An Essential Role of Factor VIII-Mediated Hemostasis in the Absence of von Willebrand Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3101-3101
Author(s):  
Yoshihiko Sakurai ◽  
Midori Shima ◽  
Shogo Kasuda ◽  
Shoko Omura ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Essential Role ◽  
Bolus Administration ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The replacement therapy with plasma-derived factor VIII (FVIII)/von Willebrand factor (VWF) concentrates is the first line treatment for the patients with type 3 von Willebrand disease (VWD). However, development of anti-VWF alloantibodies (inhibitor) is a major problem since the inhibitor neutralizes the VWF activity and may cause anaphylactic reactions. As an alternative treatment, the usage of FVIII concentrates has been reported but the mechanism of the hemostatic effects remains to be elucidated. Objectives: The purpose of this study is to address the role of FVIII in the hemostatic mechanism in the absence of VWF by in vitro and ex vivo analysis in the treatment for type 3 VWD with recombinant FVIII (rFVIII). Patient/Methods: The patient is a 55-year-old male with type 3 VWD. Blood samples were obtained before and 30 min after bolus administration. Rotating thromboelastometry (ROTEM) assay was performed to examine global interactions in hemostasis. To elucidate the effect on platelet activation, α-thrombin- and shear-induced platelet aggregation studies were performed. Further, α-thrombin-induced [Ca2+]i rise was assessed using fura2-AM loaded platelets. Results and Implications: The patient underwent two surgical procedures of multiple teeth extractions successfully with minimal bleeding by bolus administration of rFVIII (50 IU/kg) before procedure and followed by continuous infusion at rate of 10 IU/kg/h for 15 hours. FVIII:C was elevated from 1.0% to 20~30% 30 min after bolus infusion and maintained ~15% after 12 h-continuous infusion. ROTEM analysis showed that infusion of rFVIII shortened clotting time (preinfusion 2083.8±784.3 sec vs. post-infusion 1022.0±191.5 sec) and clot formation time (pre 1267.3±455.4 sec vs. post 705.8±261.8 sec) and increased α (pre 8.5±7.4 degree vs. post 23.5±4.4 degree). The α value and CFT indicate the rate of increase of elastic shear modulus. Addition of rFVIII to preinfusion blood in vitro corrected ROTEM parameters and thrombin-induced aggregation dose-dependently. Infusion of FVIII enhanced thrombin-induced platelet aggregation (% maximal aggregation: pre 26.3% vs. post 98.2%) as well as low shear-induced platelet aggregation (% maximal aggregation: pre 18% vs. post 52%). Furthermore, infusion of rFVIII meliorated thrombin-induced intracellular calcium flux of washed platelets (thrombin 10 nM, Ca flux: pre 414.0 nM vs. post 620.6 nM). Recently, the cell-based model of hemostasis provides a solid foundation for the relation between platelet and coagulation. Although coagulation initiation occurs normally via the extrinsic pathway, amplification mediated by the intrinsic pathway is seriously disturbed in type 3 VWD due to the marked decrease in FVIII. Therefore, correction of FVIII could result in the improvement of hemostasis. Our data demonstrated the effectiveness of FVIII in the surgical treatment for type 3 VWD and further suggested that FVIII molecules are incorporated into platelet phospholipids to facilitate platelet activation as well as act directly to intrinsic pathways to normalize coagulation. Conclusions: Our observations suggested that FVIII plays an essential role in hemostasis in the absence of VWF and provided the rationale for the usage of rFVIII in the hemostatic management of type 3 VWD.

Adenovirus Induced Thrombocytopenia: The Role of P-Selectin and von Willebrand Factor in Virus- Mediated Platelet Clearance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 222-222 ◽  
Author(s):  
Maha Othman ◽  
Andrea Labelle ◽  
Ian Mazzetti ◽  
David Lillicrap
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Critical Role ◽  
Flow Cytometric Analysis ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Acute thrombocytopenia has been consistently reported following IV administration of adenoviral vectors (Ad) but the mechanism responsible for this phenomenon has not been elucidated. Thrombocytopenia appears 24 hours after IV administration of Ad and is vector dose dependent. In this study, we have assessed the potential roles of the adhesive proteins P-selectin and von Willebrand Factor (VWF) on the aggregation and clearance of platelets following virus administration. We have addressed the question of whether the thrombocytopenia is due to a direct effect of the virus on platelets or an indirect effect related to interaction of platelets with other proteins or cells modified by the virus. We assessed platelet count in a group of Balb/c and C57Bl/6 mice over 1 week period following Ad administration and performed a detailed examination of the events within the first 24 h after Ad injection, the period that precedes the appearance of thrombocytopenia. We examined the effect of Ad on expression of the platelet activation marker P-selectin and the formation of platelet leukocyte aggregates (PLA) by means of flowcytometry after incubation of adenovirus with mouse platelets in vitro, and following Ad administration in vivo. To assess the role of VWF in Ad-induced thrombocytopenia we measured plasma VWF levels one hour after injection of Ad. Further investigations involved comparison of platelet counts, platelet activation, and the formation of PLA in a group of VWF KO mice. All studies have been performed with a replication deficient E1/E3-deleted Ad 1x 1011 viral particles/mouse. Our in vitro studies have shown that Ad directly activates mouse platelets as shown by increased expression of P-selectin. The average index of platelet activation for platelets stimulated by Ad was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. Our in vivo studies have confirmed the development of significant thrombocytopenia in both Balb/c as well as C57Bl/6 WT mice (n=8, p=0.00001, n= 6, p=0.002) 24 hours following Ad administration. Significant P-selectin expression was documented in both strains (n=4,p=0.0003; n=3, p=0.0008 respectively) as well as significant PLA one hour following Ad (n=4, p=0.01; n=3, p=0.007). The VWF KO mice showed non-significant thrombocytopenia (n= 6, p=0.063) at 24 hours following Ad, significant P-selectin expression (n=3, p=0.0003), but no significant PLA formation at one hour (n=3 p=0.12) relative to pre-injection levels. Plasma VWF levels were significantly elevated in both Balb/c and C57Bl/6 WT mice one hour following administration of the virus (n= 3, p=0.02; n= 3, p= 0.001). The average plasma VWF levels were 48.1 U/mL at 1h compared to 5.7 U/mL pre injection in Balb /c mice and 85.9 U/mL compared to 6.1 U/mL in C57Bl/6 mice. These studies have shown that Ad can act as an inducer of mouse platelet activation and as a promoter for platelet-leukocyte association both in vitro and in vivo. We have demonstrated a role for Ad in stimulating VWF release from the endothelium, and have shown that VWF has a critical role in platelet activation and clearance following Ad administration. We conclude that P-selectin and VWF proteins are directly involved in interactions between endothelial cells, platelets and leukocytes, a complex interaction that can explain at least in part the mechanisms underlying Ad-mediated thrombocytopenia.

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.

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.

Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

1981 ◽  
Author(s):  
F Fauvel ◽  
Y J Legrand ◽  
N Gutman ◽  
J P Muh ◽  
G Tobelem ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Human Artery ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
Aggregation Of Platelets

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.

scholarly journals Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin

2001 ◽  
Vol 134 (5) ◽  
pp. 1104-1112 ◽  
Author(s):  
Paul Jurasz ◽  
Michael W Stewart ◽  
Anna Radomski ◽  
Fadi Khadour ◽  
Marek Duszyk ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Tumour Cell ◽  
Differential Regulation ◽  
Von Willebrand ◽  
Willebrand Factor

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1535-1535
Author(s):  
Suzana M. Zorca ◽  
Emma C. Josefsson ◽  
Viktoria Rumjantseva ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Lectin Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation. The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

Filamin A binding to the cytoplasmic tail of glycoprotein Ibα regulates von Willebrand factor–induced platelet activation

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2122-2129 ◽  
Author(s):  
Shuju Feng ◽  
Julio C. Reséndiz ◽  
Xin Lu ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Fusion Protein ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Filamin A ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibα (GpIbα) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)–induced platelet activation. To begin, we examined filamin binding to GpIbα in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbα's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbα's filamin A–binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbα cytoplasmic domain fusion protein. One peptide (residues 557-575; designated “A4 peptide”) inhibited filamin A binding to the GST-GpIbα cytoplasmic domain fusion protein and competed with GpIbα for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbα and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbα regulates proaggregatory tyrosine kinase signaling.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

2012 ◽  
Vol 107 (06) ◽  
pp. 1107-1121 ◽  
Author(s):  
Thomas Vanassche ◽  
Alexandre Kauskot ◽  
Jan Verhaegen ◽  
Willy Peetermans ◽  
Joanne van Ryn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Platelet Aggregation ◽  
Typical Feature ◽  
Human Platelets ◽  
Fibrin Scaffold ◽  
Immunoglobulin Binding ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryInteractions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

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