Immunohistochemical Analyses on Angiogenesis in a Porcine Von Willebrand Disease Model

Abstract Objectives: Women with von Willebrand Disease (VWD) type 3 may develop miscarriage during pregnancy. There is evidence that besides its role in coagulation von Willebrand Factor (VWF) is also involved in angiogenesis and defects of this pathway could play a role in miscarriage. For this reason, VWF as well as proteins with known functions in angiogenesis were analysed by immunohistochemistry of several tissues in a pig model comprising all VWD type 3 genotypes (VWD, heterozygous carriers, wildtype). Methods: Uterus tissue samples including the endometrium were collected from a total of 6 pigs. Of these, two were affected by VWD type 3, two were heterozygous carriers, and the remaining two were wildtype individuals, which served as controls. Hematoxylin-eosin-staining was implemented for morphological evaluation of the tissues, especially with regard to possible differences in structure and pattern of blood vessels. In a second step immunohistochemical analyses comparing the expression of angiogenic factors including VWF were performed in VWD-animals, genetic carriers and wildtype pigs. Results: Hematoxylin-eosin-staining of histological sections of the porcine uteri comprising the different genotypes revealed differences in the vascular architecture within the lamina propria. Compared to non pregnant wild type pigs where blood vessels in the lamina propria of the endometrium usually are of smaller caliber (mostly categorized as arterioles, venules and capillaries), the lamina propria of VWD type 3 animals contained multiple large arteries and veins (of the type usually found in the uterine wall). The immunohistochemical analysis for VWF showed almost no VWF expression in the pigs affected by VWD. In wildtype and heterozygous pigs, expression was obvious in the endothelium. A narrow band of VWF expression apically on the epithel cells as well as partial expression in glands was seen only in wildtype pigs, but not in heterozygous pigs. For some, but not all further angiogenic factors analysed, expression differences were present among the porcine VWD genotypes. Conclusion: The results of our study confirm effects of VWF on the uterine vascular architecture and on the expression of specific angiogenic factors. Pigs affected by VWD showed almost no expression of VWF and the blood vessels within the uterine lamina propria had a surprisingly high percentage of large arteries and veins indicating a crucial role of vWF for uterine angiogenesis. Expression differences among the genotypes were obvious for specific angiogenic factors. Disclosures Von Depka: Octapharma: Other: Study Investigator, Speakers Bureau.

Download Full-text

Characterization of a Porcine Model for Von Willebrand Disease Type 1 and 3 Regarding Expression of Angiogenic Mediators in the Nonpregnant Female Reproductive Tract

Comparative Medicine ◽

10.30802/aalas-cm-19-000003 ◽

2019 ◽

Vol 69 (5) ◽

pp. 401-412 ◽

Cited By ~ 1

Author(s):

Hanna Allerkamp ◽

Stefanie Lehner ◽

Mahnaz Ekhlasi-Hundrieser ◽

Carsten Detering ◽

Christiane Pfarrer ◽

...

Keyword(s):

Gene Expression ◽

Animal Model ◽

Growth Factor ◽

Reproductive Tract ◽

Porcine Model ◽

Angiogenic Factors ◽

Von Willebrand Disease ◽

Female Reproductive Tract ◽

Type 3 ◽

Von Willebrand

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ3 from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

Download Full-text

Clinical manifestations and complications of childbirth and replacement therapy in 385 Iranian patients with type 3 von Willebrand disease

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02507.x ◽

2000 ◽

Vol 111 (4) ◽

pp. 1236-1239 ◽

Cited By ~ 129

Author(s):

M. Lak ◽

F. Peyvandi ◽

P. M. Mannucci

Keyword(s):

Replacement Therapy ◽

Clinical Manifestations ◽

Von Willebrand Disease ◽

Type 3 ◽

Von Willebrand ◽

Iranian Patients

Download Full-text

A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650564 ◽

1996 ◽

Vol 76 (02) ◽

pp. 253-257 ◽

Cited By ~ 14

Author(s):

Takeshi Hagiwara ◽

Hiroshi Inaba ◽

Shinichi Yoshida ◽

Keiko Nagaizumi ◽

Morio Arai ◽

...

Keyword(s):

Missense Mutation ◽

Novel Mutation ◽

Point Mutations ◽

Japanese Patients ◽

Von Willebrand Disease ◽

Homozygous Mutation ◽

Missense Mutations ◽

Reaction Products ◽

Type 3 ◽

Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

Download Full-text

Evaluation of the Abnormal Platelet Function in von Willebrand Disease by the Blood Filtration Test

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650600 ◽

1996 ◽

Vol 76 (03) ◽

pp. 460-468 ◽

Cited By ~ 7

Author(s):

Francesco I Pareti ◽

Marco Cattaneo ◽

Luca Carpinelli ◽

Maddalena L Zighetti ◽

Caterina Bressi ◽

...

Keyword(s):

Platelet Function ◽

Relevant Information ◽

Von Willebrand Disease ◽

Type I ◽

Cumulative Number ◽

Primary Hemostasis ◽

Normal Platelet ◽

Filter Test ◽

Type 3 ◽

Von Willebrand

SummaryWe have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillarysized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with l-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VUI/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ibα and Ilb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.

Download Full-text

Toward Personalized Treatment for Patients with Low von Willebrand Factor and Quantitative von Willebrand Disease

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1722864 ◽

2021 ◽

Vol 47 (02) ◽

pp. 192-200

Author(s):

James S. O'Donnell

Keyword(s):

Von Willebrand Factor ◽

Bleeding Risk ◽

Von Willebrand Disease ◽

Personalized Treatment ◽

Treatment Plans ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

AbstractThe biological mechanisms involved in the pathogenesis of type 2 and type 3 von Willebrand disease (VWD) have been studied extensively. In contrast, although accounting for the majority of VWD cases, the pathobiology underlying partial quantitative VWD has remained somewhat elusive. However, important insights have been attained following several recent cohort studies that have investigated mechanisms in patients with type 1 VWD and low von Willebrand factor (VWF), respectively. These studies have demonstrated that reduced plasma VWF levels may result from either (1) decreased VWF biosynthesis and/or secretion in endothelial cells and (2) pathological increased VWF clearance. In addition, it has become clear that some patients with only mild to moderate reductions in plasma VWF levels in the 30 to 50 IU/dL range may have significant bleeding phenotypes. Importantly in these low VWF patients, bleeding risk fails to correlate with plasma VWF levels and inheritance is typically independent of the VWF gene. Although plasma VWF levels may increase to > 50 IU/dL with progressive aging or pregnancy in these subjects, emerging data suggest that this apparent normalization in VWF levels does not necessarily equate to a complete correction in bleeding phenotype in patients with partial quantitative VWD. In this review, these recent advances in our understanding of quantitative VWD pathogenesis are discussed. Furthermore, the translational implications of these emerging findings are considered, particularly with respect to designing personalized treatment plans for VWD patients undergoing elective procedures.

Download Full-text

68. Hemorrhagic shock due to ruptured hepatic adenoma secondary to oral norethindrone acetate in a patient with type 3 von Willebrand disease: a case report

Journal of Pediatric and Adolescent Gynecology ◽

10.1016/j.jpag.2021.02.072 ◽

2021 ◽

Vol 34 (2) ◽

pp. 266

Author(s):

Preetha Nandi ◽

Kimberly Huhmann ◽

Erin Fee ◽

Andrea Zuckerman

Keyword(s):

Case Report ◽

Hemorrhagic Shock ◽

Von Willebrand Disease ◽

Hepatic Adenoma ◽

Type 3 ◽

Von Willebrand ◽

Norethindrone Acetate

Download Full-text

A common 253-kb deletion involving VWF and TMEM16B in German and Italian patients with severe von Willebrand disease type 3

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2007.02460.x ◽

2007 ◽

Vol 5 (4) ◽

pp. 722-728 ◽

Cited By ~ 26

Author(s):

R. SCHNEPPENHEIM ◽

G. CASTAMAN ◽

A. B. FEDERICI ◽

W. KREUZ ◽

R. MARSCHALEK ◽

...

Keyword(s):

Disease Type ◽

Von Willebrand Disease ◽

Type 3 ◽

Von Willebrand

Download Full-text

Phage Display Broadly Identifies Inhibitor-reactive Regions in von Willebrand Factor

10.1101/2021.03.08.434464 ◽

2021 ◽

Author(s):

Andrew Yee ◽

Manhong Dai ◽

Stacy E. Croteau ◽

Jordan A. Shavit ◽

Steven W. Pipe ◽

...

Keyword(s):

Phage Display ◽

Von Willebrand Factor ◽

Gene Deletion ◽

Von Willebrand Disease ◽

Response To Treatment ◽

Phage Library ◽

Incomplete Removal ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

SummaryBackgroundCorrection of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD).ObjectiveLocate inhibitor-reactive regions within VWF using phage display.MethodsWe screened a phage library displaying random, overlapping fragments covering the full length VWF protein sequence for binding to a commercial anti-VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma-derived VWF. Immunoreactive phage clones were identified and quantified by next generation DNA sequencing (NGS).ResultsNGS markedly increased the number of phage analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma-derived VWF replacement products.ConclusionThese results demonstrate the utility of phage display and NGS to characterize diverse anti-VWF antibody reactivities.

Download Full-text

Rare Occurrence of Inhibitors in Von Willebrand Disease: A Case Report

Frontiers in Medicine ◽

10.3389/fmed.2021.807664 ◽

2022 ◽

Vol 8 ◽

Author(s):

Bipin P. Kulkarni ◽

Kirti Ghargi ◽

Chandrakala Shanmukhaiah ◽

Shrimati D. Shetty

Keyword(s):

Correct Diagnosis ◽

Severe Form ◽

Von Willebrand Disease ◽

Rare Occurrence ◽

Standard Assay ◽

Type 3 ◽

Von Willebrand ◽

High Degree ◽

Willebrand Factor ◽

Inhibitor Titer

Introduction: Type 3 Von Willebrand Disease (VWD) is the least common but the most severe form of a disease, with a prevalence of about 0. 5 to 1 per million in Western countries. The prevalence of type 3 VWD in the developing countries, with a high degree of consanguinity, is about 6 per million. Moreover, due to underdiagnosis of the milder cases, the prevalence of type 3 VWD is about 50% of the cases. Rarely, some patients develop the Von Willebrand Factor (VWF) inhibitors, which may subsequently develop severe anaphylactic reactions on further exposure to the VWF containing factor replacement therapy. The prevalence of inhibitor development in patients with type 3 VWD has been shown to be in the range of 5.8 to 9.5%. In the absence of a gold standard assay for the quantitation of VWF inhibitors, a correct diagnosis and management of these patients are often challenging.Objectives: The objective of this study is to standardize the Bethesda assay for the VWF inhibitors and to estimate the VWD inhibitor titer in two cases of congenital type 3 VWD, which developed the VWF inhibitors.Results and Conclusions: We could successfully standardize the Bethesda assay for the quantitation of VWF inhibitors in two patients with congenital type 3 VWD with inhibitors.

Download Full-text