A Murine Model of Bernard-Soulier Syndrome and the Development of Atherosclerosis.

Platelet glycoprotein (GP) Ib-IX is a key receptor in arterial platelet adhesion and thrombus formation. A growing body of evidence suggests the inherent adhesive nature of platelets contribute to pathological events such as inflammation and atherosclerosis. However, the molecular mechanisms by which platelets might participate in these processes remain unknown. In the current study, we used a mouse model of platelet receptor GPIb-IX deficiency (mBSS), an equivalent to the human Bernard-Soulier syndrome, to determine whether the development of atherosclerotic lesions is influenced by normal platelet adhesion. Our hypothesis is that mBSS platelets might have a protective role in the development of atherosclerosis given their abnormal function and lack of a critical platelet adhesion receptor. To test this hypothesis we first generated congenic mBSS mice by backcrossing for 10 generations with wild-type C57BL/6J mice. C57BL/6J-GPIb^−/− mice were then crossed to the well-characterized murine model of hypercholesterolemia, LDL-receptor deficient animals (C57BL/6J-LDLr^tm1Her). Following genotypic screening and second generation breeding we established a colony of double knockout animals (C57BL/6J-LDLr^{tm1 Her}-GPIb^−/−) lacking both the gene for the LDL receptor and platelet GPIbα. Experimental animals were divided into 3 groups:

1. double knockouts (C57BL/6J-LDLr^tm1Her-GpIb^−/−);

2. animals only missing the LDL receptor gene (C57BL/6J-LDLr^tm1Her); and

3. wild-type controls (C57BL/6J). At 5 weeks of age mice were weaned and placed on a Western style atherogenic diet composed of 0.2% cholesterol.

We have examined aortas following exposure to this high fat diet for 18 and 24 weeks. Six mice from each group were euthanized and the aortas were dissected. The hearts were perfused with 10 ml of phosphate-buffered saline and aortas were opened longitudinally from the heart to the iliac bifurcation. The specimens were stained with Sudan IV to identify the gross presence of fatty streaks in the intimal layer of aortas. Digital images of the stained aortas were captured. All double knockout animals and LDL receptor-deficient mice developed extensive atherosclerotic lesions throughout the aorta with especially pronounced lesions in the area of the aortic arch and arterial branching points. No lesions were observed in control C57BL/6J aortas. Sudan IV-positive lesions were counted visually and they ranged from 21–23 in double knockout aortas and 16–21 in LDL receptor deficient mice. The lesions were more pronounced at earlier time points in male animals. We also observed a correlation between the development of advanced aortic lesions and clinically sound manifestations, such as skin trophic changes and limb self-amputation in some animals. Overall, these findings do not support our initial hypothesis. In fact, we observed that double knockout mice appear to have exaggerated development of atherosclerotic lesions. This finding has led us to a biochemical analysis of mBSS platelets where we have observed phosphorylated resting platelet proteins not present in resting normal mouse platelets. Thus, we conclude in this model of atherosclerosis the BSS platelet may be functioning as a “pseudo-activated” form of a platelet and might be the molecular basis for the apparent support rather than inhibition of arterial atherogenesis.