Development of monoclonal antibodies that inhibit platelet adhesion or aggregation as potential anti-thrombotic drugs.

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.

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.

New approaches for antithrombotic antiplatelet therapies.

Cardiovascular diseases are one of the major causes of mortality in the western world. As platelet dependent thrombosis is of central importance in their pathophysiology, several successful strategies, targeting a specific platelet function or interaction, have been developed to prevent or treat these disorders. However, as the current antiplatelet strategies are limited in efficacy and safety, and often influence normal haemostatic functions, new compounds are being developed with improved characteristics. This review deals with the development of novel antiplatelet compounds for which evidence is available on their antithrombotic action in vivo. In a first part, these compounds, their targets and their potential applicability are discussed. The second part of this review focuses on BT tests and bleeding models and their usefulness for determination and/or prediction of the safety of novel antiplatelet compounds.

A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations.

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.

Inhibition of platelet adhesion to collagen as a new target for antithrombotic drugs.

Platelet adhesion to a damaged blood vessel is the initial trigger for arterial hemostasis and thrombosis. Platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib/V/IX (GPIb), an interaction especially important under high shear conditions[1]. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (alpha(2)beta(1), GPVI, ) in addition[2] resulting in platelet activation. This leads to the conformational activation of the platelet alpha(IIb)beta3 receptor, fibrinogen binding and finally to platelet aggregation. Over the past decades, modulation of platelet function has been a strategy for the control of cardiovascular disease. Lately, drugs have been developed that target the fibrinogen receptor alphaIIbbeta3 or the ADP receptor and many of these promising compounds have been tested in clinical trials. However the development of products that interfere with the first step of hemostasis, i.e. the platelet adhesion, has lagged behind. In this review we want to discuss (i) the in vivo studies that were performed with compounds that target proteins involved in different adhesion steps i.e. the VWF-GPIb-axis, the collagen-VWF axis and the collagen-collagen receptor axis and (ii) the possible advantages these putative new drugs could have over the current antiplatelet agents.

Selection of phages that inhibit vWF interaction with collagen under both static and flow conditions.

Phages from a pentadecamer phage display library were selected for binding to vWF by affinity panning. Bound phages were selectively eluted with human collagen type I. After the third round of panning 95% of individual phage clones bound to vWF. The B8-phage inhibited the binding of collagen to vWF with an IC50 of 0.6 x 10(10) phages/ml, and of vWF to collagen with an IC50 of 1.0 x 10(10) phages/ml at 0.5 microg/ml vWF. Under flow conditions, 1.5 x 10(11) B8-phage/ml nearly completely inhibited platelet deposition on a human collagen type I coated surface at a shear rate of 1200 s(-1), while phages without an insert had no effect. The peptide corresponding to the one displayed on the B8-phage competed with the phage for binding to vWF with an IC50 of 30 microg/ml (16 microM). The peptide furthermore inhibited vWF-binding to collagen with a maximum of 40% at a concentration of 1.25 mg/ml (650 microM), higher concentrations of peptide could not improve this. We thus have selected phages that are potent vWF-binders and that can be used as tools to detect vWF, to inhibit vWF-collagen interaction and to further analyse the role of vWF-collagen binding.

Combination chemotherapy with vindesine-ifosfamide-cisplatin (VIP) in locally advanced unresectable stage III and in stage IV non-small cell lung cancer: a phase II trial.

UNLABELLED: The efficacy and toxicity of a regimen adding ifosfamide to the more classical cisplatin-vindesine combination was studied in patients with advanced non-small cell lung cancer. Sixty-four good performance patients with inoperable stage III or stage IV were treated with VIP: vindesine 3 mg/m2 days 1 and 8, ifosfamide 1200 mg/m2 and platinum 30 mg/m2 days 1, 2 and 3, repeated every 4 weeks, up to a maximum of six cycles. Response rate, clinical data and radiological tests were rigourously reviewed by a panel. Overall response rate was 39% (95% confidence interval, 27%-51%) with three patients achieving a complete response; response rate in stage III was 48%. Median survival was 9 months. Toxicity consisted mainly of bone marrow toxicity and nausea/vomiting, but was manageable. There was no renal toxicity greater than grade 2, four severe infections, but no treatment-related deaths. CONCLUSION: VIP as mentioned above is very active in good performance patients with advanced non-small cell lung cancer. Its activity, together with its manageable toxicity--without severe renal or pulmonary toxicity--makes it an attractive candidate for induction chemotherapy.