| Platelet activation by VWF appears to involve a complex set of pathways that require a variety of receptors including integrins, feedback loop(s), interplay between signal transduction pathways and a multitude of agonists. Delineation of both the signal transduction pathway for a single factor in such a complex environment is a major challenge. The availability of genetically modified mice offers a powerful tool to elucidate the signaling pathway for an agonist. However, a suitable strategy should first be developed to elicit VWF interaction with the murine GpIb complex. This proposal aims at developing a murine model to study VWF signaling. The strategy involves production of a recombinant murine VWF with a mutation that mimics one in human type 2B von Willebrand disease, the A1 domain of murine VWF may give it the property of spontaneous binding to the GpIb receptor on murine platelets.;Cloning the murine VWF cDNA revealed that the gene is highly conserved in relation to other species. Recombinant full-length wild-type and mutant VWF were expressed in COS-7 cells in a serum-free system to avoid contamination from VWF in the serum. A reliable method for purification of the recombinant proteins was established involving differential ammonium sulfate precipitation. Purified proteins were characterized by Western blot in conjunction with SDS-PAGE and SDS-agarose gel electrophoresis methods. Results obtained by these methods strongly indicate that the recombinant proteins are fully processed and that they undergo proper post-translational modifications. Functional studies were performed by monitoring the agglutination of washed murine platelets upon stimulation by the recombinant proteins. The marine V1316M mutant induced appreciable agglutination of marine platelets in the presence of 1 mg/ml ristocetin whereas the wild-type murine VWF did not cause any agglutination. The mutant induced agglutination in a concentration-dependent manner. Also, a modest agglutination (about 5% of the maximal) was observed with the mutant VWF at a low concentration of ristocetin (0.2 mg/ml), whereas the wild-type VWF did not induce agglutination even at a higher concentration of ristocetin (1 mg/ml). Pre-incubation of VWF with heparin inhibited the agglutination. These findings indicate that VWF is responsible for the agglutination and suggest that its primary receptor, GpIb, plays an essential role. Moreover, blocking the function of Gp IIb/IIIa with RGDS or EGTA did not effect the agglutination induced by the mutant VWF, confirming the primary role of GpIb. The agglutination caused by the mutant VWF did not induce serotonin release or thromboxane A 2 generation, suggesting that the interaction of VWF with GpIb might be too weak to provoke, activation of the platelets. There is no evidence of spontaneous activity with the mutant, but these data clearly illustrate that a point mutation in the A1 domain, mimicking human type 2B VWD, confers on murine VWF the ability to interact with its platelet receptor in presence of ristocetin. |
