Production and Characterization of Novel Neutralizing mAbs Against vWF-A3 Domain, an Inhibitor of Binding of vWF to Collagen and Influence of Interaction between vWF and Platelet GPIbα.

The interaction of collagen-vWF-GPIb is the first instance of the adherence, aggregation and activation of platelets and also is a new target of antithrombotic agent. At sufficiently elevated shear forces, platelets bind to collagen through a multistep process, involving both vWF-independent and soluble large vWF-dependent interactions. The vWF-A3 domain mediates vWF binding to collagen, after which vWF-platelet interactions occure via the vWF-A1 domain (platelet GPIb/IX/V receptor recognization ). Therefore, production of novel neutralizaing anti-vWF-A3 mAbs blocking binding of vWF to collagen is new antithrombotic target of preventing arterial thrombosis.

AIM: To produce novel neutralizing anti-vWF-A3 mAbs and study its effect on binding of vWF to collagen.

METHODS: Balb/c mices were immunized by purified recombinant vWF-A3( rvWF A3). After fusion, screening with rvWF-A3 and cloning. Hybridomas for inhibition of vWF binding to collagen were selected with ELISA. The McAbs were purified from murine ascites on a protein G-Sepharose 4B column. The recognition of mAbs with rvWF-A3 and reduced human vWF was studied by Western-blot. Inhibition of binding of purified human vWF (3μg/ml for calf skin collagen III-coated plates; 1.5ug/ml for human placenta collagen III-coated plates) or diluted normal plasma ( human and Rhesus monkey plasma :1/20 on human collagen III and 1/10 on calf skin collagen III; Beagle dog plasma : 1/10 on human collagen III and 1/5 on calf skin collagen III) to collagen by anti-vWF-A3 McAbs was studied with collagen binding test . Platelets aggregation was tested with the inducers of ristocetin (1.25mg/ml), collagen (2μg/ml), ADP(2μM).

RESULTS: A group of 30 anti-vWF-A3 mAbs was obtained, from which 2 clones were selected and identified as inhibitory. They were designated 1B9 and 3D2. Both of them are IgG1. It was shown that 1B9 and 3D2 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that 1B9 and 3D2 can recognize a band at the 27 KDa of rvWF-A3 and 2 bands at 250 KDa and 170KDa of reduced human vWF. 1B9 and 3D2 dose dependently inhibited the binding of purified human vWF to human type III collagen (IC₅₀= 0.055 ± 0.008 and 0.15 ± 0.004μg/ml, respectively) and to calf skin collagen III (IC₅₀= 0.48 ± 0.06 and 0.51 ± 0.07 μug/ml, respectively). Furthermore, 1B9 and 3D2 showed similar inhibitory potency against the binding of human, Rhesus monkey, and Beagle dog plasma vWF to these collagens. Platelets aggregation tests showed that 1B9 and 3D2 dose dependently inhibited platelets aggregation induced by ritocetin (IC₅₀=4.54 ± 0.62 and 8.21 ± 1.02 μg/ml, respectively), but they failed to inhibit platelets aggregation induced with collagen or ADP.

COUNCLUSION: These data demonstrate that 1B9 and 3D2, as novel neutralizing mAbs against vWF-A3 domain, are potent inhibitors of vWF-dependent platelets adhesion to collagen and at the same time, they influence the interaction of binding of vWF to platelets. Therefore, They may have therapeutic potential as antithrombotic agent.