Until recently, its function was completely undefined and it was known merely as a platelet glycoprotein with a molecular mass of approximately 62 kDa under reducing condition. The average from 10 non-overlapping images was used to determine percentage surface area coverage.
Once a clot has developed, continued flow of blood past the clot is likely to break it free from its attachment.
Unadhered platelets were washed with phosphate buffered saline and the adhered platelets were fixed with glutaraldehyde 0. A partial amino acid sequence of the individual chains may be determined and degenerate oligonucleotides may be generated for each chain according to the method of Lathe et al.
In one embodiment, the GPVI-specific antibody is humanized with human J1 and K constant regions using the expression vectors described by Sun etal. Conversely, when the antigen- specific domains are rendered monovalent by enzymatic removal of linking Fc domain, the resulting Fab fragments completely abolished collagen-induced aggregation of normal platelets and inhibited platelet-collagen adhesion.
Platelet rich plasma PRP was obtained by centrifugation of whole blood at xg for minutes at room temperature. GPVI-deficient animals may be generated by a number of methods known in the art.
Human and humanized antibodies are thus preferred for clinical use.
As a consequence, a clot forms at the site of trauma, plugging any breach in the vessel wall and providing a substrate for fibroblast invasion and repair.
Anti-GPVI antibodies may also be generated using the phage display technology as described in Ventor et al. More recently, in vivo technologies have been developed that utilize animals either engrafted with human immune cells or those that have been introduced with the entire human immunoglobulin loci.
Okuma has graciously made this rare serum available to the scientific community. It is understood that many techniques could be used to generate antibodies against GPVI polypeptides and peptides and that this embodiment in no way limits the scope of the invention.
In an embodiment of the invention, the host animals are Armenian hamsters.
Comparisons of the amino acid sequences of the variable regions of IgG show that most of the variability resides in three regions called the complementarity determining regions CDRs. Studies with GPVI-deficient human platelets or platelets blocked with anti-GPVI Fab fragments obtained from a patient serum clearly demonstrated a lack of platelet interaction with immobilized collagen under high and low shear rates and reduced firm adhesion to immobilized vWf under high shear.
Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A or G Sepharose column Pharmacia. In addition, the invention comprises biosynthetic GPVI antibody binding sites, as described by Huston etal.
The divalent nature of convulxin is critical to the aggregatory effect.Archived Content is only available to SHOOT members. CLICK HERE TO PURCHASE MEMBERSHIPMemberships can be purchased for as little as $ per month and can be cancelled at any time.
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