SYNTHESIS AND SECRETION OF VON-WILLEBRAND-FACTOR AND FIBRONECTIN IN MEGAKARYOCYTES AT DIFFERENT PHASES OF MATURATION

Our goals have been to define the biochemical characteristics of megak aryocytes during maturation that are critical for platelet assembly an d release into the circulation and to introduce biochemical markers fo r megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive protein s that are synthesized by megakaryocytes, stored in alpha granules, an d thought to have a fundamental role in hemostasis. The study demonstr ated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldi n A, which blocks the exit of proteins from the rough endoplasmic reti culum (RER), inhibited the formation of vWF multimers but did not affe ct the synthesis of monomers and dimers in mature megakaryocytes. Thes e 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 synthe sis of both the 260-kD and 275-kD pro-vWF was detected. However, the s ynthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident afte r 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 secrete d by mature and immature megakaryocytes, respectively. In contrast, th e synthesis of FN monomers and dimers was established in immature mega karyocytes, and their synthesis in mature megakaryocytes was very simi lar. Constitutive secretion of FN was not seen in megakaryocytes. Bref eldin 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 sy nthesized at different phases of megakaryocyte maturation and that onl y vWF is constitutively secreted by megakaryocytes provides new inform ation relevant to alpha granule formation and possibly bone marrow mat rix assembly.