. However, limited data exist to explain the mechanism(s) of von Willebrand factor degradation during left ventricular assist device support. We investigated whether left ventricular assist device-related shear stress and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13, the von Willebrand factor protease) altered von Willebrand factor metabolism. Whole blood was collected from patients (n=8) with a left ventricular assist device. von Willebrand factor multimers and degradation fragments were characterized with electrophoresis and immunoblotting. To investigate mechanisms, an invitro model was developed to generate the supraphysiologic shear stress of a continuous-flow left ventricular assist device. Normal human blood (n=8) was cycled in a laboratory vortexer (∼2400 rpm, shear stress ∼175 dyne/cm(2), 4 hours) to reproduce the pathologic degradation of von Willebrand factor that occurs during left ventricular assist device support. To investigate the specific mechanistic roles of shear stress and ADAMTS-13 in von Willebrand factor degradation, purified von Willebrand factor protein ± ADAMTS-13 protease were exposed to supraphysiologic shear stress in the vortexer. von Willebrand factor multimers and 11 von Willebrand factor degradation fragments were characterized with electrophoresis and immunoblotting. Left ventricular assist device support reduced large von Willebrand factor multimers and significantly increased 10/11 von Willebrand factor degradation fragments (P<.05). Normal human blood exposed to supraphysiologic shear stress in the vortexer demonstrated the same profile of von Willebrand factor degradation fragments as in a patient with a left ventricular assist device. Supraphysiologic shear stress alone caused modest mechanical demolition of large von Willebrand factor multimers into smaller multimers but did not greatly generate von Willebrand factor fragments. In the presence of supraphysiologic shear stress, ADAMTS-13 completely eliminated large von Willebrand factor multimers and generated statistically significant amounts of 11/11 von Willebrand factor degradation fragments (P<.05). The profile of von Willebrand factor fragments generated was identical to the profile that was observed invivo in patients with a left ventricular assist device. Supraphysiologic shear stress alone causes physical demolition of large von Willebrand factor multimers into smaller von Willebrand factor multimers. In the setting of supraphysiologic shear stress, ADAMTS-13 cleaves large von Willebrand factor multimers into von Willebrand factor degradation fragments. ADAMTS-13 may be a therapeutic target to reduce von Willebrand factor degradation and bleeding complications in patients with a left ventricular assist device.

Reduced von Willebrand factor secretion is associated with loss of Weibel–Palade body formation

von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel-Palade body (WPB)-like granules. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild-type VWF. Expression was also examined in HEK293 cells that form WPB-like granules when transfected with wild-type VWF. Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB-like granules, while R2663C was capable of WPB-like granules. Heterozygous expression of VWF variants had a negative impact on wild-type VWF with a reduction in elongated WPB-like granules in co-transfected cells. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB-like granules.

Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

Pulmonary and cardiovascular effects of traffic-related particulate matter: 4-week exposure of rats to roadside and diesel engine exhaust particles

Traffic-related particulate matter (PM) may play an important role in the development of adverse health effects, as documented extensively in acute toxicity studies. However, rather little is known about the impacts of prolonged exposure to PM. We hypothesized that long-term exposure to PM from traffic adversely affects the pulmonary and cardiovascular system through exacerbation of an inflammatory response. To examine this hypothesis, Fisher F344 rats, with a mild pulmonary inflammation at the onset of exposure, were exposed for 4 weeks, 5 days/week for 6 h a day to: (a) diluted diesel engine exhaust (PMDEE), or: (b) near roadside PM (PM2.5). Ultrafine particulates, which are largely present in diesel soot, may enter the systemic circulation and directly or indirectly trigger cardiovascular effects. Hence, we assessed the effects of traffic-related PM on pulmonary inflammation and activity of procoagulants, vascular function in arteries, and cytokine levels in the heart 24 h after termination of the exposures. No major adverse health effects of prolonged exposure to traffic-related PM were detected. However, some systemic effects due to PMDEE exposure occurred including decreased numbers of white blood cells and reduced von Willebrand factor protein in the circulation. In addition, lung tissue factor activity is reduced in conjunction with reduced lung tissue thrombin generation. To what extent these alterations contribute to thrombotic effects and vascular diseases remains to be established. In conclusion, prolonged exposure to traffic-related PM in healthy animals may not be detrimental due to various biological adaptive response mechanisms.