ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences.

BACKGROUND: The recently discovered plasma enzyme ADAMTS-13 cleaves the A2-domain of von Willebrand factor (VWF). A defective cleaving protease results in unusually large VWF multimers, which cause thrombotic thrombocytopenic purpura (TTP). AIM: Analysis of the ADAMTS-13 antigen levels in TTP patients compared with normal donors. METHODS: An antigen ELISA test was built, based on high affinity anti-ADAMTS-13 monoclonal antibodies, which were generated using genetic immunization. RESULTS: Specificity of the ADAMTS-13 antigen test was confirmed, as (i) plasma from a patient with acquired TTP but presenting without inhibitor did not contain antigen and (ii) the binding of recombinant ADAMTS-13 was inhibited by increasing amounts of normal plasma. The assay is sensitive as it can detect antigen levels as low as 1.6% of normal. The concentration in normal pooled human plasma was determined (1.03 +/- 0.15 microg mL(-1)) and arbitrarily set to 1 U mL(-1). The antigen levels in congenital TTP samples (34 +/- 21 mU mL(-1), n = 2), as well as in samples from patients with acquired TTP (231 +/- 287 mU mL(-1), n = 11), were clearly reduced when compared with normal Caucasian donors (951 +/- 206 mU mL(-1), n = 16). Remarkably, normal Chinese donors have a significantly lower antigen titer (601 +/- 129 mU mL(-1), n = 15), when compared with normal Caucasians. CONCLUSIONS: Our results show that acquired TTP patients suffer mainly from ADAMTS-13 antigen depletion, thereby indicating the importance of ADAMTS-13 antigen determination in diagnosis and patient follow-up.

The von Willebrand factor self-association is modulated by a multiple domain interaction.

BACKGROUND: Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. OBJECTIVES: As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. RESULTS: Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. CONCLUSION: We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.

Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha.

The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibalpha (GPIbalpha) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbalpha were identified through the characterization of 5 new inhibitory anti-GPIb monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb mAbs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 area by the use of canine-human chimeras. These antibodies, however, had little or no effect (approximately 40% inhibition) on the binding of vWF induced by either botrocetin or ristocetin. On the other hand, the anti-GPIb mAbs 24G10 and 6B4, which blocked GPIb-vWF binding under all conditions examined, bound to 2 different regions of GPIbalpha, aa 1-81 and aa 201-268, respectively. The epitope for 6B4 was further narrowed by phage display revealing 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIbalpha, the gain-of-function platelet-type von Willebrand disease (PT-vWD) mutations have been identified. Alignment was partially confirmed because the binding of 6B4 to recombinant GPIbalpha fragments carrying either one of the PT-vWD mutations was considerably impaired but not completely abolished. In contrast, mAb 24G10 bound more strongly to mutant PT-vWD GPIbalpha. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbalpha. In conclusion, 2 functionally important areas within GPIbalpha were identified: one localized within the leucine-rich repeat N-terminal aa 1-59 area and one composed of residues aa 1-81 in close contact with aa 201-268. Moreover, further support is provided for the existence of an intramolecular interaction between the N-terminal flanking (aa 1-81) and C-terminal flanking (aa 201-268) regions. (Blood. 2001;98:652-660)

Fc-receptor dependent platelet aggregation induced by monoclonal antibodies against platelet glycoprotein Ib or von Willebrand factor.

In this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ibalpha to the platelet Fc-receptor (FcgammaRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbalpha, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcgammaRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbalpha to the FcgammaII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs IC1E7 and B724, both known to enhance vWF binding to GPIbalpha in the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIb MoAb, indicating that again the platelet activation results from signaling through FcgammaRII crosslinked to vWF bound to GPIbalpha. In addition, both the anti-GPIb MoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIb and anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIb and FcgammaRII, suggesting a role for the FcgammaII-receptor in GPIb-related signaling.

Occurrence of the Asn45Ser mutation in the GPIX gene in a Belgian patient with Bernard Soulier syndrome.

Bernard Soulier Syndrome (BSS) is a rare inherited bleeding disorder caused by a defect in the glycoprotein (GP)Ib/IX/V complex. A patient with a bleeding problem was diagnosed as having BSS based on the prolonged bleeding time, the absence of ristocetin induced platelet aggregations, thrombocytopenia and the presence of giant platelets. Analysis of the platelets of the propositus, a 39-year-old Belgian female, by flow cytometry revealed a decreased expression of the GPIb/IX polypeptides. Western blotting confirmed these results and showed moreover that there was a decreased disulfide bridge formation between GPIb alpha and GPIb beta. After sequence analysis of the GPIb alpha, GPIb beta and GPIX genes, only a mutation in the GPIX gene at position 1826 (A-->G) was identified, changing Asn45-->Ser. Restriction analysis with Fnu4H1 demonstrated that the patient was homozygous for this mutation. As this Asn45-->Ser mutation in the GPIX gene was already found in four unrelated families, i.e. in a British, Austrian, Swedish and Finnish one, the occurrence of this mutation in a Belgian patient supports the hypothesis of Koskela et al. (1999) that the Asn45Ser mutation in GPIX appears to be an ancient mutation shared by northern and central European populations. Our present observation of a decreased disulfide bridge formation between GPIb alpha and GPIb beta shows that GPIX is not only needed for the correct assembly of the complex but might also be needed for the disulfide bridge formation between GPIb alpha and GPIb beta.

Structural determinants within platelet glycoprotein Ibalpha involved in its binding to von Willebrand factor.

Platelet adhesion to subendothelial structures upon injury to a vessel wall is one of the first steps in a sequence of reactions critical for the formation of a haemostatic plug, or in diseased vessels for the development of an arterial thrombus. This adhesion process is mediated by an interaction between the glycoprotein (GP) Ib-V-IX complex on the platelet surface with von Willebrand Factor (vWF), associated with collagen on the subendothelial surface. After this initial adhesion, platelets will activate, resulting in recruitment of additional platelets and adherence to each other to form the platelet plug or developing thrombus. Several studies to date have attempted to identify the regions of the GPIb-V-IX complex that are critical for binding to vWF. The vWF binding site is contained in the 45 kDa N-terminal domain of the GPIbalpha chain. This N-terminal domain is characterized by a structural motif consisting of 7 leucine-rich repeats (LRRs), followed by a double disulphide-bonded loop and an anionic sulphated region. This review summarizes recent research efforts elucidating the characteristics of the GPIb-vWF interaction. Potential mechanisms that regulate the GPIb-vWF function are discussed, and advances in identifying functional sequences within GPIba involved in the binding to vWF are reviewed.

Sequence alignment between vWF and peptides inhibiting the vWF-collagen interaction does not result in the identification of a collagen-binding site in vWF.

We previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-binding to collagen (1). The sequence of those peptides could be aligned with the sequence in vWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF. We mutated the 1129-1136 VWTLPDQC sequence in vWF to VATAPAAC. Expressing this mutant vWF (7.8-vWF) in a fur-BHK cell line resulted in well processed 7.8-vWF containing a normal distribution of molecular weight multimers. However, binding studies of this mutant vWF to rat tail, human and calf skin collagens type 1, to human collagen types III and VI, revealed no decrease in vWF-binding to any of these collagens. Thus, although the N- and Q-peptides did inhibit the vWF-collagen interaction, the resulting alignment with the vWF sequence did not identify a collagen binding site, pointing out that alignments (although with a high percentage of identity) do not always result in identification of binding epitopes. However, suprisingly removal of the A3-domain or changing the vWF sequence at position 1129-1136 resulted in an increase of vWF-binding to human collagen type V1 and to rat tail collagen type 1, implying that these changes result in a different conformation of vWF with an increased binding to these collagens as a consequence.

Characterization of murine anti-glycoprotein Ib monoclonal antibodies that differentiate between shear-induced and ristocetin/botrocetin-induced glycoprotein Ib-von Willebrand factor interaction.

Platelet adhesion to vascular subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein (GP) Ib-von Willebrand Factor (vWF) interaction. The aim of this study was to characterize the murine monoclonal antibodies (MoAbs) 27A10 and 28E6, both raised against purified GPIb. The MoAb 27A10 is a potent inhibitor of shear-induced platelet adhesion to collagen type I in a flow chamber at shear rates of 1,300 and 2,700 s(-1). 20 microg/ml of MoAb 27A10, furthermore, could completely block shear-induced aggregation in a modified Couette viscometer at shear rates of 1,000 and 4,000 s(-1). On the other hand, MoAb 27A10 had a negligible effect on botrocetin-induced GPIb-vWF binding and is only a poor inhibitor of the ristocetin-dependent interaction. In contrast, MoAb 28E6 did abolish both the ristocetin- and botrocetin-induced GPIb-vWF binding, whereas it did not block the shear-induced interaction. Thus, we identify here two anti-GPIb MoAbs 27A10 and 28E6 that either preferentially inhibit the shear-induced or the ristocetin/botrocetin-induced platelet-vWF interaction. With these tools it should be possible to more clearly define the mechanisms by which platelets bind to vWF in vivo.

Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates.

Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(ab')(2) fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm(2)), at a wall shear rate ranging from 700 to 1000 s(-1). Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 microgram/kg Fab fragments significantly reduced (111)In-labeled platelet deposition onto the collagen surface by approximately 43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 microgram/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.

A reliable and reproducible ELISA method to measure ristocetin cofactor activity of von Willebrand factor.

The ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIb alpha-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory anti-GPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand's disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter- and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 microl).

Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen.

A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.