Recombinant vWF Research Articles

Complement Component C3 Binds to the A3 Domain of von Willebrand Factor.

von Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (∼280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.

Prophylaxis in von Willebrand Disease: Coming of Age?

Although in most cases von Willebrand disease (VWD) is a mild disorder, a subgroup of patients experience frequent bleeding. In contrast to severe hemophilia in which prophylaxis is the accepted standard of care, this is less frequently used in VWD. Most type 1 VWD patients can be adequately managed with episodic desmopressin and tranexamic acid. In patients with more severe disease, especially those with type 3 VWD, joint bleeds, epistaxis, menorrhagia, and gastrointestinal bleeding are problematic and usually require treatment with von Willebrand factor/factor VIII (VWF/FVIII) concentrate. While in the past these patients were managed with on-demand VWF/FVIII concentrate, several recent reports have demonstrated the value of prophylactic treatment. Despite some uncertainties about the economic impact of treatment of severe VWD, prophylaxis with VWF concentrate should now be considered as the standard of care for the more severe end of the spectrum of affected individuals. The recent introduction of recombinant VWF concentrate is likely to improve the acceptability of prophylaxis in VWD.

Quantitative Measurement of VWF Binding to the Platelet Integrin αIIbβ3 - Abnormalities Identified in Individuals with VWD

Most studies of VWF function have focused on VWF interactions with platelet GPIb, FVIII, and specific sites and types of collagen. Early studies carried out by Ruggeri and Montgomery demonstrated that in addition to binding GPIb on platelets, VWF also bound to the GPIIb-IIIa receptor (also known as integrin αIIbβ3), but only in the absence of fibrinogen. Ruggeri and other groups have demonstrated that under flow, VWF interaction with GPIIb-IIIa is also critical to VWF-platelet biology. A large systematic study of VWF binding to GPIIb-IIIa (αIIbβ3) has not been undertaken previously in a cohort of type 1 and 2 VWD subjects. Studies were carried out on 619 VWD index cases (both type 1 and type 2 VWD) and 84 normal controls enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) using a novel assay of αIIbβ3 binding of VWF.Zhu and coworkers expressed the soluble αIIbβ3 headpiece and previously characterized this in the open position when bound to ligand. This αIIbβ3 headpiece was used for our studies. An antigen capture ELISA was developed using the αIIbβ3 headpiece to capture VWF and to compare binding to the VWF:Ag as a ratio of VWF-αIIbβ3/VWF:Ag. ELISA plates were coated with the GPIIIa-specific monoclonal antibody (MAb), AP3 and used to capture the αIIbβ3 headpiece. Since fibrinogen blocks VWF binding to αIIbβ3, plasma samples were heated at 56°C to precipitate fibrinogen, and then centrifuged to remove the precipitate. The resulting heat defibrinated plasma was utilized in the assay. Bound VWF was quantified using biotin-labeled AVW15, a MAb to VWF. The reference was the ISTH SSC Plasma Standard. As specific positive and negative controls, we used wild-type recombinant VWF containing the normal RGDS sequence at position 2507-2510, and mutant VWF containing the RGDE sequence at the same position. As an additional control, the monoclonal antibody 7E3, known to block aggregation by binding to β3 was used to block αIIbβ3/VWF interactions. While binding of wt-VWF to αIIbβ3 was robust, no binding of the RGDE mutant was observed (<1% of wt). 7E3 completely inhibited binding of VWF to αIIbβ3. Interestingly, two other MAbs known to block the binding of Fg to αIIbβ3, AP2 and 10E5, did not inhibit VWF binding. The 84 normal controls had a mean VWF-αIIbβ3/VWF:Ag binding ratio of 0.87 with a 5% and 95% confidence limit of 0.53-1.39. 152 type 1 VWD (VWF <30IU/dL) samples had a modestly reduced binding ratio of 0.62 (0.36-0.85) with a significance of p<0.0001. The binding ratios of 55 type 2A and 41 2B VWD samples were markedly reduced with a ratio of 0.293 (0.12-0.67) and 0.40 (0.23-0.81) both with a significance of 0.0001. Since both of these latter VWF variants have abnormal VWF multimers, we tested recombinant D-pro VWF (no VWF propeptide present) and Y87S mutant VWF (absent VWF multimerization). No binding to αIIbβ3 was observed with VWF from either of these constructs. Among our ZPMCB-VWD index cases, we also had individuals with low VWF (VWF:Ag 30-50 IU/dL) that had an intermediate reduction between the normal controls and type 1 subjects. Type 2N VWD samples demonstrated no abnormal binding to αIIbβ3 and type 2M subjects had minimal differences.While VWF binding to αIIbβ3 has been recognized previously, it has not been systematically studied in subjects with VWD. We identified a modest decrease in αIIbβ3 binding of VWF in type 1 VWD and in clinical subjects with low VWF. Furthermore, type 2A and 2B subjects have a much more profound reduction in αIIbβ3 binding and suggest an importance of normal multimeric VWF to the functional binding of VWF to αIIbβ3. Mutating the RGDS sequence in VWF, or adding 7E3 to block αIIbβ3, abrogate this VWF binding. To date we have not identified any of our enrolled ZPMCB-VWD subjects with mutation of the RGDS sequence or in the C4 domain of VWF, although mutations in this region recently have been reported by Legendre et al. at the ISTH2015 meeting. Such mutations are not identified with current VWF functional screening assays, but specific assays of αIIbβ3-VWF interaction, such as the one described here, can be used and suggest there are specific potential mutations and variants with abnormal multimers that have abnormal αIIbβ3 binding. The qualitative and quantitative assessment of VWF function continues to be complicated. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Efficacy of Anfibatide in a Murine Model of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 activity. Plasma infusion or exchange is the mainstay of treatment. However, complications associated with the administration of blood products and with the placement of a central venous line remain to be a major concern. To address the unmet clinical need, we test hypothesis that anfibatide, a novel snake venom-derived glycoprotein (GP) Ib antagonist, may be efficacious in treating TTP. Here, we show that purified anfibatide from snake venom inhibited murine platelet agglutination induced by botrocetin in the presence of recombinant murine VWF in a concentration-dependent manner. At 6-9 micro grams per ml (final concentrations), anfibatide nearly completely abolished botrocetin-induced murine platelet agglutination (Fig. 1A). As expected, anfibatide at the same concentrations showed little or no inhibitory effect on the protease-activating receptor 4 (PAR4) agonist (AYPGKF)-induced murine platelet aggregation. In vivo, an intravenously infused radiolabeled anfibatide exhibited a half-life of 5-7 hours depending on the initial loading dose. This information provided pharmacokinetic basis for therapeutic strategy for TTP in the murine model. An infusion of purified anfibatide at the dose of 60-90 ng per gram of body weight twice daily significantly reduced the rate of thrombocytopenia in Adamts13-/- mice triggered by intravenous infusion of a bacterial toxin, i.e. shigatoxin-2 (Stx2) (250 pg per gram body weight). As shown, 5/5 (100%) Adamts13-/- mice after being challenged with Stx2 and but treated with normal saline (control) exhibited severe thrombocytopenia (defined by 50% reduction of platelet counts from the baseline). Similarly, treatment of Adamts13-/- mice with anfibatide at the dose of 30 ng per gram body weight twice daily had little or no effect in preventing thrombocytopenia. However, treatment of the same mice with anfibatide at doses of 60-90 ng per gram body weight twice daily resulted in significantly reduced rate of severe thrombocytopenia to 11%-37.5% (Fig. 1B). Our ongoing effort is to determine the efficacy of anfibatide in treatment of acquired TTP associated with inhibitors. We conclude that anfibatide when given at the optimal doses and interval can efficiently prevent Stx2-induced TTP syndrome in the murine model. These findings support a rationale for further development of anfibatide as a novel therapeutic for TTP in humans. [Display omitted] DisclosuresLi:3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong: Employment. Dai:4Zhaoke Pharmaceutical Co. Limited, Hefei, Anhui, China: Employment. Li:3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong, China: Employment.

ADAMTS13 Secretion and Residual Activity among Patients with Congenital Thrombotic Thrombocytopenic Purpura with and without Renal Impairment.

Acute renal impairment is observed in 11%-23% of patients with congenital thrombotic thrombocytopenic purpura (TTP) and deficiency of a disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13, a metalloprotease that cleaves von Willebrand factor [VWF] multimers), a substantial percentage of whom develop CKD during follow-up. Here we investigated whether, in 18 patients with congenital recruited from 1996 to 2013 who fulfilled inclusion criteria, acute renal involvement occurred during bouts segregated with lower secretion and activity levels of ADAMTS13 mutants. We performed expression studies and a sensitive recombinant VWF (rVWF) A1-A2-A3 cleavage test (detection limit, 0.78% of normal ADAMTS13 activity). A higher risk of acute renal impairment during bouts was observed in patients with childhood (<18 years) onset (odds ratio [OR], 24.6 [95% confidence interval (CI), 1.11 to 542.44]) or a relapsing (≥1 episode per year) disease (OR, 54.6 [95% CI, 2.25 to 1326.28]) than in patients with adulthood onset or long-lasting remission, respectively. Whatever the age at onset, patients with acute renal impairment had mutations different from those in patients without renal involvement. Moreover, mutations in patients with acute renal impairment compared with those in patients without renal involvement caused lower in vitro rADAMTS13 secretion (1.33% versus 12.5%; P<0.001) and residual activity (0.11% versus 3.47%; P=0.003). rADAMTS13 secretion ≤3.75% and residual activity ≤0.4% best discriminated patients with renal impairment (receiver-operating characteristic curve sensitivity, 100% and 100%; specificity, 100% and 83.3%, respectively; logistic regression OR, 325 [95% CI, 6 to 18339] and 91.7 [95% CI, 3.2 to 2623.5], respectively). All mutations found in patients with childhood onset or relapsing disease were associated with acute renal impairment during bouts, confirming the link between acute renal impairment and early onset or a relapsing course. ADAMTS13 activity levels in vivo, measured in patients' serum by rVWF A1-A2-A3 cleavage test, correlated with in vitro rADAMTS13 mutant activity (r=0.95; P<0.001). In congenital TTP, renal impairment and relapsing disease might be predicted by measurements of in vitro rADAMTS13 secretion and activity levels and in vivo serum ADAMTS13 activity.

Domain Specificity and Mechanism of Action of Anti-ADAMTS13 Antibodies at Presentation and Relapse in Acquired Thrombotic Thrombocytopenic Purpura (TTP)

Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease due to autoantibodies (Auto-Ab) against ADAMTS13, resulting in a severe deficiency in enzyme activity (<10%). Little is known about the pathogenicity of anti-ADAMTS13 IgG directed against different domains of the enzyme, or how domain specificity alters between presentation, remission or TTP relapse. We developed novel assays to explore domain specificity and inhibitory potential of anti-ADAMTS13 IgG using samples from the UK TTP Registry.Full-length ADAMTS13 and ADAMTS13 fragments (MD, MDTC and MDTCS - N-terminal domains; TSR2-8 and CUB1/2 - C-terminal domains) were expressed in HEK293T cells and purified. Microtitre plates were coated with ADAMTS13 or ADAMTS13 fragments, incubated with TTP plasmas and bound IgG detected. Domain specificity was further analysed by preincubation of patient samples with ADAMTS13 fragments, assessing the ability of fragments to compete for full-length ADAMTS13 binding on the plate. From a cohort of 92 acquired TTP patients at presentation, 100% (92/92) contained auto-Ab that recognised ADAMTS13 N-terminal domains (MDTCS). Of these, 39% (36/92) had auto-Ab recognising the N-terminal domains alone, 22% (20/92) also had anti-TSR2-8, and 39% (36/92) had anti-CUB1/2 auto-Abs. There was no correlation between auto-Ab domain specificity and mortality/disease severity (anti-ADAMTS13 IgG titre, plasma exchanges to remission, platelet count and cardiac involvement).There was no evidence for auto-Ab recognising MD or MDTC domains in 25 patients tested, suggesting immunoreactivity for MDTCS was primarily mediated through the spacer domain. Consistent with this, TTP IgG inhibited proteolysis of VWF115 (a recombinant VWF A2 domain fragment), but failed to inhibit VWF106 proteolysis (VWF A2 domain fragment lacking the spacer domain binding exosite).To examine the inhibitory potency of anti-ADAMTS13 auto-Ab, IgG isolated from TTP patient samples were titrated in reactions measuring MDTCS-mediated proteolysis of FRETS-VWF73. Twenty eight out of 43 presentation samples tested had inhibitory IgG (IC50 ranging from <0.2-5μM total IgG). Addition of excess of MDTC to 19/19 of these samples did not alter the IC50, confirming inhibition in these samples is mediated solely by anti-spacer antibodies. Intriguingly, 15/43 patients had no evidence of inhibitory auto-Ab, suggesting the pathogenic mechanism in these cases may be enhanced clearance of ADAMTS13. We therefore determined ADAMTS13 antigen concentrations by ELISA. All patients at presentation exhibited markedly reduced ADAMTS13 antigen levels (n=91, median 58ng/ml (range 0-450) vs. normal n=67, median 951ng/ml (515-1829); p<0.0001). Interestingly, patients with predominantly C terminal auto-Ab and no evidence of inhibitory auto-Ab had significantly lower ADAMTS13 antigen levels at first presentation than the rest of the cohort (median 2ng/ml (0-141) vs. 57ng/ml (0-356); p=0.006).Cases where rituximab failed to normalise ADAMTS13 activity despite clinical remission were associated with the persistence of inhibitory anti-N-terminal auto-Ab. Two patients with persistently high anti-ADAMTS13 IgG but normal ADAMTS13 activity and antigen levels during remission had non-pathogenic anti-TSR2-8 antibodies that seemingly had no effect upon ADAMTS13 function or clearance. Nine out of 16 patients exhibited altered auto-Ab domain specificity upon relapse, with either loss or development of anti-C-terminal auto-Ab.In conclusion, from our functional studies ADAMTS13 inhibition appears to be mediated exclusively by anti-spacer antibodies, with no evidence for the presence of auto-Ab that recognise/inhibit other N terminal domains. Importantly, we demonstrate ADAMTS13 antigen depletion is a crucial pathogenic mechanism in acquired TTP, accounting for a large proportion of the reduced ADAMTS13 activity detected in patient samples. Our results suggest that auto-Ab directed against the C-terminal domains of ADAMTS13 may be particularly important in mediating this clearance. There is continual development of the autoimmune response during treatment of TTP patients that can result in altered domain specificity at relapse. DisclosuresNo relevant conflicts of interest to declare.

Management of VWD.

VWD is the most common inherited bleeding disorder known. It is caused by a deficiency or dysfunction of the VWF molecule. Bleeding risk varies between modest increases in bleeding seen only with procedures to major risk of spontaneous hemorrhage depending upon the type of VWD. The treatment approach to VWD has changed little in the past 2 decades, but there are numerous subtleties in optimal management. Management includes the prevention or treatment of bleeding by raising endogenous VWF levels with medications such as desmopressin or providing exogenous VWF concentrates. Fibrinolytic inhibitors and topical hemostatic agents are also effective adjunctive measures. Bleeding specific to women presents a special challenge because of heavy menstrual bleeding and pregnancy. Successful management of pregnancy in patients with VWD involves coordination with obstetrics, anesthesia, and the coagulation laboratory monitoring VWF:RCo and FVIII:C levels. Prophylactic treatment with VWF concentrates is emerging as an effective preventive therapy in patients with severe disease. Antibodies to VWF present a special challenge in the management of rare patients with type 3 disease. New therapies on the horizon include recombinant VWF, anti-VWF aptamers, and medications such as IL-11 to raise VWF levels. The key to effective treatment of VWD is an accurate diagnosis of the specific type and selection of hemostatic products appropriate for the clinical situation.

Effects of increased von Willebrand factor levels on primary hemostasis in thrombocytopenic patients with liver cirrhosis.

In patients with liver cirrhosis procoagulant and anticoagulant changes occur simultaneously. During primary hemostasis, platelets adhere to subendothelial structures, via von Willebrand factor (vWF). We aimed to investigate the influence of vWF on primary hemostasis in patients with liver cirrhosis. Therefore we assessed in-vitro bleeding time as marker of primary hemostasis in cirrhotic patients, measuring the Platelet Function Analyzer (PFA-100) closure times with collagen and epinephrine (Col-Epi, upper limit of normal ≤165 s) or collagen and ADP (Col-ADP, upper limit of normal ≤118 s). If Col-Epi and Col-ADP were prolonged, the PFA-100 was considered to be pathological. Effects of vWF on primary hemostasis in thrombocytopenic patients were analyzed and plasma vWF levels were modified by adding recombinant vWF or anti-vWF antibody. Of the 72 included cirrhotic patients, 32 (44.4%) showed a pathological result for the PFA-100. They had mean closure times (± SD) of 180±62 s with Col-Epi and 160±70 s with Col-ADP. Multivariate analysis revealed that hematocrit (P = 0.027) and vWF-antigen levels (P = 0.010) are the predictors of a pathological PFA-100 test in cirrhotic patients. In 21.4% of cirrhotic patients with platelet count ≥150/nL and hematocrit ≥27.0%, pathological PFA-100 results were found. In thrombocytopenic (<150/nL) patients with cirrhosis, normal PFA-100 results were associated with higher vWF-antigen levels (462.3±235.9% vs. 338.7±151.6%, P = 0.021). These results were confirmed by multivariate analysis in these patients as well as by adding recombinant vWF or polyclonal anti-vWF antibody that significantly shortened or prolonged closure times, respectively. In conclusion, primary hemostasis is impaired in cirrhotic patients. The effect of reduced platelet count in cirrhotic patients can at least be partly compensated by increased vWF levels. Recombinant vWF could be an alternative to platelet transfusions in the future.

A two-step approach (Enzyme-linked immunosorbent assay and confirmation assay) to detect antibodies against von Willebrand factor in patients with Acquired von Willebrand Syndrome

BackgroundAcquired von Willebrand Syndrome is a rare bleeding disorder, which arises in individuals with no personal or family history of bleeding, associated with lymphoproliferative and myeloproliferative disorders or other diseases. AimTo develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS. MethodsAVWS definition: negative personal or family history of bleeding diathesis, VWF below normal range and recent history of bleeding manifestations. Twenty-three consecutive patients affected by AVWS were enrolled. An ELISA assay (first step) using recombinant VWF protein immobilized on plates and sheep/goat polyclonal anti-human IgG or IgM labelled with peroxidase was developed. A group of 40 healthy subjects was tested to calculate the floating cut point value. A confirmation assay (with addition of purified VWF vs buffer) was performed (second step). ResultsTwenty–one patients (93%) had an associated disease, two patients had idiopathic AVWS. Anti-VWF autoantibodies were detected in 9 patients (39%). Of these, eight (89%) had VWF:RCo levels <10%, but none of them resulted positive using Bethesda assay (neutralizing antibodies). The confirmation test confirmed the positive results obtained with ELISA and resulted negative in those patients with negative results and in the controls. ConclusionWith the present two-step approach assay nine out of 23 (39%) patients affected with AVWS resulted positive for anti-VWF autoantibodies. This ELISA assay might be used as an additional confirmation tool in the diagnostic procedure in patients affected by AVWS or in the follow-up of congenital and acquired patients exposed to replacement therapy.

ADAMTS13 Expressed In Glioblastoma and Astrocytoma Cells Can Modulate Endothelial Cell Migration

ADAMTS13, the von Willebrand factor (vWF) cleaving protease, regulates platelet aggregation and microthrombi formation by cleaving high-molecular weight vWF multimers. It is expressed primarily in hepatic stellate cells, but is also found in endothelial cells. Recently, ADAMTS13 was reported to be expressed and regulated in astrocytes, microglial, neuroblastoma, and adult human brain endothelial cells. Previous in vitro studies by our group with human umbilical vein endothelial cells (HUVEC) showed that ADAMTS13 can promote angiogenesis via upregulating the secretion of VEGF and phosphorylation of VEGFR2, suggesting that ADAMTS13 may also be involved in physiological processes unrelated to vWF cleavage (Lee, M., et al. Microvasc Res. 2012, 84, 109-115). Herein, we report an additional possible role of ADAMTS13 secreted by brain tumor cells to modulate tumor cell angiogenesis.Using a human ADAMTS13 immunoassay, we detected ADAMTS13 in U-87 and LN-229 glioblastoma cell lysates, SW-1088 astrocytoma cell lysates, as well as in the supernatants of all cell lines (> 2.0 ng/mL). Co-incubation of U-87, LN-229, and SW-1088 tumor cell conditioned media with recombinant vWF indicated that brain tumor-secreted ADAMTS13 is biologically active in cleaving vWF multimers (measured by ELISA). Secretion of VEGF was upregulated in LN-229 and SW-1088 cell lines by ADAMTS13. 939 pg/mL and 674 pg/mL of VEGF were measured in LN-229 and SW-1088 cell lysates, respectively, after incubation with 100 ng/mL ADAMTS13. Incubation of LN-229 glioblastoma cells with 10 – 500 ng/mL rh-ADAMTS13 or 50 ng/mL VEGF165 did not affect tumor cell proliferation. No change in tumor cell proliferation was observed when LN-229 cells were incubated with a polyclonal antibody against ADAMTS13 in serum free media supplemented with 10 ng/mL ADAMTS13 or media supplemented with 5% FBS, suggesting that ADAMTS13 secreted by brain tumor cells may be involved in extracellular signaling of endothelial cells.Brain tumor cell secreted-ADAMTS13 induced HUVEC migration in a Matrigel invasion assay. Using a modified Boyden chamber fitted with a Matrigel-coated polycarbonate membrane, LN-229 glioblastoma cells increased HUVEC migration by 83%. LN-229 cells supplemented with 10 ng/mL ADAMTS13 further increased HUVEC migration by 190%, suggesting that tumor cell-secreted ADAMTS13 may modulate EC migration (Fig. 1). Combined with our previous findings suggesting that recombinant ADAMTS13 modulates EC angiogenesis, brain tumor-secreted ADAMTS13 may also be a regulator for tumor vasculature angiogenesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A New Microfluidic-Based Platelet Rolling Assay to Better Characterize VWD Phenotypes

Abstract 3386 Background:Von Willebrand disease (VWD), the most frequently diagnosed bleeding disorder, is characterized by variable expressivity and incomplete penetrance. Bleeding severity in type 1 VWD does not always correlate with plasma VWF levels, except in cases of severe deficiency. The purpose of this study is to determine whether the degree of type 1 VWD mutant subunit incorporation into VWF multimers affects quantitative as well as qualitative defects.While one of the major functions of VWF is to capture platelets from flowing blood at the site of an injury, most clinical diagnostic assays of the platelet glycoprotein (GP) Ibα-VWF interaction are performed under static conditions, either in the presence of ristocetin or with a gain-of-function GPIbα construct. Platelet function analyzer-100 (PFA-100®) closure time is used to measure primary hemostasis in the presence of shear stress and is sensitive to low plasma VWF levels, but it can be influenced by variables such as hematocrit and platelet count.The lifetime of the GPIbα-VWF bond is prolonged with increasing shear stress to a certain threshold (catch bond), thus comprehensively studying this interaction requires using a range of shear stresses [Coburn et al, Biophys J, 2011]. We have developed an assay to examine platelet rolling on whole, recombinant (r)VWF in a microfluidic chamber. Methods:HEK293T cells were co-transfected with equal amounts of mutant VWF in a vector that appends a MycHis (mH) tag to the C-terminus, and untagged WT VWF. R1374C, a type 2M VWD mutation which interferes with the binding of VWF A1 domain to GPIbα, and R782Q, a type 1 VWD mutation, were studied here. rVWF expression was measured with ELISA, multimer composition was analyzed using SDS-agarose gel electrophoresis and Western blot, and intracellular location of rVWF was determined by immunofluorescent staining of VWF and organelle markers. FVIII binding of rVWF was performed using the Chromogenix Coatest SP4 FVIII assay.In order to study platelet-VWF interactions, recombinant VWF (10 μg/mL) from supernatants was concentrated then incubated over a surface of anti-VWF mAb, AvW-1, which we used to determine rVWF concentrations in our ELISA. Washed human platelets (6 × 108/mL) were perfused across the rVWF surface at shear stresses of 2–50 dyn/cm2 and movies were captured at 15 frames/sec. Platelet rolling velocities were manually determined during the first second that cells interacted with the surface. Differences in rolling velocity were considered significant for p<0.05 using the Mann-Whitney test. Results:Analysis of platelet rolling velocities on rVWF demonstrated a catch-slip behavior of the platelet-VWF interaction: on all rVWF surfaces, platelet rolling velocities decreased with increasing shear stress until approximately 10 dyn/cm2, above which rolling velocities increased with further increases of shear stress. Minimal platelet rolling was observed in the presence of the GPIbα-blocking antibody, AK2, or on AvW-1 surfaces incubated with concentrated supernatants from mock transfections.Compared to platelet rolling on our control surface, WT mH/WT VWF, rolling velocities on R1374C mH/WT VWF were significantly higher at shear stresses of 2, 5 and 50 dyn/cm2.R782Q mH/WT VWF exhibited pseudo Weibel-Palade body formation in HEK 293 cells, supported full multimerization and bound FVIII similarly to WT VWF. Platelet rolling velocities were significantly higher compared to WT mH/WT VWF at 50 dyn/cm2, but statistically similar at lower shear stresses. Discussion:Our microfluidic-based platelet rolling assay is able to demonstrate the catch-slip nature of the platelet GPIbα-VWF interaction. We observed distinct differences in platelet rolling velocities between a heterozygous type 2M mutant and WT VWF at a variety of shear stresses. Additionally, while individuals with the type 1 VWD mutation, R782Q, have a VWF:RCo/VWF:Ag ratio of close to 1 [Zhang et al, Hum Genet, 1995], our studies may identify a possible platelet binding defect for this mutation at high shear stresses. Thus studying the platelet-VWF interaction at a range of shear stresses may add to the completeness of VWD classification, especially in cases where VWF:RCo/VWF:Ag is close to 1. Further studies using a range of mutant:WT co-transfection ratios for type 1 VWD mutants may elucidate a role for the degree of mutant subunit incorporation into multimers in the hemostatic potential of VWF. Disclosures:No relevant conflicts of interest to declare.

Functional Characterization of the Type 1 VWD Candidate VWF Gene Variants: P.M771I, p.L881R, p.P1413L, and p.Q1475X

Abstract 97Von Willebrand Factor (VWF) is a multimeric glycoprotein that maintains hemostasis in the vascular system. VWF is secreted mainly from endothelial cells. During its biosynthesis VWF undergoes a series of post-translational modifications and some of the newly synthesized protein is released constitutively. The remainder of the protein is stored for regulated release from cytoplasmic storage granules, Weibel Palade Bodies. Abnormalities in the biosynthetic pathway or increased clearance of plasma VWF are likely to contribute to decreased plasma VWF levels. Inherited partial deficiency of VWF is classified as Type I von Willebrand disease (VWD). Clinical diagnosis of Type I VWD is complicated due to incomplete penetrance and variable expressivity of the abnormal VWF phenotype. The molecular basis of Type 1 VWD remains incompletely understood. The purpose of this study is to explore the effect of four VWF missense mutations (p.M771I, p.L881R, p.P1413L, p.Q1475X), that were reported as candidate mutations in Type I VWD patients in the ISTH SSC VWF Database. The focus of these studies is on the intracellular biosynthetic processing and localization of VWF in a heterologous cell system. For this purpose, selected VWF gene variations were generated in a VWF cDNA expression vector by in vitro site directed mutagenesis. The effect of the candidate mutations on the intracellular localization were analyzed in HEK293 cells by immunoflourescence antibody staining and confocal microscopy. In addition, the effect of the mutations on the biosynthesis of the recombinant VWF was analyzed in COS-7 cells The expression vectors WT-cDNA (wild type) and the expression vector carrying the candidate mutations were transiently transfected into COS-7 cells alone or together to generate wild-type, mutant homozygous and heterozygous genotypes, the latter situation mimicking the status in type 1 VWD patients. To determine whether the mutant recombinant protein was retained within the cell or was efficiently secreted, VWF:Ag levels were assayed in cell lysates and in the conditioned media using ELISA.Confocal analysis of the transfected HEK293 cells demonstrated impaired intracellular localization of recombinant VWF having p.M771I and p.Q1475X variations. On the other hand, a normal pattern of intracellular storage was observed for the VWF variants having p.L881R and p.P1413L changes.Expression studies for the p.P1413L and p.Q1475X mutations are currently in progress. Transient transfection of COS-7 cells with the VWF expression vector carrying p.M771I mutation revealed that secretion of the recombinant VWF protein was decreased by 73% (P=0.013, n=3) in the homozygous state, and by 47% (P=0.013, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein was increased by 212% in the homozygous state (P=0.005, n=3) and by 69% in the heterozygous state (P=0.255, not significant, n=3). This study demonstrated that p.M771I mutation causes increased intracellular retention of the protein and it has a dominant negative effect on the processing of the wild type protein.Transient transfection of COS-7 cells with the VWF expression vector carrying p.L881R mutation revealed that secretion of the recombinant VWF protein was decreased by 10% (P=0.61, not significant, n=3) in the homozygous state, and increased by 47% (P=0.36, not significant, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein were increased by 58% in the homozygous state (P=0.925, not significant, n=3,) and decreased by 35% in the heterozygous state (P=0.405, not significant, n=3). This study demonstrates that in contrast to the findings with the p.M771I mutant, the p.L881R mutation has little effect on VWF biosynthesis and secretion and does not show a dominant negative effect on the wild type VWF. The pathogenetic mechanism responsible for the type 1 VWD phenotype associated with the p.L881R mutation requires additional evaluation but may involve enhanced protein clearance. Disclosures:No relevant conflicts of interest to declare.

The Effect of Unpaired Cysteine Residues and the C Domains On the Expression of Von Willebrand Factor

Abstract 1078Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein that mediates platelet adhesion under high shear stress and is the carrier molecule for FVIII. VWF contains a large number of cysteine residues that were previously thought to all participate in either intra- or inter-molecular disulphide bonds. However, recently it has been shown that VWF contains a proportion of unpaired cysteine residues, or free thiols and these may be involved in lateral self association of VWF. Initially Choi et al concluded that two free thiols are present in the D3 domain C889 and C898) and seven in the C domains (C2448, C2451, C2453, C2490, C2491, C2528 and C2533). Moreover, Ganderton et al recently showed that expression of the isolated VWF C2 domain resulted in the formation of disulphide linked oligomers and suggested that lateral self-association of VWF involved the C2431-C2453 bond located in the VWF C2 domain. When they mutated C2453 to Alanine the extent of oligomerisaton was enhanced. However it is not clear how this relates to the full length VWF molecule. In the present study we investigated the effect of mutating the predicted unpaired cysteine residues on the expression of full length VWF. Initially we used the binding of MPB to VWF to compare the relative amount of free thiol on plasma derived and recombinant VWF (rVWF). Interestingly, rVWF presented an increased free thiol content compared to purified plasma derived VWF indicating that free thiols, at least in rVWF are formed without exposure to the circulation. Next we created nine individual point mutations, based the observations of Choi et al, changing the predicted unpaired cysteine residues to alanine in full length VWF and analysed their expression in HEK293T cells. Interestingly, all of the point mutations failed to secrete from HEK293T cells, with the protein being retained within the cell lysate. A double point mutant, C2431A-C2453A, similarly failed to secrete. Analysis of the pro-VWF:mature-VWF ratio and Endo-H digestion of intracellular VWF demonstrated that all the mutants were retained within the endoplasmic reticulum (ER). Co-expression experiments with wild type VWF partially restored expression of some mutants, however co-expression with a deletion A1/A3 construct, demonstrated that the molecules containing the cysteine point mutations were retained predominately in the ER. Together these data suggest that in full length VWF, correct disulphide bonding within the ER is required for protein secretion. Since the point mutations did not express we created a series of deletion mutants to remove portions of the C-terminus of VWF. While VWF with either its A1, A2, A3 or D4 domain was expressed at comparable levels to wild type, all of the created C-terminal deletion variants: ΔC1C6(2255–2720), ΔC1C2(2255–2428), ΔC3(2431–2494), ΔC3C4-(2400–2515) and ΔC3C6-(2400–2662) also failed to secrete at significant levels. This data suggest that in the full length VWF molecule an intact sequence of C-domains is required for proper expression. To establish if the cysteine mutants could be expressed in smaller VWF constructs we introduced the same mutations into VWF molecules spanning the A2-CK, A2C6 and C3-CK domains. Interestingly, the mutants failed to express in VWF-A2CK and A2C6, again being retained in the ER, but were secreted in VWF-C3CK although to a significantly less extent than wild type C3CK. Furthermore, the A2C6 construct only expressed as monomers with very few dimers. Together these data demonstrate that correct disulphide bonding and an intact series of C domains are required for passage through the ER into the Golgi and efficient VWF secretion. The location and mechanism by which certain disulphide bonds break, forming free thiols remains to be established. Disclosures:No relevant conflicts of interest to declare.

The R1306W Type 2B Natural Mutation of Von Willebrand Factor Dramatically Enhances the Multimer Sensitivity to Shear Stress

AbstractAbstract 3306 Background.Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods.In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results.The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values <10dyn/cm2 the R1306W mutant, at variance with WT vWF, already showed an initial self-aggregation with formation of a network characterized by a roughness significantly higher than that of WT vWF. Mechanical stretching experiments, performed using the AFM cantilever, showed that R1306W vWF needs 30% less energy per length unit to be stretcted compared to WT protein (1.6 vs 2.2×10−11J/m) Conclusions.These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb. Disclosures:No relevant conflicts of interest to declare.

ADAMTS13 Variants in Atypical Hemolytic Uremic Syndrome

Abstract 490Atypical hemolytic uremic syndrome (aHUS) is a clinically defined thrombotic microangiopathic anemia (TMA) characterized by the symptom triad of renal failure, thrombocytopenia and microangiopathic hemolytic anemia in the absence of Shiga-toxin-producing bacteria as a triggering factor. Its pathogenesis is linked to dysregulation of the alternative pathway of the complement cascade, with loss-of-function mutations reported in complement regulators like factor H (CFH), membrane cofactor protein (MCP), factor I (CHI) and thrombomodulin (THBD), and gain-of-function mutations in complement activators like factor B (CFB) and complement component 3 (C3). In nearly 40% of patients, however, mutations are not identified in these genes raising the possibility of unrecognized contributory genetic causes.Genetic variants in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs) have been causally related to thrombotic thrombocytopenic purpura (TTP), another TMA characterized by the pentad of neurologic symptoms, fever, kidney failure, thrombocytopenia and microangiopathichemolytic anemia. The phenotypic similarities between aHUS and TTP can at times lead to difficulty in their clinical distinction. On this basis, we hypothesized that partial deficiency in ADAMTS13 function may coexist with abnormalities in the alternative complement pathway in patients with aHUS.To test this hypothesis, we measured ADAMTS13 functional activity in 26 patients with aHUS by fluorescence resonance energy transfer substrate-von Willebrandfactor 73mer (FRETS-VWF73) and recombinant VWF A2 domain cleavage assay. We also genotyped all patients for ADAMTS13 and alternative complement pathway genes. We transiently expressed 10 different ADAMTS13 missense variants in HEK293 cells to analyze activity and secretion of each recombinant protein.The ADAMTS13 functional activity was partially reduced (< 60%) in serum from 20 patients (77%) as measured by FRETS-VWF73 and recombinant VWF A2 cleavage assays. Genetic variants in ADAMTS13 were identified in 21 patients (81%) and included R386C, Q448E, A900V, V832M, R1060W, R7W/Q448E, A900V/Q448E, R1096H/A747V, R7W/A1033T, R7W/P618A/Q448E, R7W/P618A/A900V/Q448E, R7W/P618A/A732V/Q448E. The coexistence of both ADAMTS13 deficiency and excessive complement functional activity was present in 13 patients (50%). Activity of recombinant ADAMTS13 proteins containing the following variants was normal: R386C, Q448E, P618A, A732V, A747V, V832M, A900V, A1033T and R1060H. The R1096H variant of ADAMTS13 was associated with a reduction in functional activity by 50%. The secretion of recombinant proteins with the following variants was severely reduced: P618A, R1060H (<1%), R386C, R1096H (<10%), and A1033T (<50%) (Figures 1 and 2).This study implicates ADAMTS13 in the pathogenesis of aHUS and mandates the evaluation of ADAMTS13 in aHUS patients. Defining the role of ADAMTS13 in aHUS may aid in the development of new treatments and/or assist in clarifying whether long-term treatment with currently available anti-complement therapy is required for this disease. [Display omitted] Disclosures:Kroll:Aplagon: Membership on an entity's Board of Directors or advisory committees; Optimer: Consultancy; Leo: Honoraria, Travel Expenses, Travel Expenses Other.

The Role of Sialic Acid in Von Willebrand Function Under Shear Stress

Abstract 1079Sialic acids (SA) are negatively charged monosaccharides present on the terminus of N- and O-linked glycans. They carry out diverse functions such as protection from proteolytic degradation and modulation of protein clearance. Both the N- and O-linked glycans of VWF are decorated with SA residues, which have been shown to block recognition and clearance of VWF via the asialoglycoprotein receptor (ASGPR). It has been previously shown that VWF devoid of SA residues exhibits increased platelet binding and decreased susceptibility to ADAMTS13 proteolysis, however this has only been shown under static conditions. In this study we have investigated the role of VWF SA under the physiological conditions of shear stress.Firstly, using a plate-based ELISA assay we confirmed that desialylated (ds)VWF demonstrated enhanced sensitivity to ristocetin-mediated binding to GPIbα Interestingly, this effect was attributed to α 2–3 linked SA which are present primarily on O-linked glycans. For analysis under flow conditions, a recombinant VWF fragment spanning the D’-A3 domains was immobilised on Ibidi flow slides and perfused with washed, labelled platelets and red blood cells at a range of shear rates. This fragment contains the majority of the O-linked glycans on VWF which are clustered either side of the A1 domain. At low shear rates (400–600s−1), dsD’A3 captured more platelets than wild type (wt) D’A3; whilst a similar number of platelets was captured to both proteins at high shear rates (1000–2000s−1). Platelet translocation velocity over dsD’A3 was decreased at all shear rates (400–2000s−1) when compared to wtD’A3: however the dissociation rate constant was unaltered. Interestingly, when perfused over a collagen type III coated surface in reconstituted blood, dsVWF and VWF devoid of only α 2–3 linked sialic acid residues (α 2–3dsVWF), both mediated platelet capture to a similar extent as wtVWF. In the absence of VWF, no platelet capture was detected, confirming that platelet capture under the shear rate applied (1500s−1) was VWF dependent. Subsequently, to assess susceptibility of ds- and α 2–3ds VWF to ADAMTS13 proteolysis under shear, recombinant ADAMTS13 was added to plasma free blood supplemented with VWF and perfused over collagen. In control experiments, the presence of ADAMTS13, but not the inactive ADAMTS13 variant (M225Q), diminished platelet capture demonstrating the assay was sensitive to ADAMTS13 proteolysis of VWF. Interestingly, the decrease in platelet capture in the presence of ADAMTS13 was significantly less with dsVWF and α 2–3dsVWF than with control VWF, demonstrating that dsVWF and α 2–3dsVWF are proteolysed slower by ADAMTS13 under shear stress and confirming the relevance of the results from static assays. Furthermore, these data suggest that it is the α 2–3 linked sialic acid residues capping mainly O-linked oligosaccharides which modulate VWF sensitivity to ADAMTS13 cleavage under high shear stress.In summary, these data demonstrate that sialylation of VWF increases its susceptibility to ADAMTS13 cleavage under flow but does not otherwise affect VWF mediated platelet capture to collagen. ADAMTS13 is crucial for regulating haemostasis, as it controls the size of newly secreted ultra large VWF multimers and can cleave VWF at sites of haemostatic plug formation. It has been previously shown that sialyltransferase expression levels vary between different tissues, and sialic acid expression can change in different pathological conditions, it is therefore important to characterise how the differences in VWF sialylation status affect its key properties such as platelets binding and susceptibility to proteolysis. Disclosures:No relevant conflicts of interest to declare.

Recombinant VWF: is this the answer for treatment of von Willebrand disease?

The use of replacement protein factors for treatment of blood clotting disorders has a long history that has involved both major advances and setbacks. Replacement therapies can be viewed as the gold standard treatment for the majority of factor deficiencies and, for certain conditions, such treatments are both highly developed and well established. For the common inherited bleeding disorders, hemophilia A and B, replacement FVIII and FIX concentrate has been in clinical use for many years, with recombinant product prophylaxis now in routine use in those countries that can afford it, and further improvements relating to increasing the half-life of these products at a fairly advanced stage [1]. However, not all factor deficiencies have the same level of development applied to their replacement concentrate therapies. von Willebrand disease (VWD), which results from a qualitative or quantitative deficiency of the multimeric plasma glycoprotein VWF, is a case in point. VWD is a complex disorder with three main subtypes that respond to treatment modalities in fundamentally different ways [2,3,101]. In contrast with hemophilia A, where FVIII replacement therapy is the standard treatment in individuals without complicating factors such as inhibitors, treatment of VWD using other agents such as desmopressin is an effective approach for some subtypes, notably mild type 1 and some mild qualitative type 2 VWD cases in shortterm treatment situations. However, in cases where such alternative treatments are contraindicated, and in type 3 and severe type 1 VWD, the use of replacement VWF concentrate for many clinical scenarios is required. Whilst there are several licensed VWF concentrates on the market, their state of development is, in some respects, not as advanced as that for FVIII or FIX replacement therapies. The currently licensed products in use all involve plasma-derived VWF from pools of donors. Whilst current processing techniques to purify these products, as with other available plasma-derived concentrates, mean that the risk of significant viral transmission is currently very low, there are other practical concerns for the clinician who wishes to use these nonrecombinant VWF concentrates in real-life treatment scenarios. The majority of licensed concentrates for the management of VWD contain both VWF and FVIII, but the relative proportions of each factor vary markedly, both between different products and batches of the same product, due to differing manufacturing processes that result in variable multimer composition and size distribution [3]. High-molecular weight (HMW) VWF multimers are the most biologically active and with each licensed product containing different amounts, this may be predicted to translate into variable hemostatic potential when administered to the patient. Another complication is the lack of information supplied in some locales relating specifically to VWF activity “...it is reasonable to expect ... that the treatment of VWD will evolve further over time to include more routine use of prophylaxis and a switch to recombinant products, where both clinical indicators and economic considerations permit.”

A new mouse model mimicking thrombotic thrombocytopenic purpura: correction of symptoms by recombinant human ADAMTS13

AbstractDeficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.

Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage

von Willebrand disease (VWD) is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrafish is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of offspring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrafish vwf have been identified, and some functional conservation has been demonstrated. In this paper we clone the complete zebrafish vwf cDNA and show that there is conservation of domain structure. Recombinant zebrafish Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs) in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrafish Vwf that may further our understanding of the mechanisms of VWD.

In Vivo Cleavage of Recombinant VWF Upon Intravenous Administration in Preclinical and Clinical Settings

Abstract 1222Von Willebrand factor (VWF) is composed of a series of multimers with a molecular size ranging from 600 to 20,000 kDa. The ultra-high molecular weight (UHMW) portion of VWF multimers is more efficient in platelet interactions than the lower molecular weight portion, and as such it might have a thrombogenic potential under certain pathologic conditions. The concentration of UHMW multimers in healthy individuals' blood is low because the circulating specific metalloprotease ADAMTS13 rapidly cleaves UHMW VWF upon its release into the circulation.A recombinant VWF (rVWF) expressed in CHO cells has been developed as a new drug candidate for treating patients with VWF deficiency. Since this protein is not cleaved by ADAMTS13 during the expression phase, it has a high portion of UHMW multimers, similar to the human VWF stored in Weibel-Palade bodies of endothelial cells.During the development phase of rVWF in vitro experiments showed that when treated under mild denaturing conditions or under shear, the molecule was susceptible to ADAMTS13 of human or animal origin.Here we present data from preclinical studies where analyzing the ex vivo samples showed the ADAMTS13-mediated cleavage of intravenously applied UHMW-containing rVWF. In good correlation with the in vitro results, studies in rabbits and cynomolgus monkeys showed that the ADAMTS13-specific cleavage fragments appeared as soon as 15 minutes after application, as visualized by immunoblot analysis. The appearance of cleavage bands coincided with the decrease in multimer numbers, indicating that rVWF had been specifically cleaved by endogenous ADAMTS13 in the circulation. The effect was species-dependent, only a low or minimal cleavage was observed in different mice strains.A recently completed Phase I clinical study clearly demonstrated that rVWF administered intravenously to severe VWD patients in a dose-range of 7.5 to 50 VWF:RCo U/kg body weight was rapidly processed in the circulation, resulting in the disappearance of the UHMW multimers with visible cleavage fragments, as shown by immunoblot and high resolution multimer analysis. No thrombotic adverse events were seen during the study, underlying the suitability of the UHMW-containing rVWF for treating VWD patients. Disclosures:Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schreiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.