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CD40L-dependent von Willebrand factor-platelet string formation in the mouse microcirculation in vivo

Tahir, Sibgha

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Both physiological and pathophysiological vascular remodeling processes involve the crosstalk between vascular and immune cells. CD40 ligation on endothelial cells (EC) in vitro promotes the release and deposition of ultra-large von Willebrand factor (ULVWF) multimers on their surface. Platelets rapidly adhere, turn P-selectin positive and recruit circulating leukocytes, allowing their attachment to and subsequent diapedesis through the EC monolayer. ULVWF multimers are cleaved by the zinc metalloproteinase ADAMTS13. However, it is not yet known whether CD40-CD40L interactions play a role in microvessels in vivo. In particular, it is not known whether the shear stress dependency observed in vitro leads to ULVWF multimer-platelet string formation predominantly in arterioles being exposed to higher shear stress than venules, and whether these strings are formed in the presence of ADAMTS13. We hypothesized that CD40L serves as potent stimulator of endothelial cell von Willebrand factor (vWF) release in the microcirculation in vivo which leads to formation of ULVWF multimers providing active binding sites for surveilling platelets and, secondarily, circulating leukocytes (see graphical summary Figure 1). To test this hypothesis, multiphoton-based live cell imaging in cremaster vessels of the mouse was used to study ULVWF multimer-platelet string formation and leukocyte-EC interaction in vivo. Carboxy fluorescein diacetate (CFDA) stained platelets were injected into the circulation of both wild type and ADAMTS13 knockout mice. Leukocytes were labeled using PE-conjugated anti-mouse CD45 antibody. Then ULVWF multimer-platelet string formation together with interaction of leukocytes was observed in the microvasculature of these animals before and after CD40L stimulation. The distribution of CD40 and vWF in the vasculature was observed by using Qdot-525 labeled anti-CD40 and Qdot-565 labeled anti-vWF antibodies, respectively. Herein, it is reported that CD40L is a potent stimulator of vWF-dependent platelet string formation in the murine microcirculation in vivo. ULVWF multimer-platelet strings form both in arterioles (mean diameter = 53 µm) and venules (mean diameter = 60 µm) after CD40L treatment in vivo but are clearly more prevalent in venules in spite of the lower shear stress there. However, in the presence of ADAMTS13 their number is kept rather low. The deficiency of ADAMTS13 enhances ULVWF multimer-platelet string formation following CD40L stimulation. Leukocyte extravasation is much more prominent in venules and boosted in the absence of ADAMTS13, namely following CD40L stimulation. Therefore, low ADAMTS13 plasma levels as observed in patients with coronary heart disease may contribute to the pro-thrombotic and pro-inflammatory state of the vessel wall. ULVWF multimers are preferentially observed in venules where they co-localize with both platelets and the endothelial cell CD40 receptor. Based on these observations, we propose that pro-inflammatory leukocyte extravasation preferentially takes place at sites of vWF-induced platelet adherence, a process which is elicited by platelet-CD40L stimulation of endothelial cells.

Item Type: Dissertation
Supervisor: Hecker, Prof. Dr. Markus
Place of Publication: Heidelberg, Germany
Date of thesis defense: 23 January 2018
Date Deposited: 29 Jan 2018 09:23
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Medizinische Fakultät Heidelberg > Institut fuer Physiologie und Pathophysiologie
Subjects: 570 Life sciences
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