Synthesis and Secretion of von Willebrand Factor and Fibronectin in Megakaryocytes at Different Phases of Maturation

Abstract

Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.

The important role of von Willebrand factor in platelet‐derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies

“…Previous studies done by Schick et al . have demonstrated that VWF is primarily synthesized in mature pig megakaryocytes, 7.5 times more than in immature megakaryocytes; 14.5% and 4.6% of total synthesized VWF was secreted into culture media by mature and immature pig megakaryocytes, respectively. In their tissue culture system, approximately 13% of synthesized VWF was secreted into culture media from megakaryocytes (both mature and immature).…”

Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

“…Furthermore, examination of the role of Pyk2 as an integration point for integrin-mediated signaling in OBs revealed that the MK-mediated increase in Pyk2 levels in OBs was unaltered by treatment of cultures with integrin-blocker RGDS at doses we have previously shown to be efficacious for reducing MK-mediated OB proliferation (Figure 3). 17 This finding was unexpected since Pyk2 is known to be activated by phosphorylation downstream of integrin engagement in other cell types, including OCs, 34 MKs express and secrete fibronectin, 41–43 a β 1 -binding integrin ligand, and titration of RGDS into MK-OB cultures resulted in a significant reduction in OB proliferation. 17 It should be noted that our findings do not, however, exclude the possibility that integrin activation in response to MKs, results in an increase in the phosphorylation and therefore catalytic activity of Pyk2 in OBs.…”

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

“…17 RNA from CD34 ϩ cells was used only for quantitative real-time polymerase chain reaction (qPCR) analysis of PLAU transcription, as the quantities harvested precluded other analyses. RNA from day-7 and -13 megakaryocytes and platelets was used for qPCR analysis of PLAU, VWF (control for increased mRNA during megakaryocyte differentiation 18 ), and VCL 7 transcription. RNA from platelets was also used to evaluate CAMK2G transcription in QPD.…”