Development of antibodies that interfere with the collagen-VWF-GPIb axis as new antithrombotics.

The epitope and the antithrombotic effect of 6B4, an antibody that inhibits GPIb, the receptor for von Willebrand Factor (VWF) on blood platelets, and of 82D6A3, an antibody against VWF that prevents the binding of VWF to collagen, were characterised. By using canine-human chimeras, alanine-scans, phage display, mutant analysis and modeling both the epitope of 6B4 in the N-terminal domain of GPIb, and of 82D6A3 in the VWF-A3 domain, could be mapped. As both epitopes furthermore are part of the ligand binding sites, this at once also explained the mechanism of the inhibition by the antibodies. Next both antibodies were tested in a thrombosis model in a stenosed artery in baboons, where they showed potent antithrombotic activities, without a noteworthy prolongation of the bleeding time. With this we thus could reveal two new strategies to prevent arterial thrombosis, which presumably may be safer than the currently available antiplatelet agents.

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.

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.

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).

Acquired Bernard-Soulier syndrome: a case with necrotizing vasculitis and thrombosis.

We describe a patient with positive antinuclear antibodies, polyclonal gammopathy and high level of circulating immunocomplexes, resulting in vascular purpura. In addition, the patient had a slightly prolonged bleeding time and an isolated defect of ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). The patient's plasma also inhibited RIPA in normal PRP and in normal platelet suspension. The activity and multimeric structure of plasmatic von Willebrand factor showed no alteration. We could demonstrate an autoantibody against platelet membrane glycoprotein (GP) Ib, using an ELISA-type assay. These data suggest an acquired Bernard-Soulier syndrome. We suggest that the patient had an immunocomplex-mediated leukocytoclastic vasculitis accompanied by production of antinuclear autoantibodies as well as the presence of an autoantibody against GPIb. The titer of the anti-GPIb antibody, however, was too low to induce significant platelet-type bleeding tendency, only laboratory alterations were found. Moreover, in a later stage of her disease, she developed a severe necrotizing vasculitis which was followed by a deep venous thrombosis.

Effect of route and frequency of administration of apomorphine on growth hormone release in African catfish (Clarias gariepinus).

Apomorphine is known to stimulate growth hormone release in African catfish following an intraperitoneal (IP) injection. In the present study the effect of apomorphine (5 or 20 mg/kg body weight) on plasma GH levels was evaluated after gastro-intestinal or parenteral delivery. Apomorphine increased the plasma GH concentration regardless of the route of administration, indicating that apomorphine can be absorbed from the intestinal tract. The effect of repeated administration of apomorphine differed clearly between the tested doses. Although a single IP injection with 20 mg apomorphine/kg body weight resulted in a clear increase in plasma GH levels, a second injection given 12 hours later was ineffective. In contrast the last of 4 consecutive injections with 5 mg apomorphine/kg body weight given at intervals of 12 hours stimulated the plasma GH levels in a similar way to a single IP injection with the same dose.