High Molecular Weight Von Willebrand Factor Multimers Research Articles

A family having type 2B von Willebrand disease with an R1306W mutation: Severe thrombocytopenia leads to the normalization of high molecular weight multimers

In type 2B von Willebrand disease (2B VWD), abnormal von Willebrand factor (VWF) spontaneously binds to platelets. This leads to the clearance of the high molecular weight multimers (HMWM) of VWF and results in thrombocytopenia. Herein we report a family of 2B VWD with an R1306W mutation which caused thrombocytopenia with giant platelets. The most important finding in this study is dynamic changes in VWF values in association with platelet counts. When the proband (2 years of age) had severe thrombocytopenia, his HMWM were normal, however, hematological examination showed a low level of VWF and a lack of HMWM after platelet count recovered. His affected sister also exhibited similar phenomenona. These results suggest that the severe thrombocytopenia leads to decreased clearance of VWF HMWM and restoration of VWF HMWM in plasma. We must consider 2B VWD in the case of recurrent thrombocytopenia following infection or other stress condition.

Severely Impaired von Willebrand Factor-Dependent Platelet Aggregation in Patients With a Continuous-Flow Left Ventricular Assist Device (HeartMate II)

This study investigated the influence of the mechanical blood pump HeartMate II (HMII) (Thoratec Corporation, Pleasanton, California) on blood coagulation and platelet function. HMII is an implantable left ventricular assist device used for the treatment of heart failure. Patients treated with HMII have increased bleeding tendencies. We measured agonist-induced platelet aggregation in 16 patients on HMII support. The von Willebrand factor (vWF)-dependent ristocetin-induced platelet aggregation was impaired in 11 of the 16 patients, of which 12 had experienced at least 1 minor or major bleeding episode. The impaired ristocetin-induced platelet aggregation was associated both with decreased specific activity of plasma vWF, presumably due to lack of high molecular weight vWF multimers, as well as with attenuated function of the platelets themselves. The results imply that HMII treatment is associated with impaired platelet aggregation, which may contribute to an increased tendency to bleed.

The Montreal platelet syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation

AbstractMontreal platelet syndrome (MPS), hitherto described in only one kindred, is a hereditary thrombocytopenia associated with mucocutaneous bleeding, giant platelets, and spontaneous platelet aggregation in vitro. These are features shared with some forms of type 2B von Willebrand disease (VWD); however, the MPS kindred had not been investigated for VWD. We found that all affected MPS family members had borderline to normal von Willebrand factor antigen (VWF:Ag; 0.43-0.75 U/mL), discrepantly low ristocetin cofactor activity (VWF:RCo; 0.16-0.29 U/mL), and normal factor VIII coagulant activity (FVIII:C; 0.57-1.04 U/mL). Unaffected family members all had normal VWF:Ag, VWF:RCo, and FVIII:C levels. In addition, persons with MPS, but not unaffected family members, had loss of plasma (but not platelet) high molecular weight VWF multimers, and were heterozygous for the previously reported V1316M type 2B VWD mutation. Thus, in reevaluating this kindred, we determined that patients with MPS have type 2B VWD with the V1316M VWF mutation.

Autosomal dominant C1149R von Willebrand disease: phenotypic findings and their implications

Mutation C1149R in the von Willebrand factor (VWF) gene has been thought to cause autosomal dominant severe type 1 von Willebrand disease (VWD). Eight patients from three unrelated families with this mutation were included in the present study who had distinct VWF abnormalities, not described in earlier studies. The patients showed notably low levels of VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding (VWF:CB), and a reduced ristocetin-induced platelet aggregation (RIPA). VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios were lower than 0.7. At basal conditions, all the VWF multimers were decreased in plasma, with a clearly lower relative proportion of the high molecular weight VWF multimers (HMWM). In high-resolution agarose gels, a large decrease in the relative proportions of the satellite bands was seen. The patients had a brief good response to desmopressin (DDAVP) administration, but the released VWF half-life was shorter than normal, indicating an accelerated clearance of their VWF. Platelet VWF was abnormal. We conclude from the results obtained in these patients for plasma phenotypic data that this mutation should be classified as a VWD type 2A (IIE). DDAVP therapy may be somewhat helpful for this mutation, at least for mild to moderate bleeding. These data provide evidence that for VWD classification factors other than basal VWF, such as DDAVP response and platelet VWF, should be considered.

Von Willebrand factor/factor VIII concentrates in the treatment of von Willebrand disease

Therapy for von Willebrand disease (VWD) aims to restore the hemostatic function conferred by von Willebrand factor (VWF), which facilitates platelet adhesion and aggregation, and serves to increase potentially low coagulation factor VIII (FVIII) in plasma. In patients unresponsive to desmopressin (DDAVP), the preferred treatment is with plasma-derived VWF-containing FVIII concentrates. Only a few of the available VWF/FVIII concentrates have been licensed for use in VWD based on prospective studies. The efficacy of VWF/FVIII concentrates depends on the content and quality of VWF and FVIII. Several studies have demonstrated the variability of the VWF contents, as well as the differences in the VWF multimer patterns (including the high molecular weight VWF multimers that are most effective in restoring hemostasis), among these concentrates. Treating physicians should be aware of these disparities and the potential clinical implications for patients with different VWD subtypes. Dosing has traditionally been calculated based on the FVIII content of the products, although dosing based on VWF functional activity [e.g., VWF ristocetin cofactor activity (VWF:RCo)] addresses the primary protein deficiency in VWD patients. Several clinical studies have demonstrated the efficacy of concentrates dosed according to VWF:RCo. Dosing is generally consistent across VWD subtypes, although patients with severe phenotypes or undergoing major procedures may require more infusions or longer treatment duration. Other considerations for the use of VWF-containing concentrates include laboratory monitoring of efficacy and safety issues such as thrombosis risk and thromboprophylaxis.

Function And Activity Of Von Willebrand Factor Is Regulated By A Hierarchy Of Mechanical Forces

The von Willebrand factor (VWF) is a shear-flow sensitive multimeric protein. Under normal flow conditions VWF is in a globular state, it unfolds at high shear rates and is activated for adhesion at the blood vessel wall [Schneider et al. 2007 PNAS p7899]. The elongation of multimeric VWF results in a force pulling along the VWF length axis. Based on a model of the VWF A domain organization, we performed force probe molecular dynamics simulations. We reveal the basis of two force-sensing VWF functions, and test the results by experiments.Our results indicate a competition between VWF A2 domain and glycoprotein Ib (GPIb) for the same binding site of the VWF A1 domain. When the stretching force along VWF reaches a critical point, the A1 A2 interaction is lost. The domains remain connected by a linker that gives space for GPIb to bind to the A1 domain. We thus suggest a force-dependent platelet binding to VWF as mediated by GPIb, which is experimentally testable and represents an alternative mechanism to recently published studies [Chen et al. 2008 Biophys J p1303; Lou et al. 2008 PNAS p13847].We show how proteolysis of the VWF is activated under shear conditions. The specific proteolytic site is buried in the VWF A2 domain [Sutherland et al. 2004 J Mol Model p259]. At extreme forces as present in high molecular weight VWF multimers, the A2 domain C terminus unfolds until the ADAMTS13 cleavage site is uncovered. Introducing a disulfide bond by mutagensis prevents VWF cleavage. This explains the size regulation of VWF by ADAMTS13: larger multimers involve higher pulling forces and therefore higher unfolding rates under shear flow. Larger VWF is cleaved faster, preventing blood clots and thrombosis [in preparation].

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Force Dependent Structural Changes of the Von Willebrand Factor A2 Domain Are Required for VWF Cleavage by ADAMTS13

Background: An important regulatory mechanism of VWF functional activity is the proteolysis of the VWF high molecular weight multimers by ADAMTS13. The ADAMTS13 specific proteolytic site is located in the VWF A2 domain between Y1605 and M1606. Homology models by Sutherland et al. (1) predict the ADAMTS13 cleavage site of A2 to be covered by the helices of the domain. Thus an unfolding or opening of the domain is necessary in order to ensure cleavage by ADAMTS13. The main difference between the three neighboring VWF A domains is a disulfide bond connecting the respective N and C termini of the A1 and A3 domain, which is lacking in A2. An obvious assumption is that the disulfide bonds lock A1 and A3 in the folded state, while A2 can unfold in response to shear stress in blood vessels. To test this, we examined if the mutation N1493C in A2 enables the formation of a disulfide bond analogous to A1 and A3, thereby preventing unfolding of the A2 domain and stabilizing VWF against ADAMTS13.Methods: By in vitro mutagenesis of full length VWF we exchanged N1493 at the N-terminal site to Cysteine and C1670, one of two neighboring cysteines at the C-terminal site of the A2 domain, to Serine to allow creation of a Cysteine bonded loop (177 aa) in the A2 domain. Mutant and wt VWF were then digested by recombinant ADAMTS13 under denaturing non-reducing and reducing conditions, respectively, and analyzed by electrophoresis. Based on multiple sequence and structural alignments we created a homology model of the VWF A2 domain (residues 1488 to 1676 of human VWF) and the mutant A2 domain (N1493C and C1670S) from a human VWF A1 X-ray structure (PDB: 1AUQ). Our models cover a longer sequence (residues 1488 to 1676 of human VWF) than the model proposed by Sutherland et al.(1) The models were validated by molecular dynamics (MD) simulations. Force-probe MD simulations were applied to wildtype (wt) VWF to investigate unfolding.Results and Discussion: Successful introduction of a disulfide bond into A2 was validated by VWF multimer analysis. An additional loop due to a disulfide bond resulted in faster migration of VWF, suggesting a more compact structure. Persistence of the large VWF multimers and the lack of proteolytic products after treatment with recombinant ADAMTS13 indicated resistance of the A2 mutant to ADAMTS13. Electrophoresis of ADAMTS13 treated A2 domain mutant VWF under reducing conditions demonstrated a majority of undigested VWF monomers suggesting that proteolysis is not only prevented by creation of a disulfide bond spanning the proteolytic site but indeed by structural changes of the A2 domain, making the proteolytic site less accessible for ADAMTS13 in general. Physiological blood flow exerts forces onto VWF, stretching the multimeric protein to an elongated conformation. Force-induced unfolding of A2 is observed in force probe MD simulations by subjecting the termini of the A2 domain to a pulling force mimicking the tension in stretched VWF. The ADAMTS13 cleavage site is located at β-strand 4 (β4), which is after stepwise unfolding of α6, β6, α5, β5, and α4 exposed and therefore accessible for ADAMTS13. This explains the size regulation of VWF by ADAMTS13: larger multimers involve higher pulling forces and thereby a higher unfolding ratio at a given shear flow. As a result, larger VWF is cleaved more readily.Conclusion: Our combined experimental and theoretical approach suggests that locking of the VWF A2 domain by an artificial disulfide bond prevents a shear response and emphasizes the requirement of a force sensitive A2 domain structure for ADAMTS13 proteolysis. Respective experiments under shear conditions should confirm our hypothesis.

Idiopathic Non-Cirrhotic Intrahepatic Portal Hypertension Is Associated with Sustained ADAMTS13 Deficiency.

Idiopathic non-cirrhotic portal hypertension (NCPH) is caused by obliterative venopathy affecting the terminal radicals of the hepatic portal vein. It has been associated with areas of low socio-economic development. We observed a long history of thrombocytopenia and association with enteric disorders in a Caucasian population presenting with NCPH and postulated that the underlying mechanism could be deficiency of ADAMTS13, leading to the circulation of ultra large molecular weight von Willebrand factor (VWF) multimers and consequent platelet aggregate formation, causing occlusion of hepatic portal venules. ADAMTS13 activity was measured in all patients by a collagen binding assay, while antigen and IgG antibody were measured using ELISA kits (American Diagnostica Inc), normal ranges: 66–126%, 485–1242ng/ml and <11AU/ml, respectively. Plasma ADAMTS13 activity and antigen levels were measured in 14 patients with NCPH and 25 control patients with miscellaneous liver diseases associated with portal hypertension and Model for End Stage Liver Disease (MELD) scores indicating similar disease severity, median 12 (IQR 10–14) and 11 (8–16), for NCPH cases and controls. The platelet count was reduced in 13/14 NCPH cases median 69 x109/L (IQR 40–97) and 123 (85–193) in cases and controls, but did not correlate with MELD score or ADAMTS13 activity. ADAMTS13 activity was very low in 13/14 NCPH cases (median 12% (IQR <5–20)) and significantly lower than controls (59% (4–84), p<0.0001). In 4 cases, serial samples over several months revealed consistently low ADAMTS13 levels. There was a corresponding reduction in ADAMTS13 antigen, median 238ng/ml (IQR 186–361) and 584 (442–831), although NCPH cases showed a small excess of antigen over activity (antigen:activity ratio: median 25.7 (IQR 11.3–32.6) and 9.4 (8.1–13.8), in cases and controls, p<0.001), suggesting that an abnormal molecular species could be present. An inhibitor was not detected, although 10 cases and 15 controls had IgG anti-ADAMTS13: median levels 14.8AU/ml (8.8–20.0) and 13.4 (9.0–22.6), but these IgG levels did not correlate with activity. Non-inhibitory IgG antibodies to ADAMTS13 have been previously observed in certain other clinical groups (eg. coronary syndromes, sepsis, etc.). In NCPH, there was no correlation between ADAMTS13 level and either VWF antigen or VWF collagen binding activity. Increased very high molecular weight VWF multimers were observed in 4/11 NCPH cases and 0/22 controls. CD31 positive platelet aggregates were observed in the hepatic vessels of 7/20 (35%) NCPH cases and 1/15 (7%) controls. Our results suggest that sustained deficiency of ADAMTS13 associated with thrombocytopenia is characteristic of NCPH. It differs from other hepatic pathologies, where the decrease in ADAMTS13 has been reported to correlate with the severity of liver disease. NCPH also differs from thrombotic thrombocytopenic purpura because there is a chronic disease process, there is an absence of obvious red cell fragments and the primary organ affected is the liver. Since the predominant plasma source of ADAMTS13 appears to be hepatic stellate cells, the portal microcirculation is most at risk from pathological interactions of VWF and platelets if enteric factors further deplete circulating levels of ADAMTS13. The cause of the decreased ADAMTS13 remains to be elucidated, but could be due to decreased expression and synthesis, immune depletion (IgM and IgA anti-ADAMTS13 have not been investigated), or consumption in the circulation.

Systemic lupus erythematosus complicated by acquired von Willebrand’s syndrome

Haematological abnormalities are common in systemic lupus erythematosus (SLE). In some cases of acquired von Willebrand syndrome (AvWS), von Willebrand disease (vWD) is associated with autoimmune or lymphoproliferative disorders. In this study, we describe a 36-year-old woman with SLE and AvWS. The patient was referred to our hospital because of easy bruisability and recurrent vaginal bleeding. She had no history of bleeding tendency and no family history of bleeding diathesis, but she had a history of recurrent arthralgia, photosensitivity and sicca symptoms. Tests for antinuclear, anti-double stranded DNA, anticardiolipin and anti-beta2-glycoprotein I antibodies were all positive. Analysis of haemostatic parameters showed complete absence of von Willebrand factor ristocetin cofactor (vWF:Rco), von Willebrand antigen (vWF:Ag) and ristocetin-induced platelet aggregation (RIPA). Electrophoretic analysis of plasma showed a complete absence of high-molecular weight vWF multimer. The presence of antibody to vWF was detected by enzyme linked immunosorbent assay (ELISA). Treatment with corticosteroids improved SLE symptoms and corrected bleeding diasthesis. Also, the multimeric patterns of vWF became normalised and anti-vWF antibody disappeared. These findings indicated that this patient had SLE associated with AvWS, which was ameliorated by corticosteroid treatment.

Haemate® P/Humate‐P® for the treatment of von Willebrand disease: considerations for use and clinical experience

von Willebrand disease (VWD) is a heterogeneous bleeding disorder with symptoms in affected patients ranging from mild effects to potentially devastating haemorrhagic events. Desmopressin (DDAVP) and von Willebrand factor/factor VIII (VWF/FVIII) concentrates are the principal treatments. Haemate P/Humate-P is an intermediate-purity VWF/FVIII concentrate with extensive clinical experience in VWD. This concentrate has been shown to correct haemostatic defects of VWD, with efficacy ratings of good/excellent in nearly all patients treated for bleeding or surgical events. Haemate P/Humate-P has a high content of the high molecular weight (HMW) VWF multimer fraction, which has been shown to be very effective in achieving haemostasis. The HMW VWF multimer pattern in Haemate P/Humate-P is more similar to that of normal human plasma (94% for Haemate P/Humate-P vs. 100% for normal human plasma) than that of other VWF/FVIII concentrates and correlates with functional VWF activities including ristocetin cofactor activity (VWF:RCo) and collagen-binding activity. The recommended dosing of Haemate P/Humate-P is based preferentially on VWF:RCo activity, which is approximately twice that of FVIII:C (2.4:1). Haemate P/Humate-P has been shown to be safe; no serious adverse events or cases of thrombosis have been observed in clinical trials and no documented cases of viral transmission in nearly three decades of clinical use. While DDAVP is effective in a large proportion of VWD patients, it may not provide adequate haemostasis in all situations. In such cases, Haemate P/Humate-P is an effective replacement concentrate for all types of VWD in both adult and paediatric patients.

Decreased clearance of von Willebrand factor in a patient with type 2B von Willebrand disease following development of immune thrombocytopenia

We report a case of concurrent type 2B von Willebrand disease (VWD) and immune thrombocytopenia (ITP). The patient had characteristic loss of von Willebrand factor (VWF) high molecular weight multimers (HMWM) but a normal platelet count in the initial 8 years after diagnosis of type 2B VWD. When he developed severe thrombocytopenia, however, both his VWD indices and VWF HMWM normalized. As his platelet count increased, he again lost the HMWM and his VWD indices decreased. These results suggest that the severe thrombocytopenia led to decreased clearance of VWF, especially the HMWM.

Diagnostic workup of patients with acquired von Willebrand syndrome: a retrospective single-centre cohort study.

Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen-binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown. To analyze the capacity of laboratory tests, including point-of-care testing (POCT), for the identification of patients with AVWS. Thirty-five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers. According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA-100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio < 0.7 (26%), VWF:CB/Ag ratio < 0.7 (46%), anti-VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag < 50 IU dL(-1), VWF:RCo/Ag ratio < 0.7 and VWF:CB/Ag ratio < 0.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients. Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.

Diagnosis and treatment of von Willebrand disease: new perspectives and nuances

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

Type 2B “Malmo/New York” Is Not So Rare among Italian Patients with von Willebrand Disease: The Role of P1266L/Q Mutations of the von Willebrand Factor Gene in Seven Cases.

Background: Type 2B von Willebrand disease (VWD) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and reduced platelet counts, in most cases. Diagnosis of 2B is based on increased ristocetin-induced platelet aggregation (RIPA) in patient's platelet rich plasma (PRP). Type 2B “Malmo or New York” is caused by the mutation P1266L that is associated with increased RIPA, but with normal multimeric distribution in plasma and no thrombocytopenia.Aims: To determine the incidence and the molecular defects of this rare VWF variant among a large population of VWD patients regularly followed at two Northern Italian Hemophilia Centers.Methods: Criteria for VWD type 2B were those recommended by the ISTH-SSC-SC on VWF. RIPA and multimeric analysis were performed according to standardized methods. Bleeding severity score (BSS) was calculated in all patients enrolled in this study. Sequence analysis of the portion of exon 28 encoding for the A1 domain was performed to identify nucleotide substitutions in all cases where RIPA was <0.8 mg/ml.Results: 74 patients from 16 different families satisfied the criteria for VWD type 2B. Seven patients from 3 unrelated families clearly showed normal multimeric distribution in plasma and no thrombocytopenia also after stimuli. Clinical and laboratory parameters of the seven affected members are reported as mean values (ranges): BSS=2.5 (2–4); RIPA=0.56 (0.5–0.7) mg/ml; platelet count=255 x 10^9/L (172–353); VWF:RCo=46 U/dL (21–66); VWF:Ag=53 U/dL (30–67); FVIII=61 U/dL (54–76). Patients from family A showed five distinct substitutions (3686T>G, 3692A>C, 3735G>A, 3789G>A, 3797C>T), patient B1 two (3789G>A, 3797C>T) and patient from family C three (3692A>C, 3789G>A, 3797C>A). Two of these were silent, whereas the other caused the following amino acid changes: three in family A (V1229G, N1231T, P1266L), one in B1 (P1266L) and two in family C (N1231T, P1266Q). Mutations V1229G and N1231T have already been reported, whereas mutation P1266Q is novel. The substitutions found in families A and B clearly correspond to the pseudogene sequence, suggesting gene conversion into VWF gene. In family C, the nucleotide mutation causing P1266Q substitution is not consistent with this mechanism: moreover the patients do not show the substitution 3735G>A found in the pseudogene.Conclusion: We confirm that the substitution of proline 1266 with leucine appears to be the main cause of type 2B “Malmo/New York” phenotype while its replacements with a glutamine apparently results in lower VWF activities in family C. Based on these data, gene conversion appears to be more frequent than expected and capable to generate different set of mutations.

Pharmacokinetic Analysis of a von Willebrand Factor/Factor VIII (VWF/FVIII) Concentrate in Adults and Children with von Willebrand Disease (VWD).

Introduction: Although widely used for the treatment of VWD, optimal doses of VWF/FVIII concentrates for surgical procedures in patients with VWD need to be determined. Two prospective, multicenter studies were undertaken to evaluate the efficacy and safety in surgery of a VWF/FVIII concentrate marketed in the United States (US; Humate-P®) and the European Union (EU; Haemate-P®) in patients with VWD (Lethagen et al. JTH, 2007). Initially, all patients had pharmacokinetic (PK) studies to guide individual dosing for surgery.Results: In the US study, 41 subjects received 60 IU/kg VWF: RCo, and in the EU study 28 subjects received approximately 80 IU/kg VWF: RCo. At specified time points before and after infusion, median levels, half-life, mean change from baseline and in-vivo recovery (IVR) values were determined for VWF: RCo and FVIII: C. Collagen binding capacity (VWF: CB) was correlated with VWF: RCo; and VWF multimer analyses were also performed. In the US and EU studies, median baseline VWF: RCo levels were 13 (range[r] 6–124) and 8.1 (r 5–58) IU/dL; the highest median post-infusion values were 163 (r 84–330) and 147 (r 53–387) IU/dL. Mean change from baseline in the US study was > 100 IU/dL immediately after the infusion, decreasing to about 10 IU/dL at 48 hours post-infusion. The median terminal half-life of VWF:RCo in the US study was 11.7 (r 3.5–74.9) hours. These results are consistent with early and rapid distribution phase, followed by a much slower terminal elimination phase. The median incremental in vivo recovery (IVR) for VWF: RCo in the US and EU studies was 2.4 and 1.9 IU/dL/IU/kg respectively. In the US and EU studies, median baseline levels of FVIII: C were 39 (r 0.5 to 96) and 33 (r 2–106) IU/dL. Mean change of FVIII: C from baseline in the US study was about 60 IU/dL post-infusion, levels decreasing to slightly above 20 IU/dL at 48 hours post-infusion. Median incremental IVRs for FVIII: C in the US and EU studies were 2.7 and 2.8 IU/dL/IU/kg. Median baseline levels of VWF: CB were 13.0 (r 1.5–101) and 10.4 (r 1.0–84) IU/dL. For the US study, the highest median post-infusion VWF: CB value was 131.5 (r 60–204) IU/dL 15 minutes post-infusion. For the EU study, the highest median VWF: CB value was 147 (r 21–330) IU/dL 30 minutes post-infusion. At 48 hours, levels decreased to near baseline (median: 26.5 [r 10–136] IU/dL in the US study, and 13 [r 2–112] IU/dL in the EU study). Conclusions: VWF: CB values correlated well with VWF: RCo values. Analyses showed that high molecular weight VWF multimers were detectable up to 24 hours post-infusions in all subjects with absent multimers at baseline. The PK data suggest a possibility of slight accumulation of FVIII: C, presumably due to a dynamic stabilization of exogenously injected and endogenously released FVIII. The slow terminal elimination phase of VWF: RCo, compared to shorter distribution phase, suggests a minimal risk of VWF: RCo accumulation, easily managed by adjusting the dosing interval. For surgical coverage, an individual patient's PK results safely guided initial dosing, with subsequent doses based on their clinical and laboratory responses.

Human von Willebrand Disease Mutations in Mouse von Willebrand Factor Alter Its Cleavage by ADAMTS13.

Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers observed in the prothrombotic state of thrombotic thrombocytopenic purpura (TTP). VWF mutations leading to multimer changes have not been examined in an animal model. Although the human and mouse VWF genes are highly conserved, mutations in the two genes have not been compared experimentally. To that end, we have made mutations in the mouse VWF (mVWF) cDNA to compare the ADAMTS13 cleavage patterns seen with human VWF mutations in two in vitro assays. Recombinant multimerized full-length mVWF was digested with different concentrations of recombinant mouse ADAMTS13 (mADAMTS13) and 1.5M urea, and analyzed via multimer migration distance. The GST and histidine-tagged G1554-T1668 A2 domain region of mVWF (mVWF115) was assayed via ELISA as follows: the mVWF115 was bound to anti-GST coated plates, digested with mADAMTS13, and intact mVWF115 detected via HRP-labeled anti-histidine tag antibody. We examined R1597W (VWD Type 2A), R1306W, (VWD Type 2B), Y1584C (VWD Type 1), as well as two previously described changes that drastically lower VWF cleavage, Y1605A/M1606A, and D1614A/E1615A/K1617A. Y1584C showed a statistically insignificant 41% decrease in cleavage in the full-length assay, but a 20% increase in the mVWF115 assay. Conversely, R1597W had a 64% decrease in the full-length assay, but a 28% increase in the mVWF115 assay. Y1605A/M1606A showed a dramatic loss of cleavage, with no observable loss of high molecular weight multimers at 32 units/ml of mADAMTS13, and a 126-fold higher mADAMTS13 concentration than wild type in the mVWF115 assay. This contrasted with the 8-fold increase observed with D1614A/E1615A/K1617A. The R1306W A1 domain mutation showed a significant increase in cleavage, with 63% less ADAMTS13 necessary in the full-length assay. The triple mutant R1306W/Y1605A/M1606A did not cleave at the highest concentration, 32 U/ml, similar to that of Y1605A/M1606A, showing that the R1306W mutation was not able to increase cleavage of the Y1605A/M1606A change. These results show a strong correlation between the mouse mutations described here and previously reported human VWF mutations in their susceptibility to ADAMTS13 cleavage. These results provide a rationale for the development of mouse models of type 1, 2A, and 2B VWD, as well as mVWF mutations that might lead to a prothrombotic state similar to TTP in humans.mADAMTS13 Concentration for 50% mVWF CleavageADAMTS13 Concentration (Units/ml)Relative mADAMTS13 Concentration (to Wild Type)mVWF Multimer (n=1)mVWF115 (n=4)mVWF Multimer (n=1)mVWF115 (n=4)Wild Type0.3010.19711Y1584C0.4230.157*1.410.80*R1597W0.109**0.253*0.36**1.28*Y1605A/M1606A>32**24.8**>100**126**D1614A/E1615A/K1617A(−)1.58**(−)8.0**R1306W0.112**(−)0.37**(−)R1306W/Y1605A/M1606A>32**(−)>100**(−)*: P value <0.05, **: P Value < 0.001 in 2 tailed T test.

Association of acquired von Willebrand syndrome with AL amyloidosis

Acquired loss of functional von Willebrand factor (VWF) has been termed the acquired von Willebrand syndrome (AVWS). AVWS is a rare adult-onset bleeding diathesis that is clinically similar to congenital von Willebrand disease (VWD), and occurs with a variety of autoimmune, lymphoproliferative, or myeloproliferative disorders. We have identified four patients with AVWS in association with immunoglobulin light chain (AL) amyloidosis. These patients, lacking any pre-existing or family history of abnormal bleeding, developed cutaneous, mucosal, or gastrointestinal bleeding in the course of their disease without deficiency of clotting factor X or other factors; the activated partial thromboplastin time (aPTT) was prolonged in three out of the four cases. Despite normal VWF antigen levels, VWF ristocetin cofactor activity (VWF:RCo) was low. Electrophoresis patterns of high molecular weight (HMW) VWF multimers were abnormal in two of the four cases. Two of the patients were treated with high-dose intravenous melphalan followed by autologous stem cell transplantation (HDM/SCT) and achieved hematologic remission. In these two patients, the bleeding diathesis improved and the coagulation parameters normalized, confirming a causal relationship between the plasma cell dyscrasia and the AVWS. AVWS should be considered in AL amyloidosis patients with hemorrhagic diatheses and normal clotting factor levels.

Von Willebrand disease—Phenotype versus genotype: Deficiency versus disease

Von Willebrand disease (VWD) is classified into three primary subtypes; type 1 includes partial quantitative deficiency, type 2 (A, B, M and N) includes qualitative defects and type 3 includes virtually complete deficiency of von Willebrand factor (VWF). Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high molecular weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite normal sized VWF multimers. Finally, type 2N includes variants with markedly decreased affinity for factor VIII (FVIII). Studies have begun to address the association between particular genetic variants of VWF and the disease phenotype. For example, the recessive type 3 disease appears to be due to a variety of mutations that result in a null phenotype, while type 2N disease results from a group of recessive missense mutations that interfere with FVIII binding. Recently, the molecular pathogenesis of type 1 VWD has begun to be characterised revealing a diverse spectrum of genetic changes. In two large studies, patients with type 1 VWD were found to be heterozygous for a variety of missense, splice site and transcriptional mutations. In some patients, more than one variant sequence was present, and in a significant number of individuals no changes were evident in the VWF gene. These results suggest that the molecular correlates for type 1 VWD are complex and, in addition to a wide array of changes at the VWF locus, are likely to involve mutations in other genes.

Recombinant Human IL-11 (rhIL-11, Neumega®) Increases VWF Activity in Type 1 Von Willebrand Disease.

Von Willebrand disease (VWD) is the most common congenital bleeding disorder, affecting 1% of the population. Current treatment is suboptimal. Tachyphylaxis limits the use of DDAVP; potential transmissible agents limit the use of Von Willebrand Factor (VWF) concentrate; and both require venous access. Recombinant human IL-11 (rhIL-11, Neumega), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, has recently been shown to increase VWF activity with good tolerance in mice, dogs, and healthy volunteers. We, therefore, initiated a prospective Phase II open label, escalating dose trial of rhIL-11 in subjects with type 1 VWD. VWD was defined by the presence of a bleeding history with at least two of four abnormal tests: F.VIII:C, VWF:RCo, VWF:Ag, PFA-100, and normal distribution of multimers. A total of six subjects completed the study, three each at 25 mcg/kg and 50 mcg/kg dose levels, and all were given rhIL-1 subcutaneously daily for 7 days. On day 4 of rhIL-11, VWF:RCo increased 32% over baseline to 107±9% (mean ± SE) at the 25 mcg/kg dose, and 70% over baseline to 179±55%, at the 50 mcg/kg dose; VWF:Ag levels increased similarly, to 102±38% and 240±120%, respectively; and FVIII:C and VIII:Ag each increased similarly, by up to 143% and 200%. Intravenous DDAVP at a dose of 0.3 mcg/kg given 30 minutes after rhIL-11 on day 7, induced a further increase in VWF:RCo, to 114±48% and 359±152%, and in VWF:Ag, to 113±33% and 277±21%, each respectively at the two rhIL-11 doses; and very high molecular weight VWF multimers were detected by gel elecrophoresis on plasma samples. These data suggest that rhIL-11 acts on other than the DDAVP releasable pool of VWF. The drug was well tolerated. Minor adverse events included hypertension, fluid retention, and hypokalemia, each at less than grade 1 toxicity. There was a mild dilutional decrease in hemoglobin related to fluid retention. There was no significant increase in platelet count. In conclusion, rhIL-11 increases VWF activity to levels in the range associated with clinical hemostasis, suggesting the potential use of rhIL-11 in treatment of clinical bleeding in type 1 VWD. [Display omitted]