Low VWF Levels Research Articles

A novel multishear microdevice for studying cell mechanics

Shear stresses are known to influence the morphology, and even the fate, of many cell types, including endothelial, smooth muscle, and osteoblast cells. This paper describes a novel shear device for the study of cell mechanics. Unlike all other published shear devices, such as parallel-plate flow chambers, where a single shear stress is evaluated for a single input flow rate, the described device enables the simultaneous evaluation of 10 different shear stresses ranging over two orders of magnitude (0.7-130 dynes cm(-2), 0.07-13 Pa). Human umblical vein endothelial cells (HUVECs) were exposed to the shear stress profiles provided by the device over a 20 h perfusion period, and the secretion level of von Willebrand factor (vWF) was investigated. Confirming previous studies, increasing shear resulted in increased vWF secretion. Furthermore, changes in cell morphology, including cell and nuclear size (area) and perimeter with shear, were analysed. HUVECs under shear stresses ranging from 1-3 dynes cm(-2) (0.1-0.3 Pa) showed similar vWF content, cell and nuclear size and perimeter to static cultures, while cells under shear stresses above 5 dynes cm(-2) (0.5 Pa) showed significantly higher vWF secretion and were at least 30% smaller in cell size. We also note that cells exposed to perfusion rates resulting in a shear stress of 0.7 dynes cm(-2) (0.07 Pa) showed significantly lower levels of vWF and were 35% smaller in size than those under static conditions. Overall, the results confirm the significant utility of this device to rapidly screen cellular responses to simultaneously imposed physiologically relevant ranges of shear stress.

Characterization of a novel mutation in the von Willebrand factor propeptide in a distinct subtype of recessive von Willebrand disease

von Willebrand factor (VWF) is a plasma protein that consists of a series of multimers of which the high-molecular-weight VWF multimers are the most potent in platelet adhesion and aggregation. The propeptide of the VWF (VWFpp) is known to be essential in the process of multimer assembly. Genetic studies were performed in a patient with a phenotype of von Willebrand disease (VWD) characterized by very low plasma factor VIII and VWF levels and a VWF consisting of only a dimeric band and total absence of all multimers in plasma. The patient was found to be homozygous for the novel C570S mutation, caused by a 1709G>C transition in exon 14 of the VWF gene coding for the propeptide. Three asymptomatic relatives were found to be heterozygous. In-vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on VWF multimerization. Our findings show that the C570S mutation in the VWFpp abolishes multimerization of VWF. The mutation probably disrupts the normal configuration of the VWFpp, which is essential for correct orientation of the protomers and ultimately multimerization. The mutant amino acid is located in a region that is highly conserved across several species which underlines its critical role. This variant constitutes a distinct subtype of recessive 2A VWD with the exclusive presence of the dimeric form of VWF in plasma.

High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls

Mucocutaneous bleeding (MCB) is the main expression of inherited disorders of primary hemostasis. However, the relative prevalence of these disorders, their clinical differential diagnosis, and the proportion of patients with MCB of unknown cause (BUC) after an initial comprehensive laboratory testing are unknown. We studied prospectively 280 consecutive patients with MCB and 299 matched controls, using strict inclusion and exclusion criteria. A single physician recorded the clinical data in a bleeding score and estimated the severity of bleeding in clinical categories. Laboratory criteria for the diagnosis of von Willebrand's disease (VWD) and platelet function defects were established from reference values derived from controls. Fifty patients (17.9%) had VWD (type 1VWD=45, type 2=5). Platelet function defects and mild clotting factor deficiencies were found in 65 (23.2%) and 11 (3.9%) patients, respectively. Thirteen (11.5%) patients had combined defects. The remaining 167(59.6%) patients had BUC, with prolonged bleeding time in 18.6% as their only abnormality. All these disorders, including BUC, were clinically undistinguishable. Moreover, no relationship was found between the severity of bleeding and VWF/platelet function variables. The diagnostic efficacy of a first laboratory testing in patients with hereditary MCB is 40.4%. Most patients have a disease(s) of high prevalence but unknown pathogenesis. Concurrent bleeding disorders in the same patient are frequent. Our results support the proposal that low plasma VWF levels, but also platelet function defects, should be considered risk factors rather than unequivocal causes of hemorrhages.

The mutational spectrum of type 1 von Willebrand disease: results from a Canadian cohort study

AbstractIn order to evaluate the changes within the VWF gene that might contribute to the pathogenesis of type 1 von Willebrand disease (VWD), a large multicenter Canadian study was undertaken. We present data from the sequence analysis of the VWF gene in 123 type 1 VWD index cases and their families. We have identified putative mutations within the VWF gene in 63% (n = 78) of index cases, leaving 37% (n = 45) with no identified changes. These changes comprise 50 different putative mutations: 31 (62%) missense mutations, 8 (16%) changes involving the VWF transcriptional regulatory region, 5 (10%) small deletions/insertions, 5 (10%) splicing consensus sequence mutations, and 1 nonsense mutation. Twenty-one of the index cases had more than one putative VWF mutation identified. We were somewhat more likely to identify putative mutations in cases with lower VWF levels, and the contribution of other factors, such as ABO blood group, seems more important in milder cases. Taken as a whole, our data support a complex spectrum of molecular pathology resulting in type 1 VWD. In more severe cases, genetic changes are common within the VWF gene and are highly penetrant. In milder cases, the genetic determinants are more complex and involve factors outside of the VWF gene.

Von Willebrand Factor/Factor VIII Concentrate (Humate-P®) Is Safe and Effective in the Prevention of Perioperative Bleeding in Patients with Very Low von Willebrand Factor: Ristocetin Cofactor (VWF:RCo).

Optimal dosing to prevent excessive surgical bleeding in von Willebrand Disease (VWD) was investigated in an open-label study of replacement therapy with a von Willebrand factor/factor VIII concentrate (VWF/FVIII, Humate-P®). This analysis focused on the subset of patients with very low VWF, defined as those with baseline VWF:RCo levels of <12 IU/dL. Of a total of 35 patients, 17 had very low levels of VWF:RCo; of these 17 “severe” VWD patients, 12 had type 3 VWD, one each had types 1, 2A or 2B, and 2 had type 2M VWD. Previous pharmacokinetic data in each patient was utilized to calculate pre-operative and immediate postoperative doses to raise plasma VWF:RCo and FVIII to 80–100 IU/dL for major surgery and 50–60 IU/dL for minor and oral surgery; subsequent doses were adjusted based on VWF:RCo levels. Eleven patients underwent major surgery including orthopedic, gynecologic and plastic surgery, and multiple tooth extractions; 4 had minor surgery and 2 had single tooth extractions. Hemostatic efficacy was assessed by investigators as excellent, good, moderate/poor, or none immediately after the surgery, 24 hours after the last VWF/FVIII infusion and 14 days post-op. Expected and actual estimated blood loss (EBL) was compared, transfusions recorded and adverse events (AEs) documented. The median loading dose was higher among subjects with severe VWD types (71 IU/kg, range 39 to 135) compared with non-severe VWD (44 IU/kg, range 17 to 121). Subjects with severe VWD types also used higher total doses (median: 280 IU/kg, range 63 to 859) and had longer treatment duration (6 days, range 1–26) than subjects with non-severe VWD types (median total dose: 208 IU/kg, range 79 to 1699; and treatment duration: 4.5 days, range 2–19). Hemostasis was rated as effective (good or excellent efficacy) in 15/17 (88.2%) patients immediately postoperatively, in 17/17 (100%) patients 24 hours after the last infusion (primary endpoint), and in 17/17 (100%) patients 14 days postoperatively. A bleeding related serious AE occurred in one patient; she had hemorrhage post-hysteroscopic resection of uterine fibroids followed by hysterectomy; actual EBL exceeded expected EBL and hemostatic efficacy was considered moderate/poor immediately post-op but good/excellent at the other time points. Three other surgery-related hemorrhagic events were mild in the severe VWD types; in the non-severe VWD patients, 1 severe, 1 moderate and 2 mild hemorrhagic adverse events were reported; none of these were considered to be related to poor efficacy of the drug. No thromboembolic complications or changes in viral titers were observed in the study. We conclude that patients with very low baseline VWF levels can safely undergo both major and minor surgery with VWF/FVIII concentrate when dosing is calculated to achieve and maintain hemostatic VWF levels based on VWF:RCo monitoring. It is important to base therapeutic decisions on the severity of disease as assessed by baseline plasma VWF and FVIII levels as well as VWD type.

The mutational spectrum of type 1 von Willebrand disease: results from a Canadian cohort study

In order to evaluate the changes within the VWF gene that might contribute to the pathogenesis of type 1 von Willebrand disease (VWD), a large multicenter Canadian study was undertaken. We present data from the sequence analysis of the VWF gene in 123 type 1 VWD index cases and their families. We have identified putative mutations within the VWF gene in 63% (n = 78) of index cases, leaving 37% (n = 45) with no identified changes. These changes comprise 50 different putative mutations: 31 (62%) missense mutations, 8 (16%) changes involving the VWF transcriptional regulatory region, 5 (10%) small deletions/insertions, 5 (10%) splicing consensus sequence mutations, and 1 nonsense mutation. Twenty-one of the index cases had more than one putative VWF mutation identified. We were somewhat more likely to identify putative mutations in cases with lower VWF levels, and the contribution of other factors, such as ABO blood group, seems more important in milder cases. Taken as a whole, our data support a complex spectrum of molecular pathology resulting in type 1 VWD. In more severe cases, genetic changes are common within the VWF gene and are highly penetrant. In milder cases, the genetic determinants are more complex and involve factors outside of the VWF gene.

External Peer Review Quality Assurance Testing in von Willebrand Disease: The Recent Experience of the United States College of American Pathologists Proficiency Testing Program

The U.S. College of American Pathologists (CAP) has conducted a focused study of the proficiency testing for von Willebrand disease (vWD) analysis from 2003 to 2005. This report summarizes the findings regarding the accuracy and precision of the various assays at different analyte levels, as well as the influence of the reference material used to construct the assay standard curve. The results show that testing of von Willebrand factor (vWF):antigen (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) is reasonably accurate, with all-method mean values falling within 3.2 and 5.6%, respectively, of the International Society on Thrombosis and Haemostasis Secondary Coagulation Standard (lot 2) assigned values. vWF:Ag measurements are reasonably precise (all-method coefficients of variation [CVs] = 10.7 to 15.1%), even at lower levels of vWF. The highest precision was observed for immunoturbidometric assays (CVs, 6.3 to 9.7%). vWF:RCo measurements are less precise (all-method CVs, 23.3 to 30.9%). The reference materials used in the standard curves for immunoturbidometric vWF:Ag assays appear to have accurately assigned vWF values for the majority of commercial suppliers.

Disfunción endotelial y desequilibrio hemostásico en el síndrome X cardiológico

Introduction The possible presence of endothelial dysfunction and hemostatic imbalance in cardiological syndrome X, frequent in the postmenopause, remains controversial. Objective To compare the biomarkers von Willebrand factor (vWF), fibrinogen (Fb), total tissue factor pathway inhibitor (TFPI-t) and plasminogen activator inhibitor (PAI-1) between patients without coronary artery disease (cardiological syndrome X, mixed angina and vasospastic angina) and patients with coronary artery disease. Material and methods These biomarkers were determined in 10 patients with cardiological syndrome X, 13 patients with mixed angina, 15 patients with vasospastic angina, 10 patients with coronary arterial disease and 20 healthy controls. Results Compared with healthy controls, all patients had elevated levels of Fb [3.1 (2.7-3.6) vs 2.8 (2.2-3.2) g/l; P=.003], vWF [211.9 (165-266) vs 116.5 (91-155)%, TFPI-t [122.3 (101-136) vs 86 (72-100) ng/ml; P<.001] and PAI-1 [45.9 (32-61) vs 21 (11-26) ng/ml; P<.001]. However, patients without coronary artery disease had lower levels of vWF [200.5 (151-249) vs 255.2 (221-277) %; P=.01)] and TFPI-t [110.5 (100-127) vs 136.7 (121-148) ng/ml; P=.02)] than those with coronary artery disease. Conclusion The presence of endothelial dysfunction and hemostatic imbalance is similar in patients with cardiological syndrome X and those with mixed or vasospastic angina, although these disorders are less severe than in patients with coronary artery disease.

Reassessment of ABO Blood Group, Sex, and Age on Laboratory Parameters Used to Diagnose von Willebrand Disorder

We reassessed the influence of ABO blood group, sex, and age on plasma levels of von Willebrand factor (vWF) antigen, vWF:ristocetin cofactor, vWF:collagen binding assay, and factor VIII coagulant (FVIII:C). Data show that levels of vWF and FVIII:C increase with increasing age (P < .001 for all parameters) and that the ABO blood group influences plasma levels such that O group levels are significantly less than non-O group levels. There was no significant association with sex and Rh status. The selection of normal ranges based on ABO blood groups may influence the clinical diagnosis of von Willebrand disease (vWD) but might not be clinically relevant or help identify people at increased risk of bleeding. Differences in ABO-related ranges were more extensive at the high end of the ranges. This is of particular interest because high levels of vWF and FVIII are associated with thrombosis risk, and an ABO relationship also has been described. O group individuals may or may not be at greater risk for bleeding (they have lower levels of vWF and FVIII:C) and are more likely to be diagnosed with vWD. It also is possible that O group status may be protective for thrombosis.

High Dose Intranasal Desmopressin (DDAVP) Therapy in Type 2M Von Willebrand Disease (VWD): A Prospective Open Label Trial.

DDAVP is effective treatment for VWD patients with adequate biologic response to administration of a trial dose. Intranasal therapy offers the advantage of ease of administration. Few prospective data evaluating response to intranasal DDAVP exist, especially in less common types such as type 2M VWD. As part of a study of a large Amish kindred of VWD in Northern Indiana, we conducted a prospective open label trial to assess response rate to a single dose of high concentration intranasal DDAVP in 11 affected individuals. Stimate® Nasal Spray (1.5 mg/ml) with weight based dosing was used. Laboratory parameters obtained prior to and 90 minutes after administration of Stimate® to assess for response included ristocetin cofactor activity (VWF:RCo), factor VIII activity(VIII:C), and VWF antigen (VWF:Ag) values. VWF:RCo ≥ 40% at 90 minutes post Stimate® administration was defined as adequate response. Of 212 evaluated members, 54 affected members were identified as defined by abnormally low VWF levels (VWF:RCo levels <35%) and confirmed by presence of C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene. Multimer analysis in a subset of the affected individuals showed a normal pattern. Eleven adult Amish patients (5 males, 6 females), ranging in age from 20 to 56 years, were enrolled. Baseline VWF:RCo values ranged from 10% to 14%. Ten patients reported no active bleeding symptoms at the time of initiation of the trial, while one had ongoing problems with gingival bleeding. All patients weighed >50 kg; hence two intranasal Stimate® spray puffs were administered to each patient. Five of eleven patients had response to intranasal DDAVP as evidenced by ≥ 40% VWF:RCo levels 90 minutes after receiving Stimate® (Response range: 4.0 fold to 4.86 fold rise). The remaining patients did not meet definition of response (Response range: 1.6 fold to 3.8 fold rise). There were no major adverse effects. Nine patients had mild facial flushing alone; one patient had flushing and headache; one patient was asymptomatic. After Stimate® administration, the patient with gingival bleeding reported complete cessation of bleeding sustained for 24 hours. This is the first and largest report to date assessing response to intranasal DDAVP therapy in 2M VWD patients. High concentration intranasal DDAVP may be used for minor bleeding episodes in those patients with adequate response to a trial dose. It is easily self administered as home based first line hemostatic therapy. Intranasal DDAVP is well tolerated with minimal adverse effects. Prospective clinical correlation of efficacy is underway in this population.RESULTS OF STIMATE® TESTINGAGE/SEXBASELINE FVIII:C (%)BASELINE VWF:AG(%)BASELINE VWF:RCO(%)90 MINUTE FVIII:C(%)90 MINUTE VWF:AG(%)90 MINUTE VWF:RCO(%)56/M623011164784225/M5230<101821114847/M5731141961086827/M4620<10114552848/F66291289382025/M351911156935241/F5423<10106563520/F2715<1096524024/F2712<1091492420/F3419<10146163834/F4216<101125824

Identifying novel genetic determinants of hemostatic balance.

Incomplete penetrance and variable expressivity confound the diagnosis and therapy of most inherited thrombotic and hemorrhagic disorders. For many of these diseases, some or most of this variability is determined by genetic modifiers distinct from the primary disease gene itself. Clues toward identifying such modifier genes may come from studying rare Mendelian disorders of hemostasis. Examples include identification of the cause of combined factor V and VIII deficiency as mutations in the ER Golgi intermediate compartment proteins LMAN1 and MCFD2. These proteins form a cargo receptor that facilitates the transport of factors V and VIII, and presumably other proteins, from the ER to the Golgi. A similar positional cloning approach identified ADAMTS-13 as the gene responsible for familial TTP. Along with the work of many other groups, these findings identified VWF proteolysis by ADAMTS-13 as a key regulatory pathway for hemostasis. Recent advances in mouse genetics also provide powerful tools for the identification of novel genes contributing to hemostatic balance. Genetic studies of inbred mouse lines with unusually high and unusually low plasma VWF levels identified polymorphic variation in the expression of a glycosyltransferase gene, Galgt2, as an important determinant of plasma VWF levels in the mouse. Ongoing studies in mice genetically engineered to carry the factor V Leiden mutation may similarly identify novel genes contributing to thrombosis risk in humans.

Bleeding prophylaxis for major surgery in patients with type 2 von Willebrand disease with an intermediate purity factor VIII–von Willebrand factor concentrate (Haemate-P)

The parameters to diagnose von Willebrand disease (vWD) include factor VIII coagulant activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), von Willebrand factor ristocetin cofactor activity (vWF:RCo), and von Willebrand factor collagen binding activity (vWF:CB). Type 2 vWD is associated with a moderate bleeding diathesis due to low levels of vWF:RCo and vWF:CB as compared with near normal or normal values for FVIII:C and vWF:Ag. As the factor VIII/von Willebrand factor (vWF) concentrate, Haemate-P, is featured by a vWF:RCo/FVIII:C ratio of about 2.2, the recommended loading dose of 50 U/kg FVIII:C followed by 25 U/kg FVIII:C every 12 h for several days for bleeding prophylaxis in type 2 vWD patients undergoing major surgery demonstrated a predicted significant over-treatment reaching vWF:RCo levels above 2 U/ml. Therefore, we restricted Haemate-P substitution for major surgery to one loading dose of 40-50 U/kg FVIII:C (88-110 U/kg vWF:RCo) followed by 15-20 U/kg FVIII:C (33-44 U/kg vWF:RCo) every 12 h for several days and evaluated this strategy in a prospective pharmacokinetic and efficacy study for bleeding prophylaxis in five type 2 vWD patients. Pre-treatment and peak levels (1 h after Haemate-P loading dose) rose from 0.43-0.66 to 1.5-2.5 U/ml for FVIII:C, from 0.23-0.45 to 1.5-2.5 U/ml for vWF:Ag, from 0.10-< 0.20 to 1.5-2.5 U/ml for vWF:RCo, and from < 0.05-0.10 to 1.0-2.0 U/ml for vWF:CB. Mean in vivo recoveries per transfused IU FVIII:C/kg body weight were 3.2% for FVIII:C, 3.9% for vWF:RCo, and 2.8% for vWF:CB. Mean in vivo recoveries per transfused IU vWF:RCo/kg were 1.45% for FVIII:C, 1.7% for vWF:RCo and 1.25% for vWF:CB. The biological half-life times after transfused Haemate-P were about 12 h for both vWF:RCo and vWF:CB. Based on these pharmacokinetic data, we propose to adapt the loading dose factor VIII/vWF concentrate (Haemate-P) to 60-80 U/kg vWF:RCo followed by 30-40 U/kg vWF:RCo every 12 h for no longer than several days (less than 1 week) for bleeding prophylaxis during major surgery or trauma, and to one loading dose of 40-60 U/kg vWF:RCo for minor surgery, trauma or mucotaneous bleedings in patients with type 2 vWD unresponsive to DDAVP.

Laboratory Diagnosis of von Willebrand Disorder

A survey of 44 laboratories was conducted to evaluate current testing proficiency in the diagnosis of von Willebrand disorder (vWD) and to assess recent changes in test practices. Laboratories performed their usual panel of tests for vWD and interpreted results for the likelihood of vWD and potential subtype. Samples were as follows: normal plasma; borderline normal or abnormal levels of von Willebrand factor (vWF) and factor VIII; type 3 vWD; type 2A vWD; and 2 samples from a healthy person, processed after handling at 22°C and 4°C, respectively. Interassay and within-method coefficients of variation were similar for all assays (approximately 15%–25%). Most laboratories reported test values consistent with expected findings and made correct interpretations, although discrepant results for 5% to 10% of responses are of concern. For the sample stored at 4°C, all laboratories detected low or borderline levels of vWF and factor VIII coagulant, and no laboratory identified this sample as from a healthy person. In contrast, for the sample stored at 22°C, most laboratories reported normal results. Compared with previous results, performance of some assays has declined while that of others has increased. Laboratories generally are proficient in tests for vWD, and transport of samples at 4°C before processing may lead to false identification of vWD, suggesting that NCCLS guidelines should be reviewed.

Laboratory diagnosis of von Willebrand disorder. Current practice in the southern hemisphere.

A survey of 44 laboratories was conducted to evaluate current testing proficiency in the diagnosis of von Willebrand disorder (vWD) and to assess recent changes in test practices. Laboratories performed their usual panel of tests for vWD and interpreted results for the likelihood of vWD and potential subtype. Samples were as follows: normal plasma; borderline normal or abnormal levels of von Willebrand factor (vWF) and factor VIII; type 3 vWD; type 2A vWD; and 2 samples from a healthy person, processed after handling at 22 degrees C and 4 degrees C, respectively. Interassay and within-method coefficients of variation were similar for all assays (approximately 15%-25%). Most laboratories reported test values consistent with expected findings and made correct interpretations, although discrepant results for 5% to 10% of responses are of concern. For the sample stored at 4 degrees C, all laboratories detected low or borderline levels of vWF and factor VIII coagulant, and no laboratory identified this sample as from a healthy person. In contrast, for the sample stored at 22 degrees C, most laboratories reported normal results. Compared with previous results, performance of some assays has declined while that of others has increased. Laboratories generally are proficient in tests for vWD, and transport of samples at 4 degrees C before processing may lead to false identification of vWD, suggesting that NCCLS guidelines should be reviewed.

Von Willebrand factor collagen-binding (activity) assay in the diagnosis of von Willebrand disease: a 15-year journey.

The correct diagnosis and classification of von Willebrand disease (vWD) is crucial because the presenting biological activity of von Willebrand factor (vWF) determines both the hemorrhagic risk and the subsequent clinical management. A variety of laboratory assays may be employed, not necessarily restricted to assessments of vWF. This article discusses the relative strengths and limitations of various functional or discriminatory vWF assays with a special focus on the vWF:collagen-binding activity (vWF:CBA) assay. This is a functional vWF assay that relies on the property of vWF adhesion to collagen. The vWF:CBA was first described approximately 15 years ago. The journey from that time point has been an interesting one, and the vWF:CBA is now gaining more widespread acceptance. There are now many published studies confirming the superiority of the vWF:CBA over the vWF ristocetin cofactor (vWF:RCof) activity as a functional screening diagnostic test process for vWD. However, both tests may be required in order to appropriately diagnose all forms of vWD. The relationship of these assays with multimer analysis is also discussed. In summary, an optimized vWF:CBA detects primarily high-molecular-weight (HMW) vWF forms and probably only about 30% of the total plasma vWF pool detected by vWF antigen (vWF:Ag). Because these HMW vWF forms are missing in types 2A and 2B vWD, the vWF:CBA is extremely useful in the detection of these qualitative vWD subtypes. In addition, however, concordance of vWF:CBA with vWF:Ag in unison with low vWF levels may alternatively suggest a type 1 vWD, and an absence of both vWF:Ag and vWF:CBA will suggest type 3 vWD. The vWF:CBA is also being investigated in various disease states, as is its usefulness as an effective marker of functional HMW vWF in both desmopressin (DDAVP) and factor-concentrate therapy in vWD.

Conformational changes in the D′ domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment

We report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D′ domain. These results show that the native conformation of the D′ domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

Conformational changes in the D′ domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment

AbstractWe report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D′ domain. These results show that the native conformation of the D′ domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

Laboratory Testing for von Willebrand’s Disease: An Assessment of Current Diagnostic Practice and Efficacy by Means of a Multi-laboratory Survey

We report an evaluation of current laboratory practice for the diagnosis of von Willebrand's disease (VWD) by means of a multilaboratory survey. This assessment was undertaken with the RCPA Quality Assurance Program (QAP) in Haematology, which covers a wide geographic area encompassing Australia, New Zealand and Asia. A total of 25 laboratories actively involved in testing for VWD were selected to participate in a sample testing assessment exercise. Samples comprised 10 plasmas: (i) a normal plasma pool (in duplicate), (ii) this pool diluted to 50% (in duplicate), (iii) a normal individual (X1), (iv) severe Type 1 VWD (X1), (v) Type 2B VWD (x2 unrelated donors), (vi) Type 3 VWD (x1), (vii) Type 2A VWD (x1). Laboratories were asked to perform all tests available to them in order to establish a laboratory diagnosis of VWD, and then to comment on the possibility or otherwise of VWD. Overall findings indicated a wide variation in test practice, in the effectiveness of various test procedures in detecting VWD, and in the ability of various composite test panels to identify type 2 VWD subtypes. Firstly, while all laboratories (n = 25) performed tests for FVIII:C activity, von Willebrand factor 'antigen' (VWF:Ag) and a functional VWF assay [using the ristocetin cofactor assay (VWF:RCo; n = 23) and/or the collagen binding assay (VWF:CBA; n = 12)], only three laboratories carried out VWF:Multimer analysis. Secondly, for the three quantitative VWF assays, 10/25 (40%) laboratories performed all three, whereas 15/25 (60%) performed only two [VWF:Ag and VWF:RCo (n = 13); VWF:Ag and VWF:CBA (n = 2)]. Thirdly, a variety of assay methodologies were evident for VWF:Ag [ELISA, electro-immuno diffusion (EID), latex immuno-assay (LIA), and VIDAS assay] and VWF:RCo (platelet agglutination/'aggregometry' and a 'functional VWF:RCo-alternative' ELISA assay). Between method analysis for the quantitative VWF assays showed that the VWF:RCo yielded the greatest degree of inter-laboratory assay variation, and had the poorest overall performance with respect to sensitivity to low levels of VWF. The VWF:CBA also performed better than the VWF:RCo in terms of ability to detect functional VWF 'discordance' (i.e. Type 2 VWD). Within VWF:Ag method analysis showed that the EID assay procedure was associated with the greatest variation in assay results, while the EID and LIA test methods showed poorer sensitivity at low VWF levels compared to the ELISA method. Within the VWF:RCo assay procedure, greatest variation in assay results and poorest sensitivity to low VWF levels was obtained using the agglutination method; however, the agglutination procedure showed better performance than the 'functional VWF:RCo-alternative' ELISA assay in identifying Type 2 VWD plasma samples. Finally, despite identified variations, most laboratories appeared to understand the complexities involved in the VWD-diagnostic process, and made appropriate diagnostic predictions regarding tested samples. From a total possible 246 interpretation events, laboratories in most cases correctly identified normal samples as normal (67/75 events = 89%), and VWD samples as derived from individuals with VWD (117/121 events = 97%). Moreover, when VWD was suggested by laboratory findings, laboratories usually correctly predicted the general subtype of VWD present (96/109 events = 88%). When 'misinterpretations' occurred, these could often be linked to the test panels utilised by laboratories. That is, laboratories using the VWF:Ag and VWF:RCo combination were more likely to incorrectly identify samples derived from Type 2 VWD patients as being Type 1, Type 1 VWD patients as being Type 2, and normal plasma samples as potentially derived from patients with VWD, compared to those using the VWF:Ag and VWF:CBA.

Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity.

Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.

Effects of hormone-replacement therapy on hemostatic factors, lipid factors, and endothelial function in women undergoing surgical menopause: Implications for prevention of atherosclerosis

Women with premature menopause are at high risk for vascular compications associated with thrombogenesis and atherogenesis. The use of hormone-replacement therapy (HRT), however, may protect against these complications. Hemostatic abnormalities and endothelial function are closely related to the processes of thrombogenesis and atherogenesis. The purpose of the study was to evaluate the effects of premature menopause on markers of hemostasis, platelet function, and endothelial function and the effects of starting HRT. This is a prospective longitudinal study of premenopausal women undergoing surgical menopause in whom estrogen HRT is started. We measured sequential changes in plasma levels of the hemostatic factors (fibrinogen, fibrin d -dimer, and plasminogen activiator inhibitor [PAI]), markers of platelet function (soluble leukocyte adhesion molecule P-selectin) and endothelial function (von Willebrand factor [vWf], soluble thrombomodulin [sTM], and tissue plasminogen activator [TPA]), and serum lipid levels, including lipoprotein A. Twenty-seven premenopausal women (mean age 43.6 ± 6.5 years) undergoing hysterectomy and bilateral salpingo-oophrectomy were studied. In the postsurgical menopausal state (visit 2), there was a significant elevation in sTM levels (paired Wilcoxon test, p = 0.008). There was also a trend toward higher median soluble P-selectin, PAI, and mean TPA levels and lower vWf levels. After 6 weeks of HRT (visit 3), there was a significant reduction in mean vWf (paired Wilcoxon test, p = 0.0026), sTM ( p = 0.039), and TPA levels ( p = 0.02) compared with premenopausal levels. There were no significant changes in plasma fibrinogen, fibrin d -dimer, and PAI levels at visit 2 or visit 3 compared with premenopausal levels. There was a significant increase in serum lipoprotein A (paired Wilcoxon test, p = 0.008), cholesterol, and triglyceride levels after surgical menopause (paired t test, p < 0.01). Lipoprotein A and cholesterol levels after HRT (visit 3) were not significantly different from prehysterectomy levels, although triglyceride levels were increased further. HRT results in a significant reduction in vWf, sTM, and TPA levels, suggesting beneficial effects on endothelial function and atherogenesis. Although there was a significant increase in serum lipoprotein A and cholesterol levels after surgical menopause, lipoprotein A and cholesterol levels after HRT were not significantly different from presurgery levels. These observations are consistent with the beneficial effects of HRT in cardiovascular hemodynamics and cardiovascular disease. (Am Heart J 1997;134:764-71.)