Regulation of proplatelet formation and platelet release by integrin αIIbβ3

Abstract

Mature megakaryocytes form structures called proplatelets that serve as conduits for platelet packaging and release at vascular sinusoids. Since the megakaryocyte expresses abundant levels of integrin ␣ IIb ␤ 3 , we have examined a role for fibrinogen in proplatelet development and platelet release alongside that of other matrices. Primary mature murine megakaryocytes from bone marrow aspirates readily formed proplatelets when plated on fibrinogen at a degree that was significantly higher than that seen on other matrices. In addition, ␣ IIb ␤ 3 was essential for proplatelet formation on fibrinogen, as megakaryocytes failed to develop proplatelets in the presence of ␣ IIb ␤ 3 antagonists. Interestingly, inhibition of Src kinases or Ca 2؉ release did not inhibit proplatelet formation, indicating that ␣ IIb ␤ 3 -mediated outside-in signals are not required for this response. Immunohistochemical studies demonstrated that fibrinogen is localized to the bone marrow sinusoids, a location that would allow it to readily influence platelet release. IntroductionMegakaryocytes are the immediate cellular precursors to platelets, and are largely understood to give rise to nascent platelets by forming long, branching filaments called proplatelets (reviewed in Hartwig and Italiano 1 and Schulze and Shivdasani 2 ). Proplatelets have long been observed in bone marrow preparations via histological staining and electron microscopy 3,4 and in cultures of primary megakaryocytes. 5,6 Both the early studies and more current research have observed the consistent presence of small, plateletsized swellings that reside along the length of the proplatelet, presumably representing the developing platelet. Recent studies have identified the underlying architecture of proplatelets, consisting of microtubules that comprise the shaft of the proplatelet and actin filaments that allow for extensive proplatelet branching. 7 The microtubules also delineate the nascent platelet boundary and serve as a mechanism for delivery of granules and organelles to the distal end of the proplatelet, providing the forming platelets with the necessary components for hemostatic function. 7,8 While the architecture of the proplatelet has been well established, the conditions that induce proplatelet formation are not yet well understood, particularly in vivo. A number of studies have begun to address this deficiency by investigating the signaling pathways that contribute to proplatelet formation. For example, plasma from thrombocytopenic rabbits stimulates proplatelet formation, suggesting that soluble factors are capable of inducing proplatelet growth in response to platelet depletion. 9 In addition, the use of soluble inhibitors has indicated that protein kinase C, serine/threonine phosphatases, and mammalian target of rapamycin are necessary for normal proplatelet development. [10][11][12] Genetic approaches have also provided valuable insight into the intracellular factors that contribute to proplatelet growth. Deletion of the megakaryocyte transcription factors...

Measuring and interpreting platelet-leukocyte aggregates

“…While the use of antibodies against these proteins enables clear identification of platelets interacting with other cells, some antibodies affect platelet function under certain conditions. For example, high dose of anti-CD41 monoclonal antibody affects platelet aggregation and inflammatory responses in mice (66), and proplatelet formation of megakaryocytes in vitro (67). Alternatively, reporter mouse lines expressing fluorescent proteins in platelets (55), or platelet subsets (65) can be used for visualising PLA dynamics in different organs.…”

The serum response factor (SRF)/megakaryocytic acute leukemia (MAL) network participates in megakaryocyte development

“…Myosin is an important structural component of Mks, PPs and platelets cytoskeleton. Impairment of its activity may alter the structure (demarcation membrane and cytoplasm 14 ), maturation 15,16 and function of Mk; it may also affect the intracellular cytoskeleton tensions that control Mk morphology. In line, the drosophila MAL-D/SRF complex regulates cell tension and is required for a robust actin cytoskeleton during cell migration.…”

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

“…1 Recent observations suggest that α IIb β 3 is also involved in proplatelet formation. 2,3 Integrin α IIb β 3 function depends on two different signal transduction pathways: inside-out and outside-in signaling. Under resting conditions, α IIb β 3 is expressed on platelets in a bent, inactive conformation.…”