Joseph E. Italiano

Dynamic Visualization of Thrombopoiesis Within Bone Marrow

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.

Platelets actively sequester angiogenesis regulators

Clinical trials with antiangiogenic agents have not been able to validate plasma or serum levels of angiogenesis regulators as reliable markers of cancer presence or therapeutic response. We recently reported that platelets contain numerous proteins that regulate angiogenesis. We now show that accumulation of angiogenesis regulators in platelets of animals bearing malignant tumors exceeds significantly their concentration in plasma or serum, as well as their levels in platelets from non-tumor-bearing animals. This process is selective, as platelets do not take up a proportional amount of other plasma proteins (eg, albumin), even though these may be present at higher concentrations. We also find that VEGF-enriched Matrigel pellets implanted subcutaneously into mice or the minute quantities of VEGF secreted by microscopic subcutaneous tumors (0.5-1 mm(3)) result in an elevation of VEGF levels in platelets, without any changes in its plasma levels. The profile of other angiogenesis regulatory proteins (eg, platelet-derived growth factor, basic fibroblast growth factor) sequestered by platelets also reflects the presence of tumors in vivo before they can be macroscopically evident. The ability of platelets to selectively take up angiogenesis regulators in cancer-bearing hosts may have implications for the diagnosis and management of many angiogenesis-related diseases and provide a guide for antiangiogenic therapies.

Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis.

An association between platelets, angiogenesis, and cancer has long been recognized, but the mechanisms linking them remains unclear. Platelets regulate new blood vessel growth through numerous stimulators and inhibitors of angiogenesis by several pathways, including differential exocytosis of angiogenesis regulators. Herein, we investigated the differential release of angiogenesis stimulators and inhibitors from platelets. Activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF, but not endostatin whereas, thromboxane A(2) (TXA(2)) released endostatin but not VEGF. Platelet releasates generated by activation with ADP promoted migration and formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in in vitro angiogenesis models. Conversely, TXA(2)-stimulated platelet releasate inhibited migration and formation of capillary structures. Because tumor growth beyond 1-2 mm(3) is angiogenesis-dependent, we hypothesized that cancer cells preferentially stimulate platelets to secrete their pro-angiogenic payload. In support of this, the breast cancer cell line MCF-7 stimulated secretion of VEGF and a pro-angiogenic releasate from platelets. Furthermore, the antiplatelet agent aspirin inhibited platelet-mediated angiogenesis after exposure to ADP or MCF-7 cells providing a potential mechanism for how aspirin may impact malignancy. Manipulation of differentially mediated release of angiogenic factors from platelets may provide a new modality for cancer treatment.

The incredible journey: From megakaryocyte development to platelet formation.

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.

A lineage-restricted and divergent β-tubulin isoform is essential for the biogenesis, structure and function of blood platelets

BACKGROUND Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.

Cytoskeletal mechanics of proplatelet maturation and platelet release

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

Clinical relevance of microparticles from platelets and megakaryocytes

Purpose of reviewPlatelet microparticles were identified more than 40 years ago and are the most abundant circulating microparticle subtype. Yet fundamental questions about their formation and role in human disease are just beginning to be understood at the cellular and molecular level. This review will address mechanisms of platelet microparticle generation and evaluate our current understanding of their clinical relevance. Recent findingsNew evidence indicates that the majority of CD41+ microparticles circulating in healthy individuals derive directly from megakaryocytes. CD41+ microparticles also form from activated platelets upon loss of cytoskeleton–membrane adhesion, which occurs in a multitude of disease states characterized by elevated platelet microparticle levels. More recent studies have demonstrated that platelet microparticles function as a transport and delivery system for bioactive molecules, participating in hemostasis and thrombosis, inflammation, malignancy infection transfer, angiogenesis, and immunity. The mechanism of platelet microparticle participation in specific disease entities such as rheumatoid arthritis has been elucidated. SummaryContinued research into how platelet microparticles are generated and function as a transcellular delivery system will advance our basic understanding of microparticle physiology and may enable new strategies for treatment of select disease entities.

Megakaryocytes and beyond: the birth of platelets

Summary.  Megakaryocytes are highly specialized precursor cells that differentiate to produce blood platelets via intermediate cytoplasmic extensions known as proplatelets. Recent advances in the understanding of megakaryocyte differentiation and platelet formation rely on a combination of genetic and cell biological studies with detailed structural analysis of cultured cells. Visualization of sequential steps in endomitosis has expanded our views on how megakaryocytes acquire polyploid DNA content, whereas studies in mouse models of platelet disorders provide clues into transcriptional pathways and those leading to the assembly of platelet‐specific secretory granules. The experimental findings forge stronger links between cellular processes and molecular mechanisms, while observation of the underlying morphologic events in beginning to yield insights into the cytoskeletal mechanics of proplatelet formation. Here we review salient aspects of the emerging appreciation of the cellular and molecular basis of thrombopoiesis.

Normal ranges of angiogenesis regulatory proteins in human platelets

Platelets sequester angiogenesis regulatory proteins early in tumor growth, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA‐based method for accurate and reproducible measurement of vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin‐1 (TSP‐1), fibroblast growth factor, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 ± 0.37 pg/106 platelets); PDGF (23 ± 6 pg/106); PF4 (12 ± 5 ng/106); TSP‐1 (31 ± 12 ng/106); bFGF (0.44 ± 0.15 pg/106); and endostatin (5.6 ± 3.0 pg/106). There was an excellent correlation (R2 = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet‐poor plasma by factors of: VEGF (215‐fold); PDGF (914‐fold); PF‐4 (516‐fold); TSP‐1 (813‐fold); and bFGF (17‐fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5‐week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP‐1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet‐derived measurements were accurate and reproducible with minimal biovariability. Am. J. Hematol., 2010.

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.