The Blood Platelet as a Model for Regulating Blood Coagulation on Cell Surfaces and Its Consequences

Platelets actively participate in regulating thrombin production following physical or chemical injury to blood vessels. Injury to blood vessels initiates activation of the large numbers of platelets that appear in the subendothelium where they become exposed to tissue factor and to molecules adhesive for platelets and normally found in the extracellular matrix. The complex of plasma factor VIIa with extravascular tissue factor both initiates and localizes thrombin production on platelets and on extravascular cells. Thrombin production at these sites in turn enhances platelet activation and the subsequent hemostatic plug formation to minimize bleeding. Thrombin production and platelet activation also initiate the process of wound healing requiring thrombin-dependent cell activation and platelet-dependent formation of new blood vessels (angiogenesis). Activated platelets release from their storage granules several proteins and other factors that regulate local thrombin formation and the responses of blood vessel cells to injury to assure hemostasis and effective wound healing. Failure to localize and adequately regulate thrombin production and/or platelet activation can have pathological consequences, including the development and propagation of atherosclerosis and enhancement of tumor development. The primary basis for the pathological consequences of the failure to adequately regulate thrombin production is that the multi-functional thrombin activates several types of cells to initiate their mitogenesis. Mitogenesis precedes many of the undesirable consequences of poorly regulated thrombin production and platelet activation. In addition, activated platelets release a variety of products which influence the functions of several cell types to the extent that inadequate regulation of platelet activation (by excessive thrombin production) could contribute to the pathogenesis of acute and chronic arterial thrombosis and to tumor development. Activated platelets participate in tumor development by releasing several factors that positively (and negatively) regulate blood vessel formation.     

Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids

Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.     

A few immobilized thrombins are sufficient for platelet spreading

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.     

Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets

One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.     

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.     

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.     

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.     

Proteome changes in platelets activated by arachidonic acid,collagen, and thrombin

Background  

Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS.     

Differential expression of CD42b and CD9 proteins in platelets and extracellular membrane vesicles during platelet-concentrate storage

Platelet concentrate is used to restore and maintain hemostasis in patients with a reduced number or activity of platelets. Platelet concentrate changes its properties when stored, which leads to a weakening of the therapeutic effect, as well as the occurrence of transfusion side effects. One of the processes that alter platelet concentrates during storage is the secretion of several types of membrane vesicles. Despite the fact that platelet-derived membrane vesicles affect homeostasis and transmit signals of intercellular communication, mechanisms of their formation, features of regulatory activity and molecular composition are still poorly understood. In this paper, the results of analysis of CD42b and CD9 membrane proteins expression in platelets and platelet-derived membrane vesicles during platelet-concentrate storage are presented. Populations of membrane vesicles of different sizes were isolated from the platelet concentrates and characterized. Aggregation and morphological alteration of platelets are observed during storage; the protein composition of platelets, as well as membrane vesicles, changes, and there is a significant increase in the levels of CD42b and CD9 proteins in fractions of membrane vesicles. The results obtained indicate that platelet concentrates contain different populations of membrane vesicles, the molecular composition of which varies during storage.     

Finite platelet size could be responsible for platelet margination effect

Blood flows through vessels as a segregated suspension. Erythrocytes distribute closer to the vessel axis, whereas platelets accumulate near vessel walls. Directed platelet migration to the vessel walls promotes their hemostatic function. The mechanisms underlying this migration remain poorly understood, although various hypotheses have been proposed to explain this phenomenon (e.g., the available volume model and the drift-flux model). To study this issue, we constructed a mathematical model that predicts the platelet distribution profile across the flow in the presence of erythrocytes. This model considers platelet and erythrocyte dimensions and assumes an even platelet distribution between erythrocytes. The model predictions agree with available experimental data for near-wall layer margination using platelets and platelet-modeling particles and the lateral migration rate for these particles. Our analysis shows that the strong expulsion of the platelets from the core to the periphery of the blood vessel may mainly arise from the finite size of the platelets, which impedes their positioning in between the densely packed erythrocytes in the core. This result provides what we believe is a new insight into the rheological control of platelet hemostasis by erythrocytes.     

Effect of soluble fibrin on the blood coagulation process and platelets aggregation

The accumulation of soluble fibrin (SF) in the blood plasma causes acceleration of the final stage of blood coagulation. It increases functional activity of a hemostasis system platelet link, that is the precondition of thrombotic complication. Accumulation of SF in the blood plasma is accompanied by proportional reduction of coagulation time in ancistron and thrombin time tests, and also the intensification of platelets aggregation process. A conclusion was drawn that for early diagnostics of the DIC-syndrom it is expedient to carry out complex estimation of the hemostasis system with obligatory definition of the blood SF content, performance of ancistron and thrombin time tests, and also study of platelets aggregation.     

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.     

Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy.

The redistribution of platelet constituents during activation is essential for their physiological function of maintaining hemostasis. We report here about real time investigations of the activation of native human platelets under physiological conditions from the initial formation of filopodia to the fully spread form by atomic force microscopy. We followed the trafficking of granules and their interaction with the plasma membrane within single cells. Our results show movement of certain granula towards the lamellipodia. Analysis of this rearrangement and the subsequent enlargement of the platelet surface reveals details of the membrane spreading process. Images of living cells are presented that show the distribution of cytoskeletal components and membrane-bound filaments at a resolution of better than 50 nm. The local minimum forces between the tip and the platelets were estimated to be smaller than 60 pN. A model for the elastic contributions of the glycocalix to the tip/membrane interaction was developed using the theory of grafted polymers.     

Influence of cysteine proteinase inhibitors on platelet and plasma components of blood coagulation system

Factor XIIIa plays an important role in stabilization of formed fibrin clot during blood coagulation. Recent studies proved that factor XIIIa affects formation of coated platelets, which are highly procoagulant and characterized by a high level of alpha-granular proteins on their surface and expose surface phosphatidylserine after platelet activation. The ability of newly found cysteine proteinase inhibitors (CPIs) from plants to affect thiol group of the factor XIIIa active centre was recently discovered. Here, the effect of CPIs on the formation of coated platelets and activity of plasma components during blood coagulation process was investigated. It was found that CPIs dose-dependently decreased the fraction of coated platelets in the total platelet population during platelet activation and decreased endogenous thrombin potential (ETP) by 40% for thrombin generation in platelet-rich as well as in platelet-poor plasma. Such decrease of ETP could not be explained by the CPIs influence on factor XIIIa. Investigation of the effects of these inhibitors on factor Xa and thrombin activity has shown that CPIs dose-dependently inhibited their activity and might cause an ETP decrease. Thus, the obtained data indicated that CPIs affected both platelet and plasma components of blood coagulation system.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

Thrombin as a Regulator of Inflammation and Reparative Processes in Tissues

Activation of blood coagulation and thrombin formation accompany inflammation, wound healing, atherogenesis, and other processes induced by endothelial injury. Systems of hemostasis and inflammation play an important role in the pathogenesis of acute coronary syndromes. This paper reviews thrombin functions involved in its interaction with PAR family receptors, activation of platelets, endothelial cells, leukocytes, smooth muscle cells, and mast cells. Mechanisms of regulatory effects of thrombin on mast cells associated with nitric oxide release are discussed.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.