الفهرس 
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Abstract
Atomic force microscopy (AFM), along with other complementary biophysical techniques, has been used to study several important aspects of the blood clotting process. High resolution imaging modes were used to observe single monomers of fibrinogen, through to oligomers and initial stage proto-fibrils at sub-molecular resolution in order to understand the molecular basis of fibrin polymerisation. Time resolved images of the developing clot were also taken at lower resolution appropriate to the scale of the features. The structures observed were correlated with turbidity measurements to plot the course of the reaction. In addition to topographic measurements, the AFM can also provide mechanical information using force spectroscopy. Force curves may be mapped across a surface to produce an image of compliance, a “force volume”. Most studies in the field of fibrin clot elasticity have focused on measuring the final clot using a torsion pendulum, or a single fibre within a clot using optical tweezers. Part of the aim of this project is to develop a new technique using the AFM to quantitatively measure the elasticity of individual fibres in the early stages of polymerisation, with a well defined geometry, correlating with the polymerisation mechanism revealed by high resolution imaging, and then relate the initial changes in molecular structure to the final structure and function of the clot. However, a major problem with this project is eliminating experimental uncertainties, and in developing the appropriate model for extracting data from the force volumes. Various models were tested and their results compared. A nanospring model from suspended fibre force measurements was found to be the most appropriate model, agreeing well with (limited) previous data. The results also suggest the Hertzian model is inappropriate for detecting the elastic modulus of biological samples. Unexpected stain-hardening behaviour was observed when stretching suspended single fibres, and this could have important implications in the understanding of ultimate clot elasticity, explained as a deformable network of semi-flexible polymer rods Fibrinogen and a variant, DesB1-42, lacking in a particular binding site was compared. The trinodular structure of the fibrinogen molecule was observed at high resolution images. Differences in nodule size were investigated by measuring the thickness of all the domains using the AFM. We found that the E region was smaller in Des Bβ(1-42) than normal fibrinogen (1.2nm ± 0.3 vs. 1.5nm ± 0.2), whereas there were no significant differences between the D-regions (1.9nm ± 0.4 compared with 1.7nm ± 0.3 for Des Bβ(1-42) compared with normal fibrinogen). This is attributed to deletion of a sequence on the E-domain, combined with decoupling of the c domains. Imaging with the AFM showed that the rate of polymerisation of normal fibrinogen is higher than that of Des Bβ(1-42), and the mechanism of polymerisation was also different, where the monomers of DesBβ interacted mostly via the D regions only (on top of each other) but for normal fibrinogen the monomers formed half staggered overlapping protofibrils binding together via the E and D regions (as expected). The elasticity of the final clot was measured by the magnetic tweezers where the average G´ of Des Bβ (1-42) was found to be ~1 Pa compared with ~8Pa for normal fibrinogen, whereas the AFM force spectroscopy measurement showed that the absolute value of the elastic modulus for DesBβ (1-42) is 0.31±0.06 MPa compared to 1.78±1.05 MPa for the normal fibrinogen. These results show that there is an important role for the B(1-42) chain in polymerisation. Blood coagulation factor XIII is important in stabilizing the fibrin clot, resulting in a mechanically strong and fibrinolysis-resistant clot. Its effects on fibrin clot structure are however unclear. Previous studies have used contaminating quantities of FXIII and have shown no or little effect of FXIII on fibrin clot structure. The aim of this study was to assess whether there is an effect of FXIII on fibrin clot structure using physiological quantities of FXIII. Turbidity studies showed that adding FXIII resulted in dose dependent lower lateral aggregation rates and final turbidity compared to controls. Scanning electron microscopy (SEM) supported these findings by demonstrating that clots formed in the presence of FXIII had thin and tightly packed fibres. Atomic force microscopy was used to image the early stages of polymerisation in physiological conditions, the images showed that this polymerisation occurred within the first 3 min of fibrin clot formation and that the presence of FXIIIa leads to the formation of short oligomers. The elasticity of the final clot was measured by the Plazek torsion pendulum; it was found that after addition of FXIII, clots were 3x more stiff than without addition of FXIII. This compares with previous studies showing an increase of between 3-5xfold in stiffness in FXIII containing clots. These results show that presence of FXIII produces shorter protofibrils in early stages of polymerisation so the FXIII alters the blood clot structure via an effect on cross linking in the earliest stages of fibrin clot formation, and not only at later lateral aggregation stage