PFA-100 monitoring of von Willebrand factor (VWF) responses to desmopressin (DDAVP) and factor VIII/VWF concentrate substitution in von Willebrand disease type 1 and 2

Dose-response relationship was studied between PFA-100 closure times (PFA CTs) and factor (F)VIII-von Willebrand factor (VWF) parameters in patients with von Willebrand disease (VWD) type 1 and type 2 before and after treatment with DDAVP (n=84) or FVIII/VWF concentrate (n=38). DDAVP treatment of patients with VWD type 1 normalised the PFA CTs by increasing VWF levels to normal. Of the 14 patients with VWD type 2, PFA CTs did not normalize in eight. Haemate-P substitution in patients with VWD type 1 induced a less favourable response as compared to DDAVP, because PFA CTs did not correct in all patients. Of 12 patients with VWD type 2 treated with Haemate-P, six showed a correction of PFA CTs (<250 sec), which correlated with the normalisation of the VWF CB/Ag ratio. In-vitro studies were performed by using whole blood of patients with VWD and adding various amounts of FVIII/VWF concentrate. Addition of Haemate-P induced an increase of the VWF CB/Ag ratio from 0.30 to 0.70 in blood of patients with VWD type 2 with correction of the PFA CTs. Immunate did not result in an increase of VWF CB/Ag ratio in blood of VWD type 2 patients, and the PFA CTs remained prolonged. We conclude that PFA-100 might be an adequate instrument not only for diagnosis but also for monitoring of DDAVP responses and FVIII/VWF substitution of patients with VWD type 1 and 2, but this is dependent upon the type of VWD and the concentrate used.     

In vitro studies, pharmacokinetic studies and clinical use of a high purity double virus inactivated FVIII/VWF concentrate (Immunate) in the treatment of von Willebrand disease

Patients with type 2 and 3 von Willebrand disease (VWD) are treated with factor VIII/VWF concentrate in case of bleeding or surgery. Immunate (Baxter, Vienna, Austria) is a double virus inactivated FVIII/VWF concentrate and is registered in several countries for patients with VWD with reduced FVIII levels. We performed an in vitro, a pharmacokinetic and a clinical study to evaluate Immunate in VWD. In vitro studies showed a significant variation in VWF levels in 9 different batches. The median (range) values (in IU/mL) were 1.10 (0.98-1.30) for FVIII:C, 1.34 (0.95-1.61) for VWF:Ag, 0.60 (0.27-1.08) for VWF:CBA and 0.73 (0.59-0.94) for VWF:RCo. The relatively low VWF activity is mainly due to the lack of high molecular weight multimers (HMWM), as determined by electrophoresis. A pharmacokinetic study showed, based on a content of FVIII:C of 1 U/mL, in vivo recoveries (%) of 106 (56-150) (median and range) for FVIII:C, 105 (62-187) for VWF:Ag, 25 (7-41) for VWF:CBA and 43 (11-76) for VWF:RCo. Half-lives were 14.1 h (7.4-36.9) for FVIII:C, 10.8 h (7.7-26.2) for VWF:Ag, 15.3 h (7.8-44.6) for VWF:CBA and 16.4 h (4.2-26.5) for VWF:RCo. In a clinical study efficacy was determined after infusion given before surgery or dental extractions in ten patients. In two patients the hemostatic response was classified as inadequate. In conclusion, there is a wide variability in VWF concentration and activity in various batches of Immunate. In the clinical study in which the dosage was based on FVIII:C contents of the concentrate, two out of ten patients had an insufficient haemostatic response. Therefore dosing of Immunate dosing should not be based on FVIII:C levels, but should be based on VWF activity of the individual batches. Future studies using a VWF activity-guided dosage regimen have to be performed to establish the efficacy of Immunate in the treatment of von Willebrand disease.     

Comparison of the pharmacokinetics of two von Willebrand factor concentrates [Biostate and AHF (High Purity)] in people with von Willebrand disorder. A randomised cross-over, multi-centre study

Plasma-derived factor concentrates are important in the management of von Willebrand disorder (VWD). In our geographic locality, a single viral inactivation step concentrate (AHF [High Purity]), has been replaced with one using a double viral inactivation step (Biostate). The aim of this study was to compare the pharmacokinetics of von Willebrand factor (VWF) and factor VIII (FVIII) after administration of AHF (High Purity) and Biostate. This study was a single-blind, randomised cross-over, multi-centre investigation in twelve people with VWD, comprising four type 3, two type 2B, one type 2M and five type 1 VWD. The subjects received a single infusion of 60 IU/kg ristocetin cofactor activity (VWF:RCo) of either AHF (High Purity) or Biostate, and after a minimum 15-day wash-out period they received the alternative product. Blood samples were collected for up to 48 hours after each dose for assay of FVIII coagulant activity (FVIII:C) and VWF by VWF:RCo, collagen binding capacity (VWF:CB) and antigen (VWF:Ag). As a measure of delivered VWF 'functionality' we calculated the area-under-the-concentration-time-curve (AUC) ratios of VWF:RCo to VWF:Ag and VWF:CB to VWF:Ag. The effect on platelet adhesiveness by PFA-100 closure times (CTs) was measured prior to and 30 minutes post infusion. VWF multimers were also assessed pre and post infusion. Pharmacokinetic parameters after AHF (High Purity) and Biostate were in close agreement for VWF:RCo (confirming dosing equivalence). Parameters for other study markers were also similar, although Biostate tended to yield relatively lower VWF:Ag and higher VWF:CB levels. Although AHF (High Purity) and Biostate resulted in similar levels of high-molecular-weight (HMW) multimers post-infusion, the relative level of HMW to low-molecular-weight (LMW) multimers were determined to be higher following Biostate. The relative levels of functional VWF (i.e. VWF:CB and VWF:RCo) to VWF:Ag were also higher in Biostate compared to AHF (High Purity). With both study products, PFA-100 CTs 30 minutes post infusion showed minor improvement for only some subjects. In conclusion, the pharmacokinetics of FVIII:C and VWF are not significantly different after administration of AHF (High Purity) and Biostate. Study parameters considered as 'in-vitro' markers of VWF 'functionality' or potential clinical efficacy (i.e. VWF:CB and VWF:RCo relative to VWF:Ag, level of HMW VWF relative to LMW-VWF) were determined to be higher for Biostate than AHF (High Purity). PFA-100 CTs did not adequately reflect changes in these VWF parameters. Based on these results, one would expect Biostate to be at least as effective, if not superior to AHF (High Purity) for the treatment of VWD.     

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.     

Altered serum factor VIII-related antigen (VIII : AGN)/von Willebrand factor (VIII : vWf) in haemophiliacs with inhibitors to factor VIII procoagulant activity (VIII : C)

Inhibitors to factor VIII (anti-F VIII) developing in patients with classic haemophilia have apparent specificity for the factor VIII procoagulant activity (VIII : C), rather than the factor VIII-related antigen (VIII : AGN) and von Willebrand factor (VIII : vWf) regions of the factor VIII complex. Since procoagulant function is absent following in vitro clotting, but serum retains VIII : AGN/vWf properties, we searched for differences in VIII : AGN and VIII : vWf of inhibitor serum that might relate to the presence of anti-F VIII. Rocket immunoelectrophoresis and the washed platelet ristocetin assay were performed on the plasma and serum of nine haemophiliacs with inhibitors, 23 non-inhibitor haemophiliacs and six normal subjects. Unlike normal and non-inhibitor haemophilic sera, that from five of nine inhibitor patients demonstrated absent VIII : vWf and significantly lower VIII : AGN (p less than 0.05). Furthermore, VIII : AGN of faster mobility was detected on crossed immunoelectrophoresis of the sera of three inhibitor patients. Thrombin clotting of plasma from haemophiliacs with high titer anti-F VIII was associated with a greater loss of VIII : vWf than seen with non-inhibitor haemophilic plasma. This effect was independent of the presence of platelets. These data indicate that in vitro clotting is associated with alteration in the serum VIII : AGN/vWf of some haemophiliacs with anti-F VIII.     

Factor VIII (Willebrand) antigen and ristocetin-Willebrand factor in pigs with von Willebrand's disease.

The Willebrand antigen and the ristocetin-Willebrand factor were virtually undetectable in pigs with a severe form of von Willebrand's disease. These findings establish that porcine von Willebrand's disease is essentially the same as the human counterpart. The Willebrand antigen and ristocetin-Willebrand factor were reduced approximately 60% in pigs which are heterozygous. Furthermore these two new tests can be done on capillary blood, permitting earlier identification of the disease.     

Plasma ADAMTS13, von Willebrand factor (VWF) and VWF propeptide profiles in patients with DIC and related diseases

ADAMTS13, endothelial von Willebrand factor (VWF) and related proteins are involved in the pathogenesis of some life threatening systemic thrombotic coagulopathies. Changes of plasma ADAMTS13 activity in thrombotic thrombocytopenic purpura (TTP) is well known but is also involved in septic disseminated intravascular coagulation (DIC). Here we investigated the ADAMTS13 activity, VWF and VWF propeptide (VWFpp) antigens in 69 patients with DIC, 143 with non-DIC, 21 with thrombotic thrombocytopenic purpura (TTP) and 23 with atypical hemolytic uremic syndrome (aHUS) for diagnosis of DIC.The plasma ADAMTS13 activity was significantly low in patients with DIC, and the plasma levels of VWF and VWFpp antigens, were the highest in these patients, but there were no significant differences in the plasma VWFpp levels between the patients with DIC and those with aHUS. The difference in the plasma ADAMTS13 activity, the VWF and VWFpp antigens between DIC and non-DIC cases was significant in those with infectious and malignant diseases, but the difference in the VWFpp/ VWF ratio were significant only in subjects with infectious diseases. As an indicator for prognosis, the plasma levels of VWFpp were significantly higher in non-survivors than in survivors. Then, VWFpp/ VWF ratio and VWFpp/ADAMATS13 ratio will be potent informative indicators in DIC.These findings suggest that ADAMTS13/VWF profiles may have important roles in the pathogenesis of DIC, and that ADAMTS13 and VWFpp are useful indicators for the diagnosis and prognosis of DIC.     

Immunologic studies in von Willebrand's disease: alteration of factor VIII/von Willebrand protein after transfusion with plasma concentrates in patients with von Willebrand's disease.

Two patients with a severe von Willebrand's disease characterized by no detectable factor VIII related antigen in their plasma received transfusions of cryoprecipitate. The bleeding time was corrected for a short period of time and returned to its pretransfusional value although the other parameters of the disease were still corrected. Electrophoretic and immunologic properties of factor VIII related antigen infused were determined serially after transfusion. Modifications of these properties occurred progressively after transfusion. The half disappearance time of F. VIII R.A. was determined and found to be considerably shorter than in hemophilic recipients. This study suggests an alteration in vivo of F. VIII R.A. infused into von Willebrand recipients.     

Cross-reacting material in congenital factor VIII deficiencies (Haemophilia A and von Willebrand's disease)

Using a heterologous specific and precipitating anti human Factor VIII antiserum, the presence of cross-reacting material (C.R.M.) was detected in cryoprecipitate as well as in plasma of 83 patients with Haemophilia A (49 without and 34 with a circulating Factor VIII antibody). The ratio of Factor VIII activity to antigen was lower in known carriers of Haemophilia than in normal women. In 15 patients with von Willebrand's disease, C.R.M. was absent or seemed only to be proportional to the amount of biologically active Factor VIII.     

Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes.

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wildtype from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.     

Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen

We report the identification of a new mutation of von Willebrand Factor (VWF) gene within exon 30 occurring in two related patients (mother and daughter) with a hemorrhagic syndrome. A T-->A transvertion at nucleotide 5441 was found changing the serine 968 to threonine of the mature VWF subunit (S1731T of the preproVWF). The Ser968Thr mutation is located within the VWF A3 domain which interacts with type I and III collagens. Both patients were found to be heterozygous for the mutation. The propositus (daughter) exhibited a slightly prolonged bleeding time, levels of VWF:Ag and VWF:RCo at the lower limit of normal, contrasting with normal levels of VIII:C. Her mother exhibited borderline bleeding time and moderately decreased levels of VWF and VIII:C. In both patients multimeric structure of VWF and ristocetin- as well as botrocetin-induced binding of VWF to GPIb were normal; however both patients repeatedly showed decreased binding of VWF to collagen. The Ser968Thr substitution was reproduced by site-directed mutagenesis on the full-length cDNA of VWF. The mutated recombinant VWF (rVWF), T968rVWF, and the hybrid S/T968rVWF were transiently expressed by COS-7 cells. Both rVWF exhibited normal multimeric pattern and normal ristocetin- as well as botrocetin-induced binding to GPIb. T968rVWF showed significantly decreased binding to collagen while the hybrid S/T968rVWF bound to collagen in a similar way to that of the patients' plasma VWF. Thus, our data demonstrate that the Ser968Thr mutation of the VWF A3 domain is clearly responsible for the abnormal binding of VWF to collagen observed in both patients. The Ser968Thr substitution of the VWF is the first mutation identified in two patients leading to a decreased affinity of VWF for collagen and a normal multimeric structure.     

Analysis of the -1185A/G von Willebrand factor (VWF) gene polymorphism in two Brazilian ethnic groups and its effect on the plasma VWF levels

Both genetic and environmental factors influence the variation in von Willebrand factor (VWF) levels between individuals, the main genetic variable known to be involved in differences in VWF levels being the ABO blood group. The −1185A/G polymorphism in the 5′-regulatory region of VWF gene has been associated with plasma VWF levels in a normal population. The objective of our study was to examine the relationship between the −1185A/G polymorphism and plasma VWF levels in a total of 420 individuals from two Brazilian ethnic groups. The −1185A/G genotypes were identified using polymerase chain reaction (PCR) amplification of the 864-bp VWF promoter region followed by AccII restriction digestion. Allele and genotype frequencies were significantly different between Afro-Brazilians and Euro-Brazilians. The −1185A allele frequency was 62% in Afro-Brazilians and 41% in Euro-Brazilians. Although Afro-Brazilians had higher plasma VWF levels than Euro-Brazilians, the ethnic differences were not significant. No association was observed between −1185A/G genotypes and VWF plasma levels in either ethnic groups. The present data indicate that there are no ethnic differences in VWF levels. The −1185A/G polymorphism showed significantly different frequencies between Afro-Brazilians and Euro-Brazilians, but in our study, it did not appear to play a role in the determination of VWF levels.     

A new variant of von Willebrand disease (type II I) with a normal degree of proteolytic cleavage of von Willebrand factor.

A variant of type II von Willebrand disease (vWd) is described in a young woman and her mother with severe lifelong bleeding histories. On electrophoresis with low-resolution agarose gels the plasma of the proband lacked large and intermediate-size multimers of von Willebrand factor (vWF) but the platelet multimeric structure was normal. On high-resolution gels, smaller multimers could be resolved into a broader central band and four satellite bands, which were much fainter than in normal plasma. In the proband plasma, the relative concentrations of proteolytic fragments of the vWF subunit were within the normal laboratory range. Since this variant of vWd appears to differ from those reported hitherto, the designation of type II I is proposed.     

Discrimination of von Willebrands disease (VWD) subtypes: direct comparison of von Willebrand factor:collagen binding assay (VWF:CBA) with monoclonal antibody (MAB) based VWF-capture systems

Discrimination of von Willebrand's Disease (VWD) subtypes is important since it influences management. Qualitative [ie Type 2A, 2B, 2M] defects exhibit von Willebrand factor (VWF) discordance and give high VWF:Ag to VWF:'activity' ratios. Classically, VWF:'activity' is assessed using the VWF:RCof assay. The VWF:CBA is an ELISA-based VWF-functional adhesive assay which has consistently proved to be superior to VWF:RCof. A commercially available monoclonal antibody (MAB) based ELISA assay system claimed to mimic a VWF:RCof-like activity has also been recently described ('SE'), as has the production and characterisation of a large number [n = 10] of locally generated anti-VWF MAB. In the current study, we have adapted these MAB to in-house ELISA assays to assess their utility for VWD diagnosis and subtype discrimination, and to compare them with other assay systems. Thus, the VWF:CBA, VWF:RCof by agglutination, the SE assay, and in-house MAB based assays have been directly compared for their ability to discriminate Type 1 [n = 9] from Type 2 VWD samples [phenotypes 2A and 2B; n = 11]. In summary, MAB-based systems can be used to measure VWF and confirm a diagnosis of VWD, as well as exhibiting some VWD-subtype-discriminatory capabilities. However, better evidence of VWF-discordance was usually achieved using the VWF:RCof (agglutination) assay, while the greatest degree of VWF-discordance was consistently observed using the VWF:CBA assay. In conclusion, the VWF:CBA assay proved to offer the best diagnostic predictive tool for a Type 2 VWD defect, while MAB-based systems appear to be less effective in this regard.     

Safety and efficacy of continuous infusion of a combined factor VIII-von Willebrand factor (vWF) concentrate (Haemate-P) in patients with von Willebrand disease

We studied the safety and efficacy of treatment with continuous infusion of a von Willebrand factor (vWF) concentrate Haemate-P in patients with von Willebrand disease (vWD). Three patients with mild and 5 patients with severe forms of vWD, were treated with continuous infusion of Haemate-P by minipump. The indications for treatment were: to prevent bleeding during 9 surgical procedures or 1 vaginal delivery in 6 patients and to treat 2 bleeding episodes in 2 patients. The patients were monitored daily for factor VIII (FVIII:C) and ristocetin cofactor (vWF: RCo) levels and the infusion rate was adjusted to maintain the desired therapeutic level of vWF:RCo. The treatment was effective in preventing surgical bleeding and controlling bleeding episodes. All factor VIII:C and most of the vWF:RCo levels measured during the study period were above the target therapeutic levels. A significant decrease in clearance of FVIII:C and vWF:RCo was observed over the treatment period. Haemate-P consumption averaged 24.3+/-7.9 vWF:RCo U/kg/day which is approximately half the expected dose had intermittent bolus injections been used. We suggest that continuous Haemate-P infusion is superior to intermittent bolus injections for the treatment of vWD patients by virtue of its efficiency, simplicity and considerable savings.     

Long-term secondary prophylaxis in children, adolescents and young adults with von Willebrand disease. Results of a cohort study

In patients with von Willebrand disease (VWD) replacement therapy with factor VIII/von Willebrand (VWF) concentrates is increasingly applied as prophylactic regimen. Since 2000, 82 consecutively enrolled patients with clinically relevant bleeding episodes (spontaneous, peri- or postoperative) were diagnosed with VWD [type 1: 42/82; type 2: 24/82; type 3: 13/82; acquired: 3/82]. In all patients, decision for initiating prophylaxis was based on a bleeding score > 2 prior to diagnosis, concomitant with recurrent bleeds associated with anaemia in patients with on-demand VWD therapy. We report results on secondary prophylactic VWF replacement therapy applied in 32 patients [children n=13; adolescents n=7; adults n=12] with VWD [type 1: 4; type 2: 15; type 3: 13], 15 of which were females, and nine of these at the reproductive period. Eight patients were treated with Humate P? or Wilate? (n=24). Median [min-max] dose [vWF:RCo] was 40 [20-47] IU/kg, 23 patients were given substitution therapy twice weekly, seven patients three times a week, and two children four times per week. Within a 12-month-period haemoglobin concentrations returned to normal values. Median duration of prophylaxis was three years. Recurrent bleeding episodes stopped in 31 of 32 patients, whereas inhibitors developed in one. Following a 12-month observation period the monthly bleeding frequency and the bleeding score was significantly reduced [3 vs. 0.07; 3 vs. 0: p< 0.001], compared to the pre-prophylaxis/pre-diagnostic values. The use of secondary prophylactic VWF replacement therapy is an effective tolerated treatment modality, highly beneficial for patients with VWD, who present with recurrent bleeding events during on-demand therapy.     

Effects of arginine vasopressin (AVP) infusions on circulating concentrations of platelet AVP, factor VIII: C and von Willebrand factor

To study the possible role of arginine vasopressin (AVP) in the control of haemostasis AVP infusions at 3 doses (0.1, 0.2 and 0.3 mU/kg/min) were performed in 6 male volunteers. Both plasma and platelet AVP concentrations rose in a dose-related manner. At doses of 0.2 and 0.3 mU/kg/min there was an increase in the plasma concentrations of both plasma Factor VIII and von Willebrand factor. The data support the hypothesis that AVP, by interacting with platelets and stimulating factor VIII and von Willebrand factor release, plays a role in the control of haemostasis.     

Collagen binding assay for von Willebrand factor (VWF:CBA): detection of von Willebrands Disease (VWD), and discrimination of VWD subtypes, depends on collagen source

A large number of different collagen preparations [n = 21] have been assessed for their ability to both detect von Willebrands Disease (VWD), and discriminate different VWD subtypes. Collagen preparations were tested at a range of concentrations and included: Type I, III and IV, and various mixtures of these, as aqueous supplied preparations and/or reconstituted from bulk lyophilised stock. Tissue sources for collagens ranged from human placenta to calf skin to equine tendon. Three of the collagen preparations tested did not support von Willebrand factor (VWF) binding in an ELISA process (therefore unable to detect VWD). The ability of the remaining preparations to detect VWF was variable, as was their ability to discriminate VWD subtypes. Detection of VWF and discrimination of VWD subtypes was not mutually inclusive. Thus, some collagen preparations provided excellent detection systems for VWF, but comparatively poorer discrimination of Type 2 VWD, while others provided good to acceptable detection and discrimination. Subtype discrimination was also dependent on the collagen concentration, and some batch to batch variation was evident with some preparations (particularly Type I collagens). Overall, best discrimination was typically achieved with Type I/III collagen mixtures, or Type III collagen preparations (where effectiveness was highly dependent on concentration). Good discrimination was also achieved with a commercial Type III collagen based VWF:CBA kit method. Results of the various 'VWF:CBA assays' are also compared with those using the Ristocetin Cofactor (VWF:RCof) assay (by platelet agglutination) and that using a commercial 'VWF:RCof-alternative/activity' ELISA procedure. These latter methodologies tended to be less sensitive to VWF-discordance when compared to that detected by the majority of the VWF:CBA procedures.