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Platelet‐Activation Mechanisms and Vascular Remodeling

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ABSTRACT

This overview article for the Comprehensive Physiology collection is focused on detailing platelets, how platelets respond to various stimuli, how platelets interact with their external biochemical environment, and the role of platelets in physiological and pathological processes. Specifically, we will discuss the four major functions of platelets: activation, adhesion, aggregation, and inflammation. We will extend this discussion to include various mechanisms that can induce these functional changes and a discussion of some of the salient receptors that are responsible for platelets interacting with their external environment. We will finish with a discussion of how platelets interact with their vascular environment, with a special focus on interactions with the extracellular matrix and endothelial cells, and finally how platelets can aid and possibly initiate the progression of various vascular diseases. Throughout this overview, we will highlight both the historical investigations into the role of platelets in health and disease as well as some of the more current work. Overall, the authors aim for the readers to gain an appreciation for the complexity of platelet functions and the multifaceted role of platelets in the vascular system. © 2017 American Physiological Society. Compr Physiol 8:1117‐1156, 2018.

Figure 1. Figure 1. An overview representation of the four main roles of platelets in normal hemostasis. Platelets can adhere to exposed subendothelial proteins at which point they become localized to a damaged site. Platelets can release both hemostatic mediators and participate in coagulation helping to further localize proteins and cells needed at the damaged site. During coagulation, fibrin can be generated through the actions of thrombin; the presence of fibrin helps to form platelet aggregates. Finally, more recent work has elucidated the role of platelets as inflammatory mediators. We highlight some of the sections that we will discuss in detail in this overview article and how the four main platelet functions interact with/lead to the highlighted sections (note that not all sections are illustrated in this figure). Note that abnormal functioning of these processes can lead to pathological events, that these processes can occur in parallel and that the goal of this overview is to discuss these processes in details. Scanning electron micrographs of an inactive platelet, an activated platelet (with multiple small pseudopods) and a group of active platelets forming a network are shown.
Figure 2. Figure 2. A representation of the coagulation cascade highlighting the major biochemical reactions that occur to generate insoluble fibrin from soluble fibrinogen. Normal bleeding progresses through the extrinsic pathway, whereas the intrinsic pathway occurs only under special cases in vivo (deficiencies in clotting factors within the intrinsic pathway do not alter normal clotting). The intrinsic pathway can be observed in a laboratory setting. Inactive precursor forms of the enzyme are conventionally labelled as a roman numeral. Active forms of the enzyme are conventionally labeled as the same roman numeral followed by a lower case “a.” Note that positive and negative feedback biochemical mechanisms are not included within this representation and that some of the cofactors are not included. When included, cofactors are listed underneath the horizontal arrows.
Figure 3. Figure 3. A representation of a platelet and two of the major granules structures. The dense granules sequester calcium via the actions of a SERCA pump, which pumps calcium from the cytosol (low concentration) into the dense granule (high concentration). Upon activation responses, which are discussed in detail in this overview article, calcium is released from the dense granules into the cytosol. Increased intracellular calcium leads to many cellular responses that are related to platelet functions. In this figure, we have highlighted actin polymerization (which relates to platelet shape change), membrane flipping (which relates to support of coagulation reactions), protein expression/activation (which relates to adhesion, aggregation and inflammation), and α granule release (which relates to platelet positive feedback mechanisms, platelet activation, and secretion functions). This figure is intended as an overview and specific details of these processes can be found within the text.
Figure 4. Figure 4. Chemical structure of two platelet membrane phospholipids; phosphatidylcholine (PC) and phosphatidylserine (PS). PC has a net neutral charge while PS has a net negative charge (the charges associated with these phospholipids are circled on the chemical structure). This negatively charged phospholipid can associate with calcium ions, which helps to localize γ‐carboxyglutamic acid amino acid residues on protein chains. Most coagulation proteins contain approximately 10 γ‐carboxyglutamic acid residues within the beginning of their protein chain. γ‐carboxyglutamic acid residues are modified from glutamic acid residues using a vitamin‐K‐dependent liver carboxylation mechanism.
Figure 5. Figure 5. Pictorial representation of platelet adhesion (inactive platelet) and platelet aggregation (activated platelet), with accompanying scanning electron micrograph images of an inactive platelet (with typical discoid morphology) and active platelets (with altered and elongated morphology). Inactive platelets have active GPIb/IX expressed on their cell membrane that can associate with collagen via the molecular bridge, von Willebrand factor (vWF). In fact, platelet GPIb/IX can associate with vWF in the absence of collagen, but the binding kinetics strongly favor association in the presence of collagen. This process is termed platelet adhesion. At this point, GPIIb/IIIa is expressed on the cell membrane in an inactive form and it is not able to associate with fibrinogen (illustrated as the receptor turned inward). Upon various activation signals, GPIIb/IIIa becomes active (illustrated as turned outward) and can now associate with available fibrinogen forming interplatelet adhesion events. This process is termed platelet aggregation. Note that normally there is a massive shape change during platelet aggregation; we did not include this in the illustration for simplicity. Platelet shape change is associated with a change from a discoid shape to a more circular shape with many far‐reaching pseudopods that can increase the probability of interacting with adhesive ligands.
Figure 6. Figure 6. A representation of the composition of fibrinogen. Each fibrinogen molecule is composed of six protein chains (two of each Aα, Bβ, and γ) that associate with each other at numerous locations (represented by black rectangles). Thrombin has enzymatic activity on fibrinogen along the A and B chain only (the location that thrombin cleaves the fibrinogen chains is depicted with black arrows). Removal of these sections of the amino terminal causes a transition in the solubility of fibrinogen. Not depicted in these figures are the RGD sequences that allow for binding between fibrinogen and GPIIb/IIIa. More details regarding the molecule shifting of soluble fibrinogen to insoluble fibrin can be found in the text. Note also that the cleaved fibrinopeptides play a role in coagulation regulation, inflammation, and angiogenesis.
Figure 7. Figure 7. A flow chart illustrating the physiological relationships between factor XII, prekallikrein, and high molecular weight kininogen (HMWK). Additionally, we highlight some of the downstream responses that occur due to the activation of these factors. The purpose of this figure is to illustrate one of the relationships between platelets, coagulation, and inflammation. Note that there are other factors that are involved in these processes that are not shown in the figure.
Figure 8. Figure 8. A schematic flow chart illustrating the complexity of the GPIb/IX complex with von Willebrand factor (vWF) and collagen and the downstream intracellular signaling. Many elegant studies have illustrated that upon more stable association kinetics (e.g., in the presence of collagen and lower shear profiles) GPIb/IX can induce cytoskeletal reorganization, relocalization of proteins, and calcium release, through the actions of many intracellular signaling molecules. Once calcium enters the cytoplasm, many of the platelet responses that we have discussed can initiate.
Figure 9. Figure 9. Schematic representation of protease activation receptor (PAR) 1 platelet receptor. This is a seven‐transmembrane (TM) channel with four intracellular domains (ID) and four extracellular domains (ED). Some of the important amino acid sequences are highlighted on this schematic including the thrombin cleavage site, the tethered ligand, the ligand‐binding site, and the docking site for particular G‐proteins. Other highlighted sequences provide flexibility to the amino acid chain or proposed association sites that aid in thrombin recruitment/stability. Upon activation, G‐protein subunits can activate various signaling pathways that lead to various common platelet activation signals. PAR4 has a similar function/structure with the exception that PAR4 has a different thrombin cleavage site and different tethered ligand sequence. The text highlights these differences and discusses each of the important sites in more detail.
Figure 10. Figure 10. Schematic representation of the platelet GPVI receptor and the downstream signaling that elicits platelet functional responses. This glycoprotein forms a complex with FcRγ to form a functional protein. There are many signaling proteins that are involved with GPVI activity, that either associate within the cell membrane (e.g., CD148 or the adapter protein LAT; linker for activation of T‐cells) or to the intracellular tail of GPVI or FcRγ. One of the final products of this signaling mechanism is the activation of PLCγ2, which can directly induce calcium mobilization within the platelets.
Figure 11. Figure 11. Schematic representation of the protein structure of the platelet αIIbβ3 integrin receptor. This is perhaps one of the most important platelet receptors since it interacts with fibrinogen and plays a role in platelet activation and aggregation responses. Note that while many of the important domains are highlighted in the figure, the discussion surrounding the function of those domains can be found in the text. Also, the association of the two receptor protein chains is not illustrated on this figure, but can be found in the text. Normally, talin induces inside‐out activation of this integrin; although outside‐in activation is possible, it does not normally occur under physiological conditions.
Figure 12. Figure 12. Schematic representation of how an intact endothelium can inhibit platelet functions. There are three main pathways involved in these processes; the nitric oxide pathway, the prostacyclin pathway, and the CD39 pathway. Endothelial nitric oxide synthase (eNOS) present on activated endothelial cells or activated platelets can produce nitric oxide (NO). NO freely diffuses across the platelet cell membrane inducing the production of cyclic adenosine monophosphate (cAMP) or cGMP. In parallel, endothelial prostacyclin synthase (PGIS) produces prostacyclin (PGI2), which can interact with platelet prostacyclin receptors (IP receptor). IP receptor activity also induces the production of platelet cAMP. Finally, platelet‐produced ADP can interact with endothelial CD39, which hydrolyzes ADP into AMP. AMP can then be further hydrolyzed into adenosine by endothelial CD73. Adenosine can then interact with platelet adenosine receptors (A2 receptors) that induce the production of cAMP. Increased cAMP can inhibit platelet adhesion responses and platelet aggregation responses to limit clot formation.


Figure 1. An overview representation of the four main roles of platelets in normal hemostasis. Platelets can adhere to exposed subendothelial proteins at which point they become localized to a damaged site. Platelets can release both hemostatic mediators and participate in coagulation helping to further localize proteins and cells needed at the damaged site. During coagulation, fibrin can be generated through the actions of thrombin; the presence of fibrin helps to form platelet aggregates. Finally, more recent work has elucidated the role of platelets as inflammatory mediators. We highlight some of the sections that we will discuss in detail in this overview article and how the four main platelet functions interact with/lead to the highlighted sections (note that not all sections are illustrated in this figure). Note that abnormal functioning of these processes can lead to pathological events, that these processes can occur in parallel and that the goal of this overview is to discuss these processes in details. Scanning electron micrographs of an inactive platelet, an activated platelet (with multiple small pseudopods) and a group of active platelets forming a network are shown.


Figure 2. A representation of the coagulation cascade highlighting the major biochemical reactions that occur to generate insoluble fibrin from soluble fibrinogen. Normal bleeding progresses through the extrinsic pathway, whereas the intrinsic pathway occurs only under special cases in vivo (deficiencies in clotting factors within the intrinsic pathway do not alter normal clotting). The intrinsic pathway can be observed in a laboratory setting. Inactive precursor forms of the enzyme are conventionally labelled as a roman numeral. Active forms of the enzyme are conventionally labeled as the same roman numeral followed by a lower case “a.” Note that positive and negative feedback biochemical mechanisms are not included within this representation and that some of the cofactors are not included. When included, cofactors are listed underneath the horizontal arrows.


Figure 3. A representation of a platelet and two of the major granules structures. The dense granules sequester calcium via the actions of a SERCA pump, which pumps calcium from the cytosol (low concentration) into the dense granule (high concentration). Upon activation responses, which are discussed in detail in this overview article, calcium is released from the dense granules into the cytosol. Increased intracellular calcium leads to many cellular responses that are related to platelet functions. In this figure, we have highlighted actin polymerization (which relates to platelet shape change), membrane flipping (which relates to support of coagulation reactions), protein expression/activation (which relates to adhesion, aggregation and inflammation), and α granule release (which relates to platelet positive feedback mechanisms, platelet activation, and secretion functions). This figure is intended as an overview and specific details of these processes can be found within the text.


Figure 4. Chemical structure of two platelet membrane phospholipids; phosphatidylcholine (PC) and phosphatidylserine (PS). PC has a net neutral charge while PS has a net negative charge (the charges associated with these phospholipids are circled on the chemical structure). This negatively charged phospholipid can associate with calcium ions, which helps to localize γ‐carboxyglutamic acid amino acid residues on protein chains. Most coagulation proteins contain approximately 10 γ‐carboxyglutamic acid residues within the beginning of their protein chain. γ‐carboxyglutamic acid residues are modified from glutamic acid residues using a vitamin‐K‐dependent liver carboxylation mechanism.


Figure 5. Pictorial representation of platelet adhesion (inactive platelet) and platelet aggregation (activated platelet), with accompanying scanning electron micrograph images of an inactive platelet (with typical discoid morphology) and active platelets (with altered and elongated morphology). Inactive platelets have active GPIb/IX expressed on their cell membrane that can associate with collagen via the molecular bridge, von Willebrand factor (vWF). In fact, platelet GPIb/IX can associate with vWF in the absence of collagen, but the binding kinetics strongly favor association in the presence of collagen. This process is termed platelet adhesion. At this point, GPIIb/IIIa is expressed on the cell membrane in an inactive form and it is not able to associate with fibrinogen (illustrated as the receptor turned inward). Upon various activation signals, GPIIb/IIIa becomes active (illustrated as turned outward) and can now associate with available fibrinogen forming interplatelet adhesion events. This process is termed platelet aggregation. Note that normally there is a massive shape change during platelet aggregation; we did not include this in the illustration for simplicity. Platelet shape change is associated with a change from a discoid shape to a more circular shape with many far‐reaching pseudopods that can increase the probability of interacting with adhesive ligands.


Figure 6. A representation of the composition of fibrinogen. Each fibrinogen molecule is composed of six protein chains (two of each Aα, Bβ, and γ) that associate with each other at numerous locations (represented by black rectangles). Thrombin has enzymatic activity on fibrinogen along the A and B chain only (the location that thrombin cleaves the fibrinogen chains is depicted with black arrows). Removal of these sections of the amino terminal causes a transition in the solubility of fibrinogen. Not depicted in these figures are the RGD sequences that allow for binding between fibrinogen and GPIIb/IIIa. More details regarding the molecule shifting of soluble fibrinogen to insoluble fibrin can be found in the text. Note also that the cleaved fibrinopeptides play a role in coagulation regulation, inflammation, and angiogenesis.


Figure 7. A flow chart illustrating the physiological relationships between factor XII, prekallikrein, and high molecular weight kininogen (HMWK). Additionally, we highlight some of the downstream responses that occur due to the activation of these factors. The purpose of this figure is to illustrate one of the relationships between platelets, coagulation, and inflammation. Note that there are other factors that are involved in these processes that are not shown in the figure.


Figure 8. A schematic flow chart illustrating the complexity of the GPIb/IX complex with von Willebrand factor (vWF) and collagen and the downstream intracellular signaling. Many elegant studies have illustrated that upon more stable association kinetics (e.g., in the presence of collagen and lower shear profiles) GPIb/IX can induce cytoskeletal reorganization, relocalization of proteins, and calcium release, through the actions of many intracellular signaling molecules. Once calcium enters the cytoplasm, many of the platelet responses that we have discussed can initiate.


Figure 9. Schematic representation of protease activation receptor (PAR) 1 platelet receptor. This is a seven‐transmembrane (TM) channel with four intracellular domains (ID) and four extracellular domains (ED). Some of the important amino acid sequences are highlighted on this schematic including the thrombin cleavage site, the tethered ligand, the ligand‐binding site, and the docking site for particular G‐proteins. Other highlighted sequences provide flexibility to the amino acid chain or proposed association sites that aid in thrombin recruitment/stability. Upon activation, G‐protein subunits can activate various signaling pathways that lead to various common platelet activation signals. PAR4 has a similar function/structure with the exception that PAR4 has a different thrombin cleavage site and different tethered ligand sequence. The text highlights these differences and discusses each of the important sites in more detail.


Figure 10. Schematic representation of the platelet GPVI receptor and the downstream signaling that elicits platelet functional responses. This glycoprotein forms a complex with FcRγ to form a functional protein. There are many signaling proteins that are involved with GPVI activity, that either associate within the cell membrane (e.g., CD148 or the adapter protein LAT; linker for activation of T‐cells) or to the intracellular tail of GPVI or FcRγ. One of the final products of this signaling mechanism is the activation of PLCγ2, which can directly induce calcium mobilization within the platelets.


Figure 11. Schematic representation of the protein structure of the platelet αIIbβ3 integrin receptor. This is perhaps one of the most important platelet receptors since it interacts with fibrinogen and plays a role in platelet activation and aggregation responses. Note that while many of the important domains are highlighted in the figure, the discussion surrounding the function of those domains can be found in the text. Also, the association of the two receptor protein chains is not illustrated on this figure, but can be found in the text. Normally, talin induces inside‐out activation of this integrin; although outside‐in activation is possible, it does not normally occur under physiological conditions.


Figure 12. Schematic representation of how an intact endothelium can inhibit platelet functions. There are three main pathways involved in these processes; the nitric oxide pathway, the prostacyclin pathway, and the CD39 pathway. Endothelial nitric oxide synthase (eNOS) present on activated endothelial cells or activated platelets can produce nitric oxide (NO). NO freely diffuses across the platelet cell membrane inducing the production of cyclic adenosine monophosphate (cAMP) or cGMP. In parallel, endothelial prostacyclin synthase (PGIS) produces prostacyclin (PGI2), which can interact with platelet prostacyclin receptors (IP receptor). IP receptor activity also induces the production of platelet cAMP. Finally, platelet‐produced ADP can interact with endothelial CD39, which hydrolyzes ADP into AMP. AMP can then be further hydrolyzed into adenosine by endothelial CD73. Adenosine can then interact with platelet adenosine receptors (A2 receptors) that induce the production of cAMP. Increased cAMP can inhibit platelet adhesion responses and platelet aggregation responses to limit clot formation.

 

Teaching Material

D. A. Rubenstein, W. Yin. Platelet-Activation Mechanisms and Vascular Remodeling. Compr Physiol 8: 2018, 1117-1156.

Didactic Synopsis

Major Teaching Points:

  1. Understanding the function of platelets is critical to learning about normal hemostatic and thrombotic processes and to many cardiovascular disease processes.
  2. Elucidating the role of platelets as inflammatory cells and inflammatory mediators:
    1. As a means of recruiting cells to inflammatory sites
    2. As a mechanism to activate inflammatory cells
    3. As an integral component of the inflammatory process
  3. Platelet activities are regulated by both chemical and mechanical factors:
    1. Chemical mediators can interact with multiple platelet receptors using multiple mechanisms
    2. Mechanical mediators can interact with platelets using multiple mechanisms
    3. Chemical and mechanical mediators can act in concert to modulate the response both positively and negatively
  4. Determining how platelet receptors are responsible for platelets interacting with the external environment:
    1. Platelets have multiple types of receptors that interact with the external environment using multiple mechanisms
    2. Many platelet signaling pathways converge on calcium release leading to many physiological changes
    3. Many platelet receptors, their signaling pathways, and how these pathways are antagonized are not completely characterizedc.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1 This figure illustrates the main role of platelets in normal hemostasis. Traditionally, this has been divided into adhesion, activation, and aggregation, but more recently the role of platelets as immune cells has been elucidated. Scanning electron micrographs of platelets are also included.

Figure 2 This figure illustrates the biochemical reactions that occur within the coagulation cascade. The coagulation factors are listed as roman numerals. Important cofactors are listed (small font underneath the horizontal lines).

Figure 3 This figure illustrates an overview of how calcium flux within the platelet can relate to functional changes, such as participation in coagulation reactions, shape change, and aggregation.

Figure 4 This figure illustrates the chemical structure of important platelet membrane phospholipids and the chemical structure of an important amino acid residue contained within many coagulation factors. Glutamic acid amino acids (Glu) can be modified to γ-carboxyglutamic acid (Gla) residues in the liver. Calcium can act as a bridge between the negatively charged phospholipids and the amino acid residues.

Figure 5 This figure illustrates the difference between platelet adhesion and platelet aggregation. Platelets can adhere to collagen, via GPIb/IX (glycoprotein Ib/IX) and von Willebrand factor (vWF) in both inactive and activated forms. In contrast, platelets can only aggregate to fibrinogen after platelets undergo activation responses.

Figure 6 This figure illustrates the chemical composition and the association of the different protein chains within a fibrinogen molecule. Black rectangles denote locations where the peptide chains associate with each other and black arrows depict locations where thrombin cleaves fibrinogen.

Figure 7 This figure illustrates a relationship between the coagulation cascade and inflammatory processes. Additionally, we have included some of the downstream effects of these activation products.

Figure 8 This figure illustrates the signaling pathways related to GPIb/IX association with von Willebrand factor and collagen. We have included some of the major signaling molecules within this pathway that lead to particular platelet-activation functional changes.

Figure 9 This figure illustrates the molecular mechanism of the platelet-protease-activated receptor 1 (PAR1), which is one of the two major platelet thrombin receptors. Some of the major downstream signaling molecules and the responses that they elicit are also highlighted in this figure.

Figure 10 This figure illustrates the structural organization and molecular signaling mechanisms associated with platelet GPVI. This protein can associate with collagen and induce platelet-activation responses due to increased calcium mobilization.

Figure 11 This figure illustrates the structural organization of the platelet αIIbβ3 integrin and the important structural domains of each of the protein chains.

Figure 12 This figure illustrates the three primary mechanisms by which endothelial cells can inhibit activated platelets to limit clot size.

 


Related Articles:

Angiogenesis
Platelets and Their Interactions with Other Immune Cells
Teaching Material

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How to Cite

David A. Rubenstein, Wei Yin. Platelet‐Activation Mechanisms and Vascular Remodeling. Compr Physiol 2018, 8: 1117-1156. doi: 10.1002/cphy.c170049