Vascular PG‐M/versican variants promote platelet adhesion at low shear rates and cooperate with collagens to induce aggregation

Abstract

We have identified a novel von Willebrand factor/fibrinogen/selectin-independent, platelet adhesion-promoting function of vascular PG-M/versicans that may be relevant in normal venous thrombosis and critical in atherosclerotic conditions. A purification scheme was devised to obtain vascular versicans, which by biochemical, immunochemical, and ultrastructural means were asserted to be 1) composed primarily of isoforms V1 and V2; 2) free of contaminants; 3) prevalently substituted with chondroitin-4-sulfate and dermatan sulfate (DS) chains; and 4) capable of binding hyaluronan to form link protein-stabilized ternary complexes. Real-time analysis of human platelet perfused under diverse shear forces showed that they largely failed to bind to several vascular and nonvascular proteoglycans (PGs). In contrast, they bound in a dose- and shear rate-dependent manner to vascular versicans, exhibiting a unique attachment-detachment kinetics and establishing a firm substrate tethering characterized with no significant aggregation. Digestion of these PGs with lyases and competition experiments with purified glycosaminoglycans revealed that platelet adhesion to vascular versicans was primarily mediated by their DS chains. Incorporation of the versicans into fibrillar collagen substrates augmented their adhesive activity and strongly promoted platelet aggregation at low and high shear rates. Affinity chromatography of platelet surfaces on DS columns identified a 120-140 kDa polypeptide complex that behaved as a specific vascular versican binding membrane ligand in solid-phase binding assays. These findings indicate that selective versican variants of the subendothelium may serve as ancillary GPIbalpha/integrin/selectin-independent platelet ligands in healthy and diseased vascular beds and may be directly responsible for the platelet accruing after rupture of atherosclerotic plaques.

Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc

“…Details of the antibodies used and epitope retrieval steps required for detection are provided in table 1 and suppliers are provided below. Monoclonal antibody (Mab) 5-D-5 to versican G3 domain [1,30,48] was a gift from Dr Firoz Rahemtulla, University of Alabama, Birmingham, AL, USA. Mab A7L6, rat anti perlecan domain IV [14,15] and MAb CS-56 to native CS [23] were purchased from Abcam, Cambridge, UK.…”

Distribution of PG-M/Versican Variants in Human Tissues andde Novo Expression of Isoform V3 upon Endothelial Cell Activation, Migration, and Neoangiogenesis in Vitro

“…Specificity of these mAbs has partially been described elsewhere (3,37) and further details about their reactivities were determined here by enzyme-linked immunosorbent assay, dot-blot assays, and immunoblotting on intact and enzyme-digested PGs, including versicans from various bovine and human sources, aggrecans from cartilage tissues of various animal species, fibromodulin, decorin, and biglycan. An antiserum produced against a recombinant fragment corresponding to the N-terminal, hyaluronan-binding globular domain of versican (named anti-Vc; Ref.…”

“…Solid-phase Binding Assays and TEM/Rotary Shadowing-Bovine aorta versicans were isolated as previously described (37,41). Enzymelinked immunosorbent assay with the various antibodies and enzyme digestions was perfomed as described in previous studies (3).…”

Ovarian cancer stem cells and inflammation

“…In addition, some TLRs, such as TLR-2, and -4 respond to endogenous "stress" proteins, such as heat shock protein (Hsp 60), hyaluronan and fibrinogen. 22,23 It should be noted that most of these endogenous ligands are released as part of cellular debris following cell death. 24 Most TLRs signal through a common pathway since they possess a common intracellular domain known as the Toll/IL-1R homology region (TIR).…”