Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow

We have identified type VI collagen (Col VI) as a primary subendothelial ex tracellular matrix component responsible for von Willebrand factor (vWF)-de pendent platelet adhesion and aggregation under high tensile strength. Inta ct tetrameric Col VI was the form of the collagen found to be capable of pr omoting vWF-mediated platelet adhesion/aggregation under this shear conditi on, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digest ed Col VI to support any platelet interaction at high flow was because of t he failure of the A3(vWF) domain to bind to this form of collagen, suggesti ng a stringent requirement of a tridimensional conformation or of intactnes s of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with t he cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A 1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha 3(VI) chain. Soluble recom binant A7(VI) poly-peptide strongly perturbed the vWF-mediated platelet adh esion to Col VI under high shear rates, without affecting the binding of th e vWF platelet receptor glycoprotein Ib alpha to its cognate ligand A1(vWF) , The findings provide evidence for a concerted action of the A1(vWF) and A 3(vWF) domains in inducing platelet arrest on Col VI, This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha 3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen, The data further emphas ize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hem ostatic process triggered upon damage of the blood vessel wall.