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Hepatic ischaemia–reperfusion (I/R) injury is a serious liver damage that critically influences the clinical outcome of liver surgery or transplantation. Since recent studies indicated the critical involvement of von Willebrand factor (VWF) in reperfusion injuries of brain and myocardium, we hypothesized that VWF-dependent thrombotic or inflammatory responses also play a role in hepatic I/R injury. Using a mouse model of hepatic I/R injury, we explored the functional relevance of the VWF–ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) axis in this pathologic condition. Time-course studies during hepatic I/R revealed significantly lower alanine aminotransferase (ALT) values, as well as greater hepatic blood flow, in VWF gene-deleted (KO) mice in comparison with wild-type (WT) mice. Histological analysis revealed a significantly lesser extent of neutrophil infiltration and hepatocellular necrosis in liver tissues of VWF-KO mice. Human recombinant ADAMTS13 significantly improved the impairment in ALT values and hepatic blood flow and decreased neutrophil infiltration within the liver tissue of WT mice. Real-time intravital imaging successfully visualized significantly reduced leukocyte–vessel wall interactions in I/R liver of VWF-KO mice. Taken together, our results indicate that VWF promotes neutrophil recruitment in ischaemic mouse liver, critically aggravating reperfusion injury, and suggest that functional regulation of VWF by ADAMTS13 represents a promising therapeutic option for hepatic I/R injury.

Upshaw-Schulman syndrome (USS) is a thrombo-hemorrhagic disease caused by congenital deficiency of ADAMTS13 due to ADAMTS13 gene mutations. USS is characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond dramatically to infusions of fresh frozen plasma. There are two phenotypic expressions of USS: one is the early-onset type and the other, the late-onset type, is asymptomatic during childhood with the first bout of thrombotic thrombocytopenic purpura (TTP) developing after adolescence or during adulthood. We found that gravida with the latter phenotype developed thrombocytopenia and hemolytic anemia during the second or third trimesters, often followed by thrombotic microangiopathies (TMAs). These phenomena suggest that elevated plasma von Willebrand Factor (VWF) might be crucial because plasma levels of VWF antigen usually increase by 200-500% during this period of gestation. Here, we performed platelet function assays using a mixture of anti-coagulated blood from normal volunteers, human VWF, anti-ADAMTS13 monoclonal antibody A10, and purified plasma-derived ADAMTS13 to investigate the effect of plasma VWF levels on platelet thrombus formation in the context of deficient ADAMTS13. In vitro studies showed that mural thrombus formation and platelet aggregation under high shear stress were markedly augmented by increasing the amounts of exogenously added VWF when ADAMTS13 activity was deficient, as may be the case in the in vivo circulation of gravida with USS. These results suggest that highly elevated plasma VWF might accelerate platelet thrombus formation not only in the circulation but also on the surface of vascular endothelial cells in the setting of ADAMTS13 deficiency in USS.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Von Willebrand factor (VWF)-cleaving protease (ADAMTS13) cleaves ultralarge VWF (ULVWF) secreted from endothelium and by which is regulating its physiologic function. An imbalance between ULVWF secretion and ADAMTS13 level occurs in sepsis and may cause multiple organ dysfunction. We evaluated the association between the VWF-propeptide (VWF-pp)/ADAMTS13 ratio and disease severity in patients with severe sepsis or septic shock. In 27 patients with severe sepsis or septic shock and platelet count less than 120,000/μL, we measured plasma VWF, VWF-pp, and ADAMTS13 levels on hospital days 1, 3, 5, and 7. The VWF-pp/ADAMTS13 ratio was increased greater than 12-fold in patients with severe sepsis or septic shock on day 1 and remained markedly high on days 3, 5, and 7 compared with normal control subjects. The VWF-pp/ADAMTS13 ratio significantly correlated with Acute Physiology and Chronic Health Evaluation II score on days 1 and 5; Sepsis-related Organ Failure Assessment score on days 1, 3, and 5; maximum Sepsis-related Organ Failure Assessment score and tumor necrosis factor α level on days 1, 3, 5, and 7; and creatinine level on days 1, 5, and 7. Patients with greater than stage 1 acute kidney injury had significantly higher VWF-pp/ADAMTS13 ratio than patients without acute kidney injury. In summary, the VWF-pp/ADAMTS13 ratio was associated with disease severity in patients with severe sepsis or septic shock and may help identify patients at risk for multiple organ dysfunction by detecting severe imbalance between ULVWF secretion and ADAMTS13 level.

SummaryCoagulation factor VIII (FVIII) plays an essential role in haemostasis. To date, physiologic activity of FVIII circulating in the bloodstream (S-FVIII) is evaluated by classic coagulation assays. However, the functional relevance of FVIII (-von Willebrand factor complex) immobilised on thrombogenic surfaces (I-FVIII) remains unclear. We used an in vitro perfusion chamber system to evaluate the function of I-FVIII in the process of mural thrombus formation under whole blood flow conditions. In perfusion of either control or synthetic haemophilic blood, the intra-thrombus fibrin generation on platelet surfaces significantly increased as a function of I-FVIII, independent of S-FVIII, under high shear rate conditions. This I-FVIII effect was unvarying regardless of anti-FVIII inhibitor levels in synthetic haemophilic blood. Thus, our results illustrate coagulation potentials of immobilised clotting factors, distinct from those in the bloodstream, under physiologic flow conditions and may give a clue for novel therapeutic approaches for haemophilic patients with anti-FVIII inhibitors.

Aim: Patients with severe aortic stenosis (AS) may have bleeding episodes due to the loss of high-molecular-weight (HMW) von Willebrand factor multimers (VWFMs). The absence of HMW-VWFMs and bleeding tendency are usually corrected after aortic valve replacement (AVR). To investigate the process of VWFM recovery and symptoms in patients with severe AS, we analyzed changes in VWF antigen (VWF:Ag), ADAMTS13 activity (ADAMTS13:AC), and platelet thrombus formation under high shear stress conditions. Methods: Nine patients with severe AS undergoing AVR were analyzed. Results: Evident deficiency of HMW-VWFMs was observed in six patients before surgery, which was rapidly restored within 8 days after AVR. Median levels of VWF:Ag before surgery, on postoperative days (PODs) 1, 8, 15, and 22, and one year after AVR were 78.1%, 130%, 224%, 155%, 134%, and 142%, respectively. In contrast, ADAMTS13:AC was 50.5%, 35.5%, 25.5%, 25.1%, 30.3%, and 84.6%, respectively. Preoperative thrombus formation but not surface coverage was significantly lower than that on POD 22, which was considered as normal level in each patient. Compared with preoperative levels, thrombus volume was significantly lower on POD 1, but rapidly increased by POD 8. Conclusion: Bleeding tendency and loss of HMW-VWFMs observed in patients with severe AS before surgery was rapidly corrected after AVR. Instead, patients were in a VWF-predominant state between POD 8 and 22.

Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib–von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13−/− and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13−/− mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13−/− compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a function-blocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWF-cleaving activity of ADAMTS13, provided visual evidence for a shear rate–dependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis.

Sepsis is a systemic inflammatory response syndrome caused by severe microbial infection. Uncontrollable activation of both inflammatory and coagulation pathways may result in multiple organ failure, a leading cause of death in intensive care units even in developed countries. An adhesive protein

Acquired haemophilia A (AHA) is a life-threatening haemorrhagic disorder that occurs with various underlying conditions such as autoimmune disease, drug reactions, lymphoproliferative diseases, solid tumours and pregnancy/postpartum status. However, in half of all reported cases, the underlying disease is unknown. Most AHA cases develop in adults; paediatric/adolescent cases are extremely rare. The main clinical symptom is bleeding into the skin, muscles, soft tissues and/or mucous membranes. Here, we report the case of an otherwise healthy 12-year-old girl who presented with prolonged bleeding postexodontia. After being diagnosed with AHA, she was successfully treated with recombinant activated factor VII infusion and oral prednisolone. To avoid such unanticipated bleeding when performing dental extraction, preoperative haemostatic screening tests are recommended.

Hemophilia A is an inherited bleeding disorder caused by a deficiency of the procoagulant cofactor VIII (FVIII). Currently, the patients with hemophilia A are being treated through repeated intravenous injection of FVIII concentrates. Since FVIII is a secreted protein and the tight regulation of its expression is not necessary, ex vivo gene therapy strategies for hemophilia A might be good for the therapeutic alternative. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et.al. PLoS One 2013 8(12):e83280). To enhance the viability of the implanted BOECs which boost the FVIII expression, double-layered cell sheets transplantation was performed by placing the recovered BOECs sheet onto the fibroblast sheet directly. FVIII transduced BOECs and non-transduced fibroblasts were cultured on temperature-responsive culture dish independently. Both cells were recovered as a contiguous cell sheet. Subcutaneous co-transplantation studies of BOEC and fibroblast sheet indicated that all hemophilia A mice treated with cyclophosphamide expressed between 40-50 mU/mL levels of FVIII without developing anti-FVIII inhibitory antibodies over 200 days. In contrast, the hemophilia A mice without any prior immuno-suppressive treatment developed neutralizing anti-FVIII antibodies between 5-10 Bethesda units. These therapeutic FVIII levels were two times higher than the group of hemophilia A mice that were transplanted BOECs sheet alone. Histological examination in the part of the mice revealed that the combination of BOEC and fibroblast sheet was structured as flat clusters without any cells infiltration. In conclusion, tissue engineering approach using double-layered cell sheets are viable for persistent tissue survival and providing therapeutic values. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A and merits further assessment.

Summary Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Sa (PSa) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A fi G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A fi G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient’s family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A fi G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A fi G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A fi G mutation itself might influence phenotypic expression of PS status in different individuals.

To explore the mechanisms that underlie the bleeding tendency in type 2A and 2B von Willebrand disease (VWD), we analyzed the mural thrombus generation process on a collagen surface under physiologic blood flow in a perfusion chamber using whole blood from these VWD patients. At a low shear rate (50 s−1), thrombus generation in all type 2A and 2B VWD patients was comparable to that of healthy controls. At a high shear rate (1500 s−1), thrombus generation was impaired in all type 2A patients, whereas that in type 2B VWD patients varied from normal to significantly defective, as judged by epifluorescence microscopy of thrombus surface coverage. However, in type 2B patients who showed normal thrombus generation at 1500 s−1, the height and volume of thrombi was significantly reduced, albeit with the normal surface coverage, compared with control thrombi, and von Willebrand factor (VWF) was poorly distributed within the type 2B thrombus mass when analyzed in detail by confocal laser scanning microscopy. Addition of purified VWF to patient blood completely reversed the defective spatial thrombus growth in type 2B VWD. Thus, our results confirm the impaired thrombus generation in type 2B VWD, which has never been demonstrable in previous in vitro soluble-phase platelet aggregation assays, and point to the critical function of larger VWF multimers in the proper spatial growth of mural thrombi under high shear rate conditions.

Recent studies have revealed that the platelet adhesive process under flow is tightly regulated by multiple ligand-receptor interactions. However, platelet morphological changes during this process, particularly its physiological relevance, remain unknown under blood flow conditions. Using epifluorescence and scanning electron microscopy, we evaluated the real-time changes in platelet morphology during a platelet adhesive process on a von Willebrand factor-coated surface under physiological high shear flow in a perfusion chamber. Here, we show that dynamic platelet shape changes occurring during distinct phases of the adhesive process are precisely regulated by “inside-out” and “outside-in” integrin signals and are also a key regulatory element in successful platelet thrombogenesis opposing rapid blood flow in vivo.

We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of ‘active PAI’ in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s(-1)). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (>/=1, 210 s(-1)) but normal thrombus formation under low shear (

To know the role and initiation mechanism of the interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF), we examined the effect of shear stress levels on platelet binding to vWF-coated plates using a cone-and-plate type viscometer capable of loading various levels of shear stress. The extent of platelet binding to immobilized vWF reached a plateau at the shortest period tested (20 seconds) under high shear stress (90 dyne/cm2), whereas 9 to 12 minutes was necessary for saturable platelet binding under static conditions. This shear effect, which was found to be dependent on the vWF-GP Ib interaction, was observed even under the lowest shear stress (1.5 dyne/cm2) examined. In contrast with the high shear effect previously reported to initiate the interaction of GP Ib with soluble vWF, these results indicate that relatively low levels of shear stress can promote the interaction of GP Ib with surface- immobilized vWF. This effect of shear stress was observed regardless of the manner in which vWF was immobilized, suggesting that immobilization itself and not, as previously hypothesized, a conformational change in vWF induced by direct adsorption to the surface is responsible for the enhanced GPIb binding. Thus, the present findings suggest that the vWF- GP Ib interaction contributes optimally to rapid platelet cohesion on a thrombogenic surface when vWF is in a static state and when platelets are moved by an appropriate rheological force such as low shear stress.

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

Hepatic ischemia-reperfusion (I/R) injury is a liver damage occurring during liver surgeries such as hepatic resection or transplantation, and denotes the major basis for graft dysfunction after transplantation. Although cellular and molecular mechanisms of hepatic I/R injury are complex and remain to be clarified, an excessive inflammatory response is assumed to play a role in this regard. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. In this context, we assumed that VWF may also be involved in the pathophysiology of hepatic I/R injury, in which VWF-dependent platelet activation or inflammatory responses could play a role at liver sinusoidal milieu. To test this hypothesis, we have used a mouse experimental model of hepatic I/R injury. Mice were anesthetized with sodium pentobarbital and a midline laparotomy was then performed on a heating pad. Blood supply for left lateral and median lobes of liver (approximately 70% of the liver mass) was interrupted by cross-clamping the hepatic artery and portal vein with a microvascular atraumatic clip for 90 min. Then a clip was taken off to provoke the reperfusion of hepatic blood flow, which was monitored on the surface of left lateral lobe by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The hepatic blood flow was measured again 24 h after reperfusion and mice were then sacrificed for blood collection and histological analysis of liver tissue. We compared 16 wild-type (WT) and 12 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 8-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. As compared to WT mice, restoration of hepatic blood flow was significantly greater in VWF-KO mice at 24 h after reperfusion (WT; 61± 17% vs. KO; 87 ± 17%, expressed as the percentage of pre-ischemic value). Consistent with the hepatic blood flow, the time-course analysis of serum alanine aminotransferase (ALT) at several time points after reperfusion revealed the lesser liver damages of KO mice (WT; 6898 ± 3270 and 1313 ± 621 IU/L vs. KO; 3043 ± 1320 and 478 ± 330 IU/L, at 3 h and 24 h after reperfusion, respectively). In addition, histological analysis confirmed that neutrophil infiltration in the liver tissue of KO mice was significantly reduced as compared to WT mice at 24 h after reperfusion. These impaired hepatic blood flow and ALT values as well as intensified neutrophil infiltration in WT mice were significantly improved to an extent comparable to those of KO mice by the bolus injection of recombinant human ADAMTS13 (3 μg/mouse equivalent to 2800 U/kg, n=12) just prior to the I/R operation. Our results altogether indicate that VWF-dependent inflammatory responses with neutrophil recruitment at ischemic sites are involved in pathophysiology of hepatic I/R injury, and functional regulation of VWF by ADAMTS 13 may serve as a promising therapeutic option for hepatic I/R injury. Disclosures No relevant conflicts of interest to declare.

Preclinical and clinical studies using adeno-associated viral (AAV) vectors for hemophilia B showed that the safety profile is vector dose-dependent and that immune responses to AAV-capsid proteins with subsequent hepatocyte toxicity required transient immunosuppression for sustained transgene expression. However, there still remain concerns over the safety of systemic vector injection. Potential side effects include adverse immunological reactions, vector-mediated cytotoxicity, germ-line transmission, and insertional oncogenesis. Moreover, especially in hemophilia A, an alternative transgene delivery approach may be necessary due to the large size of the factor VIII (FVIII) cDNA. Blood outgrowth endothelial cells (BOECs) are considered to be an attractive candidate to treat hemophilia A, because BOECs express von Willebrand factor, which is known to act as a carrier protein for FVIII and prevents its proteolytic degradation. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et. al. PLoS One 2013 8(12):e83280). While this new technology is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of cell sheet implantation in canine models as the first step toward gene therapy in clinic. GFP-transduced BOECs were cultured on temperature-responsive poly (N-isopropylacrylamide) (PIPAAm)-grafted dish to engineer BOECs sheet. This dish allows for simple detachment of the cultured cells without the use of proteolytic enzymes such as trypsin and enables us to harvest the cell sheet as a contiguous monolayer that retains its native intercellular communications and intracellular microstructure, which are indispensable for normal cellular function. When the cultured BOECs reached confluency, the cultured cells were detached from the PIPPAm dish as a uniformly connected tissue sheet by lowering the culture temperature to 20°C for 30 minutes. Under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the operation table. To exteriorize the greater omentum, an abdominal ventral midline incision was made and 20 BOECs sheets in total were implanted. Upon completion, the omentum was placed back into the abdomen and the incision was closed. For the assessment of GFP expression, the liver samples were collected by open liver biopsy both 30 and 120 days after sheet implantation. We confirmed that implanted BOECs were differentiated into mature endothelial cells and contribute to new blood vessel formation by histological examination. In conclusion, tissue engineering approach by omental endothelial cell sheet implantation are viable for persistent tissue survival and providing therapeutic values in canine model. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A. Now, we are conducting FVIII gene transfer by cell sheet implantation in canine hemophilia A model.

We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.

Cilostazol is an anti-platelet drug that reversibly inhibits phosphodiesterase III (PDE-III), which is ubiquitously expressed in platelets and various tissues. PDE-III converts cyclic adenosine monophosphate (cAMP) to 5'-AMP and up-regulates the intracellular concentration of cAMP, a potent inhibitor of platelet aggregation. Unlike other anti-platelet drugs, cilostazol is unique because patients receiving this drug do not have a significantly prolonged bleeding time, but the reasons for this difference are still unknown. In this study, we have examined how cilostazol inhibits platelet thrombus formation using anti-coagulated normal whole blood in which the platelets were labeled with a fluorescent dye in comparison with the anti-GPIIb/IIIa agent, tirofiban. We used an in vitro assay to examine mural platelet thrombus growth on a collagen surface under a high-shear rate flow in the absence of ADAMTS13 activity. These experimental conditions mimic the blood flow in patients with thrombotic thrombocytopenic purpura. Using this model, we clearly determined that cilostazol down-regulates the height of mural platelet thrombi formed on a collagen surface in a dose-dependent manner, without affecting the surface coverage. The concentration of cilostazol used in this study was relatively high (60–120 μM) compared to clinically relevant concentrations (1–3 μM), which may be due to the in vivo synergistic effects of PDE-III present in other tissues aside from platelets. Cilostazol does not affect the initial formation of platelet thrombi, but does inhibit the height of thrombi. These results showed a sharp contrast to tirofiban, and address why cilostazol does not significantly prolong bleeding time, despite its strong anti-platelet activity.

SummaryIn addition to lowering cholesterol, the 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have a range of pleiotropic effects that help reduce the risk of adverse cardiovascular events. We sought to understand the molecular mechanisms by which statins could exert anti-platelet actions under physiologic whole blood flow conditions. Using an in vitroperfusion chamber system, we examined the anti-platelet effects of pravastatin under whole blood flow conditions with high or low shear rates. We determined that pravastatin significantly suppressed platelet activation-dependent procoagulant activity, decreasing P-selectin membrane expression, tissue factor accumulation, and thrombin binding within platelet thrombi generated on a von Willebrand factor-surface under high shear rate conditions. These effects resulted in reductions of intra-thrombus fibrin deposition. These antithrombotic properties of pravastatin, which were comparable to those of atorvastatin, could be abrogated by mevalonate. Our experimental approach revealed a novel mechanism mediating the anti-platelet action of statins. Shear rate-dependent antithrombotic activity may explain the favourable effect of pravastatin on the reduction in cardiovascular events that typically occur in vivounder whole blood flow conditions with high shear rates.

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombogenic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain-specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mammalian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond formation into a dimeric adhesive domain without contributions from other regions of vWF, including propeptide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains.

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and its mortality remains high even in the developed countries. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. Recent mouse model studies demonstrated that VWF-mediated thrombotic and inflammatory responses could play a role in the disease progression of myocardial infarction or brain stroke. Thus, we assumed that VWF may also be involved in the pathophysiology of AKI, the major cause of which could be an insufficient renal circulation and/or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the relevant role of VWF in AKI in mouse model of acute ischemia-reperfusion (I/R) kidney injury. All mice used were male, 8-12 weeks of age, healthy and whose right kidney was surgically removed by the standard mouse nephrectomy procedure 1 week prior to the kidney I/R experiment. The preliminary experiments confirmed that surgical removal of mouse right kidney did not affect their general conditions including renal functions. Mice were anesthetized with inhaled isoflurane and then placed in an abdominal position on a heating pad. Surgical incision was given on the left side of back and the left kidney was brought out and kept outside during the operation. Both renal artery and vein were clamped at the renal hilus by a clamping clip for 30 min ischemia. Then a clip was taken off to provoke the reperfusion of renal blood flow, which was monitored by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The kidney was then put back in a body and skin incision was closed. The renal blood flow was measured again 30 h after reperfusion and mice were then sacrificed for blood collection. We compared 15 wild-type (WT) and 16 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME). Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. Although no difference was seen immediately after reperfusion, significantly (p < 0.05) higher renal blood flow at 30 h after reperfusion was confirmed in VWF-KO mice, as compared to WT (KO; 24.0±2.3 vs. WT; 15.1±1.46 ml/min/100g of kidney weight, and the reperfusion/base flow ratio: KO; 1.0±0.07 vs. WT; 0.6 ±0.07). Consistent with the renal blood flow data, the serum creatinine value at 30 h after reperfusion were significantly (p < 0.05) lower in VWF-KO mice than WT (KO; 2.77±0.11 vs. WT; 3.15±0.11 mg/dl). Our results suggest that VWF does play a role in the pathogenesis of AKI, in which VWF-dependent thrombotic or inflammatory responses may trigger thrombotic ischemia or endothelial damages of vascular bed in the kidney. Thus, proper functional regulation of VWF would be beneficial for better microcirculation and vessel functions in the kidney, suggesting a novel therapeutic potential against AKI. Disclosures No relevant conflicts of interest to declare.

Currently, hemophilia A patients are treated with plasma-derived or recombinant factor VIII (FVIII) concentrates. These treatments have substantially improved the management of bleedings in hemophilia and more recently long-acting recombinant FVIII concentrates are available in clinic, but hemophilia A patients still require the repeated intravenous injections frequently 2-3 times per week. Therefore, gene and cell-based therapy is an attractive strategy for the treatment of the disease because continuous expression of FVIII levels as low as 1-5 % of normal have been shown to ameliorate the bleeding phenotype as well as improve the quality of life. Although several attempts have been made for hemophilia A gene therapy, systemic delivery of viral vectors into liver cells in vivo, however, resulted in a transient and low-level expression of FVIII, because the large size of FVIII cDNA prohibited the efficient delivery of conventional viral vectors. Moreover, there remain concerns over the safety of these approaches such as adverse immunological reactions or virus-mediated cytotoxicity. Hydrodynamic gene delivery (HGD) is considered to be an alternative candidate to viral vectors. Previously, we tested the HGD in hemophilia A mouse model with the combination of non-viral piggyBac transposon vectors which can transfer the full-length FVIII transgene. As a result, we confirmed the sustained FVIII expression for over 300 days without any immune responses (Matsui H, et.al. PLos One 9(8) e104597, 2014). While HGD system is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of liver-target HGD in dogs as the first step toward hydrodynamic gene therapy in clinic. Hemophilia A carrier dog was provided from Dr. Lillicrap group at Queen's University and we have already developed hemophilia A dog colony in our institution. We used both normal and hemophilia A dogs in this study. In brief, under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the table of fluoroscopy machine. A 18G peripheral catheter was directly inserted into the saphenous vein followed by insertion of a 0.038 inch guide wire, a long sheath (5Fr) and an injection balloon catheter. The balloon catheter was placed and inflated just below the branch of hepatic vein using the image-guided interventional radiology technique. Liver-targeted HGD was performed into 4 liver lobes, the right lateral, right medial, left medial, and left lateral lobe, separately using piggyBac transposon vector expressing either GFP or FVIII. An injection of saline containing plasmid DNA were performed at the speed of 10mL/second sequentially into 4 liver lobes through the hepatic veins with 20 minutes interval between each injections.Various physiological parameters including heart rate, blood pressure, oxygen saturation were continuously monitored and recorded before, during and after each injection. For the assessment of tissue damage, blood samples were collected at the various time points. For the assessment of GFP expression, the liver samples were collected under echo-guided liver biopsy both 5 and 120 days after HGD. Although, transient 5-10 fold increase in hepatobiliary enzymes, including AST, ALT, were observed, there is no significant impact on physiological parameters, such as systemic circulation, respiration and cardiac function.By histological examination of liver samples, approximately 70-80% of hepatocytes in all 4 lobes were positive with GFP both 5 and 120 days after HGD. These results suggest that HGD system combined with interventional radiology technique is capable for the large animal with safe, efficient and less invasive. Now, we are conducting FVIII gene transfer by HGD in canine hemophilia A model. Disclosures No relevant conflicts of interest to declare.

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Salpha (PSalpha) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A--G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A--G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient's family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A--G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A--G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A--G mutation itself might influence phenotypic expression of PS status in different individuals.

Using a perfusion chamber and confocal laser scanning microscopy, we analyzed the interplay of von Willebrand factor (VWF) and fibrinogen during thrombus growth on a collagen surface under physiologic high shear rate conditions. During initial thrombogenesis, platelet thrombi were constructed totally by VWF, not by fibrinogen. Fibrinogen accumulated predominantly inside the growing thrombi as a function of time, whereas the thrombus surfaces directly exposed to flow were occupied constantly by VWF throughout the observation period. In perfusion of afibrinogenemia (AF) blood lacking both plasma and platelet fibrinogen, the final height and volume of thrombi were significantly reduced compared with controls, albeit the area of surface coverage was normal. The impaired thrombus growth in AF was only partially corrected by the addition of purified fibrinogen to AF blood, whereas the addition of purified VWF to blood of severe von Willebrand disease (VWD) completely normalized the defective thrombus growth in this disease. Thus, the initial 2-dimensional thrombus expansion involves only VWF, whereas the time-dependent accumulation of fibrinogen, released from activated platelets, acts as a core adhesive ligand, increasing thrombus strength and height and resulting in 3-dimensional thrombus development against rapid blood flow.

von Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, is stored in the alpha-granule of platelets and in the Weibel-Palade body of endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in both hemostasis and pathological intravascular thrombosis, in which vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple adhesive functions for the platelet membrane receptors, glycoprotein Ib-IX-V complex, integrin alphaIIbbeta3, heparin, various types of collagen, and coagulation factor VIII. Among various functions, the most characteristic feature of vWF is its determinant role on platelet thrombus formation under high-shear-rate conditions. Indeed, at in vivo rheological situations where platelets are flowing with high speed in the bloodstream, the only reaction that can initiate mural thrombogenesis is the interaction of vWF with platelet glycoprotein Ibalpha. The recent x-ray analysis of the crystal structure of various functional domains and functional studies of this protein under experimental flow conditions have rapidly advanced and revised our knowledge of the structure-function relationships of vWF, a key protein for hemostasis and arterial thrombosis.

Sepsis is a serious inflammatory response syndrome, in which systemic activation of both inflammatory and coagulation pathways are provoked by severe microbial infection. In addition to the known hemostatic functions, von Willebrand factor (VWF) is recently assumed to participate in a cross-talk between inflammation and thrombosis. Indeed, recent mouse model studies demonstrated that proper functional regulation of VWF-dependent inflammatory responses by ADAMTS13 considerably improved the disease progression of brain stroke or myocardial infarction. However, little is known about the detailed mechanisms or relevant role of VWF functions in inflammation. We therefore studied the physiologic relevance of VWF-dependent inflammatory responses in a mouse model of experimental sepsis by cecal ligation and puncture (CLP). The mouse CLP was performed according to the established standard protocol (Rittirsch D, et al., Nat Protoc, 2009). Briefly, mouse cecum was ligated distal to the ileocecal valve under laparotomy, punctured with 18 gauge needle and gently pressed until a small drop of stool appeared. The cecum was returned to the peritoneal cavity and 200 μL of saline was injected into the cavity to avoid dehydration before body wall and skin incision were closed with a 4-0 Sofsilk. We compared 17 wild-type (WT) and 17 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 10-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all (WT or KO) mice during the CLP operation. Kaplan-Meier analysis revealed the significantly (p < 0.05) lower survival rate of KO mice than that of WT (KO; 23.5% vs. WT; 58.8% at the Day 7 of CLP). The impaired survival rate of KO mice was restored by the bolus administration of human VWF (n=21, 2.5 U/mouse) to an extent comparable to that of WT. Peripheral blood analysis of KO mice at 24 hours after CLP showed the severe leukocytopenia with sharp decrease of neutrophils in blood, as compared to WT. In addition, formation of walled-off abscess was confirmed at the peri-cecal space in all the WT and KO mice alive even at the Day 7 of CLP, while such focal abscess formation was not found in mice died before the Day 3 of CLP. Thus, our results altogether indicate that VWF could play a role on the recruitment or accumulation of neutrophils for microbial killing at the local inflammatory focus. VWF-mediated platelet aggregate formation in peripheral capillary vessels then could shut down the local microcirculation, thereby blocking systemic microbial expansion as a crucial biological defense mechanism. Disclosures Shima: Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Modern plasma-derived clotting factor concentrates are produced using various virus-inactivation protocols and are assumed to be safer than they were previously with regard to the risk for transmitting viral infections such as human immunodeficiency virus, hepatitis B, and hepatitis C. The risks from viruses that are relatively resistant to the current inactivation procedures remain uncertain. Patient: A 7-year-old boy with mild hemophilia A who had not been previously infused with any blood products was treated with a plasma-derived, monoclonal antibody-purified factor VIII concentrate to cover orthopedic surgery after traumatic fracture of his left arm. Results: A typical primary human parvovirus (HPV)-B 19 infection was observed associated with transient hypoplastic anemia. Retrospective studies including serologic examination and polymerase chain reaction analysis confirmed that the HPV-B19 infection was transmitted by the factor VIII concentrate. Conclusions: Clotting factor concentrates for the treatment of hemophilia retain a risk for HPV-B 19 contamination. HPV-B 19 viral infection might induce hypoplastic anemia in these patients, particularly during enhanced hemopoiesis after acute blood loss.