Increased von Willebrand factor antigen and high molecular weight multimers in sickle cell disease associated with nocturnal hypoxemia

We evaluated vWF profiles in children and adolescents with SCD and sleep hypoxemia. Mean vWF:Ag levels were significantly elevated in the SCD-hypoxemia group when compared with SCD-normoxia and control groups (p=0.007); and correlated inversely with pulse oximetry (r=-0.54, p=0.01). Densitographic analyses of vWF multimer distribution also showed an inverse correlation between %HMW-multimers and oxygen saturation (r=-0.62, p=0.03). The previously reported association between nocturnal desaturation and SCD vascular complications, including stroke, may be influenced by hypoxemic modulation of vWF as noted in this study.     

Allelic frequencies of three VNTRs in intron 40 of the human von Willebrand factor gene in types 1, 2, and 3 von Willebrand disease patients and controls of a Brazilian population

Intron 40 of the human von Willebrand factor (vWF) gene contains a polymorphic region with three variable-number tandem repeats (VNTRs), type (ATCT)_n. In the present report, we evaluated the allelic frequencies of these three VNTRs in a population constituted by 51 Brazilian Caucasian and 25 Types 1, 2, and 3 von Willebrand disease (vWD) patients, and performed segregation analysis in eight families affected by vWD Types 1 and 2. Three pairs of primers were used to amplify independently nucleotides 1640–1794 (VNTR 3), 1890–1991 (VNTR 1), and 2215–2396 (VNTR 2) from intron 40. The observed heterozygosities (0.86, 0.66, and 0.66 for VNTRs 3, 1, and 2, respectively) were in accordance with the expected heterozygosities derived from the allele frequencies (0.81, 0.64, and 0.70, respectively). Although the three VNTRs were highly polymorphic, VNTR 3 showed the highest values of heterozygosity [Haemostasis 25 (1995) 264; Hum. Mol. Genet. 1 (1992) 287.].     

Towards improved diagnosis of von Willebrand disease: Comparative evaluations of several automated von Willebrand factor antigen and activity assays

Introduction

von Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing has important management implications and requires a wide range of tests, including VWF activity and antigen, and involves differential identification of qualitative vs quantitative defects.

Methods

We have assessed several VWF antigen and activity assays (collagen binding [VWF:CB], ristocetin cofactor [VWF:RCo] and the new Siemens INNOVANCE assay [VWF:Ac], employing latex particles and gain of function recombinant glycoprotein Ib to facilitate VWF binding and agglutination without need for ristocetin) using different instrumentation, including the new Sysmex CS-5100, with a large sample test set (n = 600). We included retrospective plus prospective study designs, and also evaluated desmopressin responsiveness plus differential sensitivity to high molecular weight VWF.

Results

VWF:Ag and VWF:RCo results from different methods were respectively largely comparable, although some notable differences were evident, including one high false normal VWF:Ag value (105 U/dL) on a type 3 VWD sample, possibly due to heterophile antibody interference in the latex-based CS-5100 methodology. VWF:Ac was largely comparable to VWF:RCo, but VWF:CB showed discrepant findings to both VWF:RCo and VWF:Ac with some patients, most notably patients with type 2M VWD.

Conclusions

(a) VWF:Ag on different platforms are largely interchangeable, as are VWF:RCo on different platforms, except for occasional (some potentially important) differences, and manufacturer recommended methods may otherwise require some assay optimization; (b) VWF:RCo and VWF:Ac are largely interchangeable, except for occasional differences that may also relate to assay design (differing optimizations); (c) VWF:CB provides an additional activity to supplement VWF:RCo or VWF:Ac activity assays, and is not interchangeable with either.     

Assessment of a cohort of primarily pediatric patients with a presumptive diagnosis of type 1 von Willebrand disease with a novel high shear rate, non-citrated blood flow device

Background

A precise approach to the diagnosis of von Willebrand disease (vWD) remains elusive. One important reason is that vWD is a blood flow‐related disorder: a vW Factor‐platelet GPIb binding defect exists in this condition under the high shear‐rate (> 1000 sec‐1 in whole blood; > 3000 sec‐1 in PRP) conditions of physiologic blood flow which exist in the arterioles of mucous membranes, from which most bleeding in vWD occurs.

Methods

We therefore studied 28 patients (mean 18.9 yrs) with vWD, diagnosed according to the 2007 NHLBI clinical guidelines, and 26 healthy controls (mean 17.5 yrs). Blood was collected into a plastic tube containing 4 U/ml FC dalteparin, 1.75 μg/ml of the Tab (anti‐CD41) monoclonal antibody directed against platelet GPIIb, and 1.0 μg/ml of an ALEXA 555‐conjugated rabbit anti‐mouse second antibody. Within 30-90 min, the blood was then withdrawn at 667 and 1330 sec^− 1 through a special flow chamber allowing for real-time epifluorescence digital videomicroscopy of platelets interacting with a microfibrillar collagen substrate. With MetaMorph software (Universal Imaging) we quantified the percent area (PA) covered by and total volume (TV) of adherent platelet aggregates within a 435 μm × 580 μm field of view.

Results

At 667 sec^− 1 after 1 min PA and TV were similar for patients and controls, but at 1330 sec^− 1 PA was 9.32 ± 4.21 (mean ± SD) for patients, a value lower (p < 0.001) than the 12.8 ± 3.39 for controls. TV was (1.43 ± 0.91)x10⁴ for patients, a value also lower (p < 0.001) than the (2.22 ± 0.77)x10⁴ for controls. PA or TV was below the 2.5th percentile for controls in 10 patients (36%) and both PA and TV were below the 2.5th percentile in eight.

Conclusions

The novel flow device found that PA and TV were significantly reduced under high shear stress in vWD patients compared to normal controls. However, there was some overlap between the vWD and the control group, suggesting that some vWD patients had normal platelet adhesion/aggregation under the conditions studied. Further study with a higher shear rate appears indicated.     

Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: Differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?

We performed a retrospective audit of desmopressin (DDAVP) usage to assist in the functional characterisation of von Willebrand disease (VWD). Data was evaluated for 208 patients, comprising those with VWD (Type 1 [n = 160], Type 2A [n = 19], Type 2M [n = 10]), plus 19 individuals with haemophilia or carriers of haemophilia. Laboratory testing comprised pre- and post-DDAVP evaluation of factor VIII (FVIII:C), von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo) activity, VWF collagen binding (VWF:CB) activity, and in one laboratory an alternate VWF activity assay. In brief, combined usage of VWF:RCo and VWF:CB appears to provide improved functional characterisation and/or ‘classification’ of VWD types, in particular better differentiation of Type 2A and 2M VWD, and clearer validation of a Type 1 VWD diagnosis. Thus, (i) Type 1 VWD displayed generally good absolute and relative rises in all test parameters, although relative rises were greatest for FVIII:C and VWF:CB, and CB/Ag ratio increases overshadowed those for RCo/Ag; (ii) Type 2A VWD patients showed good absolute and relative rises in both FVIII:C and VWF:Ag, but poor absolute rises in both VWF:CB and VWF:RCo; although small rises in both CB/Ag and RCo/Ag were also observed, both ratios tended to remain below 0.7; (iii) finally, Type 2 M VWD patients generally showed good absolute and relative rises in FVIII:C, VWF:Ag and VWF:CB, but a poor absolute and relative rise in VWF:RCo; thus, there were good rises in CB/Ag ratios but little change in RCo/Ag, which tended to remain below 0.7. Future multi-centre prospective investigations are warranted to validate these findings and to investigate their therapeutic implications.     

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.     

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Introduction

VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.

Methods

The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.

Results

The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.

Conclusions

The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.     

Thrombotic thrombocytopenic purpura in the brain: possible prognostic impact of von Willebrand factor physiology

We report three patients with CNS involvement in thrombotic thrombocytopenic purpura (TTP). The first two cases presented with fluctuating levels of consciousness and lateralized neurologic deficits. Repeated brain CT scans were normal in case 1, and showed small parietal lucencies in case 2. Both patients recovered to only minor residues after combined treatment of plasma exchange, corticosteroids, immunoglobulins, vincristine (case 1), and cyclophosphamide (case 2), respectively. Case 3 with multiple infarctions in brain CT died of heart failure. Autopsy showed myocardial infarction and multiple hemorrhages in various organs including cerebral cortex. Excess deposition of von Willebrand factor antigen within hyaline thrombi in small and medium-sized arteries of the cerebral cortex is shown for the first time immunohistologically.     

Binding of porcine von Willebrand factor to human platelets in the presence of ristocetin

The interaction of porcine von Willebrand factor (vWF) with human platelets in the presence of ristocetin was examined. Binding was rapid, specific, saturable and dependent on ristocetin concentration with half-maximal stimulation occuring at a ristocetin concentration of 0.5mM. Assuming vWF to be a tetramer with a MW of 9.5×10^?5, approximately 94,000 vWF binding sites per platelet were found with an average binding constant of 2.1×10^?8M. Humam vWF competed with the porcine protein for this site only in the presence of ristocetin. Binding of porcine vWF to platelets was found to be less sensitive to reduction with dithiothreitol than platelet agglutinating activity.     

Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging

Introduction

Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps.

Materials/Methods

VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed.

Results

The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas.

Conclusions

The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.     

A new method measuring the interaction between von Willebrand factor and coagulation factor VIII

Introduction

There is a need for more reliable methods measuring the binding of coagulation factor VIII (FVIII) to von Willebrand factor (VWF) in plasma samples, for use in the clinical routine. We have developed such a method measuring FVIII binding in plasma, utilizing an ELISA system.

Materials and Methods

Microtiter plates were coated with a monoclonal antibody (ESH-8), reacting with the C2 domain in FVIII. Thereafter the wells were treated with recombinant FVIII (Kogenate Bayer®). After washing, diluted plasma samples were added and incubated for 1 h. Then HRP-conjugated antibodies against VWF were added and used for quantification of bound VWF.

Results

A strong signal to VWF concentration response was obtained. Plasma from patients with different types of von Willebrand disease gave frequently diminished responses. However, after correction for the VWF antigen levels, by calculation of FVIII binding/VWF antigen ratio, only the patients with known von Willebrand disease type 2 N (n = 4) had clearly abnormal results. The FVIII binding in 40 healthy individuals was determined as 1.08 ± 0.48 U/mL (SD). After correction for the VWF antigen levels the result was 0.94 ± 0.15. Thus, the SD declined substantially by this correction. The within-series CV and between-series CV were determined as 6.8 and 11.3%, respectively.

Conclusions

We have established a simple and reliable method to detect decreased binding of FVIII to von Willebrand factor in plasma samples. The method can conveniently be used to study large populations, as well as finding minor binding defects in patients.     

The interaction between collagens and factor VIII/von Willebrand factor: Investigation of the structural requirements for interaction

The blood protein Factor VIII/von Willebrand factor (FVIII/VWF) has been shown to bind to a variety of collagen polymers including (i), the native-type fibres (of collagens types I and III), (ii), segment-long-spacing (SLS) aggregates (of collagens types I, III, IV and V), (iii), the insoluble polymer obtained by random cross-linking of the type I monomer and (iv), the non-striated fibril (of type I) produced by alcohol precipitation. Relatively little binding of FVIII/VWF to the amorphous, non-fibrillar form of collagen (type I) produced by salt precipitation from acid solution was observed. No significant binding either to elastin or to the insoluble polymer derived by random cross-linking of bovine serum albumin was noted. The absorption of FVIII/VWF to collagens was affected by ionic concentration and FVIII/VWF was only totally bound at relatively low ionic strength. Binding of radiolabelled FVIII/VWF could be largely inhibited by an excess of the unlabelled protein. The interaction of FVIII/VWF with collagen fibres was inhibited in a concentration-dependent manner by monomeric collagen when present at relatively high concentrations. Gelatin did not appear to inhibit binding significantly. The structural requirements of collagen for binding to occur appear to resemble those required for collagen-induced platelet aggregation in which collagen quaternary structure rather than collagen type $\text{per se}$ is the important factor.Loss of secondary or higher orders of structure of FVIII/VWF as a result of heat denaturation or reduction of disulphide bonds decreased or prevented binding. In accord with the association of biological activity with FVIII/VWF aggregates, optimal binding appeared to require the presence of aggregated FVIII/VWF.     

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

Background

Acquired 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.

Aim

To develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS.

Methods

AVWS 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).

Results

Twenty–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.

Conclusion

With 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.     

Surface-dependent expression in the platelet GPIb binding domain within human von Willebrand factor studied by atomic force microscopy

Adsorption of plasma proteins such as von Willebrand factor (vWF) on thrombogenic surfaces can induce conformational changes in tertiary structure so that the prothrombotic functional epitopes are exposed for interactions with platelets, resulting in platelet adhesion and thrombus formation. Thus, understanding platelet binding following changes in the structure of vWF is critical in understanding the mechanisms of thrombogenesis. The present study examined the accessibility of platelet binding epitopes within vWF adsorbed on two different thrombogenic surfaces, a hydrophobic synthetic surface and collagen VI coated substrates, under physiological buffer conditions using atomic force microscopy (AFM) in combination with immunogold labeling. Our results demonstrated that the glycoprotein Ib (GPIb) binding domain in vWF undergoes changes when adsorbed on collagen VI compared to vWF on a hydrophobic synthetic surface. This study provides a basis for a novel approach to understand the molecular mechanisms of surface-induced thrombosis by directly examining the structure–function relationships of plasma proteins involved in the thrombus formation.     

Prospective study of low-dose ristocetin-induced platelet aggregation to identify type 2B von Willebrand disease (VWD) and platelet-type VWD in children

Type 2B von Willebrand disease (VWD2B) and platelet-type von Willebrand disease (PT-VWD) are rare bleeding disorders characterised by an increased ristocetin-induced platelet aggregation (RIPA) at low dose of ristocetin. It was the objective of this study to detect children with VWD2B and PT-VWD using RIPA at low dose of ristocetin (0.5 mg/ml) in the screening evaluation of bleeding disorders, and to analyse the phenotypic data along with the molecular findings. Over a 14-year period, 641 children with personal and family bleeding symptoms or bleeding from birth with previously uncharacterised haemostatic disorders were prospectively studied. Six unrelated patients (0.93%) showed RIPA at low dose of ristocetin. RIPA-based mixing studies identified that the plasma of the six probands and at least one parent from five unrelated families induced aggregation of normal platelets with the addition of low-dose ristocetin. None of the probands' platelets showed aggregation with cryoprecipitate. Low ristocetin cofactor activity/VWF antigen ratio with absent collagen binding activity or thrombocytopenia were detected respectively in only two patients. Molecular analysis of exon 28 of the VWF gene identified mutations in only three patients. No mutation in the GP1BA gene was found. In this large prospective paediatric study, the screening approach including RIPA at low dose of ristocetin permitted the detection of patients with VWD2B that would otherwise have been missed. No patient with phenotype or genotype of PT-VWD was identified. Heterogeneity of bleeding symptoms and phenotypic parameters were found among members of the same family.     

Filter bleeding time: a new in vitro test of hemostasis. II. Application to the study of von Willebrand disease and platelet function inhibitors

The effect of platelet function inhibitors and von Willebrand (vW) disease on hemostasis was evaluated with a new method, the Filter Bleeding Time (FBT) test which does not require a skin incision. This method is based on the progressive slowing of blood flow associated with plugging of a woven Dacron filter by platelet clumps during passage of blood through the filter. Antiplatelet agents studied were acetyl salicylic acid (ASA), prostaglandin I₂ (PGI₂), and a thromboxane synthetase inhibitor, UK-37, 248-01 (UK). Parameters measured were FBT (time required for the drop rate interval to exceed 30 sec), bleeding volume (BV; total number of drops in FBT), initial bleeding rate (IBR; number of drops during the first minute), and percent platelet count reduction (PCR) during passage of blood through the filter. ASA (650 mg p.o. in normal human subjects) caused an increase in FBT from 4.94±2.97 ($\text{x}$±SD) to 10.28±3.27 min (p = 0.003) and BV (34±19 to 69±31 drops, p = 0.01), and a decrease in PCR (29.4±7 to 12.5±2%, p = 0.006). PGI_p (0.1 uhf added to normal human blood) caused an increase in,FBT (4.76±2.33 to 14.44±3.39, p = 0.0006) and BV (41±18 to 94±35, p = 0.001) and a decrease in PCR (29.6±6.4 to 7.1±7.8, p = 0.004). IBR did not change significantly in these studies. UK (3 mg/kg p.o. in normal dogs) caused no significant change in any of these parameters despite the drug's inhibition of platelet aggregation by ADP and collagen. Values in 4 pigs with vW disease were abnormal compared to normal pigs: FBT, 23.83±20.47 vs 5.09 min, p = 0.04; BV, 168±154 vs. 30±24 drops, p = 0.04; PCR, 7.1±1.0 vs 28.5±10.7%, p = 0.002; and IBR, 29.7±7 vs. 10±6 drops/min, p = 0.002. These studies suggest that IBR is a measure of platelet adhesion, while FBT, BV and PCR reflect the totality of platelet reactions including platelet adhesion and aggregation. FBT is considered useful in the study of the mechanisms and efficacy of platelet function inhibitors, and in the investigation of platelet disorders.