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The principal role of Von Willebrand Factor (VWF) is platelet capture at sites of vascular injury, via interaction of its A1 domain with platelet glycoprotein Ibα (GPIbα). The GPIbα binding site overlaps with a binding site for heparin, the functional significance, and physiological ligand, of which remains unknown. Previous studies have demonstrated heparin inhibition of VWF-dependent platelet binding, a secondary effect distinct from heparin's anticoagulant role. Using a physiologically representative flow assay, this secondary effect has been demonstrated, with unfractionated heparin (UFH) exerting the greatest inhibition. This antithrombotic action of heparin is a potential mechanism for unpredictable bleeding complications during cardiopulmonary bypass surgery (CPB) when high concentrations of UFH are needed to maintain the extracorporeal circuit. A prospective cohort study to determine the changes in VWF antigen activity and function, occurring pre- and post-UFH in CPB demonstrates a significant rise in the VWF antigen (VWF:Ag) level by the end of surgery, with a relative fall in VWF collagen binding. The VWF Ristocetin Cofactor (VWF:RCo) fell but not significantly following UFH, with recovery seen in the absolute VWF:RCo after UFH reversal with protamine sulphate (PS) but not in the VWF:Ag/VWF:RCo. Both heparin and PS sulphate affect many components of haemostasis and these off-target effects, alongside the consumptive and dilutional coagulopathy during CPB make prediction of bleeding difficult. Recombinant VWF heparin binding site variants demonstrate impaired platelet capture on a collagen surface, suggesting a greater degree of overlap between the two binding sites or that the heparin-binding site of VWF has a role in the GPIbα interaction, identifying a potential physiological ligand. Heparan sulphate (HS) within the endothelial glycocalyx has been suggested as the physiological ligand for the VWF heparin-binding site. Removal of HS from the glycocalyx of human umbilical vein endothelial cells is a promising model to investigate this further.

Von Willebrand Factor and Factor XII have well established roles in haemostasis and coagulation. While VWF is a mediator of platelet capture to sites of vascular injury under high shear stress, FXII initiates the intrinsic pathway of coagulation. Interestingly, multiple studies have shown that absence or inhibition of FXII does not impair haemostatic function but has a thrombo-protective effect, rendering it a potentially safe anticoagulant target. In this study, I investigated a novel binding interaction between VWF and FXII. Using substrate based activation assays, I showed that VWF is a potent and previously unrecognised activator of FXII. Additionally, I demonstrated that both inactive FXII and activated FXII bind to VWF but not to VWF lacking the A1 domain suggesting that the FXII binding site lies within the A1 domain. By carrying out further binding studies using static binding assays and surface plasmon resonance with isolated and full-length VWF A1 domain mutants, I identified amino acids E1452 and D1459 on the α-6 helix of the A1 domain as the FXII binding site in VWF. The physiological relevance of the VWF/FXII interaction was examined using plate-based thrombolysis as well as perfusion assays investigating the formation of platelet rich blood clots under conditions of flow. Using adapted thrombolysis assays, I showed that clot lysis times were significantly reduced when either VWF, FXIIa or activity of both proteins was blocked. Furthermore, I demonstrated that the interaction between VWF and FXIIa facilitates VWF mediated platelet capture under conditions of high shear and that interference with FXII/VWF binding has a prohibitive effect on overall thrombus formation. Based on these results, I hypothesise binding of VWF to FXII is an important component of thrombus formation and inhibition of this interaction might be a novel and safe way to prevent thrombosis without increasing the risk of bleeding side effects.

The primary aim of this research is to study in detail the soil pattern and erosion characteristics of a small representative catchment in the Wither Hills, and to determine the interrelationship between soil properties, both field and laboratory, on the initiation of tunnel erosion and its sequential development into open gullying. The basic steps of this approach to the problem of tunnel-gully erosion are briefly outlined as follows. 1. A survey of the soils of the representative catchment in order to delimit the problem. It was noticed from cursory examination of the Wither Hills that tunnel-gully erosion was not prevalent over every section of the landscape. The lower spurs and ridges seemed to be relatively free of tunnel gullies, although minor ones were apparent in isolated localities. The main ridges and higher slopes of the hills were definitely devoid of this erosion form although scars of former slips were evident. 2. A detailed study of a toposequence on those soils where the tunnel-gully problem was most acute. This involved intensive field and laboratory determinations in an effort to relate pedological, chemical and physical properties to the incidence and severity of erosion. In addition this detailed study was used to characterise these soils as typical of the dry-subhygrous, central yellow grey earth subgroup, on the basis of their morphology, chemistry, mineralogy and physical properties. 3. To fully describe the process of tunnel-gully formation as it occurs in the field. 4. To compare the tunnel-gullied soils of the Wither Hills with: (a) soils from other regions showing similar erosion forms, (b) with soils in similar environments but not liable to tunnel-gully erosion. It was thought that such a comparison using morphological properties and selected laboratory determinations would help explain the susceptibility of some soils to tunnel-gullying and also the actual process of tunnel formation. Considerable time was devoted to the soil survey to gain as much information as possible on the geomorphology and pedology of the representative catchment so as to relate slope development and soil formation which were seen to be interdependent. Hence, an attempt to understand the genesis of these soils is an essential part of the work as not all yellow grey earths have exactly the same pedogenic history. The ultimate aim of the whole study was to elucidate the tunnel-gully erosion pathway on the Wither Hills, and to provide such data that practical methods could eventually be devised to control existing tunnel-gullies.

Thrombosis is common in Behçet’s Syndrome (BS), and there is a clear need both for a better understanding of causation and for biomarkers to enable thrombotic risk assessment. I set out to determine whether plasma microparticles (MP), particularly tissue factor expressing (TF+ MP) were increased in BS. Additionally, I investigated whether MP expressing tissue factor pathway inhibitor (TFPI) were also evident. Finally, I investigated whether these MP had a functional consequence role. MP were prepared from 88 BS patients and 72 healthy controls. The BS group contained 21 patients with a history of thrombosis (Th+) and 67 without (Th-). MP were identified by size and annexin V binding using flow cytometry, and were further analysed with antibodies to surface antigens. Assays used to assess the function of MP were an in-house coagulation lysis assay, calibrated automated thrombin generation assay and Factor Xa generation assay. Total MP numbers were increased in BS compared to HC, as were MP expressing TF and TFPI (all p < 0.001). Amongst BS patients, the Th+ group had increased total and TF positive MP (both p < 0.001) compared to the Th- group, but a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI to TF MP counts (TFPI/TF) was significantly lower in Th+ versus Th- BS patients (p < 0.001), with no history of clinical thrombosis in BS patients with a TFPI/TF MP ratio > 0.7. Functional assays revealed no correlation with MP count. I conclude that MP expressing TF are increased in BS, more so in patients with thrombosis. An imbalance between microparticulate TF and TFPI may be pathophysiologically important for thrombosis in BS and may allow improved identification and appropriate treatment of thrombotic risk.