New steps in the process of conversion of proplatelet extensions from megakaryocytes into mature platelets are defined.

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.

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...

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This article is available online at http://www.jlr.org Health consequences associated with low intakes of the long-chain, marine omega-3 (n3) FAs have become a central issue in nutritional lipid research. The United States Department of Agriculture's 2010 Dietary Guidelines recommend consumption of 8 ounces per week of fi sh, providing an average of 250 mg eicosapentaenoic acid (20:5n3) and docosahexaenoic acid (22:6n3) per day for prevention of heart disease ( 1 ). Moreover, public awareness regarding the potential health benefi ts of n3 FAs has spurred an increase in fatty fi sh and fi sh oil consumption ( 2 ).Although the mechanisms by which n3-HUFAs improve health are still being explored, it is clear that increasing n3-HUFA intake can decrease the risk of cardiovascular disease (CVD) in at-risk individuals ( 3-5 ). In hyperlipidemic subjects, treatment with high doses of n3-HUFAs lowers triglycerides ( 6 ) and improves total:HDL cholesterol ratios ( 6, 7 ), a surrogate marker associated with a reduction in CVD risk. The n3-HUFAs 20:5n3 and 22:6n3 also reduce infl ammatory responses in a range of conditions ( 8-10 ). A well-accepted effect of n3-HUFA supplementation is a reduction in thrombin-stimulated platelet aggregation due to decreased platelet cyclooxygenase metabolism ( 11 ). More recently, a cyclooxygenase-independent diminution of platelet sensitivity to collagen has been reported after P-OM3 treatment ( 12 ). Thus, an increase in the anti-infl ammatory n3-HUFAs, which lowers the more proinfl ammatory n6-HUFAs ( 13-15 ), at least partially explains the health benefi ts of n3-HUFA consumption ( 8-10 ). 5306-51530-019-00D (J.W.N.), National Institute of Food and Agriculture National Needs Fellowship grant 2008-38420-04759 (A.H.K.), and by grant LVZ112860 from GlaxoSmithKline (G.C.S.) Abbreviations: CVD, cardiovascular disease; HUFA, highly unsaturated fatty acid; P-OM3, prescription omega-3 acid ethyl esters; RBC, red blood cell.

During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1-deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1-deficient MKs in response to a gradient of stromal cell-derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1 ؊/؊ MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be ex- IntroductionMegakaryocytopoiesis involves proliferation and differentiation of megakaryocyte (MK) progenitors to a large, terminally differentiated cell with a multi-lobulated, polyploid nucleus. Nuclear maturation, a process known as endoreplication, proceeds in concert with cytoplasmic maturation and expression of platelet surface markers including the glycoprotein receptors IIb/IIIa, GPIb, GPIX, and GPVI. As the MK matures and differentiates, it migrates to sinusoidal bone marrow endothelial cells where it forms transendothelial projections called proplatelets that release 1000 to 5000 platelets per MK into the intravascular space. [1][2][3][4][5] Thrombopoietin (TPO) participates in the humoral regulation of thrombopoiesis. TPO is produced constitutively in the liver and by bone marrow stromal cells and its levels are regulated by binding to the receptor c-Mpl expressed on platelets. 6 This has the net effect of reducing the concentration of TPO in the circulation and thereby inhibiting differentiation of progenitor cells along the MK lineage. Thus, megakaryocytopoiesis and platelet count are directly regulated by circulating TPO. 2 A key step in thrombopoiesis is migration of maturing MKs from the proliferative osteoblastic niche within the bone marrow microenvironment, where hematopoietic stem cells reside, to the capillary-rich vascular niche, where proplatelets are formed. 6 This process is regulated by a variety of chemokines and cytokines, as well as by adhesive interactions with interstitial cells and extracellular matrix proteins. For example, the chemokine SDF1 directs movement of MK progenitors through its receptor CXCR4 from the proliferative "osteoblastic niche" to the "vascular niche," where platelets are formed. 6 SDF1 therefore acts in combination with TPO to promote differentiation of MK progenitor cells to mature MKs. 7,8 In addition, SDF1 promotes the interaction and transmigration of mature MKs through bone marrow endothel...

In response to agonist stimulation, the αIIbβ3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of αIIbβ3 is believed to occur in part via engagement of the β3 cytoplasmic tail with talin; however, the role of the αIIb tail and its potential binding partners in regulating αIIbβ3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the αIIb tail, is an endogenous inhibitor of αIIbβ3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced αIIbβ3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to αIIbβ3, thus providing a model for tightly controlled regulation of αIIbβ3 activation.

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin ␣ IIb ␤ 3 activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcR␥ but independent of another platelet collagen receptor, ␣ 2 ␤ 1 . Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y 12 and not the P2Y 1 receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y 1 or the P2Y 12 -associated G-protein, G␣i 2 , indicate that human and murine platelets also have a significant P2Y 12 -independent component of GPVImediated Rap1 activation. The P2Y 12 -independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y 12 -dependent and -independent components are also observed in GPVImediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVImediated platelet activation and implicate Rap1 as a key signaling protein in GPVI IntroductionCollagen is a major component of the subendothelial matrix and atherosclerotic plaques and is a potent platelet agonist that contributes to thrombus formation upon plaque rupture. Collagenmediated platelet activation results from a complex set of signals initiated by the coordination of platelet surface proteins, including the immunoglobulin superfamily receptor glycoprotein (GP) VI, the integrin ␣ 2 ␤ 1 , and most recently, GPV. [1][2][3] Activation of GPVI causes the assembly of distinct proximal signaling pathways. GPVI is constitutively associated with FcR␥, a protein containing an immunoreceptor tyrosine-based activation motif. 4,5 Upon collagen ligation to GPVI, FcR␥ becomes phosphorylated by a Src family kinase, 6,7 which subsequently allows the recruitment of other signaling proteins such as Syk, Bruton tyrosine kinase, and phospholipase C␥. [6][7][8][9][10][11] GPVI-mediated signaling is absolutely required for collageninduced platelet aggregation. Human platelets lacking GPVI, but expressing ␣ 2 ␤ 1 , fail to aggregate in response to collagen. 12 In addition, GPVI antagonists inhibit collagen-mediated aggregation. 13 Although human platelets lacking ␣ 2 ␤ 1 also fail to aggregate in response to collagen, 14 fibrillar collagen-mediated platelet aggregation still occurs in ␤ 1 -deficient murine platelets that express GPVI. 15,16 Additionally, GPVI signaling itself can activate ␣ 2 ␤ 1 function. 15 This finding suggests that not only is GPVI a key signaling component of collagen-mediated platelet activation, ...