Sequence analysis of human antiphospholipid antibodies (aPL) has shown that their binding properties are derived from the accumulation of replacement mutations and basic residues in their complementarity determining regions (CDRs). This thesis describes the effects of particular CDR motifs, often sites of somatic mutation and/or containing arginine residues, on the binding to cardiolipin (CL) of two human IgG monoclonal aPL, IS4 and CL24. Following sequence analysis of IS4 and CL24 a transient expression system was used to express whole IgG containing variants of IS4 and CL24 produced by chain shuffling experiments with three other monoclonal antibodies, UK4, B3 and 33H11. The light chain sequences (VL) of four of these five mAbs were encoded by the same germline VL gene and hence differed only in their pattern of somatic mutations. The heavy chain (Vh) of IS4 was dominant in conferring the ability to bind CL in a direct ELISA, whilst the identity of the paired Vl was important in determining the strength of CL binding. Computer-generated models revealed surface exposed arginine residues in the CDRs of IS4Vh and those VL sequences that were particularly favourable for binding to CL. Seven new Vl sequences and six new Vh sequences were produced by using CDR exchange and site-directed mutagenesis to alter the patterns of somatic mutation and arginine residues in the wild-type VL and Vh sequences of these antibodies. Alteration of specific arginine residues in B3VL CDR1 and IS4VH CDR3 led to a significant reduction in CL binding. It was concluded that it is not just the presence but precise locations of specific arginine residues in the CDRs of pathogenic aPL, which are important in determining binding affinity for CL. In order to produce larger quantities of these expressed Vh/Vl combinations, a stable expression system was developed. Three stable cell lines were produced, expressing IS4VH with three different Vl sequences. The IgG produced showed the same CL binding characteristics as those produced in the transient system.

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Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder of unknown aetiology, characterized by sustained thrombocytosis and megakaryocyte hyperplasia. Three strategies were used to investigate disease pathology. Firstly, PCR-based assays using fluorescently labelled primers to measure X-chromosome inactivation patterns were evaluated and clonality status shown to be stable in 14 patients with polyclonal and 8 patients with clonal myelopoiesis studied over a median of 54 months (range 10-102) and 52 months (range 5-97) respectively. Secondly, mutations and polymorphisms in cytokines implicated in thrombopoiesis were investigated in ET patients. No mutations in the 5' untranslated region of the thrombopoietin gene, previously described in hereditary thrombocythaemia, were found. Genotypes and gene frequencies for polymorphisms previously shown to affect circulating cytokine levels in the transforming growth factor pi and interleukin-6 genes did not differ between patients and controls. Thirdly, representational difference analysis was used to compare global gene expression in platelet mRNA from an ET patient with monoclonal myelopoiesis with that from a normal control. Three genes were identified as differentially expressed: RANTES, CD32 (FcylIRA), and FLP. CD32 expression was further investigated using a multiplex semi-quantitative RT-PCR assay. Platelet CD32 expression was increased in 52 ET patients compared to normal controls (median CD32:GAPDH ratio 21, range 2-73 and 1, range <l-3 respectively, p=0.005). However, expression levels were not significantly different between 15 polyclonal and 18 clonal patients (median ratios 23, range 2-73, and 25, range 8-60, respectively, p=0.281), nor between 11 polycythaemia vera patients and eight patients with reactive thrombocytosis (median ratios 30, range 7-63, and 10, range 2-25, respectively). Therefore, whilst this cohort of ET patients has raised levels of CD32 mRNA, this cannot currently be used as a diagnostic marker, or to identify patients at higher risk of thrombotic complications.

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We carried out the first genome-wide linkage analysis in British families for VWF levels. ABO and VWF loci were specifically examined. The results showed that VWF levels are highly heritable 42% ( P>0.000001) with age and C-reactive protein (CRP) the main covariates. The ABO locus was strongly associated with VWF levels ( P=2x10-18), but linkage was modest (LOD = 1.6). VWF gene marker showed no significant association or linkage with phenotype. Genome-wide linkage analysis, conditioned on age and ABO genotypes, revealed regions with potential linkage. Highlighted regions were on chromosomes 2 and 3 (LOD = 1.78 & 1.08 respectively) and two areas on chromosome 12 (LOD = 1.5 & 1.3). Inflammatory biomarkers concentrations were also investigated. Heritabilities of CRP and tumour necrosis factor-alpha (TNF-alpha) were significantly high 38% and 47% respectively, while interleukin-6 was not heritable. VWF levels showed significant genetic correlation with CRP. As ABO group has a major role in determining VWF levels, if the VWF stroke association is causal, blood groups should be associated with stroke (Mendelian Randomisation). ABO genotype frequencies in stroke patients were investigated by a case-control study (503 objectively diagnosed cases and 327 controls). Non-O groups were not significantly over-represented in stroke cases (OR 1.1, CI95 0.83--1.47). Monte Carlo method, taking into account ABO effect on VWF levels, showed no association between ABO genotypes and stroke risk. This was reinforced by the findings of the systematic review we conducted on ABO groups and stroke. In contrast to results obtained from venous TE systematic review, where almost a two fold increased risk was found with non-O groups vs. O group.

Paradoxical embolism of fat, thrombus and bone marrow during orthopaedic surgery can be associated with confusion, stroke and death. A patent foramen ovale (PFO) is the most common venousto arterial circulation shunt (v-aCS) permitting paradoxical embolism. Transcranial Doppier ultrasound (TCD) is capable of detecting emboli during surgery and quantifying a shunt but the reproducibility of this technique and the association between paradoxical embolism and organ dysfunction is unclear.