Forms Of Von Willebrand Factor Research Articles

Prevalence of Acute and Chronic Migraine Among Patients with Von Willebrand Disease

Background: The prevalence of migraine in the general population is approximately 18% in females and 6% in males (Lipton, et. al. Headache 2001;41:646). Among migraineurs, 7-8% experience chronic migraines (CM) (Adams AM, et. al. Cephalalgia 2015;35:563). Von Willebrand factor (VWF), secreted from endothelial cells, is reported to be elevated in migraineurs and in particular during the course of migraine, although its role is unclear (Cesar, et. al. Acta Neurol Scand 1995;91:412; Tietjen, et. al. Neurology 2001;57:334; Tietjen, et. al. Cephalalgia 2018;38:511). Patients with Von Willebrand Disease (VWD) have low levels of or a defective form of VWF, which has led some to hypothesize a lower prevalence of migraine in the VWD population. Conversely, a retrospective case control study found that migraine in VWD patients (n=197) was 6 times more prevalent when compared with age- and sex-match healthy controls (n=197) (18% vs. 3%, respectively)(Hassan, et. al. Intl Journal of Hem, Blood Diseases and Disorders 2014;2:23-5). The current investigation was performed to further explore the relationship between migraine and VWD. Methods: A cross-sectional study was conducted in March 2021 using the InCrowd digital survey tool in which patients with self-reported diagnosis of VWD were invited to participate. The study included 75 VWD patients ≥18 years of age. Survey questions detailed VWD sub-type, gender, clinical diagnosis of migraine, medical specialty diagnosing migraine, migraine treatments, and age of diagnosis. The Lipton Identify Chronic Migraine (ID-CM) tool was used within the survey to identify patients who had experienced chronic migraine(Lipton, et al. Cephalalgia. 2016;36:203). Criteria for designation of chronic migraine from the ID-CM was either the combination of Frequency of Migraine and Symptoms or the combination of Use of Medication and Activity Avoidance. Results: The gender distribution of the 75 participants was 83% female and 17% male. The sub-type of VWD diagnosed was: Type 1 (57%), Type 2 (28%), Type 3 (15%). Fifty-seven percent (57%) of all VWD patients reported being diagnosed with migraine. Rates did not vary significantly by VWD sub-type. Diagnosed migraine was only slightly more common in females (60%, n=62) compared with males (46%, n=7). Average age of diagnosis was 22 years with a median of 17 years (n=43). Two clear clusters defined the age of diagnosis with migraine. The first was between ages 10 and 20 (n=24, 56% of total sample reporting age) and the second was approximately age 30 [28-32] (n=8, 19% of total sample reporting age). Migraine diagnosis was established by primary care physicians (44%), neurologists (44%) hematologists (9%), and headache specialists (2%). Only 4% of surveyed patients met the ID-CM criteria for chronic migraine, yet 71% of patients met the criteria for migraine symptoms. Chronic migraine diagnosis using the ID-CM tool requires a combination of headache frequency (at least half of the past 30 or 90 days), multiple migraine symptoms (present >rarely in the past 30 days), medication use for headache as well as interference with activities (≥10 of the past 30 days) and a headache that either interfered with plans or caused worry for making plans (>rarely in the past 30 days). Frequency of migraine was mentioned by 11% of patients and symptoms by 71%, medication use by 31%, activity limitation by 15%, and changing plans by 25%. Conclusions: This survey illustrates that migraine is significantly more common in people with VWD than in the general population, regardless of VWD sub-type. However, the age-associated incidence pattern follows a similar bimodal distribution as observed in the general population(Victor, et al. Cephalalgia. 2010;30:1065). Notably, the prevalence of diagnosed migraine in VWD males approaches the prevalence in VWD females and exceeds that of females in the general population (Lipton RB, et. al. Headache 2001;41:646). These results suggest a distinct etiology of VWD that makes affected patients more susceptible to migraine. Notably, migraine treatment options may be more limited for these patients, given that traditional NSAIDs are commonly used for fast-acting migraine relief, but their antiplatelet effects and potential for gastrointestinal bleeding make them undesirable in VWD. Further investigation of both the etiology and treatment for VWD migraine is warranted. DisclosuresMacgregor: Tremeau Pharmaceuticals Inc.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Helm: Tremeau Pharmaceuticals Inc.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Cerasoli: RX Medical Dynamics LLC: Consultancy, Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Boice: Tremeau Pharmaceuticals Inc.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Sidonio: Novo Nordisk: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Catalyst: Consultancy; Genentech: Consultancy, Research Funding; Biomarin: Consultancy; Guardian Therapeutics: Consultancy; Octapharma: Consultancy, Research Funding; Takeda: Consultancy, Research Funding.

Idiopathic Thrombotic Microangiopathy with ChAdOx1 nCov-19 Vaccination in a Middle Aged Male: A Clinical Challenge in the Covid Era

Thrombotic thrombocytopenic purpura (TTP) is defined as thrombotic microangiopathy occurring in the context of severe ADAMTS13 deficiency (<10%) which is a normal enzyme in plasma that cleaves large forms of von Willebrand factor into smaller functional subunits. We report a case of a middle aged male with de novo iTTP ( idiopathic thrombotic thrombocytopenic purpura) after vaccination with ChAdOx1 nCov-19. He presented with severe abdominal pain, increased bruising, hematuria along with episodes of transient ischemic attacks, 5 days from the first dose of the antiCOVID-19 vaccine. Laboratory data revealed a very severe ADAMTS13 deficiency, however, autoantibody titer could not be measured due to the failure of refrigerating facility in our reference laboratory. Patient was successfully treated with serial plasma exchange and immunosuppression with steroids and rituximab.

Traumatic injury results in prolonged circulation of ultralarge von Willebrand factor and a reduction in ADAMTS13 activity.

Increases in plasma von Willebrand Factor (VWF) levels, accompanied by decreases in the metalloprotease ADAMTS13, have been demonstrated soon after traumatic injury while downstream effects remain unclear. A cohort of 37 injured trauma patients from a randomized control trial investigating the use of prehospital plasma transfusion were analyzed for activity and antigen levels of ADAMTS13 and VWF at 0 and 24 hours after admission. Relevant clinical data were abstracted from the medical records. Trauma patient plasma was analyzed via agarose gel electrophoresis to evaluate the effects of injury on VWF multimer composition compared to healthy controls. von Willebrand factor levels were elevated at presentation (189% [110%-263%] vs. 95% [74%-120%]), persisting through 24 hours (213% [146%-257%] vs. 132% [57%-160%]), compared to healthy controls. Ultralarge VWF (UL-VWF) forms were elevated in trauma patients at both 0 and 24 hours, when compared to pooled normal plasma (10.0% [8.9%-14.3%] and 11.3% [9.1%-21.2%], respectively, vs. 0.6%). Circulating plasma ADAMTS13 activity was decreased at 0 hours (66% [47%-86%] vs. 100% [98%-100%]) and at 24 hours (72.5% [56%-87.3%] vs. 103% [103%-103%]) in trauma patients. ADAMTS13 activity independently predicted the development of coagulopathy and correlated with international normalized ratio, thromboelastography values, injury severity, and blood product transfusion. Traumatic injury is associated with acute coagulopathy that is characterized by increased UL-VWF multimers and reduction in ADAMTS13, which correlates with blood loss, transfusion requirement, and injury severity. These findings suggest the potential for future trials targeting ADAMTS13 repletion to enhance clearance of VWF multimers.

A Murine Model of Type 2N VWD Was Developed By CRISPR/Cas9 Gene Editing and Recapitulates Human Type 2N VWD

Von Willebrand Disease (VWD) is caused by either an inherited deficiency of von Willebrand factor (VWF) protein or synthesis of a dysfunctional von Willebrand factor (VWF) that is referred to as a type 2 variant. One of the important functions of VWF is to serve as a carrier protein for FVIII - prolonging FVIII's plasma half-life from <2 hrs without VWF to 10-12 hrs with VWF. A variant form of VWF was identified in 1982 in which VWF did not bind FVIII resulting in FVIII's rapid clearance and a phenotype similar to moderate hemophilia and currently referred to as type 2N VWD. Type 2N VWD is caused by an autosomal recessive variant of VWF in which mutations in the D'D3 region of VWF cause decreased or absent binding of FVIII to VWF resulting in rapid FVIII clearance similar to that seen in the absence of VWF. These individuals may have a type 2N mutation on each of their VWF alleles or, more commonly, have 1 allele with a 2N mutation and their 2nd allele contains a null mutation (producing no VWF). In each of these scenarios, only functionally abnormal VWF is synthesized, resulting in reduced or absent FVIII binding, rapid FVIII clearance, and a marked reduction in plasma FVIII. While Lillicrap and coworkers have demonstrated 2N VWF expression by hydrodynamic transfection in a VWF-/- mouse, their approach represents a model of plasma 2N VWF dysfunction but does not recapitulate human 2N VWD where the abnormal VWF is not only in plasma, but also in endothelial cells and platelets.We selected 8 different human type 2N mutations, and mutated murine VWF at these positions and expressed the mutant VWF in HEK293T cells in vitro. Binding of each of the 2N mutants of mouse VWF to both murine and human FVIII was tested. The G785E 2N mutation had the most severe defect with negligible binding to both human (rhF8) and mouse FVIII (mF8) and was therefore chosen for further study. Using CRISPR/Cas9 gene editing, we generated two lines of VWF2N mice (termed VWF2N1/2N1 and VWF2N2/2N2) each with a 2N causative 2354G>A (G785E) mutation on the C57BL6 background. Plasma VWF levels were measured by ELISA and FVIII was measured by chromogenic assay. VWF binding to FVIII was determined by FVIII binding to monoclonal captured VWF or by VWF binding to monoclonal captured FVIII. For the remainder of this abstract, we will only discuss the VWF2N1/2N1 mice and refer to them as wt VWF+/+, heterozygous VWF2N/+ or homozygous VWF2N/2N mice. The plasma VWF in VWF+/+, VWD2N/+ or VWF2N/2N mice was 109±31, 100±27, and 115±29 U/dL. The plasma FVIII levels in homozygous VWF2N/2N mice were markedly reduced to 4.9±2.2 U/dL compared to either wt (84±21U/dL) or heterozygous (82±18U/dL) controls. VWF binding to rhF8 was reduced to <2% in VWF2N/2N mice and 57±9% in VWF2N/+ mice when compared to wt controls.To assess the clinical phenotype of VWF2N/2N mice, tail bleeding times were determined demonstrating prolongation to 3.1±0.4 hrs in VWF2N/2N mice in contrast to 2.4±0.4 in VWF2N/+ and 2.1±1.0 hrs in wt controls. ROTEM assessment of whole blood clotting time (wbCT) and whole blood thrombin generation assays (wbTGA) were performed. The wbCT was 10.7±1.1 min in VWF2N/2N mice compared to 8.3±0.5 min in VWF2N/+ heterozygotes. In wbTGA, the Lag Time and Peak Time in the VWF2N/2N group were 18.53±5.57 and 34.15±8.37 min, respectively, which were significantly longer than those obtained in the VWF2N/+ group (8.3±0.5 and 17.83±2.33 min, respectively). Peak Thrombin, Endogenous Thrombin Potential, and Thrombin Generation rate in the VWF2N/2N group were significantly lower than those obtained in the VWF2N/+ group.In human type 2N VWD, FVIII synthesis is normal but plasma levels are reduced because of increased FVIII clearance in the absence of VWF binding. Infusion of rhVWF (no FVIII) into the VWF2N/2N mice demonstrated rhVWF peaking by 30 minutes with a rescue of the endogenous mouse FVIII (44±1.7 U/dL) at 4-6 hrs post infusion. This demonstrates improved FVIII survival in the presence of normal VWF.In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in Type 2N patients. Plasma levels of VWF are normal in our VWF2N/2N mice. This VWF is incapable of binding FVIII at neutral pH, but is otherwise fully functional. This is a unique model of 2N VWD that can be used to investigate the biological properties of VWF/FVIII association. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule.

Wall shear rates are critical for a broad variety of fluidic phenomena and are taken into account in nearly every experimental or simulation study. Generally, shear rates are not observable directly but rather derived from other parameters such as pressure and flow, often assuming somehow idealized systems. However, there is a biological system which is able to constantly measure the wall shear as a part of a regulatory circuit: The blood circulation system takes advantage of shear rate sensor (protein)molecules (multimeric forms of von Willebrand Factor, VWF), which are dissolved in the blood plasma and dramatically change their conformation under shear conditions. The conformational changes are accompanied by several functional variations and therefore interplay with the regulation of the coagulation system. In this study, we use a recombinantly produced and therefore well-defined multimeric form of VWF as a sensor which directly responds to shear rates. Shear rates, up to 32.000 s-1, were obtained using a kind of micro-plate-to-plate rheometer capable of adsorbing shear-stretched VWF oligomeric molecules on a surface to conserve their differently stretched conformation and so allow detection of their elongation by atomic force microscopy. The laminar flow in this geometrically simple device has been characterized by adopting classical fluid dynamical models, in order to ensure well-known, stable shear rates which could be correlated quantitatively with an observed stretching of sensor molecules.

Recombinant von Willebrand factor (vonicog alfa) in von Willebrand disease: a guide to its use

Vonicog alfa (VONVENDI), a human recombinant form of von Willebrand factor (rVWF), is a valuable addition to the treatments available to treat bleeding episodes in adults with von Willebrand disease (VWD). It is a highly purified and homogenous concentrate that contains all sizes of VWF multimers, including high-molecular weight and ultra-large multimers. In patients with VWD, intravenous rVWF provides bleeding control and sustained stabilization of endogenous factor VIII activity, is well tolerated and overcomes some of the inherent limitations associated with the use of plasma-derived VWF concentrates.

Advantages and limits of ADAMTS13 testing in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare disease (5–10 cases per million persons per year) characterised by the massive formation of platelet rich-thrombi in the microcirculation of multiple organs1,2. It affects both sexes, although the incidence is two to three times higher among females3. TTP was first described by Moschowitz in 1924 in a 16-year old girl who presented with fever, anaemia, thrombocytopenia and focal changes in the central nervous system and kidneys4. Several reports of TTP followed this initial description leading to a definition based upon the following pentad of symptoms: fever, mechanical haemolytic anaemia, thrombocytopenia, central nervous system abnormalities and renal impairment5. However, fever, neurological abnormalities and renal impairment are not constant symptoms, especially during the early stage of the disease, thus leading to the currently accepted definition of TTP consisting of the association of mechanical haemolytic anaemia with fragmented erythrocytes and thrombocytopenia (platelet count <100 × 109/L) without alternative causes6. In parallel to the evolution of clinical criteria, the pathophysiological mechanisms of TTP were elucidated when Joel Moake and his colleagues observed that the plasma of a patient with recurrent TTP contained very high molecular weight [so-called ultralarge (UL)] multimers of von Willebrand factor (VWF), a multimeric adhesion glycoprotein contained in endothelial cells, platelets and plasma7. Once released from the abnormally stimulated endothelial cells, these ultralarge forms of VWF, present in the endothelium but not in plasma in physiological conditions, promote intravascular aggregation of platelets and the consequent microvascular thrombosis and haemolytic anaemia caused by mechanical damage, particularly in blood flow conditions characterised by high fluid shear stress such as in the microcirculation3. Moake himself hypothesised the deficiency of a cleaving protease as being responsible for the presence of UL VWF7, but it was Furlan et al. and Tsai and Lian in 1996 who managed to isolate from human plasma a metal-dependent protease able to cleave the peptide bond between the tyrosine at position 1605 and the methionine at position 1606 in the central A2 domain of VWF. The same investigators subsequently and independently found that the VWF-cleaving protease was deficient in a retrospective cohort of patients clinically diagnosed as having TTP8,9. The protease, which is responsible for regulating the multimeric structure of VWF, was identified in 2001 by Zheng et al.10 as a new (the thirteenth) member of the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin 1 repeats) family of metalloproteases and was called ADAMTS132. While the discovery of ADAMTS13 has renewed interest in TTP, as documented by the exponential increase in the number of publications on this topic in the last few years, it has raised the issue of the diagnostic and prognostic value of ADAMTS13 testing in this condition.

Orthotopic liver transplantation (OLTx) in non-cirrhotic portal hypertension secondary to ADAMTS13 deficiency.

Non-cirrhotic intrahepatic portal hypertension (NCIPH), also called idiopathic or benign, may lead to life-threatening complications. It is a rare indication for orthotopic liver transplantation (OLTx), although it may remain underdiagnosed. Overt symptoms of portal hypertension, predominantly variceal bleedings, misleadingly suggest that these patients are cirrhotic. Non-cirrhotic intrahepatic portal hypertension can be related to obliteration of portal venous microcirculation as a consequence of ADAMTS13 deficiency, a metalloproteinase which cleaves the ultra-large molecular weight forms of von Willebrand factor (VWF). The physiological role of VWF, secreted from endothelium, is to facilitate platelet adhesion at sites of endothelial damage. Decreased ADAMTS13 activity and persistence of ultra-large VWF at the endothelial surface predisposes to platelet clumping, causing microvascular occlusion. We describe the first OLTx for ADAMTS13 deficiency-related NCIPH in Poland. A 20-year-old, previously healthy male student presented with upper gastrointestinal bleeding. Gastroduodenoscopy revealed active bleeding from oesophageal varices, which was successfully treated with endoscopic banding ligation. Physical examination showed splenomegaly, but was otherwise normal with no signs suggesting chronic liver disease. Laboratory investigations were all normal except thrombocytopenia of 40,000/ml. Viral, metabolic, and autoimmunological markers of liver disease as well as bone marrow examination were normal. Transjugular liver biopsy was performed and was essentially normal. However, the tissue sample size was rather slight and we were not able to measure hepatic venous pressure gradient (HVPG). Percutaneous liver biopsy performed later confirmed these findings. Contrast enhanced magnetic resonance imaging scan excluded portal vein thrombosis and Budd-Chiari syndrome. Laboratory investigations showed undetectable levels of ADAMTS13. The patient has been followed up over a period of 5 years, showing constant progression of his portal hypertension with continuous enlargement of his spleen (Figures 1 and ​and2)2) and frequent oesophageal/gastric variceal treatments. Follow-up liver biopsy showed progression to F2 fibrosis. He underwent OLTx with rapid recovery and remains extremely well 6 months after surgery. The histological changes in the explanted liver are shown in Figures 3 A–C. Figure 1 Magnetic resonance imaging of portal hypertension Figure 2 Computed tomography images of the liver hypotrophy and spleen enlargement Figure 3 Histopathology of the explanted liver: A – Liver fibrosis with inflammatory infiltrates. Neocholangioles, small venules, and lack of correct arterioles. Haematoxylin and eosin stain (H + E). Objective magnification 10×. B – Thick ... Non-cirrhotic intrahepatic portal hypertension secondary to ADAMTS13 deficiency is a progressive condition and in case of uncontrolled symptoms of portal hypertension may require liver transplantation. Non-cirrhotic intrahepatic portal hypertension is also called idiopathic or benign portal hypertension. The latter term is misleading as it was shown that more than half of these patients develop liver failure when observed over a median period of 88 months [1]. Thus NCIPH is considered a rare but clinically important cause of portal hypertension. The underlying problem is related to the obliteration of portal venous microcirculation, secondary to the deficiency of ADAMTS13, a metalloproteinase that cleaves the ultra-large molecular weight forms of VWF to smaller ones [2]. The physiological role of VWF, secreted from endothelium, is to facilitate platelet adhesion at sites of endothelial damage. Mutations of ADAMTS13 are seen in congenital TTP, while antibodies to ADAMTS13 are found in the majority of adult acquired cases. Decreased ADAMTS13 activity and persistence of ultra-large VWF at the endothelial surface is thought to predispose to platelet clumping, causing microvascular occlusion. The imbalance in VWF and ADAMTS13 levels in portal microcirculation, where hepatic arterial blood pressures are superimposed, with upstream relation to hepatic stellate cells, would provide a mechanism for obliteration of terminal portal venules, which is characteristic of NCIPH. In our patient plasma activity of ADAMTS13 was below 1%, and its inhibitor was within the normal ranges. He has been treated with β-blockers and remains clinically stable with appropriate endoscopic surveillance. However, in sequential MRI and computed tomography imagines, atrophy of the right lobe of the liver has been observed, with enlargement of the left lobe, periportal fibrosis, varices, and dilatation of the portal tract veins. Chronic NCIPH leads to hepatic atrophy, although the progression of the disease is usually slow. The disorder can be associated with celiac disease and ulcerative colitis, and sustained deficiency of ADAMTS13 appears characteristic of NCIPH despite preserved liver function [1]. Older age at first presentation, hepatic encephalopathy, and portal vein thrombosis were found to be significant predictors of reduced transplant-free survival with, as already mentioned, progression to decompensated liver disease and the need for liver transplantation in a significant proportion of patients [1].

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

Haemorrhagic and thrombotic diatheses in mouse models with thrombocytosis.

We studied haemostasis in two mouse models with thrombocytosis caused by different pathogenic mechanisms. In one strain (Yall;Mpl-/-) thrombocytosis is driven by a misbalance between thrombopoietin and its receptor, whereas in the other strain, thrombocytosis is caused by expressing a human JAK2-V617F transgene (FF1) that depends on activation by Cre-recombinase (VavCre;FF1, MxCre;FF1). Thrombotic responses were increased following some, but not all types of challenges. In a vaso-occlusive thrombotic model following collagen-adrenaline injection we found increased mortality in both strains. Arterial thrombosis, examined after ferric chloride-induced carotid injury, was accelerated but with little impact on maximal thrombus size. In a vena cava stasis model, clots were of similar size as in wild-type controls, but exhibited a different composition with a higher platelet to fibrin ratio. Both thrombocytosis strains displayed increased haemorrhagic tendency in a tail bleeding assay. Yall;Mpl and VavCre;FF1 displayed a lower proportion of the more reactive high-molecular-weight forms of von Willebrand factor in their plasma, mimicking essential thrombocythaemia with very high platelet counts. Bleeding could not be explained by clear defects in platelet activation, which were normal or only weakly decreased. In conclusion, these models of thrombocytosis recapitulate several features of the haemorrhagic and thrombotic diatheses in ET and PV demonstrating potentials but also some limitations to study these major complications.

Hypochlorous Acid Generated by Neutrophils Inactivates ADAMTS13: AN OXIDATIVE MECHANISM FOR REGULATING ADAMTS13 PROTEOLYTIC ACTIVITY DURING INFLAMMATION

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

IntroductionThe multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells’ surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. Material and MethodsVWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. ResultsTo investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. ConclusionsOur assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.

Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

AbstractThe interaction between platelet receptor glycoprotein Ibα and the A1 domain of von Willebrand factor (VWF) mediates tethering/translocation of platelets to sites of vascular injury. Unexpectedly, we observed platelets translocating over A1A2A3 domains protein slower than on A1 domain at high shear stress. This observation suggests an additional interaction between A domains and an adhesive receptor. We investigated vimentin because we have data showing the interaction of vimentin with the A2 domain of VWF. Moreover, vimentin is expressed on the platelet surface. This novel interaction was analyzed by using purified VWF, recombinant proteins, anti-vimentin antibodies, parallel flow chamber adhesion assays, flow cytometry, and vimentin-deficient murine platelets. The active form of VWF bound to vimentin, and the purified A2 domain blocked that binding. The interaction of a gain-of-function A1A2A3 mutant with platelet was reduced using anti-vimentin antibody. Platelet adhesion to wild-type (WT) A1A2A3 protein, collagen, and fibrin(ogen) was inhibited (32-75%) by anti-vimentin antibody under high shear stress. Compared with WT mice, platelets from vimentin-deficient mice had a reduced flow-dependent adhesion to both collagen and purified murine VWF. Last, the vimentin knockout mice had a prolonged tail bleeding time. The results describe that platelet vimentin engages VWF during platelet adhesion under high shear stress.

Von Willebrand Factor Antagonizes Nitric Oxide Synthase To Promote Insulin Resistance during Hypoxia

Hypoxic respiratory diseases or hypoxia exposures are frequently accompanied by glucose intolerance and impaired nitric oxide (NO) availability. However, the molecular mechanism responsible for impaired NO production and insulin resistance (IR) during hypoxia remains obscure. In this study, we investigated the possible mechanism of impaired NO production and IR during hypoxia in a mouse model. Mice were exposed to hypoxia for different periods of time (0-24 h), and parameters of IR and endothelial dysfunctions were analyzed. Exposure to hypoxia resulted in a time-dependent increase in IR as well as multimeric forms of von Willebrand factor (vWF) and subsequently a decrease in eNOS activity. Preincubation with plasma of hypoxia-exposed animals (different time points) or human vWF inhibited insulin-induced NO production in a dose-dependent manner; larger doses of insulin reversed the effect. In contrast, preincubation of vWF-immunodepleted plasma failed to inhibit insulin-induced NO production, whereas vWF immunoneutralization abolished the effect of hypoxia-induced IR and D-[U-(14)C]glucose uptake. Furthermore, the interactions between vWF and eNOS were studied by far-Western blotting, co-immunoprecipitation, and surface plasma resonance spectroscopy. Kinetic analyses showed that the dissociation constant (KD), inhibitory constant (Ki), and half-maximal inhibitory concentration (IC50) were 1.79 × 10(-8) M, 250 pM, and 18.31 pM, respectively, suggesting that vWF binds to eNOS with a high affinity and greater efficacy for activator (insulin) inhibition. These results indicated that vWF, an antagonist of eNOS, inhibits insulin-induced NO production and causes IR.

Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

Upshaw-Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, causing intravascular platelet clumping and thrombotic microangiopathy. Our patient, a 26-year-old man, had attacks of thrombotic thrombocytopenic purpura (TTP) with thrombocytopenia and a urine dipstick positive for hemoglobin (4+), often as the only sign of hemolytic activity. He had ADAMTS13 activity of <1% of normal plasma without the presence of inhibitors of ADAMTS13. ADAMTS13 deficiency was caused by two new mutations of the ADAMTS13 gene: a deletion of a single nucleotide in exon17 (c. 2042 delA) leading to a frameshift (K681C fs X16), and a missense mutation in exon 25 (c.3368G>A) leading to p.R1123H. This case report confirms the importance of the analysis of the ADAMTS13 activity and its inhibitor in patients who have episodes of TTP, with a very low platelet count and sometimes without the classic biochemical signs of hemolysis.

High Density Lipoprotein and Apolipoprotein A-I Bind Von Willebrand Factor and Prevent Its Self-Association Into Thick Fibers

Von Willebrand factor (VWF) is a large plasma protein secreted constitutively or upon activation by endothelial cells. On activated endothelium, a portion of the VWF molecules remain attached to the surface, where they self-associate to produce long hyperadhesive strands and fibers capable of capturing platelets and other blood cells if not removed by the plasma metalloprotease ADAMTS13. Failure to clear these fibers underlies the pathophysiology of many microangiopathies, most prominently thrombotic thrombocytopenic purpura, in which accumulation of VWF-platelet aggregates in the microvasculature leads to ischemia and organ failure. VWF self-association also plays a major role in the adsorption of purified VWF to surfaces. We observed that adsorption of VWF to surfaces began with contact and direct binding of VWF to the surface via hydrophobic interactions. After saturating the binding sites on the surface, VWF continued to bind to and associate with the immobilized VWF molecules via hydrophilic VWF-VWF interactions, depositing multiple layers of VWF molecules on the surface until essentially all VWF molecules were depleted from the fluid phase. Using the method of Magnani and coworkers, we eluted VWF molecules that were bound to the surface via VWF-VWF interactions with the ionic detergent SDS, and then eluted VWF molecules that were bound to the surface via VWF-surface interactions with the zwitterion detergent CHAPS. Quantification of the eluted VWF showed that self-association of VWF onto the surface accounted for >80% of the adsorptive loss. Using the disappearance of purified VWF from the fluid phase as a measurement of self-association, we fractionated human plasma by heat and identified that apolipoprotein A-I (ApoAI), the major apolipoprotein component in high density lipoprotein (HDL) particles, which is stable to heat at 100° C, stabilized and prevented VWF surface adsorption by interfering with VWF self-association. Commercial preparations of ApoAI and HDL, prepared without exposure to heat, similarly prevented adsorption of purified VWF to surfaces. Half-maximal stabilization occurred at a molar ratio of eight ApoAI molecules to each VWF subunit. We assessed the role of HDL in VWF self-association that leads to the assembly of ultra-large VWF (ULVWF) strings on the surface of phorbol myristyl acetate-stimulated endothelial cells in flow chambers. We observed that HDL reduced the number and length of platelet-decorated ULVWF strings on the endothelial surface, consistent with the ability of HDL to interfere with VWF self-association and ULVWF assembly. We also studied the role of ApoAI in the recruitment of fluid-phase VWF to hyperadhesive ULVWF fibers in a synthetic microvessel system. We observed that ApoAI directly bound to hyperadhesive transluminal ULVWF fibers under flow, and this binding completely blocked the recruitment of fluid phase VWF molecules to the immobilized ULVWF fibers. These results showed that ApoAI or HDL interacted with hyperadhesive forms of VWF and modified the adhesive properties of the ULVWF strings and fibers. Consistent with its antithrombotic properties, the level of ApoAI in patients with hyperadhesive forms of VWF, such as thrombotic thrombocytopenic purpura and sepsis, was significantly reduced. These results suggest that regulation of VWF self-association may be another mechanism by which HDL protects against cardiovascular disease and extends its protective effects from large arteries to the microvasculature. Disclosures:No relevant conflicts of interest to declare.

Distinct Role of von Willebrand Factor Triplet Bands in Glycoprotein Ib-Dependent Platelet Adhesion and Thrombus Formation under Flow

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer have one shorter or longer N-terminal peptide sequence, respectively. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Therefore, platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, whereas preparation B predominantly contained larger triplet bands. Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb but an unchanged affinity for collagen type III when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Thus, VWF forms lacking larger-size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.

Desmopressin-related myocardial infarction in a patient with Wegener's granulomatosis: A case report and review of the literature

Desmopressin is a synthetic vasopressin analog that increases the plasma levels of coagulation factor VIII, von Willebrand factor, and tissue plasminogen activator. This hemostatic agent, which can be administered either parenterally or intranasally, has been approved for use in the prevention and treatment of hemorrhagic events during surgery in patients with hemophilia A, in cases of prolonged idiopathic bleeding, and for complications associated with platelet antiaggregant therapy. This case report describes cardiac toxicity associated with desmopressin administered according to the recommended indications: a 55-year-old woman diagnosed with Wegener's granulomatosis (WG) was treated with desmopressin to improve hemostasis and shorten bleeding time before a planned renal biopsy. She developed cardiac arrest within 60 minutes of the desmopressin injection. Cardiopulmonary resuscitation began immediately and was successful, although the patient subsequently died of WG-associated complications. Desmopressin administration thus appears, in some cases, to be associated with a high risk of thrombotic events, possibly by stimulating the rapid release of endothelial factors such as an abnormal multimeric form of von Willebrand factor, which might cause platelet aggregation. Clinicians should be aware of the possible occurrence of this little-known but potentially serious cardiac event associated with desmopressin administration and be prepared to initiate cardiopulmonary resuscitation immediately if needed.

The Co-Influence of VWD Type 2B and Type 2M Mutations and Platelet GPIbα On the Susceptibility of VWF to ADAMTS13 Under Shear Stress

Abstract 1108 Introduction:Von Willebrand Factor (VWF) is one kind of vital proteins involved in thrombosis, which exists in the form of multimeric glycoprotein in plasma of human. The size of VWF multimer is down regulated by metalloproteinase ADAMTS13. This physiological process is modulated by multiple factors. It was reported that the binding of platelets or GPIbα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to recombinant A1A2A3 segment in the condition of static state. Due to the mutations, VWF synthesized by patients of von Willebrand disease (VWD) type 2B and type 2M may enhance and decrease the binding of platelet membrane glycoprotein Ib (GPIb), respectively. This study aims to observe the influence of 2B/2M VWD mutations and recombinant GPIbα active fragment on the susceptibility of VWF to ADAMTS13, in the condition of high shear stress. Materials and Methods:We constructed and expressed different forms of recombinant full-length human VWF (rhVWF) including three type 2B mutations (P1337L, H1268D, R1308C), three type 2M mutations (M740I, R1205H, D1302G) and wide-type (WT) in Hela cells. Recombinant human ADAMTS13 (rhADAMTS13) and GPIbα (H1-V289) were both from our own lab. The different types of rhVWF were characterized by multimeric analysis and ristocetin-induced platelet aggregation (RIPA). We use rhADAMTS13 to digest seven full-length rhVWF in the reaction Buffer (20 mM Tris-HCl, 150mM NaCl, pH7.4, 1% bovine serum albumin) in the absence and presence of GPIbα (H1-V289). To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of rhVWF by rhADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics arterial fluid shear stress (30 dynes/cm2). The 176kDa fragment resulting from the cleavage of rhVWF at the Y1605-M1606 bond by rhADAMTS13 was visualized by 5% SDS-PAGE under reducing conditions and Western blot analysis. Results:SDS-agarose gel electrophoresis showed that all rhVWF mutants were in the similar multimeric distribution to the rhVWF-WT. In the RIPA assay, type 2B rhVWF exhibited clearly enhanced RIPA responsiveness with ristocetin at 0.5 mg/ml (R1308C > P1337L > H1268D > WT), while type2M rhVWF showed decreased RIPA responsiveness with ristocetin at 1.0 mg/ml (WT > M740I > R1205H > D1302G). At physiological salt concentration (150nM NaCl), type 2B mutants exhibited more susceptibility to rhADAMTS13 than WT and type 2M mutants in the absence of GPIbα (H1-V289). In the presence of GPIbα (H1-V289), the higher susceptibility of mutants to rhADAMTS13 was detected, which decreased in the following order: type 2B > WT > type 2M, whereas no obvious enhancement happened to the two type 2M mutants (R1205H and D1302G). Conclusions:Our study further confirmed that platelet GPIbα binding to VWF A1 domain could increase the susceptibility to ADAMTS13 at physiological conditions under high stress. The three type 2B mutations are all localized in the A1 domain and these mutations alone may also increase the susceptibility to ADAMTS13. While type 2M mutations M740I, R1205H and D1302G are localized in the D2, D3 and A1 domain, respectively. It is concluded that type 2M mutations localized close to or in the A1 domain may exhibit significantly lower susceptibility to ADAMTS13 in the presence of GPIbα (H1-V289). It may partially give the explanation that some VWD type 2M may display larger than normal VWF multimers in plasma. Disclosures:No relevant conflicts of interest to declare.

Differential sensitivity of von Willebrand factor (VWF) ‘activity’ assays to large and small VWF molecular weight forms: a cross‐laboratory study comparing ristocetin cofactor, collagen‐binding and mAb‐based assays

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura.