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Abstract
Uncontrollable haemorrhage remains one of the leading causes of death in both civilian and military trauma patients. Due to a poor understanding of the mechanism of haemostasis, the applications of current haemostatic control agents are limited. Platelet adhesion to damaged blood vessel walls is accepted as the first step in the healing process of a vascular injury, which is promoted by circulating serum proteins, exposed extracellular matrix proteins and platelet activating compounds. Proteoglycans (PG) are one such group of molecules which are also found in platelet α-granules associated with various factors that modulate the coagulation cascade such as platelet factor 4 (PF4). The aims of this thesis were to characterise the PGs in platelets, and investigate the interactions between platelets and the PGs in vascular extracellular matrices (ECM).
Three types of PGs were identified in human platelets, serglycin, betaglycan and syndecan-1. Serglycin was found to be a heterogeneous population with molecular weights between 50 and 250 kDa, as well as a band at 10 kDa. Betaglycan was also found to be a heterogeneous population with molecular weights between 70 and 250 kDa, whereas syndecan-1 was detected as discrete bands of 30 and 66 kDa. The core of serglycin was demonstrated to be 25 kDa, and was decorated with chondroitin sulfates (CS) and dermatan sulfates (DS).
Serglycin was found in complex with PF4 through its CS/DS chains. The serglycin bound PF4, however, could be easily removed from the CS/DS chains by the HS chains on endothelial perlecan. It is speculated that the difference in the affinities for the GAGs establishes gradients of PF4, which control its biological activities and affect processes where PF4 has well established roles such as in wound healing.
Perlecan was found to be anti-adhesive for platelets; however after removal of the HS chains, the platelets adhered and aggregated to the protein core via interactions involving the integrin α2β1 and αIIbβ3. Antibodies specific for either domain I or IV had no effect whereas antibodies against domain III or V significantly reduced platelet adhesion. Both integrin α2β1 and αIIbβ3 were shown to mediate platelet adhesion to recombinant domain V of perlecan. It is suspected that these two integrins also mediate platelet adhesion to domain III of perlecan.