Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunologically savvy than we once thought.

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro-and antiangiogenic proteins are separated in distinct subpopulations of ␣-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct ␣-granules. These observations motivated the hypothesis that distinct populations of ␣-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH 2 ) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH 2 ) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of ␣-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis. IntroductionAngiogenesis, the process of new vessel development, plays an essential role in embryogenesis, but postnatal angiogenesis is limited to sites of abnormal vascular surface. An activated vascular endothelium can be induced by tissue injury or wound healing, by hormonal cycling such as in pregnancy and ovulation, or by tumor-induced vessel growth. In all of these circumstances, platelets act as the initial responder to vascular change and provide a flexible delivery system for angiogenesis-related molecules. [1][2][3][4] The process of postnatal angiogenesis is regulated by a continuous interplay of stimulators and inhibitors of angiogenesis, and their imbalance contributes to numerous inflammatory, malignant, ischemic, and immune disorders. 5 There is a revived interest in the overlap between angiogenesis and platelets 6 because several clinical trials have now shown that anticoagulation can improve cancer survival 7,8 beyond the benefit derived from the treatment of deep vein thrombosis alone.It is known that platelets stimulate endothelial cells in culture and can promote the assembly of capillary-like structures in vitro. 9,10 Platelets may modulate angiogenesis by releasing promoters such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and matrix metalloproteinases (MMPs). 1,6,[11][12][13][14][15][16][17][18] The repertoire o...

Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

Platelets are generated from megakaryocytes (MKs) in mammalian bone marrow (BM) by mechanisms that remain poorly understood. Here we describe the use of multiphoton intravital microscopy in intact BM to visualize platelet generation in mice. MKs were observed as sessile cells that extended dynamic proplatelet-like protrusions into microvessels. These intravascular extensions appeared to be sheared from their transendothelial stems by flowing blood, resulting in the appearance of proplatelets in peripheral blood. In vitro, proplatelet production from differentiating MKs was enhanced by fluid shear. These results confirm the concept of proplatelet formation in vivo and are consistent with the possibility that blood flow-induced hydrodynamic shear stress is a biophysical determinant of thrombopoiesis.

Circulating blood platelets are specialized cells that prevent bleeding and minimize blood vessel injury. Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets. Given the need for platelets in many pathological situations, understanding how this process occurs is an active area of research with important clinical applications.

SummaryHuman induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

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