Differential Effects of Extracellular Matrix Proteins on Proplatelet Formation: A Novel Role for Fibrinogen.
Authors: Larson Mark K. , Watson Steve P. 
Published: April 2020

Abstract
Megakaryocytes are large, polyploid cells which serve as platelet progenitors and are localized primarily in the bone marrow. Mature megakaryocytes are capable of forming microtubule-rich filamentous structures called proplatelets that are thought to serve as conduits for platelet packaging and release at the vascular sinusoids within the bone marrow. Previous research has determined that soluble factors within the bone marrow are critical for parasinusoidal megakaryocyte localization and normal platelet counts (Avecilla et al., Nat. Med.10:64, 2004). Since the bone marrow microenvironment is also rich in extracellular matrix proteins, we hypothesized that matrix/megakaryocyte interactions may also play a significant role in regulating proplatelet formation and subsequent platelet release. We discovered that uncultured, mature murine megakaryocytes from bone marrow aspirates readily form proplatelets when plated on fibrinogen, with an average of 19% of the megakaryocytes displaying proplatelets. Megakaryocytes on laminin also form proplatelets, with an average of 9% of the megakaryocytes displaying proplatelets. Interestingly, megakaryocytes were able to adhere to fibronectin, Horm collagen, and vWF with botracetin, but were unable to induce proplatelets, suggesting that only specific matrices such as fibrinogen and laminin are capable of regulating proplatelet formation. Further, neither αIIb −/− megakaryocytes nor megakaryocytes treated with the αIIbβ3 antagonist lotrafiban were capable of forming proplatelets on fibrinogen, due in part to a dramatic reduction in megakaryocyte adhesion. Additionally, αIIb −/− mice were thrombocytopenic, suggesting that the loss of the αIIbβ3/fibrinogen interaction may have a direct effect on platelet number. However, lotrafiban-treated megakaryocytes were able to form proplatelets on laminin, suggesting that αIIbβ3 is not the only cell surface receptor that can induce proplatelet formation. Given the importance of the αIIbβ3/fibrinogen interaction in proplatelet formation, we then determined whether there was a contribution from downstream αIIbβ3 integrin signaling events. Inhibition of Ca++ release with dimethyl BAPTA-AM did not affect proplatelet formation, a finding complemented by the normal level of proplatelets seen in PLCγ2 −/− megakaryocytes. Conversely, inhibiting Src family kinases with the soluble antagonist PP2 doubled the percentage of megakaryocytes with proplatelets, consistent with recently published findings demonstrating that Src inhibitors can induce megakaryocyte polyploidization (Lannutti et al., Blood105:3875, 2005). Since platelet release is thought to arise at vascular sinusoids, we then investigated whether fibrinogen is localized at these sites within the bone marrow. Confocal microscopy of stained frozen femur sections indicated a partial localization of fibrinogen with CD105, a marker for vascular endothelium. Along with a previous report of laminin localization at sinusoid endothelium (Nilsson et al., J. Histochem. Cytochem.46:371, 1998), this suggests that two adhesion molecules that promote proplatelet formation are localized at sites of platelet release. These findings suggest that specific matrix interactions, particularly between fibrinogen and αIIbβ3, are a critical component of inducing proplatelet formation, and potentially, platelet release.
We are grateful to the British Heart Foundation for their support.