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.

Type 2B von Willebrand's disease in thirteen individuals from five unrelated Australian families: phenotype and genotype correlations.

Type 2B von Willebrand's disease (VWD) is due to a qualitative defect in von Willebrand factor (VWF) in which there is an increased affinity for the platelet glycoprotein Ib-IX-V receptor complex. Spontaneous binding of type 2B VWF to platelets and subsequent clearance from the plasma is thought to account for the characteristic phenotype of type 2B VWD. These gain-of-function mutations are due to single amino substitutions that are clustered within the functionally important A1 domain of VWF. We describe 13 individuals from five unrelated families in Australia with type 2B VWD, report their phenotypic abnormalities, and delineate their causative mutations. We confirm that the mutation Arg543Trp is also particularly common among families with type 2B VWD in Australia.

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation.

Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481-Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf-dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700-Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf-induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494-Leu-512; CR2, Phe-536-Ala-554; CR3, Arg-578-Glu-596; CR11 and CR15, Ala-564-Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700-Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

Use of a novel platelet function analyzer (PFA-100) with high sensitivity to disturbances in von Willebrand factor to screen for von Willebrand's disease and other disorders.

The PFA-100 is a new platelet function analyzer which uses whole blood and high shear stress blood flow to simulate primary hemostasis and assess platelet function. A small volume of blood is introduced into a disposable cartridge, and forced through a capillary tube. Platelet adhesion and aggregation is then initiated following exposure to either collagen/ADP [C/ADP] or collagen/epinephrine [C/Epi] coated membranes. Movement of blood through the capillary, and its subsequent occlusion is monitored and yields the measured endpoint (closure time [CT] in seconds). Using two approaches, we assessed the sensitivity of this system to disturbances in the function of von Willebrand Factor (VWF). Firstly, we assessed the ability of the PFA-100 to detect the presence of von Willebrands Disease (VWD). Using normal individuals (N = 18), CTs (in seconds; mean [range = mean +/- 2SD]) were (i) C/ADP, 95 [66-124], (ii) C/Epi, 128 [98-158]. A panel of 47 patients undergoing evaluation for clinical hemostatic defects inclusive of VWD were also evaluated. All samples from patients confirmed to have VWD following specific VWF studies [N = 9; 3 x Type 1, 1 x Type 3, 1 x Type 2A, 4 x Type 2B] gave prolonged CTs (>/= 200 s) for both C/ADP and C/Epi membranes; in contrast, all patients yielding normal CT values were found to yield normal VWF results (i.e., were found not to suffer from VWD). Patients with hemophilia (1 x hemophilia A, 1 x hemophilia C) gave normal PFA-100 CT, while those with clinical thrombocytopaenia (N = 3) gave prolonged PFA-100 CT. A number of other patient samples also gave abnormal CT values which in some cases could be linked to recent aspirin consumption. In the second evaluation process, and using normal blood, we have assessed the ability of various antibodies to influence the CT. Of the monoclonal antibody panel tested [N = 20], only a proportion of those against VWF [6/10] or gp1b/IX [CD42; 2/5] were found to be inhibitory (i.e., prolonged the CT). Data using polyclonal antibodies (against platelets, VWF, fibrinogen and fibronectin) is more complex but largely confirms the sensitivity of the system to VWF. On the basis of these results, we conclude that the PFA-100 is highly sensitive to disturbances in VWF and to the presence of VWD and may thus provide a valuable screening test for VWD in certain specific circumstances (i.e., acute need conditions or remote testing sites; normal CT result generally effective as negative predictor, i.e. not severe VWD). However, since abnormal CT values were obtained in clinical situations other than evident VWD, the PFA-100 cannot be used as a specific diagnostic tool to establish the presence of VWD. Thus, any abnormal PFA-100 CT result should be thoroughly evaluated by follow-up specific testing to establish the true clinical disorder affecting the individual under investigation, inclusive of appropriate VWF assays if VWD is clinically suspected.

Identification and characterization of a novel mutation in von Willebrand factor causing type 2B von Willebrand's disease.

Type 2B von Willebrand's disease (VWD) is a variant in which the structurally abnormal von Willebrand factor (VWF) has an increased affinity for the platelet glycoprotein Ib-IX-V complex. Spontaneous binding of type 2B VWF to platelets and their subsequent clearance from the plasma appear to account for the characteristic phenotype of type 2B VWD. Several type 2B mutations have been described and shown to be grouped along the amino edge of the beta sheet of the VWF A1 domain. In this report we describe a novel missense mutation, Arg543Leu, in the VWF A1 domain in three members of a family with type 2B VWD. We have expressed and characterized the corresponding recombinant mutant VWF in transiently transfected COS-7 cells. Relative to wild-type VWF, recombinant Arg543Leu VWF showed a similar multimer composition but exhibited binding to fixed platelets both in the presence and absence of ristocetin, confirming the ability of this mutation to permit spontaneous interaction of VWF with platelets. These studies are consistent with a recently proposed model in which the VWF-A1 domain exists in either 'on' or 'off' states, with type 2B mutations switching VWF to an 'on' state to facilitate GPIb binding.

The molecular mechanism of platelet adhesion.

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which vWF or the GP Ib-V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of vWF by the GP Ib-V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.

Laboratory assessment of von Willebrand factor. Use of different assays can influence the diagnosis of von Willebrand's disease, dependent on differing sensitivity to sample preparation and differential recognition of high molecular weight VWF forms.

Three separate laboratory assays for von Willebrand Factor (VWF), a standard "antigen" (antisera-ELISA-based) assay (VWF:Ag), a standard ristocetin-dependent-platelet-agglutination procedure (VWF:RCof), and an ELISA-based collagen-VWF binding assay (VWF:CBA), have been evaluated for their ability to detect alterations in VWF levels following differential processing of blood for testing, and specifically in (1) serum compared to plasma and (2) filtered plasma compared to nonfiltered plasma. Although all assays tended to detect some change, sensitivity of detection varied between assays, with the VWF:CBA most consistently able to detect large decreases in VWF levels in serum and filtered plasma. The authors propose that the increased sensitivity of the VWF:CBA assay to VWF depleted in these circumstances is that this assay selectively detects higher molecular weight forms (ie, those known to be more functionally relevant), and that assay results reflect the preferential incorporation of these forms in in the platelet-fibrin-gel during the clotting process, and onto the filter matrix during filtration. To confirm this, multimer analysis was performed and showed a reduction in high molecular weight forms of VWF in these cases. Finally, direct evidence that the VWF:CBA assay preferentially detects high molecular weight forms of VWF was obtained following fractionation of normal plasma VWF (separation according to molecular weight using size exclusion matrix; confirmed by specific multimer analysis) and assessment of eluted VWF. Using a standard VWF:Ag assay, detection of eluted VWF was unrelated to molecular size. In contrast, the VWF:CBA showed selective detection, and was able to preferentially discriminate high and intermediate forms of VWF from low molecular weight forms. The findings are of particular relevance to diagnostic pathology laboratories because filtered plasma or serum can be inappropriately (and unknowingly) provided for the clinically queried diagnosis of von Willebrand's disease (VWD). As outlined in this report, these samples can yield VWF results that closely mimic those of a Type 2A or Type 2B VWD individual, and thus, VWD may be incorrectly diagnosed.

CD13 (GP150; aminopeptidase-N): predominant functional activity in blood is localized to plasma and is not cell-surface associated.

This study is the first to report the presence of CD13/glycoprotein 150 (GP150)/aminopeptidase-N activity in cell-free plasma. We have determined that aminopeptidase-N in plasma provides, quantitatively, aminopeptidase-N's predominant functional activity within flowing blood. Thus, while aminopeptidase-N activity observed in whole blood can be partly, but significantly, blocked by the CD13 monoclonal antibody (MAB) WM15, the magnitude of such inhibition is low (< 25%) and similar to that observed using washed cell fractions selectively enriched for neutrophils (30.6% inhibition) or monocytes (21.8% inhibition). Plasma, free of cell components, possesses substantial aminopeptidase-N activity that is largely inhibitable (> 70%) by WM15. Blood collected into heparin or citrate yields similar data, while blood collected into EDTA gives rise to reduced CD13/aminopeptidase-N activity, consistent with inhibition of the known heavy-metal ion association necessary for proper functioning of this molecule. Although monocyte- and granulocyte-enriched cell fractions possess aminopeptidase-N activity significantly inhibitable by CD13 antibodies, lymphocyte-enriched cell fractions also possess aminopeptidase-N-like activity; however, in the latter case, this activity is not inhibitable by CD13 antibodies. Immunoaffinity isolation of plasma aminopeptidase-N has also been carried out; further characterization using functional studies and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis indicates that CD13 MABs can completely clear plasma of aminopeptidase-N activity and that the purified protein has similar electrophoretic characteristics to cell-derived material. These data, therefore, provide evidence for the presence within blood both of a soluble (that is, non-cell-associated) form of CD13/GP150/aminopeptidase-N localizable to plasma and of cell-associated, aminopeptidase-N-like proteins other than CD13/GP150. These findings have significant implications for our understanding of the many functions of this molecule in blood.