3P-0842 Contribution of von Willebrand factor to thrombus formation on neointima of rabbit stenotic iliac artery under high blood flow velocity

SummaryThe stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2C1 was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 μM) and chymotrypsin(10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymo-trypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.Electron microscope gold immunocytochemistry using antihuman fibrinogen IgG, anti-von Willebrand factor and anti-fibronectin showed a) that fibrinogen in the vacuoles of degranulated platelets was visible at focal points of platelet contact in the aggregates, but that large areas of platelet contact had no fibrinogen detectable between them; and b) in comparison to fibrinogen, little fibronectin or von Willebrand factor (vWf) was detectable in the platelets.Since the linkages between thrombin-degranulated platelets reaggregated by thrombin can be disrupted either by raising cAMP (thus making glycoprotein IIb/IIIa unavailable) or by proteolysis, these linkages are less stable than those formed between normal platelets. It might therefore be expected that platelets that take part in thrombus formation and then recirculate are likely to form less stable thrombi than platelets that have not released their granule contents.

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Immunohistological Evaluation of Von Willebrand Factor in the Left Atrial Endocardium and Atrial Thrombi from Cats with Cardiomyopathy

Animals ◽

10.3390/ani11051240 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1240

Author(s):

Wan-Ching Cheng ◽

Lois Wilkie ◽

Tsumugi Anne Kurosawa ◽

Melanie Dobromylskyj ◽

Simon Lawrence Priestnall ◽

...

Keyword(s):

Von Willebrand Factor ◽

Left Atrial ◽

Thrombus Formation ◽

Clinical Signs ◽

Naturally Occurring ◽

Feline Model ◽

Von Willebrand ◽

And Control ◽

Endocardial Endothelium ◽

Willebrand Factor

Aortic thromboembolism (ATE) occurs in cats with cardiomyopathy and often results in euthanasia due to poor prognosis. However, the underlying predisposing mechanisms leading to left atrial (LA) thrombus formation are not fully characterised. von Willebrand Factor (vWF) is a marker of endothelium and shows increased expression following endothelial injury. In people with poor LA function and LA remodelling, vWF has been implicated in the development of LA thrombosis. In this study we have shown (1) the expression of endocardial vWF protein detected using immunohistofluorescence was elevated in cats with cardiomyopathy, LA enlargement (LAE) and clinical signs compared to cats with subclinical cardiomyopathy and control cats; (2) vWF was present at the periphery of microthrombi and macrothrombi within the LA where they come into contact with the LA endocardium and (3) vWF was integral to the structure of the macrothrombi retrieved from the atria. These results provide evidence for damage of the endocardial endothelium in the remodelled LA and support a role for endocardial vWF as a pro-thrombotic substrate potentially contributing to the development of ATE in cats with underlying cardiomyopathy and LAE. Results from this naturally occurring feline model may inform research into human thrombogenesis.

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Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

Thrombosis and Haemostasis ◽

10.1160/th10-09-0615 ◽

2011 ◽

Vol 105 (03) ◽

pp. 435-443 ◽

Cited By ~ 12

Author(s):

Veronika Bruno ◽

Rudolf Jarai ◽

Susanne Gruber ◽

Thomas Höchtl ◽

Ivan Brozovic ◽

...

Keyword(s):

Atrial Fibrillation ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Independent Predictor ◽

Thrombus Formation ◽

Critical Role ◽

Inverse Correlation ◽

Von Willebrand ◽

Rhythm Stability ◽

Willebrand Factor

SummaryVon Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

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Abstract 2641: Plasma Von Willebrand Factor and ADAMTS13 Concentrations in Atrial Fibrillation

Circulation ◽

10.1161/circ.116.suppl_16.ii_585-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Takashi Uemura ◽

Koichi Kaikita ◽

Hiroshige Yamabe ◽

Masakazu Matsukawa ◽

Kenji Soejima ◽

...

Keyword(s):

Atrial Fibrillation ◽

Von Willebrand Factor ◽

Left Atrial ◽

Thrombus Formation ◽

Significant Negative Correlation ◽

Platelet Surface ◽

Control Subjects ◽

Number Of Patients ◽

Von Willebrand ◽

Willebrand Factor

Background : Von Willebrand factor (VWF) is released from damaged endothelium, and has a role in platelet aggregation through a receptor on the platelet surface. A metalloprotease that cleaves VWF multimers has been identified, namely, ADAMTS13. We recently reported that the serial changes in plasma VWF and ADAMTS13 antigen levels in patients with acute myocardial infarction (AMI), and that the VWF/ADAMTS13 ratio was a useful prognostic indicator of long-term thrombotic events after AMI. Although previous studies have shown raised plasma VWF in patients with atrial fibrillation (AF), little is known about the role of ADAMTS13 in the pathogenesis of AF. In the present study, we examined the relation between VWF and ADAMTS13 in AF patients. Methods and Results : We measured the plasma VWF and ADAMTS13 antigen levels by ELISA in 45 AF patients and 49 control subjects, and also performed echocardiography to examine the relations between these markers and left atrial or ventricular functions. The plasma VWF antigen levels were significantly higher in AF patients compared with controls (2017±749 vs. 1504±497 mU/ml, P=0.0002). In contrast, the plasma ADAMTS13 antigen levels were significantly lower in AF patients compared with controls (825±181 vs. 911±193 mU/ml, P=0.03). The VWF/ADAMTS13 ratio was significantly higher in AF patients compared with controls (2.59±1.20 vs. 1.75±0.76, P<0.0001). The number of patients who received aspirin and warfarin was significantly higher in AF group than control subjects, however, those medical therapy did not affect the VWF and ADAMTS13 antigen levels. There was significant positive correlation between VWF antigen levels and the left atrial dimension (n=128, r=0.228, P=0.0095). Furthermore, there was significant negative correlation between VWF antigen levels and the left atrial appendage peak flow velocity measured by transesophageal echocardiography (n=23, r=-0.611, p=0.0015). Conclusions : These findings suggest that the balance between VWF and ADAMTS13 levels may play an important role in the intra-atrial thrombus formation in AF patients. The present results would open a new therapeutic target for prevention of thromboembolic complications in AF.

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«ADAMTS13 – VON WILLEBRAND FACTOR – PLATELETS» SYSTEM IN ATYPICAL HEMOLYTIC UREMIC SYNDROME IN CHILDREN

PEDIATRIA Journal named after G N SPERANSKY ◽

10.24110/0031-403x-2021-100-4-12-19 ◽

2021 ◽

Vol 100 (4) ◽

pp. 12-19

Author(s):

Kh.М. Emirova ◽

◽

O.M. Orlova ◽

E.M. Chichuga ◽

А.L. Мuzurov ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Von Willebrand Factor ◽

Thrombus Formation ◽

Atypical Hemolytic Uremic Syndrome ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Fluorescent Substrate ◽

Uremic Syndrome ◽

Von Willebrand ◽

Willebrand Factor

Atypical hemolytic uremic syndrome (aHUS) is an orphan disease caused by hyperactivation of the alternative complement pathway. Objective of the study: to assess the state of the «ADAMTS13 – von Willebrand factor (vWF) – platelets» system in children with aHUS. Materials and methods of research: [by the FRET method (fluorescence resonance energy transfer) for the FRETSVWF73 (Peptide Institude, Inc., Japan)] hydrolysis of the fluorescent substrate and ADAMTS13 antigen [by ELISA using TECHNOZYM® ADAMTS13 5450551 ELISA (Technoclone GmbH, Austria)], vWF activity [for platelet agglutination (aggregation) in the presence of ristomycin (NPO Renam reagent kit for the ALAT-230LA-2 aggregometer, Russia)] and vWF antigen [by ELISA using the TECHNOZYM® vWF kit: Ag 5450201 ELISA (Technoclone GmbH , Austria)]. Results: there was a decrease in the activity and concentration of ADAMTS13 in 63% and 62% of patients, respectively. A decrease in vWF activity was noted in 44% of cases, an increase in its concentration – in 54% of children. Thrombocytopenia was diagnosed in 99% of children. Conclusion: the imbalance in the «ADAMTS13 – vWF – platelets» system supports the process of thrombus formation with the development of organ ischemia in aHUS under conditions of endothelial dysfunction. Reduced ADAMTS13 activity predicts the severity of the disease.

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Computational Mechanical Analysis of the Platelets Behavior in the Blood Flow Using Discrete Element Method

10.1115/imece2000-2534 ◽

2000 ◽

Author(s):

Hisako Miyazaki ◽

Hao Liu ◽

Takami Yamaguchi

Keyword(s):

Blood Flow ◽

Vessel Wall ◽

Thrombus Formation ◽

Mechanical Analysis ◽

Soluble Form ◽

Blood Constituents ◽

Fibrin Network ◽

Insoluble Form ◽

Von Willebrand ◽

Willebrand Factor

Abstract Platelets play an important role in blood coagulation, particularly in the formation of primary thrombi. It is thought that the aggregation of platelets, which initiates primary thrombi formation, is mediated by a macromolecule called von Willebrand Factor (vWF). vWF is a long chain macromolecule that exists in the blood flow as a soluble form and in the vessel wall as an insoluble form. Figure 1 schematically shows normal (a) and pathological (b) thrombus formation processes as illustrated by Ikeda (1998) In both cases, platelets adhere to the injured vessel wall and then form a thrombus in cooperation with the fibrin network, red cells, and other blood constituents. vWF is thought to play a more important role in pathological thrombosis formation than in the normal hemostatic process, particularly due to its ability to react to hemodynamic stress.

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Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice

Thrombosis and Haemostasis ◽

10.1055/s-0040-1702228 ◽

2020 ◽

Vol 120 (03) ◽

pp. 466-476

Author(s):

Sibgha Tahir ◽

Andreas H. Wagner ◽

Steffen Dietzel ◽

Hanna Mannell ◽

Joachim Pircher ◽

...

Keyword(s):

Von Willebrand Factor ◽

Knockout Mice ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Cd40 Ligand ◽

Inflammatory Conditions ◽

String Formation ◽

Von Willebrand ◽

Willebrand Factor

Abstract Background von Willebrand factor (vWF) plays an important role in platelet activation. CD40–CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. Methods and Results The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. Conclusion CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

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Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab

Haematologica ◽

10.3324/haematol.2019.235549 ◽

2019 ◽

Vol 105 (11) ◽

pp. 2631-2638 ◽

Cited By ~ 4

Author(s):

Kazuya Sakai ◽

Tatsuhiko Someya ◽

Kaori Harada ◽

Hideo Yagi ◽

Taei Matsui ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Von Willebrand Factor ◽

Plasmon Resonance ◽

Binding Sites ◽

Thrombus Formation ◽

Static Conditions ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

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