It also leads to vasoconstriction which is important during tissue injury and inflammation. After the completion of previous steps starting from the formation of platelets to successfully being triggered by several stimuli for activation, changes in shape from inactive to the activated form takes place. The original inactive shape of platelets is described as amorphous or disc-like colorless cell fragment.
But when activation takes place, it makes an even more prominent shape, where it looks like spiculated spheres with several protruding extensions. Next to platelet activation is the bindings.
The binding is a process that follows the change in shape where platelets clump or bind to each other, working with the common goal of forming a blood clot. The binding results to platelet plug formation. The binding is mainly achieved with the use of receptors.
The receptors are found in the outer layer of the activated platelets. The fibrinogen , a soluble protein and coagulation factor, is a substance that binds to its receptor in the process of clumping together. Blood coagulation is the process where the blood changes from liquid to gel in response to bleeding. When platelets successfully take an activated form and clumped together, it forms a blood clot.
This results to hemostasis, the cessation of blood loss in the damaged blood vessel. Primary hemostasis happens when platelets immediately form a plug at the site of injury. The coagulation process involves a mechanism of activation, adhesion, and aggregation of platelets.
Moreover, there are several coagulation factors or clotting factors that respond in a complex cascade for the formation of fibrin strands that strengthens the platelet plug. Along with the mechanisms initiated by platelets, blood-clotting proteins that circulate in the blood plasma are poised to participate in blood coagulation in response to the tissue injury.
The next and final step is the conversion of prothrombin to the active enzyme thrombin. The sequence is dependent on the platelet response. In combination with platelets, it completes the blood clot. The enzyme converts the blood protein fibrinogen, which is also present in plasma, to fibrin.
The resulting fibrin molecules adhere to each other and assemble long fibrils. As the developing fibrils of fibrin is formed, interspersed activated platelets are trapped, forming the clot. Blood platelets, prothrombin, regulatory membrane, blood proteins, active enzyme transformations, etc. The importance of keeping the entire process functional should always be highly considered, knowing that the inability to stop bleeding can result to a life-threatening condition.
Platelet count can be altered by several factors resulting to abnormal levels. These may either be factors that cause a low platelet count or high platelet count , or it may simply be a mild drop in platelets during pregnancy.
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Handle as if capable of transmitting infection and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Other anticoagulants can also work, however. Heparin anticoagulant activates platelets and is not recommended for measuring in vivo platelet activation. The following protocol is designed to minimize artifactual activation of platelets during blood draw.
This procedure is one example of a variety of methods that can be used to activate platelets. Fixation of the blood with paraformaldehyde prior to staining inhibits spontaneous platelet activation. For clinical testing, fixing platelets can make the assay more manageable.
Fixation has an effect on activation-dependent platelet antibodies. If fixation is not desired or possible, proceed to the next section, Direct Immunofluorescence Staining, and use fresh whole blood.
If additional fixed blood is required, prepare the appropriate number of tubes. Do not increase the volume of the blood or paraformaldehyde in the tube as this will increase the possibility of platelet aggregation. Single- or multicolor staining can be used in the assay.
With multicolor staining, one antibody conjugate can be used to threshold data acquisition to analyze only those blood cells that bind an activation-independent, platelet-specific antibody, 4,5 for example, CD61 or CD42a. Another antibody conjugated to a different fluorochrome can be used to simultaneously assess the binding of platelet-associated, activation-dependent antibodies, for example, CD62P or PAC Acquisition and analysis can be performed on scatter gating Figure 1 through Figure 4 or fluorescence gating Figure 5 through Figure 8.
Scatter gating gating on forward scatter [FSC] and side scatter [SSC] can be difficult when the platelet count is low or when there is aggregation in the sample. In both normal and disease states, and especially when activated, platelets and red blood cells can have overlapping light scatter signatures.
For scatter gating, exclude debris and background noise by setting the appropriate FSC threshold. Fluorescence gating gating on FL3 and SSC can be done on the activation-independent platelet marker, and then the light-scatter profile of the positive population can be independently analyzed. Venous blood typically demonstrates three subpopulations of particles Figure 6. The majority of the particles consist of single intact platelets. However, since platelets are much smaller than leucocytes, the events are not optimally displayed along each axis.
The following procedure uses fluorescence gating and a setup that optimally displays platelet events. This form is intended to help us improve our website experience. For other support, please visit our Contact Us page. This increases the cytosolic calcium concentration and activates specific signaling pathways.
P2Y 12 receptor sustains platelet activation in response to ADP and therefore has a central role in this process. TxA 2 produced and released by stimulated platelets also activates further platelets via GPCR, thereby promoting plug formation. Thrombin is the most strong platelet agonist and also responsible for converting fibrinogen into fibrin to stabilize the platelet plugs [ 5 , 6 , 9 , 13 ].
PAR1 mediates human platelet activation at low thrombin concentration, while PAR4 requires higher concentration of thrombin for platelet activation. Signaling via PAR4 is available for a protective mechanism in situations such as trauma to contribute to arrest bleeding. Other agonists like epinephrine, prostaglandin E 2 , and serotonin can also utilize GPCR to potentiate platelet responses [ 13 ]. All these platelet signaling events converge upon the final common pathway of platelet activation, the functional upregulation of integrin adhesion receptors [ 5 , 11 — 13 ].
This promotes further the recruitment of additional platelets to the site of vascular injury, allowing the subsequent thrombus formation. Activated platelets secret a number of inflammatory mediators that have no apparent role in hemostasis. Under hemostatic conditions, platelets generally do not bind to leukocytes. However, when activated, platelets adhere to neutrophils and monocytes, and interactions with lymphocytes have also been identified [ 2 , 5 , 6 , 8 — 12 ].
Platelets interact with the vascular endothelium and leukocytes and link inflammation, thrombosis, and atherogenesis. Recently platelet serotonin in dense granules has been shown to play an important role in neutrophil rolling and adhesion to the endothelium [ 5 ]. Binding between platelets and other cell types is primarily mediated by P-selectin also known as CD62p. P-selectin via its ligand, P-selectin glycoprotein ligand-1 PSGL-1 , has a central role in the interactions between platelets, leukocytes, and endothelial cells.
Monocytes, neutrophils, eosinophils, and hematopoietic progenitor cells have all been reported to express PSGL-1 [ 5 , 6 , 10 ]. P-selectin cross-links platelets and leukocytes and is a major mediator of platelet-leukocyte aggregate formation, thereby upregulating release of proinflammatory cytokines and adhesion to endothelium. Platelets expression of CD40L has been shown to affect dendritic cells as well as B and T lymphocytes, suggesting that it provides a communicative link between innate and adaptive immunity [ 5 , 6 , 8 — 12 ].
It also interacts with CD40 on endothelial cells to promote secretion of chemokines and expression of adhesion molecules. Furthermore, platelets are known as the predominant source of soluble CD40L sCD40L , which can induce vascular cells to express E-selectin and P-selectin and release interleukin- IL- 6 [ 5 ]. In addition to a role in thrombosis and hemostasis, PF4 has a broad range of activities related to innate immunity [ 5 , 6 , 9 ].
PF4 promotes neutrophil granule release and adhesion to endothelial cells, mediated by L-selectin and leukocyte function-associated antigen-1 LFA PF4 also prevents monocyte apoptosis, promotes monocytic differentiation into macrophages, and induces phagocytosis and generation of reactive oxygen species ROS [ 5 ].
PF4 and RANTES regulated on activation, normal T cell expressed and secreted form heterodimers, leading to promote monocyte recruitment to the endothelium. Platelets also participate in pathogen capture and sequestration. When platelets bind to neutrophils, they trigger the release of chemokines and the formation of neutrophil extracellular traps NET [ 5 , 8 — 10 , 12 ].
NET are released from activated neutrophils and comprise an extrusion of DNA, DNA-associated nuclear proteins, such as histones and serine proteases including neutrophil elastase. The association between platelets and leukocytes bringing on the release of intravascular NET provides the new opportunities for the development of diagnostic assays and prognostic indicators in inflammatory or septic conditions [ 5 , 8 — 10 , 12 ].
But flow cytometer is currently the best standardized method to study platelet function. Although flow cytometry requires sophisticated equipment and available monoclonal antibodies, it has several advantages including small volume of whole blood and independence of platelet count [ 2 , 3 , 11 ].
Flow cytometry allows the analysis of the expression of platelet activation markers and receptors on individual platelets as well as the quantitation of associates between platelets and other blood cells [ 16 — 20 ]. Granule-stored mediators include coagulation and angiogenic factors, adhesion molecules, cytokines, and chemokines. Although preformed mediators allow for rapid release following platelet activation, platelets also possess the ability to synthesize additional mediators.
Many functions of platelets result from the vast array of preformed cell membrane and soluble mediators contained within granules. The contents are known to include adhesive glycoproteins such as P-selectin, fibrinogen, and vWF, coagulation factors, mitogenic factors, angiogenetic factors, fibrinolytic inhibitors, immunoglobulins, granule membrane-specific proteins such as P-selectin and CD63, and various chemokines including PF4 CXCL4 and RANTES [ 5 , 9 , 10 ].
Dense granules store a variety of hemostatically active nonprotein molecules which are secreted during platelet activation. The third type of granule, platelets lysosomes, contain glycosidases, acid proteases, and cationic proteins that have a bactericidal activity [ 5 , 10 ]. The major effect of these cytokines is to regulate leukocyte movement, migration from the vasculature into the tissues, and other proinflammatory functions like phagocytosis and generation of ROS [ 5 , 21 ].
Platelets express numerous adhesion molecules and ligands that facilitate interactions between platelets, leukocytes, and endothelium [ 5 , 6 , 9 , 10 , 22 ].
Platelets express large amount of P-selectin, which has a key role in linking hemostasis and inflammation [ 5 , 10 ]. They mediate interactions with extracellular matrix ECM molecules and adhesion molecules on other cells [ 10 , 22 ]. This complex array of adhesion molecules and ligands allow and facilitate platelets to bind to a number of diverse cellular and structural targets under shear conditions [ 10 ].
The interaction between platelets, leukocytes, and endothelium can occur in various ways. In addition to serving as a platform to which leukocytes can adhere, platelets also have the capacity to modulate the expression and activation of adhesion molecules on other cell types such as neutrophils, monocytes, lymphocytes, and endothelium.
Platelet-neutrophil and platelet-monocyte aggregates have been detected in the blood of humans with a variety of diseases and are now considered as one of the most sensitive markers related to platelet activation [ 17 — 19 ]. They may reflect a prothrombotic state and are reported to be associated with acute coronary syndrome, systemic inflammatory conditions, and neoplastic and autoimmune diseases.
In recent years, platelets have emerged as important markers for various types of diseases. They are multifunctional blood particles and now regarded to be very important clinical targets for many disease pathophysiology. In addition to playing a central role in normal hemostasis and thrombosis, platelets can make important contributions to host inflammatory and immune responses to infection or injury. Under uncontrolled pathological conditions, they have profound roles in pathogenic processes underlying atherosclerosis and cardiovascular diseases, uncontrolled inflammation, tumor metastasis, and neurodegenerative diseases including Alzheimer's disease [ 17 — 25 ].
Upon activation, platelet surface P-selectin is overexpressed, and platelets secret their granule contents into circulation. Because these markers have relatively short detectability in the circulating blood, platelet-monocyte aggregates have emerged as markers for platelet activation [ 18 , 19 ].
Platelet-monocyte aggregates have longer persistence in peripheral blood and were shown to be more sensitive markers of in vivo platelet activation than other platelet surface markers. They serve to link coagulation and the development of atherosclerosis. Microparticles are plasma membrane-derived vesicles ranging in diameter from 0. Platelets and megakaryocytes are the primary source of microparticles in the blood circulation. They can carry nuclear and cytoplasmic components from their parent cells and transfer this information to affect nearby or distant cells [ 20 , 25 , 27 ].
Platelet microparticles contain proteins, lipids, and RNA derived from their precursor cells. Therefore, circulating platelet microparticles can be considered as biologic markers associated with platelet activation.
Previous studies have reported that the levels of circulating platelet microparticles were increased in patients with various diseases such as hypertension, atherosclerosis, and stroke [ 27 ]. A few studies have demonstrated a predictive value of platelet microparticles in thrombotic diseases [ 20 ].
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