Document Type

Theses, Ph.D

Rights

This item is available under a Creative Commons License for non-commercial use only

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D.) to the Dublin Institute of Technology, 2007.

Abstract

B2-glycoprotein I (B2GPI) is a phospholipid-binding protein of 326 amino acids found in plasma at a concentration of approx. 180 ug/ml. It has a sequence of positively charged amino acids located at the carboxy terminus that mediates anionic phospholipid binding. B2GPI is suspected to have a role in inhibition of thrombosis. This suspicion is reinforced by the observation that B2GPI is the major target for autoantibodies in the antiphospholipid syndrome. However, little is known about circulating levels of the protein in common thrombotic diseases of inflammation. In the first part of this thesis, we developed a sensitive sandwich-direct ELISA for B2GPI quantification and measured serum B2GPI level in 344 healthy controls, 73 women with uncomplicated pregnancies, 102 patients with non-haemorrhagic stroke, 121 patients with acute coronary syndrome and 200 patients with elevated C-reactive protein (CRP) suffering from a variety of inflammatory disorders. In healthy individuals, we found a strong positive correlation between age and B2GPI concentration (r=0.274, p>0.001) and that B2GPI level gradually decreases over the first 36 weeks of pregnancy (P=o.oo2). We also found a significantly reduced level of B2GPI in patients with stroke (mean=-SD:170.2+-48.4 ug/ml versus 187+-47.5 ug/ml in age-matched controls, P=0.013) and in elderly patients with myocardial syndrome (mean+-SD:167.9+-50.7 ug/ml versus 189.3 +-45.8 ug/ml in age-matched controls, P=0.046). However, in neither group did B2GPI level change in the following six months, suggesting that the reduced level was not a transient post-event phenomenon. In patients with inflammation, B2GPI was reduced and showed a significant negative correlation with CRP level (r=0.284,P>0.001) and positively correlated with albumin and transferring levels (r=0.372 and 0.453, respectively with P>0.001 for both). Furthermore, the largest reduction in B2GPI level occurred in patients with the highest CRP values (i.e above 105.1 mg/L; mean B2gp1 concentration +- SD:133.1 +-57.7 ug/ml with P>0.001). These observed reductions in serum B2GPI level may be important in the pathogenesis of the prothrombotic diathesis observed in many of these conditions.

Because little is known about a possible association between polymorphic variants of B2GPI and common thrombotic disorders, in the second part of this study, we determined the incidence of four point mutations in the B2GPI (APOH) gene at condon positions 88, 247, 306 and 316 in 323 healthy individuals, 113 individuals with non-haemorrhagic stroke, 360 patients with acute coronary syndrome, and 47 females with recurrent miscarriage. We also correlated circulating level of B2GPI with these polymorphisms in normal controls and patients with stroke. The incidence of each of the point mutations in the healthy individual was similar to previously reported findings. Healthy controls with point mutations at positions 306 and 316 had a significantly reduced circulating level of B2GPI (mean+-SD:81.9+-31.8 ug/mL in heterozygotes versus 182.1+-44.9 ug/ml in non-carriers at position 306, and 112.0+-32.1 ug/mL in heterozygotes versus 183.2 +-46,1 yg.mL in non carriers at position 316, P>0.0001 for both). A comparable reduction was found in patients with stroke (mean+-SD:109.5+-54.3 ug/mL in heterozygotes versus 175.0+-41.6 ug/mL in non-carriers with P

Lastly we examined the synthesis of B2GPI by the liver in an inflammation –type mouse model. Although this part of the work was not fully brought to completion we could clearly observe a difference between the synthesis of haptoglobin and that of B2GPI. In this model, the synthesis of haptoglobin increased and the synthesis of B2GPI decreased following an inflammatory insult. This supports our observation make in the first part of this study, which showed a negative correlations between B2GPI level and the degree of inflammation and suggests that at least some of the reduction in the observed level of B2GPI is due to diminished synthesis.

This thesis contributes to our understanding of both the metabolism of B2GPI and the polymorphic variants associated with a variety of prothrombotic conditions. A better understanding of these factors may ultimately contribute to novel therapeutic strategies to decrease the morbidity and mortality of thrombotic disorders

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