Platelet Production and Maturation
Adults have nearly a trillion platelets in circulation. These anuclear cell fragments are the smallest cellular component of blood, with an average diameter of 2 to 4 µm, and have a lifespan of approximately seven to ten days. Platelets primarily play a role in hemostasis, responding to blood vessel injury, contributing to the coagulation process, and forming thrombi. Essentially, they act as a defense mechanism when the continuity of the vasculature is compromised, with the goal of preventing blood loss. In order to carry out these functions, platelets must first go through a complex maturation in the megakaryocyte progenitor cells.
All cellular components of blood are derived from hematopoietic stem cells, found in the bone marrow. These stem cells give rise to both common lymphoid progenitors, which eventually differentiate into lymphocytes, and common myeloid progenitors (CMP). The CMPs are responsible for the production of monocytes, granulocytes, erythrocytes, and megakaryocytes. The type of cell that is produced depends on both regulatory transcription factors and environmental factors. The ex
The maturation process for megakaryocytes begins with endomitosis. Most cell types strictly regulate DNA replication and cell division so that the processes are coupled. Megakaryocytes are an exception, as they can contain as many as 128 multiples of the normal diploid chromosome content (128N) within one cell, suggesting they have evolved to decouple the two processes. Although capable of many more, most megakaryocytes undergo three rounds of endomitosis to reach a DNA content of 16N. The polyploidization of the cells allows for increased protein synthesis. The increased synthesis is part of the second step of the maturation process: expansion of the cytoplasm. During this expansion, the cytoplasm rapidly fills with not only proteins but also organelles and membrane systems that will eventually be packaged into platelets.
During the expansion phase of maturation, the megakaryocytes develop three distinct features. The first is the demarcation membrane system (DMS). It is suggested that this system of membrane channels serves as a membrane reserve for platelet formation, though its exact function remains unclear. The second feature that develops during the expansion phase is the dense tubular system, which is passed on to the platelets and is the site of platelet prostaglandin synthesis. Granules are also produced during cytoplasm expansion. The granules that are produced are packaged with platelets and are essential to platelet function.
Platelets are produced once the DMS, dense tubular system, and granules have been formed and the cytoplasmic expansion is complete, including protein synthesis. The details of platelet formation and release remain elusive. However, the proplatelet theory of formation is generally accepted as an accurate, if potentially incomplete, model of the last stage of megakaryocyte development. This model hinges on the formation of proplatelets, which are long, cytoplasmic tubules that extend from the body of the megakaryocyte. These tubules branch in order to produce more proplatelet ends. As the formation and branching of proplatelets continues, the entire cytoplasm of the megakaryocyte converts to proplatelet extensions and a smaller body containing the nuclear material. Microtubules run the length of the proplatelets and act as tracks along which organelles and granules move. Constrictions form along the extended proplatelet and the organelles and granules become trapped, giving a beaded appearance to the proplatelet. Through a mechanism that is still poorly understood, the proplatelets then fragment into individual platelets. The remaining body that contains the nuclear material undergoes apoptosis after it is released from all of the proplatelets.
Signal Transduction during Platelet Activation
Platelets activate after they are exposed to soluble platelet agonists. The agonists can be released from damaged cells or produced during coagulation and inflammation. They can also be released from platelets that are already active. Common agonists include collagen, adenosine diphosphate (ADP), von Willebrand factor (vWF), thrombin, fibrinogen, fibronectin, serotonin, platelet-activating factor (PAF), and many more. When an agonist binds to one of the receptors on the platelet surface, a three stage signaling process begins, consisting of the early platelet activation signaling, intermediate common signaling events, and, finally, integrin signaling.
The interaction of the agonist with a platelet adhesion receptor initiates the early platelet activation signaling or signal transduction. Platelets must be able to express a range of receptors so that they can bind to the varied agonists. Four of the major classes of receptors include: 1) integrins, 2) members of the Leucine-rich repeat (LRR) family, 3) seven transmembrane receptors, and 4) members of the immunoglobulin superfamily. These are by no means the only platelet receptors expressed but the majority of platelet receptors can be classified as one of these four.
Though the receptors and agonists are varied, the early platelet activation signaling, or signal transduction, occur through common mechanisms. Integrins, members of the LRR family, and members of the immunoglobulin superfamily follow similar signal transduction mechanisms. All three groups involve Scr family kinases (SFKs), phosphoinositide 3-kinases (PI3Ks), and the immunoreceptor tyrosine-based activation motif (ITAM) during the early activation signaling. Seven transmembrane receptors, on the other hand, transmit signals through heterotrimeric G proteins, as they are in the GPCR family.
Regardless of the type of receptor involved in early activation signaling, all signal transduction pathways converge during the second stage of signaling. During this intermediate stage, common intracellular signaling events occur. Following early signal transduction of either type, Phospholipase C (PLC) is activated. Activation of PLC catalyzes the release of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 is responsible for the mobilization of calcium, while DAG activates protein kinase C (PKC). Both increased calcium and PKC are required for granule secretion from activated platelets. Calcium, in particular, is known to be necessary for the cytoskeletal rearrangements that change the shape of the activated platelet.
The final stage of platelet activation signaling, integrin signaling, can be inside-out or outside-in. Inside-out signaling modulates the ligand-binding function of integrins. In the resting platelet, integrins are in the low-affinity state but change to the high-affinity state when the platelet is activated. The change in conformation is dependent upon the intermediate common intracellular signaling, which ultimately allows for the proteins talin and kindlins binding to a cytoplasmic domain of the integrin. Binding of these proteins to an intracellular domain, results in a change to the extracellular ligand-binding domain to the high-affinity state. Thus, this form of signaling is called inside-out, as intracellular changes affect extracellular functions. Inside-out signaling is initiated primarily by GPIIb/IIIa and facilitates the aggregatory function of activated platelets.
The other type of integrin signaling that occurs during this third stage of activation signaling is outside-in. Outside-in signaling is initiated solely when the agonist-bound receptor is the integrin GPIIb/IIIa. The converse of inside-out, extracellular changes effect intracellular activity during outside-in signaling. When an agonist binds to GPIIb/IIIa, changes in intracellular domains of the integrin allow a G protein subunit to bind. This amplifies the platelet’s responses to GPCR agonists, enhancing activation signaling. Outside-in signaling, then, ultimately helps promote cytoskeletal rearrangement and activated platelet function. This signaling is especially effective when receptors cluster on the surface of the platelet and bind multimeric macromolecule, like fibrinogen.