Amounts Of Von Willebrand Factor Research Articles

Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells

Weibel–Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.

Inside the Thrombus: Association of Hemostatic Parameters With Outcomes in Large Vessel Stroke Patients.

Background: Actual clinical management of ischemic stroke (IS) is based on restoring cerebral blood flow using tissue plasminogen activator (tPA) and/or endovascular treatment (EVT). Mechanical thrombectomy has permitted the analysis of thrombus structural and cellular classic components. Nevertheless, histological assessment of hemostatic parameters such as thrombin-activatable fibrinolysis inhibitor (TAFI) and matrix metalloproteinase 10 (MMP-10) remains unknown, although their presence could determine thrombus stability and its response to thrombolytic treatment, improving patient's outcome.Methods: We collected thrombi (n = 45) from large vessel occlusion (LVO) stroke patients (n = 53) and performed a histological analysis of different hemostatic parameters [TAFI, MMP-10, von Willebrand factor (VWF), and fibrin] and cellular components (erythrocytes, leukocytes, macrophages, lymphocytes, and platelets). Additionally, we evaluated the association of these parameters with plasma levels of MMP-10, TAFI and VWF activity and recorded clinical variables.Results: In this study, we report for the first time the presence of MMP-10 and TAFI in all thrombi collected from LVO patients. Both proteins were localized in regions of inflammatory cells, surrounded by erythrocyte and platelet-rich areas, and their content was significantly associated (r = 0.41, p < 0.01). Thrombus TAFI was lower in patients who died during the first 3 months after stroke onset [odds ratio (OR) (95%CI); 0.59 (0.36–0.98), p = 0.043]. Likewise, we observed that thrombus MMP-10 was inversely correlated with the amount of VWF (r = −0.30, p < 0.05). Besides, VWF was associated with the presence of leukocytes (r = 0.37, p < 0.05), platelets (r = 0.32, p < 0.05), and 3 months mortality [OR (95%CI); 4.5 (1.2–17.1), p = 0.029]. Finally, plasma levels of TAFI correlated with circulating and thrombus platelets, while plasma MMP-10 was associated with cardiovascular risk factors and functional dependence at 3 months.Conclusions: The present study suggests that the composition and distribution of thrombus hemostatic components might have clinical impact by influencing the response to pharmacological and mechanical therapies as well as guiding the development of new therapeutic strategies.

Traumatic Hyphema in a Type 1 Von Willebrand Patient: Clinical Case and Management

Von Willebrand disease (vWD) is the most common inherited congenital bleeding disorder. Type 1 is a deficiency, but not absence, of von Willebrand factor (vWF) and is the most common type. Bleeding episodes in patients with type 1 vWD vary and are related to the relative amount of von Willebrand factor produced. We report a case of a 15-year-old male with mild von Willebrand disease type 1 (vWF: 35%) who presented with a right eye grade lll hyphema and elevated intraocular pressure (IOP) following a traumatic injury. Despite medical management with Diamox, ophthalmic steroids, vWF replacement with Humate-P, the patient had a repeat bleeding event and worsening of his elevated intraocular pressure. Due to concern for corneal blood staining, the patient required surgical intervention with an anterior chamber washout. Von Willebrand factor replacement was given on presentation and prior to the procedure with goal von Willebrand factor (vWF) activity and Factor Vlll (FVlll) levels of 80-100 IU/dL. Factor replacement for a traumatic hyphema is necessary to reduce bleeding, but could lead to increased risk of thrombosis formation and increase risk of corneal staining. Balance between bleeding and thrombosis can lead to treatment challenges in patients with bleeding disorders including vWD. Disclosures Smink: Forma Therapeutics:Membership on an entity's Board of Directors or advisory committees;Highmark Insurance:Membership on an entity's Board of Directors or advisory committees.

Immune Tolerance Induction (ITI) in Paediatric and Adult Patients with Haemophilia a (HA) and High-Titre (HT) Factor VIII (FVIII) Inhibitor - Real Life Data

Introduction: In Poland, the principles for the management of rare bleeding disorders as well as financing of medication are regulated by the National Program for the Management of Haemophilia and Related Bleeding Disorders. One objective of the Program for 2012-2018 was to include the financing of ITI for all haemophilia patients with inhibitor. The qualification for ITI was performed by a physician from a Haemophilia Treatment Centre (HTC). Goal: the aim of the study is/was prospective assessment of the efficacy of ITI in real life setting. Patients and Methods: The study comprised 30 patients with severe and one with moderate HA and HT FVIII inhibitors. Factor VIII activity (FVIII:C) was measured by one-stage clotting assay and Factor VIII inhibitors by a modified Nijmegen-Bethesda assay and expressed in Bethesda Units/ml (BU/ml). Cut-off level for positive inhibitor was ≥0.5BU/ml. The following data was collected: historical peak inhibitor titre (HPT), pre-ITI titre (measured 1 month prior to ITI), peak titre recorded during the course of ITI, time between inhibitor detection and ITI start, and ITI duration. During ITI course blood samples were collected every 4 weeks. Treatment regimen with dosing and type of FVIII concentrate was at the discretion of the physician in each HTC. ITI success was defined as inhibitor titre below 0.5 BU/ml with FVIII half-life ≥6h and FVIII recovery ≥66% of normal. Partial response to ITI was defined as clinical response to FVIII therapy with no anamnestic response despite the presence of FVIII inhibitor or FVIII half-life &lt;6h and FVIII recovery &lt;66% of normal. Results: 31 patients with HA and inhibitor were divided into two groups: Group 1 comprised 12 adults with severe HA, median age 36 years (mean 40.1±15.1 and Group 2 included 19 boys, (&lt;18 years) (18 with severe, one with moderate HA), median age 8 years (mean 8.1±4.3) The median (mean) duration time between inhibitor detection and ITI start was 108 months (102±91) in Group 1 and 10 months (9±10) in Group 2. The median (mean) HPT before ITI was 68 BU/ml (156±193) and 50 BU/ml (126±242) in Group 1 and 2, respectively. ITI is still ongoing in one patient (Group 1) and in 3 boys (Group 2); these 4 patients were excluded from outcome analysis. Two adult patients (16,6%) of Group 1 and 2 boys (10,5%) of group 2 discontinued ITI therapy for a variety of reasons such as death (one patient), poor compliance or loss to follow-up; those patients were however included in the outcome analysis. The median (mean) duration of ITI was 24 months (30.8±31.7) (1-120) in Group 1 and 28 months (33.4±21.4) (6-84) in Group 2. The median (mean) peak titre during ITI was 40 BU/ml (225.7±185.7) in Group 1 and 85 (160±171.7) in Group 2. Immune tolerance to FVIII (success) was achieved in 3/11 adult (27.3%) and in 6/16 paediatric patients (37.5%). Median HPT, median time between inhibitor detection and ITI start, median peak inhibitor titre during ITI and median duration of ITI in patients who achieved immune tolerance were 380 BU/ml, 120 months, 8 BU/ml, 33 months in Group 1 and 22.5 BU/ml, 9.5 months, 205 BU/ml, 25 months in Group 2, respectively. No partial response to ITI was observed in our patients. The most common initial ITI regimen in both groups was 100 IU/kg/d of FVIII (50 - 200 IU/kg/d). In 28/31 (90.3%) patients only plasma derived FVIII concentrates were used which contained various amounts of von Willebrand factor. In two patients recombinant FVIII was administered. Recombinant, plasma derived and EHL FVIII concentrates were used in the case of one boy. In two patients immunosuppressive therapy (prednisone or rituximab) was concomitantly applied. Conclusions: it is our belief that the presented real-life data reflects i) the challenge of ITI therapy in clinical practice and ii) wide variety of approaches to ITI strategy in individual haemophilia treatment centres. Disclosures Windyga: Octapharma: Honoraria, Research Funding; Rigel Pharmaceuticals: Honoraria, Research Funding; Ferring Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Alnylam: Honoraria, Research Funding; Alexion: Honoraria, Research Funding; Werfen: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Baxalta/Shire (a Takeda company): Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Siemens: Honoraria, Research Funding; Sobi: Honoraria, Research Funding.

Quantitative analysis of desmopressin (DDAVP) response in adult patients with type 1 von Willebrand disease.

Patients with Type 1 von Willebrand disease (VWD) have reduced amounts of von Willebrand factor (VWF) in their blood. Desmopressin (DDAVP) has been used to raise the blood levels of VWF in these patients. However, not all patients with Type 1 VWD are known to respond to DDAVP therapy. We sought to compare the levels of Factor VIII, VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and fibrinogen at different time points after administration of DDAVP. A retrospective analysis was conducted at a single institution on 89 patients with Type 1 VWD who received a single intravenous dose of 0.3μg/kg DDAVP. Levels of Factor VIII, VWF:Ag, VWF:RCo, and fibrinogen were measured before and then 30, 60, 90, and 120minutes after completion of DDAVP administration. Median levels of Factor VIII, VWF:Ag, and VWF:RCo were significantly elevated (P<0.001) at 30 (175IU/dL, 111IU/dL, 127IU/dL), 60 (218IU/dL, 138IU/dL, 139IU/dL), and 90 (212IU/dL, 134IU/dL, 130IU/dL) minutes time points when compared to their respective baseline values (71IU/dL, 54IU/dL, 54IU/dL). At 60minutes, 96% of subjects were responders, but three subjects failed to respond at any time point. Measurement of levels of Factor VIII, VWF:Ag, and VWF:RCo, optimally at 60minutes after the DDAVP infusion, is sufficient to confirm a patient as a responder in a DDAVP challenge test. Curtailing the number of time points of measurement will result in significant savings in cost and time to patients and their providers.

A Rare Case of Delirium: Acquired Thrombotic Thrombocytopenic Purpura in an HIV Patient

IntroductionThrombotic thrombocytopenic purpura (TTP) is a rare disorder with incidence of one in a million. Manifestations of TTP include fever, thrombocytopenia, thrombotic microangiopathy affecting the brain, heart and kidney causing neurological symptoms, and hemolytic anemia. Not all patients with TTP will have each and every manifestation, and therefore a high index of suspicion is necessary when managing a patient with microangiopathic hemolytic anemia and thrombocytopenia. In recent years, increased incidence of TTP has been reported in patients with coexisting Human immunodeficiency virus (HIV)infection .Clinical CaseA 55 year old female with unknown past medical history presented with confusion and altered sensorium after she was found in the airport wandering. When questioned, she was oriented to self only, and gave single phrase answers.Vitals on admission were significant only for tachycardia with HR of 125 beats per minute. Rest of physical exam was normal. Lab work revealed severe anemia (Hemoglobin 5.1), thrombocytopenia ( Platelet count 6), borderline renal function( BUN 30, serum Creatinine 1.1) and Schistocytes on peripheral smear. TTP was the presumptive diagnosis. Without evidence of sepsis, she was treated with intravenous fluids, systemic steroids, blood products (pure red cell concentrate and fresh frozen plasma) and plasmapheresis. Patient was found to be HIV positive, and had acquired immunodeficiency syndrome with CD4 count of 193. ADAMTS13 inhibitor was 1.3 (nl <0.4) and ADAMTS13 activity level was <3 (normal range 68-163%).Patient's mental status improved to baseline over next 3 days and, renal failure and encephalopathy resolved. Upon further investigation, she recalled having a needle stick injury around 10 years ago. She was started on elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide (Genvoya) and prophylactic antibiotics prior to discharge.DiscussionDespite its rare nature, TTP can be fatal if one fails to make a proper timely diagnosis. If left untreated, the mortality can reach up to 90% but with empiric treatment and therapeutic plasma exchange up to 85% survival rate can be achieved.Through this case report, we hope to raise the awareness of this disorder with its particular association with HIV infection. The exact mechanism for HIV related TTP is unknown, but some proposed ones include the release of large amounts of von Willebrand factor and downregulation of ADAMTS13 and/or the production of antibodies against ADAMTS13. ADAMTS13 activity levels below 10% are seen in acquired and hereditary TTP, but not all patients with such exhibit low levels with this assay. Acquired TTP with low levels of ADAMTS13 has been increasingly reported in association with HIV infection, such as in this case. DisclosuresNo relevant conflicts of interest to declare.

JAK2V617F - Positive Endothelial Cells Display Pro-Thrombotic Characteristics

Background: Clinical complications of BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) include mainly vascular events and on longer term transformation to Myelofibrosis (MF) or acute leukemia. MPNs are also associated with neo-angiogenesis leading to extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. The most frequent molecular alteration present in hematopoietic cells is the JAK2V617F mutation, which has also been detected in hepatic Endothelial Cells (ECs) of MPN patients with Budd-Chiari syndrome (BCS) and in ECs derived from patients' splenic capillaries. The consequences of the presence of the JAK2V617F mutation into the endothelial lineage are not known although these cells play an important role in thrombosis.Methods: Isogenic JAK2 wild-type (WT) and JAK2V617F mutant induced pluripotent stem cells (iPSC) were derived from an MPN patient. The reprogrammed clonal cells were induced to differentiate toward mesoderm for 3 days and then driven toward the endothelial lineage using Stem-Pro medium, Vascular Endothelial Growth Factor (VEGF) and Forskolin. Enriched CD34+/CD144+ progenitors were then matured in VEGF-supplemented Stem-Pro medium for 5 days before being cultured in the EGM-2 specific EC medium in the presence of the TGF-β inhibitor SB-431542. Cells were harvested and tested for specific surface markers by flow cytometry before expanding them; passages 3 to 6 were used for further characterization and functional studies. Transcriptomic analysis was performed in triplicate from two independent endothelial differentiation experiments using Affymetrix HTA 2.0 gene expression arrays.Results: Phenotypically, the endothelial-like cells obtained after differentiation from both mutant and WT JAK2 iPSC displayed a classical EC morphology and were positive for CD34 and EC markers (CD144, CD309, CD31 and ICAM-1). Such characteristics were conserved for at least 6 rounds of cell passaging. TNF-α treatment increased ICAM-1 cell surface levels and induced the expression of VCAM-1, as observed in other EC models. Our iPS-derived cells were also responsive to VEGF, which induced cellular proliferation and angiogenesis demonstrated by tube formation in Matrigel with both JAK2V617F and WT ECs. As expected, JAK2V617F ECs exhibited higher levels of phosphorylated forms of JAK2, STAT3 and Akt in a serum and growth factor-reduced medium, showing that, as in hematopoietic cells, the presence of the mutation increases basal intracellular signaling. Interestingly, Weibel-Palade bodies detection by confocal microscopy revealed a more important amount of Von Willebrand factor (vWF) in JAK2V617F ECs (2-fold increase), suggesting a higher pro-thrombotic potential for these cells. Basal surface expression of the adhesion molecule ICAM-1 was also clearly increased (1.8-fold) compared to WT ECs suggesting that JAK2V617F ECs may be more prone to recruit activated leukocytes expressing LFA-1, the ICAM-1 receptor. Gene expression microarrays analysis confirmed a significantly higher expression of cell adhesion molecules involved in leukocyte or platelet adhesion in JAK2V617F vs. WT ECs (ICAM-1, VCAM-1, or P-selectin), together with several inflammatory chemokines (IL8, CXCL1, or CXCL5), chemokine receptors (CXCR4 or IL7R) and extracellular matrix modulators (Periostin, Tenascin C, Lysyl Oxidase, or MMP-10) . Finally, Gene Set Enrichment Analysis pointed out gene sets induced in situations of inflammation, response to cytokines and hypoxia. These results could be in agreement with an activated endothelium, leukocyte recruitment and vascular remodeling.Conclusion: Previous discovery of JAK2V617F-positive endothelial cells in the splanchnic vasculature of patients with BCS or MF was of particular interest because of the well-known association between thrombosis and MPN. In this work we first demonstrate that the presence of JAK2V617F doesn't impair endothelial differentiation from stem/progenitor cells. Moreover, JAK2V617F-mutant ECs display features of pro-inflammatory and pro-thrombotic EC activation, which we are currently further characterizing with additional functional studies. Finally, this iPSC model may represent an effective screening tool for molecules antagonizing the pro-thrombotic events in a JAK2V617F context. DisclosuresKiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII

It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

The Ability of Von Willebrand Factor (VWF) to Bind Factor VIII (FVIII) Can Decrease during Pregnancy

Background: The coagulation protein von Willebrand Factor (VWF) and its circulating partner protein, Factor VIII, are known to be elevated in pregnancy. However, the nature of changes in VWF which occur during pregnancy are not well understood. We previously presented results from a study of 46 healthy pregnancies in which we measured VWF antigen (VWF:Ag) and Factor VIII (FVIII determined both by activity and antigen measurements). We found that VWF:Ag and FVIII increased during pregnancy, consistent with less FVIII relative to VWF. We have also observed that VWF multimer size can decrease during pregnancy. We hypothesized that pregnancy can induced molecular changes in VWF, and that such changes could impact the interaction of VWF with its ligands, specifically FVIII.Methods: We adapted a Type 2N von Willebrand Disease (VWD) assay to test the ability of plasma VWF to bind recombinant FVIII. In brief, serial dilutions of plasma VWF were captured using a polyclonal anti-VWF antibody (DAKO), endogenous FVIII was stripped using a CaCl2 wash, recombinant FVIII (Recombinate) was applied, and bound FVIII was detected using an anti-FVIII antibody (Cedarlane). In parallel, a VWF:Ag ELISA was performed using the same anti-VWF capture antibody. The VWF-FVIII binding ratio (VWF-FVIIIb) was then determined by dividing the quantity of rFVIII detected by the amount of VWF:Ag immobilized on the plate. The range, reproducibility, and variance of the assay was first determined in a healthy blood donor repository. We then tested VWF-FVIIIb in healthy pregnancies for which 3rd trimester and non-pregnant baseline samples were available. Additional time points during pregnancy were also studied, when available. VWF-FVIIIb results for each trimester and the non-pregnant baseline were analyzed by one-way ANOVA (All Pairwise Multiple Comparison Procedures, Holm-Sidak method).Results: Forty-six healthy pregnancies with non-pregnant baseline samples were studied. Samples tested were drawn in the first (n=6), second (n=38), and third (n=43) trimesters, at 38 weeks gestational age (n=13), and at a non-pregnant time point (baseline). In non-pregnant samples, the VWF-FVIIIb was close to 1.0 (0.988 +/- 0.084), similar to a normal blood donor repository. We identified a significant decrease in VWF-FVIIIb in the second trimester (0.931 +/- 0.82; p = 0.013), which became more pronounced in the third trimester (0.899 +/- 0.75; p < 0.001) and at 38 weeks gestational age (0.88 +/- 0.072; p < 0.001). The means, quartiles, and standard deviations for each pregnancy time point are shown in Figure 1.Conclusions: These data support the hypothesis that VWF can acquire a decreased ability to interact with FVIII during pregnancy. A decreased capacity to carry FVIII could contribute to the increased VWF:FVIII ratio we and others have observed during pregnancy, although other factors, such as increased VWF production relative to FVIII, could also play a role. This work provides further evidence for a new model of acquired VWF changes in the setting of pregnancy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical use of factor VIII and factor IX concentrates.

Factor VIII (FVIII) and factor IX (FIX) are the cofactor and the pro-enzyme, respectively, acting in the tenase complex, a key mechanism of physiological haemostasis in which a phospholipide-dependent reaction produces the activation of factor X1. Mutations in the genes encoding for coagulation FVIII or IX, both located in the X chromosome, are responsible for haemophilia A and haemophilia B, respectively. These congenital bleeding disorders occur overall in approximately 1 in 5,000 male live births without racial predilection. However, the prevalence of haemophilia A is estimated 4–6 times higher than haemophilia B2–3. According to the residual endogenous FVIII/FIX concentrations, individuals with plasma factor levels 5–40 IU/dL are defined as moderate and mild haemophiliacs4. Although the bleeding phenotype is recognised to be heterogeneous and affected by many genetic and environmental factors5–6, this classification reflects rather closely the severity of clinical symptoms. Thus, patients with mild haemophilia experience excessive bleeding in the large majority of cases in response to surgery, other invasive procedures or major injuries4,7. On the other hand, patients with moderate haemophilia experience excessive bleeding after relatively minor trauma and those with severe haemophilia bleed spontaneously or after trivial trauma4,8. In the latter, haemarthrosis, particularly into hinged joints (ankle, knee, elbow), is largely the most frequently reported bleeding symptom8,9. Recurrent joint bleeding is associated with progressive joint morbidity (haemophilic arthropathy), leading to chronic pain and disability, that often warrant orthopaedic surgery and joint replacement10. Soft-tissue haematomas, mucosal, parenchimal and delayed post-surgical bleeding are less common manifestations, including life-threatening intracranial, neck-throat and gastro-intestinal haemorrhages8,9,11. Replacement therapy with intravenously delivered plasma-derived or recombinant FVIII and FIX concentrates, aimed at correcting the coagulation factor deficiency in the case of, or in order to prevent, bleeding is the cornerstone of management of patients with haemophilia A and haemophilia B, respectively. Indeed, these specific and viral-inactivated products are the source of choice of the missing proteins, in preference to fresh frozen plasma (for both factors), cryoprecipitate (for FVIII) or prothrombin complex concentrates (for FIX)11. The two forms of haemophilia have been usually considered indistinguishable from a clinical point of view and, consequently, as regards patterns of treatment. In this respect, most literature data have been achieved in cohorts of patients with haemophilia A and extrapolated to those with haemophilia B. However, according to recent findings, the bleeding tendency associated with FIX deficiency is likely to be less severe, with possible better long-term outcomes12. Plasma-derived FVIII concentrates may also contain variable amounts of von Willebrand factor (VWF), therefore some products are used in the management of those patients with von Willebrand disease with severe VWF deficiencies, in whom response to endogenous release by desmopressin is impossible or insufficient13. After a historical overview of treatment of haemophilia, this article will report the current clinical use of FVIII and FIX concentrates, also with reference to the Italian clinical scenario.

Long-Term (Postnatal Day 70) Outcome and Safety of Intratracheal Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Neonatal Hyperoxic Lung Injury

PurposeThis study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model.Materials and MethodsNewborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5×105) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated.ResultsImpaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation.ConclusionThe protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

Increased amounts of von Willebrand factor are bound to microparticles after infusion of desmopressin

Effects of desmopressin (DDAVP) in platelet disorders and primary haemostasis cannot be attributed solely to the increase in FVIII/VWF (von Willebrand factor), as VWF/FVIII concentrates have no effect in these circumstances. Microparticles (MP) can support haemostasis by expression of phospholipids, tissue factor and VWF on their surface. We hypothesized that significant amounts of VWF are bound to MP after DDAVP administration and that consequently depletion of MP should influence VWF:Ag and VWF:RCo plasma levels. Platelet-poor plasma was either obtained well from healthy controls or before and after DDAVP administration from patients with von Willebrand's disease (type 1 or possible type 1) or patients with other bleeding disorders as controls. Concentrations of MP and VWF parameters were determined before and after MP depletion by different methods (magnetic bead selection, plasma microfiltration, ultracentrifugation). Platelet MP and VWF-bearing MP were significantly increased after DDAVP. MP depletion by magnetic bead selection led to a significant reduction in VWF:Ag (-18.0%) and VWF:RCo (-27.7%) plasma levels without changes in VWF multimer composition. As results were similar for DDAVP control subjects, the amount of VWF bound to circulating microparticles was significantly higher after DDAVP administration compared with healthy controls (reduction -11.7%). DDAVP leads to a release of microparticles and increases the amount of VWF bound to microparticles which might explain the clinical efficacy of DDAVP in platelet disorders.

HIV-associated Thrombotic Thrombocytopenic Purpura — What We Know So Far

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by microvascular platelet deposition and thrombus formation in selected organs, resulting in microangiopathic haemolytic anaemia, thrombocytopenia, neurological symptoms and renal failure. Typically a very rare disorder, TTP is being seen with increased frequency in patients infected with the human immunodeficiency virus (HIV). Deficiency of the von Willebrand factor cleavage protease, ADAMTS13, has been implicated as the cause of TTP. However, the pathophysiology of HIV-associated TTP and the thrombotic potential in these patients are not known. This article provides not only an overview of the literature regarding HIV-associated TTP, but also presents new data on this disease. We propose a mechanism for the initial onset of HIV-associated TTP that includes the release of extreme amounts of von Willebrand factor and the downregulation of ADAMTS13 and/or the production of autoantibodies to ADAMTS13.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

VWF/FVIII concentrates in high‐risk immunotolerance: the RESIST study

Immune tolerance induction (ITI), through the regular infusion of coagulation factor concentrates over a time period ranging from 1 to more than 24 months, is successful in about 75% of high responders. Among the issues of ITI treatment that are still open, the choice of the product to use is one of the most difficult. In fact, common practice is to start with the same product that induced the inhibitory response, but recent findings indicated that plasma-derived products containing large amounts of von Willebrand factor (VWF) can play a positive role. Two retrospective cohorts in Germany and in France and one prospective cohort have shown a high rate of success when VWF/factor VIII (FVIII) products are used to induce ITI. For these reasons, two prospective studies have been planned to complement the international ITI study already started: an observational study in patients who had already experienced a failure with a VWF-free FVIII concentrate, called RESIST(exp) (experienced); a randomized, controlled study in patients who have never tried an ITI treatment before and at high risk to fail, called RESIST(naïve) (naïve).

A Tiered Management Program To Conserve Replacement Therapy Resources for a Rural Population of Women with Type 2M von Willebrand Disease during Labor and Delivery.

Introduction: Type 2M Von Willebrand Disease (VWD) results from a mutation in the Von Willebrand factor (VWF) gene resulting in decreased or absent binding to platelet glycoprotein Ib. Bleeding episodes in type 2M VWD are variable, but can be severe or life-threatening; effective treatment requires administration of exogenous normal VWF. Pregnant women with type 2M VWD require close monitoring to prevent adverse bleeding events. Following a fatal post-partum hemorrhage in a woman from a large kindred with type 2M VWD, routine prophylactic doses of VWF were administered both pre- and postpartum to women with this disorder. This population is uninsured and utilized product is donated for their care. To determine if the amount of VWF could be safely reduced, a tiered risk stratification and replacement therapy program was developed to manage women with type 2M vWD during labor, delivery and the peuperium. We report the results of this tiered risk stratification in terms of the total product utilized and the rate of bleeding experienced.Methods: Between Jan–Mar 2007, we followed 7 Amish women from a large kindred with type 2M VWD (mutation 4120 C-->T on exon 28). This rural community has limited access to sophisticated hematologic monitoring and is uninsured. Women were risk stratified into 1 of 3 tiers (T) based on their personal or immediate family history of bleeding with childbirth. A nurse provided peripartum monitoring via home visits on postpartum days 1, 3, 5, 10 with a portable hematocrit (Hct) machine. Women in T1 were at lowest risk and given VWF replacement only in the event of excessive bleeding or postpartum Hct <30; T2 (intermediate risk) were given a single prophylactic VWF replacement dose immediately prior to delivery then monitored as T1; T3 received prophylactic VWF dose immediately prior to delivery, at 12 hours postpartum, then monitored by serial Hct per the other tiers. Women requiring Cesarean section were placed into T3 but treated with further VWF replacement due to the surgical intervention.Results: The 7 women fell into the following tiers; T1: 4; T2: 2; T3: 1. Of the 4 women in T1, none experienced excessive bleeding and no VWF therapy was required. In the 2 women in T2, there were no bleeding events requiring additional VWF. Postpartum Hct remained >30 on all evaluations in T1 and 2. One woman was delivered by elective caesarean section for complicated twin pregnancy, and given VWF prior to surgery and once daily for three consecutive postoperative days. She had no bleeding complications, and maintained Hct >30. Compared with the prior method of required pre- and post-partum prophylactic VWF administration regardless of tier or Hct, this pilot protocol safely decreased VWF usage in women assigned to T1 and 2 without adverse bleeding outcome or the need for transfusion.Conclusions: Women with type 2M VWD can be safely managed with a tiered risk stratification system and close postpartum clinical/Hct monitoring that utilizes conservative VWF replacement. For T1 and 2 this lead to a reduction in VWF administration of 83% compared with the prior method of mandatory pre- and postpartum VWF administration. This further translated into a substantial cost savings while still achieving safe hemostatic outcomes. Women at highest risk continue to receive prophylactic VWF pre- and postpartum to prevent serious hemorrhagic events.

Polyethylene glycol and a novel developed polyethylene glycol-nitric oxide normalize arteriolar response and oxidative stress in ischemia-reperfusion

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion (P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.

Occurrence of inhibitors in previously untreated or minimally treated patients with haemophilia A after exposure to a plasma‐derived solvent‐detergent factor VIII concentrate

A solvent-detergent virus-inactivated plasma-derived FVIII concentrate (SD-pdFVIII) has been employed for treatment of Italian patients with haemophilia A for 15 years. This product is a non-monoclonally purified, high purity FVIII concentrate, containing large amounts of von Willebrand factor (VWF). A retrospective survey was carried out in Italy in order to evaluate the immunogenicity of SD-pdFVIII in previously untreated patients (PUPs) or in minimally treated patients (MTPs), i.e. previously exposed for up to 5 days only to other plasma-derived concentrates. The survey included 99 patients with ages ranging from 6 to 64 years (median=21.3) of whom 31 PUPs and 68 MTPs, the latter with a median of four exposure days (EDs; range 1-5) to other plasma products. Surveyed patients had been exposed to SD-pdFVIII for a median of 83 EDs (range 21-1580). Seven patients (three PUPs and four MTPs), all with severe haemophilia, had developed inhibitors [7.1%, 95%; confidence interval: 3-14%] after a median of 11 EDs (range 4-22). Of them, two were low responders (<or=5 BU mL-1) and five high responders. Two low responders and one high responder tolerated inhibitors spontaneously; one high responder underwent immunotolerance treatment with complete, long-standing response. These findings show that SD-pdFVIII is at low risk of inhibitor development in PUPs and MTPs with severe and moderate haemophilia, the risk of inhibitor development being similar to that previously reported for other plasma-derived VWF-containing FVIII products.

Vasovagal fainting as an evolutionary remnant of the fight against hemorrhage

Major bleeding is one of the most frequently occurring medical conditions that require the attention of physicians and surgeons. The most frequent causes of major bleeding in most hospitals are trauma, peri-operative bleeding (which can in fact be viewed as a variant of trauma), gastro-intestinal disease, and bleeding due to the use of anticoagulant agents [6]. Severe hemorrhage leads to an insufficient circulation and an inadequate supply of oxygen to tissue. Patients with serious bleeding often present with circulatory shock and collapse. Impaired brain perfusion and subsequent syncope seems an understandable sequence of events following a severe drop in systemic blood pressure as a consequence of acute blood loss. However, it is intriguing to speculate whether syncope may not only be a logical consequence of major hemorrhage but can rather be viewed as a response with distinct protective effects for the organism and as such has been carefully preserved as a beneficial mechanism throughout the evolution. In this issue of Clinical Autonomic Research, Dr. Diehl proposes an interesting explanation for the occurrence of vasovagal syncope, as a mechanism that was originally developed as a protective strategy against exsanguination upon major bleeding in mammals [3]. Dr. Diehl hypothesizes that low blood pressure and bradycardia that is associated with vasovagal syncope is beneficial for the organism that is threatened by ongoing major blood loss,when the initial physiological and compensatory responses, such as vasoconstriction and tachycardia, have failed. Indeed, a low blood pressure is likely to result in reduced blood loss during major hemorrhage and clinical studies indeed suggest that to strive for normalization of blood pressure in patients with trauma and serious bleeding by large volume intravenous infusion may do more harm than good [7]. Based on this notion, strategies that employ permissive hypotension awaiting further medical intervention are currently being investigated [8]. Dr. Diehl’s hypothesis is interesting and thought-provoking and is indeed supported by various physiological and clinical observations. As the author himself indicates, the hypothesis can be challenged by experimental studies in bleeding animals. In addition to this, we must also identify a mechanism by which fainting would not only be beneficial to slow the rate of bleeding but may be helpful in arresting the blood loss. The answer to this question may be found in the response of the coagulation system to circulatory collapse. Fainting leads to an immediate and marked increase in von Willebrand factor (and associated factor VIII), presumably due to release from Weibel Palade bodies in endothelial cells of the vessel wall [2]. Interestingly, post-fainting blood not only has a four-fold higher concentration of von Willebrand factor, but also the released von Willebrand factor occurs in unusually large multimers, which are known to be hemostatically much more potent than normal von Willebrand factor multimers. Under normal circumstances the constitutive release of large von Willebrand factor multimers is followed by enzymatic degradation by the von Willebrand factor cleaving protease (ADAMTS-13) [9]. It may be hypothesized that the immediate release of large amounts of von Willebrand factor as induced by fainting does not allow this cleaving protease to act properly, leaving the large multimers for C A R 52 M. Levi, MD ( ) Dept. Internal Medicine (F-4) Academic Medical Centre University of Amsterdam Meibergdreef 9 1105 AZ Amsterdam, The Netherlands Tel.: +31-20/5662171 Fax: +31-20/6919658 E-Mail: m.m.levi@amc.uva.nl

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001). Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.