Presence Of Von Willebrand Factor Research Articles

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies

BackgroundCOVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. ObjectivesTo determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. MethodsWe compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. ResultsIn lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. ConclusionWe provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19.

Hidden behind thromboinflammation: revealing the roles of von Willebrand factor in sickle cell disease pathophysiology.

This review provides an update on the pathophysiology of sickle cell disease (SCD) with a particular focus on the dysregulation of the von Willebrand factor (VWF) - ADAMTS13 axis that contributes to its pathogenesis. In discussing recent developments, we hope to encourage new and ongoing discussions surrounding therapeutic targets for SCD. Within the last 5 years, the role of VWF in the pathophysiology of SCD has been further elucidated and is now a target of study in ongoing clinical trials. The pathophysiology of SCD is multifaceted, as it involves systemwide vascular activation, altered blood rheology, and the activation of immune responses and coagulative pathways. The presence of VWF in excess in SCD, particularly in its largest multimeric form, greatly contributes to its pathogenesis. Understanding the molecular mechanisms that underly the presence of large VWF multimers in SCD will provide further insight into the pathogenesis of SCD and provide specific targets for therapy.

Plasma-derived FVIII/VWF complex shows higher protection against inhibitors than isolated FVIII after infusion in haemophilic patients: A translational study.

IntroductionPresence of von Willebrand factor (VWF) in FVIII concentrates offers protection against neutralizing inhibitors in haemophilia A (HA). Whether this protection is more evident in plasma‐derived (pd) FVIII/VWF or recombinant (r) FVIII concentrates remains controversial.AimWe investigated the protection exerted by VWF against FVIII inhibitors in an in vivo mouse model of HA exposed to pdFVIII/VWF or to various rFVIII concentrates.MethodsHaemophilia A mice received the different FVIII concentrates after administration of vehicle or an inhibitory IgG purified from a commercial pool of HA plasma with inhibitors and FVIII:C recoveries were measured. Furthermore, using a novel clinically oriented ex vivo approach, Bethesda inhibitory activities (BU) of a commercial pool of HA plasma with inhibitors were assessed using normal plasma, or plasma from severe HA patients, without inhibitors, after treatment with the same concentrates.Resultsin vivo studies showed that pdFVIII/VWF offers markedly higher protection against inhibitors when compared with any of the FVIII products without VWF. More importantly, in the ex vivo studies, plasma from patients treated with pdFVIII/VWF showed higher protection against inhibitors (P values ranging .05‐.001) in comparison with that observed in plasma from patients who received FVIII products without VWF, regardless of the type of product evaluated.ConclusionData indicate that FVIII+VWF complexes assembled in the circulation after rFVIII infusion are not equivalent to the naturally formed complex in pdFVIII/VWF. Therefore, rFVIII infused into HA patients with inhibitors would be less protected by VWF than the FVIII in pdFVIII/VWF concentrates.

Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

AbstractIntroduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear.Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses.Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice.Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin. DisclosuresNo relevant conflicts of interest to declare.

Thrombosis and Von Willebrand Factor in Chuvash Polycythemia

Background: In Chuvash polycythemia (CP), homozygous germline VHLR200W results in augmented hypoxia-sensing, elevated erythropoietin and hemoglobin, and increased morbidity and mortality from venous and arterial thromboses.1 In CP patients and controls from Russia's Chuvash Republic, the probability of new thrombosis in CP increased with age, smoking, and higher expression of THBS1, the gene encoding thrombospondin-1.2 In mice, hypoxia leads to upregulation of THBS1 through HIF-23 and to upregulation of VWF .4 THBS1 reportedly regulates the size of circulating von Willebrand Factor (VWF) multimers5 through inhibiting proteolytic cleavage of VWF by ADAMTS13.6 We hypothesized that upregulation of THBS1 in CP may be a risk for thrombosis through an effect on ADAMTS13 and VWF.Methods: We enrolled into an observational study 155 patients with CP and 154 controls matched by age, sex and place of residence. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. We genotyped for VHLR200W, measured baseline plasma THBS-1 concentrations2 and followed the subjects for a median of 9.4 years. Here, we report results for determining baseline circulating levels of ADAMTS13 antigen, activity and specific activity and of VWF antigen and differential multimers in up to 57 CP patients and 30 controls.Results: CP subjects had higher hemoglobin and erythropoietin concentrations and lower white blood cell and platelet counts (Table 1). They were also more likely to be current smokers. There was a trend to higher THBS-1 concentrations in the CP patients (p=0.084), which was significant after adjusting for platelet count (P=0.016). ADAMTS13 and VWF antigen levels did not differ between CP patients and controls but ADAMTS13 activity and especially specific activity were lower in CP. Plasma THBS-1 concentration correlated positively with ADAMTS13 antigen concentration (r=0.358, P=0.001) but negatively with specific activity (r=-0.363, P=0.001) (Figure 1). Eight (14.0%) CP patients (aged 27-76) years had a history of thrombosis (Table 1). The mean serum VWF antigen concentration (P=0.001) and differential VWF multimer size (P=0.090) were higher in CP patients with a history of thrombosis (Figure 2). During the prospective follow-up period, 10 CP patients (17.5%) experienced a new thrombosis at a median age of 46 (range 26-70) years. Three patients also experienced an additional thrombosis for a total of 13 new thromboses: myocardial infarction (n=5), stroke (n=3), splanchnic thrombosis (n=2), deep vein thrombosis (n=2), pulmonary embolism (n=1). Baseline serum VWF antigen levels (P=0.019) and VWF multimer size (P=0.099) were higher in subjects who developed a new thrombosis during follow-up (Figure 3), and these relationships persisted after adjusting for smoking history.Discussion: THBS1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and facilitates platelet aggregation.7 In mice, platelet-derived THBS1 modulated arterial thrombosis only in the presence of VWF.8 Our present findings suggest that CP subjects have lower ADAMTS13 specific activity than controls and that this lower activity may be related to higher plasma THBS1 concentrations. Lower ADAMTS13 specific activity suggests the presence of an inhibitor or partial inactivation of the protease. Furthermore, higher VWF antigen and multimer size in CP patients is associated with a past history of thrombosis and a greater risk for new thrombosis in the future. More broadly, our findings suggest a possible implication of VWF in thrombosis of conditions associated with an up-regulated hypoxic response. Further studies may identify new pathways of prevention and treatment with broad applicability.

Von Willebrand factor in follicular fluid in women undergoing IVF/ICSI: a “ghost” associated with oocyte and embryo quality

Introduction: The purpose of the present study to determine the presence of Von Willebrand factor (vWF) in the follicular fluid of women undergoing IVF/ICSI and to investigate the association of its concentration with oocyte and embryo quality. Methods: We conducted a prospective observational study based in women undergoing in vitro fertilization treatment. vWF concentrations in follicular fluid samples collected during transvaginal oocyte retrieval were measured using an enzyme-linked immunosorbent assay (Elisa). They were also analyzed by the VIDAS® vWF method for confirmation of the results. The latter is an automated quantitative test for the measurement of vWF in human plasma, using the ELFA technique (enzyme-linked fluorescent assay). vWF concentrations were correlated to oocyte and embryo quality. Results: Contrary to previous reports, vWF was determined in follicular fluid. A higher percentage of good quality oocytes were found in the presence of lower vWF follicular fluid levels. Similarly, better quality embryos were derived from oocytes retrieved from follicles with lower vWF follicular fluid levels. Conclusions: Contrary to existing evidence to date, vWF was found in the follicular fluid of women undergoing IVF/ICSI treatment. In addition, the quality of oocytes and embryos was adversely related to vWF follicular fluid levels. In fact, a better outcome of IVF/ICSI treatment was suggested when the levels of vWF in follicular fluid were low. In this context, vWF follicular fluid levels could be used as a prognostic factor for oocyte and embryo quality.

Population pharmacokinetics of the von Willebrand factor–factor VIII interaction in patients with von Willebrand disease

Recent studies have reported that patients with von Willebrand disease treated perioperatively with a von Willebrand factor (VWF)/factor VIII (FVIII) concentrate with a ratio of 2.4:1 (Humate P/Haemate P) often present with VWF and/or FVIII levels outside of prespecified target levels necessary to prevent bleeding. Pharmacokinetic (PK)-guided dosing may resolve this problem. As clinical guidelines increasingly recommend aiming for certain target levels of both VWF and FVIII, application of an integrated population PK model describing both VWF activity (VWF:Act) and FVIII levels may improve dosing and quality of care. In total, 695 VWF:Act and 894 FVIII level measurements from 118 patients (174 surgeries) who were treated perioperatively with the VWF/FVIII concentrate were used to develop this population PK model using nonlinear mixed-effects modeling. VWF:Act and FVIII levels were analyzed simultaneously using a turnover model. The protective effect of VWF:Act on FVIII clearance was described with an inhibitory maximum effect function. An average perioperative VWF:Act level of 1.23 IU/mL decreased FVIII clearance from 460 mL/h to 264 mL/h, and increased FVIII half-life from 6.6 to 11.4 hours. Clearly, in the presence of VWF, FVIII clearance decreased with a concomitant increase of FVIII half-life, clarifying the higher FVIII levels observed after repetitive dosing with this concentrate. VWF:Act and FVIII levels during perioperative treatment were described adequately by this newly developed integrated population PK model. Clinical application of this model may facilitate more accurate targeting of VWF:Act and FVIII levels during perioperative treatment with this specific VWF/FVIII concentrate (Humate P/Haemate P).

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition.

Type 2A von Willebrand disease (VWD) is common in type-2 group caused by qualitative deficiency of von Willebrand factor (VWF). Emicizumab is a bispecific antibody that mimics activated factor VIII (FVIIIa) cofactor function, and emicizumab prophylaxis substantially reduces bleeding in patients with haemophilia A. It is unknown whether emicizumab affects thrombus formation in type 2A VWD characterized by not only low FVIII levels but also the impaired platelet adhesion and aggregation. To examine the coagulant potential of emicizumab in type 2A VWD. Perfusion chamber experiments combined with immunostaining were performed using whole blood from 5 patients with type 2A VWD under high shear condition (2500s-1 ). The addition of FVIII to type 2A VWD whole blood did not augment thrombus formation, whilst supplementation with VWF or FVIII/VWF enhanced. FVIII appeared to contribute to thrombus height rather than surface coverage. The addition of emicizumab enhanced thrombus formation in type 2A VWD compared with FVIII, but this potency was less than the presence of VWF. The effect on thrombus formation mediated by emicizumab appeared to be more rapid than that by FVIII for non-requirement of activation step of FVIII, whilst that by FVIII showed more impact on thrombus formation at the late phase. Emicizumab-induced enhancing effects of thrombus formation, independent on VWF, may be useful as an alternative therapy for type 2A VWD patients. These results supported a critical role for the FVIII-VWF complex facilitating thrombus formation under high shear.

Pharmacokinetics, Efficacy and Safety of a Plasma-Derived VWF/FVIII Concentrate (Formulation V) in Pediatric Patients with von Willebrand Disease (SWIFTLY-VWD Study).

PurposeFormulation V (VONCENTO®) is a plasma-derived high-concentration/low-volume, high-purity von Willebrand factor (VWF)/factor VIII (FVIII) concentrate, originally indicated for von Willebrand disease (VWD) in adults and adolescents. This multicenter, open-label study (SWIFTLY-VWD) evaluated the pharmacokinetics (PK), as well as hemostatic efficacy and safety, of Formulation V in pediatric patients (<12 years) with severe VWD requiring treatment or prophylaxis of bleedings.MethodsPK investigations were performed following one dose of Formulation V at Day 1 and 180. Nonsurgical bleeds were analyzed, while hemostatic efficacy was graded as excellent/good/moderate/none. Safety assessments included adverse events, and presence of VWF and/or FVIII inhibitors.ResultsFormulation V was administered as on-demand (N=13) or prophylaxis therapy (N=4) for 12 months (<6 years, N=9; 6 to <12 years, N=8). PK parameters for VWF markers were generally comparable to adults but showed lower VWF:ristocetin cofactor (RCo) exposure. Incidence of major bleeds was lower for prophylaxis (3.3%) than on-demand therapy (27.1%); joint bleeds were also lower (3.3% vs 11.5%, respectively). Investigator-reported excellent/good hemostatic efficacy against nonsurgical bleeds was 100%. No clinically relevant differences in PK, hemostatic efficacy, or safety were observed between age-groups (<6 years and 6 to <12 years). Formulation V was well tolerated. Adverse events were mild–moderate and consistent with the adult safety profile. No cases of anaphylactic reactions or angioedema, development of FVIII/VWF inhibitors, thromboembolic events, or viral infections were reported.ConclusionThis study provides evidence for use of Formulation V to treat and prevent bleeding in pediatric patients with severe VWD, and led to the European approval of Formulation V in children.

Accumulations of von Willebrand factor within ECMO oxygenators: Potential indicator of coagulation abnormalities in critically ill patients?

Clot formation within membrane oxygenators (MOs) remains a critical problem during extracorporeal membrane oxygenation (ECMO). The composition of the clots—in particular, the presence of von Willebrand factor (vWF)—may be an indicator for prevalent nonphysiological flow conditions, foreign body reactions, or coagulation abnormalities in critically ill patients. Mats of interwoven gas exchange fibers from randomly collected MOs (PLS, Maquet, Rastatt, Germany) of 21 patients were stained with antibodies (anti‐vWF and anti‐P‐selectin) and counterstained with 4′,6‐diamidino‐2‐phenylindole. The extent of vWF‐loading was correlated with patient and technical data. While 12 MOs showed low vWF‐loadings, 9 MOs showed high vWF‐loading with highest accumulations close to crossing points of adjacent gas fibers. The presence and the extent of vWF‐fibers/“cobwebs,” leukocytes, platelet–leukocyte aggregates (PLAs), and P‐selectin‐positive platelet aggregates were independent of the extent of vWF‐loading. However, the highly loaded MOs were obtained from patients with a significantly elevated SOFA score, severe thrombocytopenia, and persistent liver dysfunction. The coagulation abnormalities of these critically ill patients may cause an accumulation of the highly thrombogenic and elongated high‐molecular‐weight vWF multimers in the plasma which will be trapped in the MOs during the ECMO therapy.

Inositol hexakisphosphate increases the size of platelet aggregates

The inositol phosphates, InsP5 and InsP6, have recently been identified as binding partners of fibrinogen, which is critically involved in hemostasis by crosslinking activated platelets at sites of vascular injury. Here, we investigated the putative physiological role of this interaction and found that platelets increase their InsP6 concentration upon stimulation with the PLC-activating agonists thrombin, collagen I and ADP and present a fraction of it at the outer plasma membrane.Cone and plate analysis in whole blood revealed that InsP6 specifically increases platelet aggregate size. This effect is fibrinogen-dependent, since it is inhibited by an antibody that blocks fibrinogen binding to platelets. Furthermore, InsP6 has only an effect on aggregate size of washed platelets when fibrinogen is present, while it has no influence in presence of von Willebrand factor or collagen. By employing blind docking studies we predicted the binding site for InsP6 at the bundle between the γ and β helical subunit of fibrinogen.Since InsP6 is unable to directly activate platelets and it did not exhibit an effect on thrombin formation or fibrin structure, our data indicate that InsP6 might be a hemostatic agent that is produced by platelets upon stimulation with PLC-activating agonists to promote platelet aggregation by supporting crosslinking of fibrinogen and activated platelets.

Identification of aggregates in therapeutic formulations of recombinant full‐length factor VIII products by sedimentation velocity analytical ultracentrifugation

Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A.Aggregates in biopharmaceutical products are an immunogenic risk factor.Aggregates were identified in recombinant full‐length factor VIII products.Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. SummaryBackgroundThe development of inhibitory anti‐factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full‐length FVIII is more immunogenic than plasma‐derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti‐drug antibodies.ObjectiveTo evaluate recombinant full‐length FVIII products for the presence of aggregates.MethodsAdvate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s 20,w values and molecular weights.ResultsThe major component of all three products was a population of ~7.5 S heterodimers with a weight‐average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer‐exchanged Advate and Kogenate. SV AUC analysis of a plasma‐derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII.ConclusionsAggregate formation has been identified in recombinant full‐length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.

Endothelial damage is aggravated in acute GvHD and could predict its development.

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n=31) and without (NoGvHD, n=13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.

Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells.The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091–2095 and 2155–2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis.These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.

Circulating platelet aggregates damage endothelial cells in culture

BackgroundPresence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). MethodsEndothelial cells were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. Results and conclusionNo ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.

Therapeutic efficacy of the platelet glycoprotein Ib antagonist anfibatide in murine models of thrombotic thrombocytopenic purpura

AbstractThrombotic thrombocytopenic purpura (TTP), a potentially fatal blood clot disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from acquired autoantibodies. Plasma exchange is the only effective initial therapy. However, the high mortality rate and the complications associated with plasma exchange therapy remain a major concern. To address unmet clinical needs, therapeutic efficacies of anfibatide, a snake venom-derived platelet glycoprotein Ib antagonist, in murine models of spontaneous thrombocytopenia and shigatoxin-induced TTP were determined. A light scattering platelet aggregometry, microfluidic shear-based assay, and murine models of TTP were used in the study. We showed that purified anfibatide inhibits ristocetin- or botrocetin-induced human or murine platelet agglutination in the presence of von Willebrand factor in a concentration-dependent manner. Anfibatide could also dramatically inhibit the adhesion and aggregation of murine and human platelets on a collagen surface under arterial shear stress, in the presence or absence of plasma ADAMTS13 activity. Most importantly, we demonstrated that an intraperitoneal administration of anfibatide at the dose of 60 ng/g body weight twice daily mitigated spontaneous thrombocytopenia and prevented shigatoxin-induced TTP in Adamts13−/− and disease-susceptible mice (CAST/Ei strain). Thus, we conclude that anfibatide, when administered at the optimal dosage, route, and interval, is efficacious in treating spontaneous and bacterial shigatoxin-induced TTP in the murine models. Our findings may provide the basis for further development of anfibatide for the treatment of acute TTP in humans.

ADAMTS13-mediated thrombolysis of t-PA–resistant occlusions in ischemic stroke in mice

AbstractRapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA–resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

Case-Control Pilot Study of the Immune Modulating Effect of FEIBA® on Patients with Haemophilia a and Inhibitors

Background: Immune tolerance therapy (ITT) is the only available therapy for eliminating the inhibitor in patients with haemophilia and high-responding inhibitors. However, it is a very expensive treatment with an efficacy of up to 60%-75%. Factors influencing the outcome of ITT are not completely understood. Some studies have suggested that plasma derived factor VIII (FVIII) concentrates might offer certain advantages in the elimination of the inhibitor when compared with recombinant derivatives. The presence of von Willebrand factor (Auerswald et al,Haematologica 2003;88:EREP05; Kreuz et al,Haematologica 2001;86:16-20) and some molecules with immunomodulatory effect like anti-idiotype antibodies, transforming growth factor-β, HLA type I molecules and soluble FAS ligand in plasma derived concentrates may be involved in such effect (Berntorp et al,Haemophilia 2001;7:109-13; Hodge et al,Br J Haematol 2001;115:376-81; Hodge et al, Haemophilia 2006;12:133-9; Ghio et al, Thromb Haemost 2003;89:365-73). The activated prothrombin complex concentrate (FEIBA®) is a plasma derived product used for bleeding control in patients with haemophilia A and high-responding inhibitors. We hypothesize that FEIBA® may have an immunomodulatory activity due to its plasmatic origin. This effect might be observed in patients with intense treatment with FEIBA® (e.g. prophylaxis treatment) by the reduction in levels of antibodies against FVIII compared with patients on demand treatment with this product.Objective: The aim of this study was to compare levels of inhibitors in patients with severe haemophilia A and inhibitors on demand or under prophylactic treatment with FEIBA®.Methods and results: This was a prospective, observational and multicenter study. A total of 12 adult patients with severe haemophilia A and high-responding inhibitors (6 patients on demand and 6 patients under prophylaxis treatment with FEIBA®) were included. All patients had normal levels of serum creatinine, serum alanine aminotransferase and CD4+ lymphocyte counts at inclusion. Levels of inhibitors were determined by Bethesda assay. Levels of inhibitors in patients on prophylaxis with FEIBA® were lower than those observed in patients on demand treatment but differences were not statistical significant. In most of the patients on prophylaxis, levels of inhibitors went down along the prophylaxis regimen.TableMedical history of patients.Patient NºAgeType of treatmentUsual dose (IU/kg)FrecuencyTiter (BU)Time frame with start of prophylaxis (months)**48010153Prophylaxis60Three times a week64*4.4804.1961.3250-1.348010258Prophylaxis55Three times a week5*3.3122.17-3.812-6.1150101UNKProphylaxis60Three times a week4*3.05-6.06-8.028010128Prophylaxis56Every other day109*7.3774.1101-8.3101-9.728010257On Demand56 x 8 dosesN/A174*N/A37N/A83N/A21N/A25N/A184N/A28010357On Demand55 x 6 dosesN/A8000*N/A64N/A5N/A21N/A28010458On Demand68 x 6 dosesN/A152*N/A28010523On Demand68 x 3 doses 40 x 6 dosesN/A30*N/A08010128On Demand50 x 5 dosesN/A5.2*N/A7.07.24.07.846010137Prophylaxis66every 48hNegative21,046010257On demand702 doses/ bleeding usually66.5*More than 20 years30000150Prophylaxis85Three times a week8*3*Titer at inclusion. **Positive values represent number of months after starting prophylaxis and negative values are referred to number of months before starting prophylaxis. For example, the patient number 480101 had 250 BU at 1.3 months before starting prophylaxis (in the table= -1.3 months) and 64 UB at month 4.4 after initiation of prophylaxis (in the table= 4.4 months). N/A: not applicable.Conclusion: Prophylaxis with FEIBA® might decrease levels of inhibitors in patients with severe haemophilia A and high-responding inhibitors. However, a broader study is needed to confirm this result. DisclosuresIruin:Baxalta: Research Funding. Bonanad:Baxalta: Research Funding. López Fernández:Baxalta: Research Funding. Altisent:Baxalta: Research Funding. García Candel:Baxalta: Research Funding. Rivas Pollmar:Baxalta: Research Funding. Canales:Baxalta: Research Funding.

Von Willebrand Factor Protects Factor VIII from Rapid Endocytosis Via LDL Receptor-Related Protein By Blocking the Initial Rate-Limiting Cell Surface Binding Step of Factor VIII

Blood coagulation factor VIII is internalized and degraded by cellular systems via an endocytic mechanism that involves low-density lipoprotein receptor-related protein (LRP). Factor VIII's chaperone protein von Willebrand factor (VWF) has a dual role in this uptake mechanism. Under conditions of low shear stress VWF is particularly effective in blocking LRP-dependent endocytosis of factor VIII, whereas VWF itself mediates the uptake of factor VIII under high shear stress conditions. This study addresses the poorly understood uptake mechanism of factor VIII and the role therein of VWF in the absence of shear stress.LRP binds its ligands via ligand-binding domains that are clustered in four regions. Cluster II, III and IV have each been demonstrated to bind factor VIII. However, one of the poorly understood aspects of the LRP dependent endocytosis of factor VIII is that the affinity of LRP for factor VIII is an order of magnitude lower compared to the affinity of LRP for other ligands like, for instance, alpha-2-macroglobulin (α2M). We and others have previously hypothesized that, in contrast to α2M, factor VIII may first require pre-concentration on the cell surface before it is transferred to LRP for effective endocytosis. Direct evidence for the existence of this two-step mechanism of endocytosis is, however, still lacking. In the present study, we compared the endocytic uptake mechanism of fluorescent labeled α2M and a factor VIII GFP variant employing a confocal microscopy study. To this end, U87MG cells were employed, which are particularly effective in internalizing ligands via the LRP-dependent pathway. The role of vWF in the LRP-mediated endocytosis of factor VIII was assessed as well.The confocal microscopy studies surprisingly showed that factor VIII traffics to early endosomes irrespective of the presence of α2M. Co-incubation of factor VIII and α2M, revealed that both proteins end up in the same endocytic compartment of the cells. This finding can be explained if LRP can simultaneously bind and endocytose both factor VIII and α2M. Taking maximal advantage of this phenomenon, factor VIII and α2M were together incubated with the cells to accurately assess whether or not there is any difference in the rate of endocytosis of these proteins. The results demonstrated that α2M was present at the cell surface after 2 minutes of incubation. After 5 minutes, α2M was distributed in a punctate pattern over the cells indicating that α2M has been internalized by the cells. Interestingly, a 15 minutes incubation of factor VIII with the cells was required before factor VIII could be detected at the cell surface and inside the cells. This finding implies that the uptake of factor VIII is delayed compared to the rate of endocytosis of α2M. This is in agreement with the hypothesis that factor VIII may first require a cell surface binding step prior to LRP mediated endocytosis. These observations also imply that the cell surface binding step is rate-limiting in the endocytic uptake mechanism of factor VIII. Compelling evidence of the two-step endocytic model was obtained when factor VIII was incubated with the cells in the presence of receptor-associated-protein (RAP), which prevents direct binding of ligands to LRP. The results of these studies showed a striated fluorescent pattern that was localized at the borders of the cells. Notably, in the presence of VWF, no factor VIII was observed on or within the cells at all. These findings imply that VWF blocks cell surface binding of factor VIII.Based on our findings, we propose that the ligand-binding clusters of LRP can mediate the binding of more than one ligand to LRP. The combined results further convincingly show that factor VIII endocytosis includes an initial cell surface binding step which is rate limiting in the uptake mechanism of factor VIII. The observations also explain why VWF is particularly effective in blocking the endocytosis of factor VIII. VWF may not only block the direct binding of factor VIII to LRP but also the rate limiting cell surface binding step prior to LRP mediated endocytosis. DisclosuresNo relevant conflicts of interest to declare.