Von Willebrand Factor-Glycoprotein Ib-Alpha Interactions Play a Role in Generating Platelet Microparticles: Data from Ex Vivo and in Vitro Studies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3920-3920
Author(s):  
Andrea Artoni ◽  
Anna Lecchi ◽  
MariaElisa Mancuso ◽  
Elena Santagostino ◽  
Augusto Federici ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Shear Stress ◽  
Tissue Factor ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand ◽  
Normal Controls

Abstract Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins such as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb might be critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in relationship with VWF levels and multimeric composition as well as shear stress exploiting ex vivo and in vitro natural models. PMP and TFMP were analyzed by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. In the first set of experiments, levels of PMP and TFMP were measured before and after DDAVP administration in 9 patients with mild hemophilia A. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma at baseline and 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed after DDAVP; peak levels were achieved after 2–8 h. Correlation was found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). We then studied 27 patients with different types of von Willebrand disease (VWD) and 15 normal controls: citrated platelet rich plasma was exposed to increasing shear stress (from 0 to 90 dyne) for 45 seconds in a cone and plate viscometer and generated PMP were then enumerated by flow cytometry. In normal controls the number of MP at 25 dyne was significantly higher than the number at 0 dyne (p<0.05). In patients with type 1 VWD and type 2B PMP significantly increased as in normal controls (p<0.05 at 25 dyne), while in type 3 patients no PMP increase was observed (p n.s. till 90 dyne). Interestingly, in type 2M patients PMP started to increased significantly at 50 dyne (p<0.05), while type 2A patients had the same behaviour of type 3 patients. This study support the hypothesis that ultralarge VWF multimers released by the endothelium interact with platelets and trigger MP generation, both in vivo and in vitro.

Clinical cases of using coagulation factor VIII with von Willebrand factor in parturient women with type III von Willebrand disease

2020 ◽  
Author(s):  
И.В. Куртов ◽  
Е.С. Фатенкова ◽  
Н.А. Юдина ◽  
А.М. Осадчук ◽  
И.Л. Давыдкин
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Coagulation Factor Viii ◽  
Vitro Fertilization ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Болезнь Виллебранда (БВ) может представлять определенные трудности у рожениц с данной патологией. Приведены 2 клинических примера использования у женщин с БВ фактора VIII свертывания крови с фактором Виллебранда, показана эффективность и безопасность их применения. У одной пациентки было также показано использование фактора свертывания крови VIII с фактором Виллебранда во время экстракорпорального оплодотворения. Von Willebrand disease presents a certain hemostatic problem among parturients. This article shows the effectiveness and safety of using coagulation factor VIII with von Willebrand factor for the prevention of bleeding in childbirth in 2 patients with type 3 von Willebrand disease. In one patient, the use of coagulation factor VIII with von Willebrand factor during in vitro fertilization was also shown.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

scholarly journals The Potential Role of Emicizuamb Prophylaxis in Severe Von Willebrand Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Assaf Arie Barg ◽  
Gili Kenet ◽  
Tami Livnat ◽  
Einat Avishai ◽  
Ivan Budnik ◽  
...  
Keyword(s):  
Factor Viii ◽  
Potential Role ◽  
Prophylactic Treatment ◽  
Ex Vivo ◽  
Peak Height ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand

Severe Von Willebrand's disease (VWD) may be associated with chronic joint damage and may require prophylactic therapy. In severe VWD, factor VIII (FVIII) levels are low due to rapid clearness. Emicizumab is a humanized bispecific antibody which mimics the function of coagulation factor VIII (FVIII). It has been approved for prophylaxis in hemophilia A. This is the first study assessing the potential role of emicizumab as an alternative prophylactic treatment in a cohort of patients with severe VWD. We present a TG model evaluating patients' hemostasis following ex vivo spiking of their plasma samples with emicizumab. We also report 24 weeks of successful emicizumab prophylaxis in a child with severe VWD and repeated hemarthroses. A cohort of twenty-four VWD patients were included in the study. Fifty-four percent of our patients were males and the cohort consisted of 14 children (≤18 years) and 10 adults. The majority of patients (96%) were of Caucasian origin. Hemarthrosis was encountered in most type 3 VWD patients, whereas none of the type 2 VWD patients had any joint bleeds. Prophylactic treatment was administered in the majority of type 3 VWD patients, whereas type 2 VWD patients largely required only intermittent on demand therapy applied for bleeding episodes or any surgical interventions. Thrombin generation analysis was carried out blindly in plasma obtained from thirteen type 3 VWD patients and eleven type 2 VWD patients. Seventeen healthy volunteers served as a control group. In plasma from type 3 VWD patients, TG was substantially lower than in plasma from type 2 VWD patients, with ETP of 765 nM×min (596-962) vs. 1954 nM×min (1483-2008) (P = 0•001) and peak height of 47 nM (36-65) vs. 262 nM (142-318) (P = 0•002) In order to examine the potential use of emicizumab as an alternative treatment option for type 3 VWD patients, an ex vivo spiking analysis comparing the effect of Haemate P and emicizumab on TG was performed. An improvement in peak height was demonstrated following spiking with both Haemate P concentrations (P = 0•001 for both) and with the higher emicizumab concentration (P = 0•011). Notably, whereas spiking with both Haemate P concentrations increased peak height to near-normal level, spiking with higher emicizumab concentration increased it to a lesser extent (the median was still lower than in normal controls (P = 0•005). Following the decision to treat our impetus patient with emicizumab prophylaxis, TG analyses were performed in the patient's plasma before and during emicizumab loading and maintenance (Figure 3). As expected, patient's initial TG was extremely low and improved following the first administration of emicizumab loading dose (at week 2 after therapy initiation), at which time emicizumab level was 21 µg/mL. Further significant improvement of TG was noted following loading period completion while emicizumab level was 62 µg/mL. Our patient has been treated with emicizumab for more than six months altogether and did not encounter any joint bleeds since the commencement of therapy. During this period, a single dose of Haemate P was administered following tooth exfoliation. Our study contributes towards a better understanding of TG as a surrogate marker of VWD patients' hemostasis. Our data suggests that some severe VWD patients could be safely and efficiently treated with emicizumab. The successful prophylaxis of our patient and our ex vivo laboratory findings should set the ground for further collaborative multicenter studies to examine the efficacy and safety of emicizumab prophylaxis in type 3 VWD patients. Disclosures Barg: roshe: Honoraria, Speakers Bureau. Kenet:PI Healthcare, CSL Behring: Honoraria; Bayer, Pfizer, Takeda, BioMarin, Novo Nordisk: Speakers Bureau; Bayer, Pfizer, Roche, Alnylam (Sanofi), Shire: Research Funding; Bayer, Pfizer, BioMarin, Takeda, Roche, Novo Nordisk, Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The use of Emicizumab in sever Von Willebrand disease

Diagnostic Fidelity of Historically Diagnosed Patients with VWD Enrolled from 27 Centers in the ZPMCB-VWD

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 381-381 ◽  
Author(s):  
Robert R. Montgomery ◽  
Pamela A. Christopherson ◽  
Sandra L Haberichter ◽  
Veronica H Flood ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Von Willebrand Disease ◽  
Blood Type ◽  
Similar Process ◽  
Central Laboratory ◽  
Type 3 ◽  
Von Willebrand ◽  
Normal Controls

Abstract Abstract 381FN2 The TS Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease (ZPMCB-VWD) is a large NIH PPG to study existing subjects with von Willebrand Disease in the United States and to contrast these with prior and ongoing studies in Canada and the UK. 569 index cases (ICs) and 1732 family members were recruited from 8 Primary Clinical Centers and 19 Secondary Clinical Center. The inclusion criteria were that the subjects had a historical diagnosis of VWD and were registered as ongoing patients in the local HTC. 247 normal controls (NCs) were studied for comparison. Data included pre-existing diagnosis, historical diagnostic VWD testing, detailed bleeding history using a modified MCMDM-1VWD QBS, and subjects had plasma and DNA drawn for studies at a central laboratory for VWD, VWF phenotyping, and full length VWF exon sequencing. ICs included 391 type 1, 105 type 2, 43 type 3, and 30 unclassified. The recent HLBI Guidelines suggested that the diagnosis of VWD be based on a VWF level of <30 IU/dL (either VWF:Ag or VWF:RCo). Using the historical data, only 39.4% of ICs had either VWF:Ag or VWF:RCo of <30. Since historic data were studied using various lower limits of normal, we found 80% of subjects had either an Ag or RCO of <60. Central Laboratory testing of NCs and ICs was performed. While 7 of 247 NCs had reduced VWF:Ag or VWF:RCo, none of the NCs had <30 by either assay. Of the NCs with low VWF, 6 of 7 were blood type O and none had an abnormal bleeding score. While 372 of 569 ICs (65.4%) had reduced VWF assays by either method, only 201 (35.3%) had VWF assays that were below the NHLBI Guidelines of <30. Among ICs the correlation between current and historic VWF levels was an r2=0.199 for VWF:RCo and r2=0.268 for VWF:Ag. The current VWF:RCo correlated with the current VWF:Ag with an r2=0.827. The VWF:Ag and VWF:RCo were also restudied in a second laboratory with an r2=0.849 for VWF:Ag and r2=0.854 for VWF:RCo. The MCMDM-1VWD QBS was normal in all normal controls. In contrast, 350 of the ICs born prior to 1996, 247 (70.5%) had an abnormal QBS. Evaluated by pre-existing diagnosis, 65% of type 1 VWD, 78.6% of type 2 VWD and 100% of type 3 VWD had abnormal QBS. Interestingly, of the 351 VWD subjects born before 1996, the QBS was abnormal in 83.5% of males and 67.7% of females. In this age group, there were 248 females and 103 males followed with the diagnosis of VWD. VWF sequencing was carried out on all ICs and compared to the VWF Database maintained at the University of Sheffield. Sequence variations were present in 100% of the 391 type 1 VWD subjects with VWF levels of <10, 96% with VWF 11–20, 77% with VWF 21–30, 70% with VWF 31–40, 39% with VWF 41–50, and 42% of those with VWF 51–60. The decrease in VWF sequence variation between those <40 and those >40 seemed to be striking. The mean VWF:Ag levels of type 1 ICs with a VWF sequence variation was significantly lower (p<0.0001) than those with normal VWF sequence. More than 20 years ago, we demonstrated that besides blood type, age had the most significant effect on level of VWF in normal blood donors. We evaluated the VWF:Ag levels in our 247 NCs and VWF:Ag levels rose about 5% for each 10 years of age from age 20 to 60. In VWD it is not known if a similar process results in reduced bleeding symptoms with age or if this increase is related to the aging process or progressive vascular pathology. Stress is a significant confounding variable in the hemostatic evaluation of children – particularly for the diagnosis of VWD, but the effect of aging has not been defined. We therefore have demonstrated that the diagnosis of VWD was not consistently substantiated in a large group of patients diagnosed with VWD – particularly if using the VWF levels recommended by the NHLBI Guidelines. Historic VWF levels do not correlate with current levels of VWF. Moreover, 35% of these VWD subjects do not have evidence even of reduced VWF levels upon retesting. A number of questions remain. Are the NHLBI recommendations too strict? Is there an effect of aging on the normal level of VWF that affects the correct diagnosis of VWD? Will more rigid initial diagnostic testing improve the fidelity of the diagnosis of VWD? Is the diagnosis of VWD applied to a group of individuals with clinical bleeding with only some of these being associated with low VWF? Further longitudinal study of these VWD subjects is important. In those with significant clinical bleeding, other hemostatic abnormalities must be sought. The fidelity of the diagnosis of VWD needs improvement. Disclosures: Montgomery: GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Biogen IDEC: Honoraria; Bayer: Consultancy; Baxter: Consultancy.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

Characterization of Bleeding in Hemophilia Carriers and Comparison to Women with Type 1 Von Willebrand Disease, Type 3 Von Willebrand Disease Obligate Carriers and Controls

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 875-875
Author(s):  
Natasha Amrita Satkunam ◽  
Johnny Mahlangu ◽  
Christoph Bidlingmaier ◽  
Maria Eva Mingot-Castellano ◽  
Meera B. Chitlur ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Von Willebrand Disease ◽  
Advisory Committees ◽  
Type 3 ◽  
Von Willebrand ◽  
Hemophilia Carriers ◽  
Normal Controls ◽  
Mucocutaneous Bleeding

Abstract Background: Hemophilia carriers report abnormal bleeding, even when factor VIII or IX levels are normal. Information comparing bleeding events between carriers and women with other inherited bleeding disorders is lacking. Purpose: The purpose of our study was to characterize bleeding in hemophilia carriers using the International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) and to compare it with bleeding in normal controls, women with Type 1 VWD and Type 3 VWD obligate carriers (OC). Method: This was a prospective, observational, cross-sectional study performed by members of GEHEP (Global Emerging HEmostasis Panel). Study participants were recruited from GEHEP members' clinics in North America (Kingston, Canada, Detroit and Philadelphia, USA), Europe (Malaga, Spain; Milan, Italy; Munich, Germany; Oslo, Norway) and South Africa (Johannesburg). Potential participants were identified through local patient databases and approached during clinic visits. All participants signed informed consent. Hemophilia carriers were defined by a documented FVIII or FIX mutation and/or by an appropriate family history (daughter of a man with hemophilia or mother of two sons with hemophilia or mother of one son with hemophilia with at least one other affected male relative). Demographic information was collected using a CRF and the ISTH-BAT was completed for each participant by study personnel. Existing ISTH-BAT data for women with Type 1 VWD, Type 3 VWD OC and age-matched female controls were used for comparison. Results: A total of 329 participants were included in this study; 168 hemophilia carriers, 83 women with Type 1 VWD, 32 Type 3 VWD OC and 46 female normal controls. Hemophilia carriers and normal controls were similar in age (40.1 vs 41.6, p=0.445). The mean overall ISTH-BAT bleeding score (BS) was significantly higher in carriers than in controls (5.7 vs 2.48, p<0.0001). Carriers reported significantly more bleeding in the categories of cutaneous, minor wounds, oral cavity bleeding, post-dental bleeding, surgical bleeding, menorrhagia, post-partum bleeding and other when compared with controls. Carriers were older than Type 1 VWD patients (40.1 vs 36.4 years, p=0.042). While women with Type 1 VWD had higher total ISTH-BAT BS (8.7 vs 5.7, p<0.0001) as well as higher scores for epistaxis, cutaneous bleeding, minor wounds, oral cavity bleeding and menorrhagia, hemophilia carriers had significantly higher scores for muscle hematomas and hemarthrosis. Carriers were younger than Type 3 VWD OC (40.1 vs 45.2 years, p = 0.02), had higher overall ISTH-BAT BS (5.7 vs 3.0, p=0.009) and reported more bleeding in the following categories: total score, epistaxis, hematuria, dental, muscle hematomas, hemarthrosis, and other. In fact, hemophilia carriers reported more musculoskeletal bleeding than all other groups. Importantly, given the concern about over-reporting of joint bleeds by hemophilia carriers because of familiarity with hemarthrosis in affected male relatives, no Type 3 VWD OC reported joint bleeds. See Table 1 for detailed results. Conclusion: In summary, our study showed that hemophilia carriers report significantly more bleeding by overall ISTH-BAT BS than age-matched female controls. Carriers experience both mucocutaneous bleeding as well as musculoskeletal bleeding. They score higher for mucocutaneous bleeding when compared with controls and when compared with Type 3 VWD OC. Overall Type 1 VWD patients experience more severe mucocutaneous bleeding than hemophilia carriers. However, hemophilia carriers report more musculoskeletal bleeding in the form of hemarthrosis and hematomas than all other groups. A comparison of overall ISTH-BAT BS between groups shows that bleeding in women with Type 1 VWD > hemophilia carriers > Type 3 VWD OC > controls. Additional research into the underlying pathophysiology of this abnormal bleeding is a critical next step in understanding and determining how to appropriately manage these patients. Disclosures Bidlingmaier: Novo Nordisk: Honoraria; Sobi: Honoraria; Pfizer: Honoraria; Biotest: Honoraria; Baxalta: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding. Mingot-Castellano:Amgen: Consultancy; Pfizer: Consultancy; Novo Nordisk: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; Novartis: Consultancy; Bayer: Consultancy, Research Funding. Chitlur:Novo Nordisk: Consultancy; Baxalta: Honoraria; Bayer: Honoraria; Biogen-Idec: Honoraria; Pfizer: Honoraria. Fogarty:Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter/Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Pfizer: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Research Funding. Cuker:T2 Biosystems: Research Funding; Genzyme: Consultancy; Biogen-Idec: Consultancy, Research Funding; Amgen: Consultancy; Stago: Consultancy. Mancuso:Bayer Healthcare: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; Sobi/Biogen Idec: Consultancy, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Consultancy. Holme:Baxalta, now part of Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Investigator Clinical Studies. Mathew:Bayer: Employment. James:CSL Behring: Research Funding; Octapharma: Research Funding; Biogen: Consultancy; Basalt: Consultancy; Bayer: Research Funding.

scholarly journals Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D′D3 and D4 domains

2020 ◽  
Vol 4 (14) ◽  
pp. 3405-3415 ◽  
Author(s):  
Monica Sacco ◽  
Stefano Lancellotti ◽  
Mattia Ferrarese ◽  
Francesco Bernardi ◽  
Mirko Pinotti ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Von Willebrand Disease ◽  
Blood Analysis ◽  
Force Microscopy ◽  
Transmission Electron ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
In Cis

Abstract We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G&gt;A and (2) c.6532G&gt;T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D′D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient’s daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C&gt;T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B–like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

scholarly journals The disulfide bond Cys2724-Cys2774 in the C-terminal cystine knot domain of von Willebrand factor is critical for its dimerization and secretion

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuxin Zhang ◽  
Fengwu Chen ◽  
Aizhen Yang ◽  
Xiaoying Wang ◽  
Yue Han ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Von Willebrand Disease ◽  
Cystine Knot ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background Type 3 von Willebrand disease (VWD) exhibits severe hemorrhagic tendency with complicated pathogenesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, however, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear. Method and results In this study, by the next-generation sequencing we found a missense mutation a c.8171G>A (C2724Y) in the CTCK domain of VWF allele in a patient family with type 3 VWD. In vitro, VWF C2724Y protein was expressed normally in HEK-293T cells but did not form a dimer or secrete into cell culture medium, suggesting that C2724 is critical for the VWF dimerization, and thus for VWF multimerization and secretion. Conclusions Our findings provide the first genetic evidence for the important role of Cys2724-Cys2774 in VWF biosynthesis and secretion. Therefore, all of the four intrachain disulfide bonds in CTCK monomer contribute to VWF dimerization and secretion.

scholarly journals Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort

2013 ◽  
Vol 110 (08) ◽  
pp. 264-274 ◽  
Author(s):  
Adel Abuzenadah ◽  
Ashley Cartwright ◽  
Nawal Al-Shammari ◽  
Rachael Coyle ◽  
Michaela Eckert ◽  
...  
Keyword(s):  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Missense Mutations ◽  
End Joining ◽  
Turkish Patient ◽  
Non Homologous End Joining ◽  
Type 3 ◽  
Von Willebrand ◽  
High Degree

SummarySeveral cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

scholarly journals Clinical Applications, Pitfalls, and Uncertainties of Thrombin Generation in the Presence of Platelets

2019 ◽  
Vol 9 (1) ◽  
pp. 92 ◽  
Author(s):  
Marina Panova-Noeva ◽  
Paola E.J. van der Meijden ◽  
Hugo ten Cate
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Coagulation Factor ◽  
Bleeding Risk ◽  
Coagulation Factors ◽  
Clinical Applications ◽  
Von Willebrand Disease ◽  
Von Willebrand

Platelet-dependent thrombin generation is a helpful tool to assess ex vivo the interaction between platelets and plasma coagulation factors in the initiation, amplification, and inhibition of thrombin generation (TG). This review article discusses the most relevant available data on the clinical applications of fluorogenic TG, the most widely used TG assay, performed in the presence of platelets, i.e., in platelet-rich plasma. With respect to prothrombotic states, arterial hypertension and obesity were the most prominent cardiovascular conditions linked to increased platelet-dependent TG. In addition, platelet-associated hypercoagulability, assessed by the TG assay, has been shown in individuals with active cancer. In terms of bleeding, platelet-dependent TG has been applied to assess bleeding risk in individuals with hemophilia, von Willebrand disease, and Glanzmann thrombasthenia as well as in subjects with other congenital or acquired coagulation factor deficiencies. In addition to risk prediction, a role of the TG assay has been suggested in monitoring antiplatelet therapy in prothrombotic conditions and replacement therapy in bleeding diathesis. Finally, for the routine clinical use and as a biomarker of disease development and progression, better standardization and clinical validation of platelet-dependent TG are still needed.

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