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1
Rinder, HM, JL Bonan, CS Rinder, KA Ault, and BR Smith. "Dynamics of leukocyte-platelet adhesion in whole blood." Blood 78, no. 7 (1991): 1730–37. http://dx.doi.org/10.1182/blood.v78.7.1730.1730.
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Abstract The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
2
Rinder, HM, JL Bonan, CS Rinder, KA Ault, and BR Smith. "Dynamics of leukocyte-platelet adhesion in whole blood." Blood 78, no. 7 (1991): 1730–37. http://dx.doi.org/10.1182/blood.v78.7.1730.bloodjournal7871730.
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The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
3
Bender, Markus, Anita Eckly, John H. Hartwig, et al. "ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing." Blood 116, no. 10 (2010): 1767–75. http://dx.doi.org/10.1182/blood-2010-03-274340.
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Abstract The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.
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Wilkins, Ngozi A., Brian Storrie, and Jeffrey A. Kamykowski. "Characterization of Platelet Alpha-Granule Dynamics." Blood 116, no. 21 (2010): 327. http://dx.doi.org/10.1182/blood.v116.21.327.327.
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Abstract Abstract 327 Background: Platelets, anucleated cells that play a critical role in blood clotting, store proteins and small molecules in alpha-granules and dense granules, respectively, for secretion. Alpha-granules contain several proteins including von Willebrand factor and fibrinogen and dense granules contain serotonin. Rab4, a marker for the early endosomes has been implicated in regulating alpha granule secretions (Sirakawa et al, 2010). Previous fluorescence microscopy mapping of alpha-granule protein distributions suggested that there are either two different alpha-granule types or subdomains within a single granule population (Storrie and Seghal, 2007; Italiano et al, 2008). More recent work based on electron tomography (Kamykowski et al, manuscript in preparation) indicates that human platelets are comprised of one alpha granule population. We hypothesized that there was a single population of alpha-granules in which all fibrinogen is similarly compartmentalized. Hence, fibrinogen endocytocized by guinea pig megakaryocytes and platelets in vivo at 4 h (short label) and 24 h (long label) would map to the same location. Aims: We carried out several experiments to form a basis for future high-resolution (5 nm) electron tomography to establish packaging of HRP-conjugated fibrinogen or nanogold conjugated fibrinogen into platelet alpha-granules. (a) Using PD-10 columns, we prepared Cy3 conjugated fibrinogen. Using an in vivo guinea pig model to test the ability of guinea pig platelets to take up fluorescently labeled fibrinogen, we injected 10 mg/ml of Cy3 conjugated fibrinogen (short label, 4 h) and 10 mg/ml of commercially purchased AlexaFluor 488 conjugated fibrinogen (long label, 28 h) into guinea pigs. Platelets were then fixed, purified and confocal microscopy performed. (b) Using triple immunofluorescence, serotonin antibody was applied to fixed and purified resting state human and guinea pig platelets and immunofluorescence microscopy was performed to provide whole platelet information on the staining pattern of the dense granules in comparison to the alpha-granules and early endosomes. (c) Preliminary Electron Microscopy fixation conditions were also tested on guinea pig platelets. Results: For the uptake experiment, spinning-disk confocal microscopy was used to collect full platelet volume image stacks which were then deconvolved, pixel shift corrected for red and green channels and analyzed. Overlap of green and red fibrinogen conjugates was observed where the fluorescently tagged fibrinogens were taken up by structures presumed to be alpha-granules. For the triple labeling experiments, the distribution of serotonin, Rab4 and von Willebrand factor was observed in resting state platelets. Using spinning-disk confocal microscopy, full platelet volume image stacks were collected, deconvolved, pixel shift corrected for red, far red and green channels and analyzed. Serotonin antibody gave an abundant punctate staining pattern in both the triple-labeled human and guinea pig platelets. In both the human platelets and the guinea pig platelets, the serotonin positive punctate granules, presumed to be dense granules, had a more similar pattern to the von Willebrand factor positive punctate alpha granules, than to the Rab4 positive punctate granules, presumed to be the early endosomes. The triple label results were unexpected because previous electron microscopy studies have indicated that the dense granules in human platelets are fewer in number than the alpha-granules and fewer than the corresponding dense granules in guinea pig platelets. Results of the electron microscopy preparations are pending. Conclusions: Our results indicate that the guinea pig model, while its platelets are a much smaller size than human platelets, is a good system for loading alpha-granules with labeled proteins for electron tomography. The serotonin distribution results together with previous electron tomography also raise the question as to whether dense granules could be a specialized form of the alpha-granules. A summary of this research will be presented at the Promoting Minorities in Hematology event during the 2010 ASH meeting. Disclosures: No relevant conflicts of interest to declare.
5
Dubois, Christophe, Laurence Panicot-Dubois, Barbara C. Furie, and Bruce Furie. "Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse." Blood 106, no. 11 (2005): 649. http://dx.doi.org/10.1182/blood.v106.11.649.649.
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Abstract Platelet accumulation at sites of vascular injury arrests bleeding but also plays a critical role in the pathogenesis of thrombosis, leading to ischemia in myocardial infarction or stroke. Intracellular calcium mobilization in platelets is a critical step in the activation of platelets and formation of the platelet thrombus. Here we show the relationship of the dynamics of intracellular calcium mobilization with platelet accumulation into the developing thrombus in a living mouse. Following injection of 100 x 106 fura-2 loaded platelets into a living mouse we used high speed intravital multi-channel digital fluorescence microscopy to monitor calcium status in circulating and thrombus-bound platelets during thrombus development. One population of platelets binds transiently to the developing thrombus but does not mobilize calcium. The mean duration of platelet-thrombus interaction for these platelets is 11 sec. Another population of platelets undergoes calcium mobilization after binding to the developing thrombus. The time interval from attachment to calcium mobilization for individual platelets varied from 1.0 to 12 sec, with a median of 3.5 sec. More than 90% of platelets that undergo calcium mobilization do so with in 5 sec of attachment. The calcium mobilization in the thrombus bound platelets is reversible. About two thirds of the platelets return rapidly to the basal Ca2+ state while the remaining thrombus bound platelets maintain an elevated Ca2+ level for an extended period. The mean duration of platelet-thrombus interaction is 35 sec with a range of 1.5 sec to 284 sec (median duration 39.5 sec) as calculated from multiple independent observations of single platelets. In each platelet studied, only one calcium peak is detected per platelet. There is a close correlation between the duration of calcium mobilization in an individual platelet and the time that the platelet remains attached to the developing thrombus, suggesting a relationship of calcium-dependent events and platelet-thrombus affinity. A population of platelets binds to the thrombus, mobilizes calcium and remains associated with the thrombus. Using widefield deconvolution techniques to obtain planar images and increased numbers of dye-loaded platelets, individual platelets could be observed undergoing sustained calcium elevation within the thrombus. As the platelet thrombus reaches maximal size at about 120 sec, calcium mobilization continues in the stable core of the thrombus for several minutes, then decreases. These studies describe thrombus formation in a living animal under conditions in which the endothelium and vessel wall, blood cells and plasma components, and flowing blood are preserved in the absence of anticoagulants. Our results indicate that stable platelet thrombus formation is dependent upon durable calcium mobilization, and that intracellular calcium regulates thrombus development and maturation in vivo.
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Falet, Hervé, Gregory Chang, Brigitte Brohard-Bohn, Francine Rendu та John H. Hartwig. "Integrin αIIbβ3signals lead cofilin to accelerate platelet actin dynamics". American Journal of Physiology-Cell Physiology 289, № 4 (2005): C819—C825. http://dx.doi.org/10.1152/ajpcell.00587.2004.
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Cofilin, in its Ser3 dephosphorylated form, accelerates actin filament turnover in cells. We report here the role of cofilin in platelet actin assembly. Cofilin is primarily phosphorylated in the resting platelet as evidenced by a specific antibody directed against its Ser3 phosphorylated form. After stimulation with thrombin under nonstirring conditions, cofilin is reversibly dephosphorylated and transiently incorporates into the actin cytoskeleton. Its dephosphorylation is maximal 1–2 min after platelet stimulation, shortly after the peak of actin assembly occurs. Cofilin rephosphorylation begins 2 min after activation and exceeds resting levels by 5–10 min. Cofilin is dephosphorylated with identical kinetics but fails to become rephosphorylated when platelets are stimulated under stirring conditions. Cofilin is normally rephosphorylated when platelets are stimulated in the presence of Arg-Gly-Asp-Ser (RGDS) peptide or wortmannin to block αIIbβ3cross-linking and signaling or in platelets isolated from a patient with Glanzmann thrombasthenia, which express only 2–3% of normal αIIbβ3levels. Furthermore, actin assembly and Arp2/3 complex incorporation in the platelet actin cytoskeleton are decreased when αIIbβ3is engaged. Our results suggest that cofilin is essential for actin dynamics mediated by outside-in signals in activated platelets.
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Patel, Dipti, Heikki Väänänen, Markéta Jiroušková, Thomas Hoffmann, Carol Bodian, and Barry S. Coller. "Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy." Blood 101, no. 3 (2003): 929–36. http://dx.doi.org/10.1182/blood.v101.3.929.
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Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.
8
Filipovic, N., M. Kojic, and A. Tsuda. "Modelling thrombosis using dissipative particle dynamics method." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 366, no. 1879 (2008): 3265–79. http://dx.doi.org/10.1098/rsta.2008.0097.
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Aim . Arterial occlusion is a leading cause of cardiovascular disease. The main mechanism causing vessel occlusion is thrombus formation, which may be initiated by the activation of platelets. The focus of this study is on the mechanical aspects of platelet-mediated thrombosis which includes the motion, collision, adhesion and aggregation of activated platelets in the blood. A review of the existing continuum-based models is given. A mechanical model of platelet accumulation onto the vessel wall is developed using the dissipative particle dynamics (DPD) method in which the blood (i.e. colloidal-composed medium) is treated as a group of mesoscale particles interacting through conservative, dissipative, attractive and random forces. Methods . Colloidal fluid components (plasma and platelets) are discretized by mesoscopic (micrometre-size) particles that move according to Newton's law. The size of each mesoscopic particle is small enough to allow tracking of each constituent of the colloidal fluid, but significantly larger than the size of atoms such that, in contrast to the molecular dynamics approach, detailed atomic level analysis is not required. Results . To test this model, we simulated the deposition of platelets onto the wall of an expanded tube and compared our computed results with the experimental data of Karino et al . ( Miscrovasc. Res. 17 , 238–269, 1977). By matching our simulations to the experimental results, the platelet aggregation/adhesion binding force (characterized by an effective spring constant) was determined and found to be within a physiologically reasonable range. Conclusion . Our results suggest that the DPD method offers a promising new approach to the modelling of platelet-mediated thrombosis. The DPD model includes interaction forces between platelets both when they are in the resting state (non-activated) and when they are activated, and therefore it can be extended to the analysis of kinetics of binding and other phenomena relevant to thrombosis.
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Suzuki, Aae, Lurong Lian, Liang Zhao, et al. "Mice That Lack RhoA In Their Platelets Have Normal Actin Dynamics, but Have Macrothrombocytopenia." Blood 116, no. 21 (2010): 549. http://dx.doi.org/10.1182/blood.v116.21.549.549.
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Abstract Abstract 549 In response to agonist stimulation, platelets undergo a rapid reorganization of their actin cytoskeleton. This process involves simultaneous disassembly and assembly of filamentous actin, and is one of the earliest phenomena seen in platelet activation. Ex vivo flow models suggest that the platelet cytoskeleton is required for platelet adhesion that can withstand the shear conditions found within the arterial vascular system. The signaling pathways that link external stimuli with actin assembly are believed to include polyphosphoinositides, small GTP-binding proteins, and actin binding proteins. Extrapolations of data, mostly derived from tissue culture cell lines, suggest that a central component of this signaling cascade is the small GTP binding protein, RhoA. A few studies using a RhoA-specific pharmacologic inhibitor, C3 exotoxin, suggest that RhoA is essential for platelet spreading and focal adhesion formation. These findings support the hypothesis that RhoA within platelets is critical for the cytoskeletal dependent processes that contribute to platelet plug formation. To determine the true in vivo role of RhoA within platelets, we utilized a murine genetic approach. Mice were genetically modified to contain conditional RhoA null mutation by inserting LoxP sites flanking exon 3. This exon encodes the P-loop and Switch 1 domains within this protein. RhoA fl/fl mice were crossed with Platelet factor 4 (PF4) expressing Cre mice. The PF4 promotor leads to Cre expression exclusively in platelets and megakaryocytes, thereby producing homologous recombination at the LoxP sites, and deletion the critical exon only within these cells. This end result of this breeding strategy produced RhoA fl/fl PF4 Cre+ mice that specifically lacked RhoA only in their platelets and megakaryocytes. RhoA fl/fl PF4 Cre+ mice were compared with their RhoA fl/fl PF4 Cre- littermates. RhoA fl/fl PF4 Cre+ mice appeared normal, but had platelet counts that were 30% +/− 3% lower than normal. The mean platelet volume was also increased by 25 % +/− 7 % in the RhoA-null platelets. Review of the peripheral blood smears confirmed that the mice had macrothrombocytopenia, but did not reveal any abnormalities in the erythrocytes or leukocytes of the mice. Examination of the bone marrows from RhoA fl/fl PF4 Cre+ mice demonstrated that they had at least as many megakaryocytes as RhoA fl/fl PF4 Cre- mice. But compared to the control cells, the RhoA-null megakaryocytes were larger, more lobulated, and had more cytoplasm. Furthermore, the thromobocytopenia is probably not due to splenic sequestration because the spleens of RhoA fl/fl PF4 Cre- mice were only minimally larger (less than 10%) than those of the control mice. These results suggest that the mechanism for thrombocytopenia is due to peripheral destruction. Platelets derived from RhoA fl/fl PF4 Cre+ mice were studied ex vivo, and were found to undergo shape change and aggregate normally in response to thrombin, collagen, and the thromboxane A2 analog, U46619. Surprisingly, platelet adhesion and cell spreading was also unaffected by the loss of RhoA. It is also remarkable that total F-actin (as assessed by phalloidin staining) was identical in the platelets derived from RhoA fl/fl PF4 Cre+ and RhoA fl/fl PF4 Cre- mice. Our results definitively refute the model that RhoA is an essential component of platelet actin dynamics and platelet adhesion. Instead our findings surprisingly indicate that loss of the platelet RhoA causes macrothrombocytopenia. Our data suggests that the development of macrothrombocytopenia is due to an intrinsic platelet abnormality that leads to a shortened platelet lifespan. Disclosures: No relevant conflicts of interest to declare.
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Patel-Hett, Sunita, Jennifer L. Richardson, Harald Schulze, et al. "Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules." Blood 111, no. 9 (2008): 4605–16. http://dx.doi.org/10.1182/blood-2007-10-118844.
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Abstract The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.
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Haley, Kristina M., Joseph E. Aslan, Garth W. Tormoen, et al. "The PAK Signaling System Links Rho Gtpase Activation to Platelet Lamellopodia Formation, Aggregation and Aggregate Stability Under Shear." Blood 120, no. 21 (2012): 1060. http://dx.doi.org/10.1182/blood.v120.21.1060.1060.
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Abstract Abstract 1060 Platelets serve as the primary mediators of hemostasis and thrombosis, circulating as surveyors for gaps in vascular integrity. As platelets encounter exposed extracellular matrix proteins, receptors on the platelet surface trigger intracellular signaling events that result in rapid platelet activation and a complex rearrangement of platelet morphology to form filopodia and lamellipodia. Rac1, a member of the Rho GTPase family, has emerged as a key regulator in platelet actin dynamics. However, the specific downstream events following Rac1 activation that mediate platelet actin cytoskeleton reorganization remain ill-defined. The Rho GTPase, Rac, supports the autocatalytic activation of the p21 activated kinases, or PAKs, to mediate actin reorganization processes in focal adhesion formation and cell migration. Upon activation by GTP-bound Rac, the PAKs phosphorylate a number of substrates to coordinate actin dynamics. Platelets express a number of PAK isoforms, and like Rac, PAK has been shown to be activated as platelets spread on collagen in a Src and PI3K dependent manner. Furthermore, the adaptor protein SLP-76 has been proposed to potentiate PAK activity downstream of Rac activation to mediate platelet lamellipodia formation. However, the specific roles of PAK in platelet function have yet to be characterized. Thus we set out to elucidate the role of PAK in platelet function and to define the connection between Rac activation, PAK, and platelet cytoskeletal reorganization. Our initial experiments with mass spectrometry revealed that following platelet activation, Rac1 associates with a set of PAK effectors, GIT1, GEFH1, LIMK1, and Merlin. We next demonstrated a co-localization of Rac1 and PAK with actin at the leading edge of spread platelets on fibrinogen. In addition, inhibition of PAK signaling by two different pharmacologic inhibitors blocked platelet focal adhesion and lamellopodia formation on both fibrinogen and collagen. Inhibition of PAK signaling abrogated intracellular calcium mobilization in platelets, prevented platelet aggregation to the GPVI-agonist, CRP, and destabilized platelet lamellipodia, resulting in the retraction of lamellipodia in spread platelets. Finally, inhibition of PAK resulted in the disaggregation of platelet aggregates formed under shear flow conditions. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation to PAK effector function and platelet lamellopodia formation, thus filling an important gap in the understanding of platelet actin cytoskeletal organization. In addition, these data characterize the integral role of PAK in platelet spreading, aggregation, and aggregate stability. Elucidating the mechanisms that mediate platelet spreading and aggregate formation may highlight important steps in the platelet activation cascade at which to pharmacologically intervene in order to inhibit or treat pathologic thrombi formation. Disclosures: No relevant conflicts of interest to declare.
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Flaumenhaft, Robert C., Secil Koseoglu, James R. Dilks, et al. "Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Secretion and Thrombus Formation In Vivo." Blood 118, no. 21 (2011): 361. http://dx.doi.org/10.1182/blood.v118.21.361.361.
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Abstract Abstract 361 Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC50 of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC50 of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca2+ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Disclosures: No relevant conflicts of interest to declare.
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JACKSON, S. P., W. S. NESBITT, and E. WESTEIN. "Dynamics of platelet thrombus formation." Journal of Thrombosis and Haemostasis 7 (July 2009): 17–20. http://dx.doi.org/10.1111/j.1538-7836.2009.03401.x.
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Tykhomyrov, A. A. "Dynamics of thrombin-induced exposition of actin on the platelet surface." Ukrainian Biochemical Journal 86, no. 5 (2014): 74–81. http://dx.doi.org/10.15407/ubj86.05.074.
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Stalker, Timothy J., Jie Wu, and Lawrence F. Brass. "Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo." Blood 124, no. 21 (2014): 96. http://dx.doi.org/10.1182/blood.v124.21.96.96.
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Abstract Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo. Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy. Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core. Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute. Disclosures Stalker: Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.
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Dunne, Eimear, Qin M. Qi, Eric S. Shaqfeh, et al. "Blood group alters platelet binding kinetics to von Willebrand factor and consequently platelet function." Blood 133, no. 12 (2019): 1371–77. http://dx.doi.org/10.1182/blood-2018-06-855528.
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Abstract Blood type O is associated with a lower risk of myocardial infarction. Platelets play a critical role in myocardial infarction. It is not known whether the expression of blood group antigens on platelet proteins alters platelet function; we hypothesized that platelet function would be different between donors with blood type O and those with non-O. To address this hypothesis, we perfused blood from healthy type O donors (n = 33) or non-O donors (n = 54) over pooled plasma derived von Willebrand factor (VWF) protein and purified blood type–specific VWF at arterial shear and measured platelet translocation dynamics. We demonstrate for the first time that type O platelets travel farther at greater speeds before forming stable bonds with VWF. To further characterize these findings, we used a novel analytical model of platelet interaction. Modeling revealed that the kinetics for GPIb/VWF binding rate are significantly lower for type O compared with non-O platelets. Our results demonstrate that platelets from type O donors interact less with VWF at arterial shear than non-O platelets. Our results suggest a potential mechanism for the reduced risk of myocardial infarction associated with blood type O.
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Italiano, Joseph E., Jennifer L. Richardson, Harald Schulze, et al. "The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array." Blood 106, no. 11 (2005): 1653. http://dx.doi.org/10.1182/blood.v106.11.1653.1653.
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Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.
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Yu, Zhiqian, Mami Ohba, Masanori Nakamura, et al. "Dynamics of platelet mobilisation into lungs in response to 5-hydroxytryptamine (serotonin) in mice." Thrombosis and Haemostasis 102, no. 12 (2009): 1251–58. http://dx.doi.org/10.1160/th08-06-0406.
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SummaryIn experimental animals, the lung rapidly removes intravenously injected 5-hydroxytryptamine (5HT), but the mechanism underlying this pulmonary 5HT removal (P-5HT-R) and the responsible cells remains unclear. 5HT reportedly induces rapid pulmonary platelet accumulation (P-PLT-A). Here, we examined the relationship between P-5HT-R and P-PLT-A in mice by comparing the platelet count in the blood with the endogenous 5HT in the tissues (a marker for platelets because the 5HT is largely contained within platelets). 5HT levels in murine blood and tissues were also examined after intravenous injection of 5HT. The data revealed that: (i) 5HT injection (at ≥ 0.04 μmol/kg) induced a transient P-PLT-A (occurring within 6 seconds), (ii) platelets rapidly took up injected 5HT, (iii) the P-5HT-R was saturated following injection of 5HT at 1 μmol/kg, (iv) ketanserin (5HT2-receptor antagonist) strongly inhibited P-PLT-A, (v) under fluoxetine (5HT-uptake inhibitor), 5HT levels at 6 seconds after 5HT injection were markedly higher in blood, but significantly lower in lung (versus fluoxetine-untreated mice), (vi) P-5HT-R was barely detectable in mutant mice with platelets lacking dense bodies, and was much reduced in platelet-depleted mice, (vii) 5HT injected intravenously at 10 μmol/kg had a half-life in the lung of < 20 seconds, and (viii) unlike 5HT, injected histamine was largely excreted by the kidney. These results demonstrate that platelets rapidly translocate into the lung upon stimulation of 5HT2 receptors, take up 5HT (and possibly swiftly metabolise it), and then return to the circulation. Hence, pulmonary platelet accumulation plays an important role in pulmonary 5HT removal in mice.
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Steiner, M. "Dynamics of Platelet Tubulin in Response to Changes in Free Sulfhydryl Groups." Thrombosis and Haemostasis 53, no. 02 (1985): 176–79. http://dx.doi.org/10.1055/s-0038-1661267.
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SummaryPools of polymerized and total tubulin were measured in human platelets as a function of free sulfhydryl groups both in acid-soluble and acid-precipitable cell fractions. Changes in free thiols were produced either by storage of platelets at room temperature or by addition of the potent oxidizing agent diazene dicarboxylic acid (diamide) and were correlated with shifts in the dynamic equilibrium between assembled and disassembled microtubules and platelet aggregation. Diamide at concentrations of 0.5 to 5 mM depleted acid soluble SH groups and reduced protein thiols while causing a progressive decrease in polymerized tubulin. Similar changes, although not as severe, were initiated by storage of platelets at room temperature. Platelet aggregation especially that induced by collagen showed a positive correlation with the pool of polymerized tubulin. Our results indicate that the state of oxidation of sulfhydryl groups especially in the acid- precipitable fraction plays an important role in determining the position of equilibrium between polymerized and depolymerized tubulin.
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Vasilev, Grigorii A., Aleksandra A. Filkova, and Anastasia N. Sveshnikova. "Study of Reversible Platelet Aggregation Model by Nonlinear Dynamics." Mathematics 9, no. 7 (2021): 759. http://dx.doi.org/10.3390/math9070759.
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Blood cell platelets form aggregates upon vessel wall injury. Under certain conditions, a disintegration of the platelet aggregates, called “reversible aggregation”, is observed in vitro. Previously, we have proposed an extremely simple (two equations, five parameters) ordinary differential equation-based mathematical model of the reversible platelet aggregation. That model was based on mass-action law, and the parameters represented probabilities of platelet aggregate formations. Here, we aimed to perform a nonlinear dynamics analysis of this mathematical model to derive the biomedical meaning of the model’s parameters. The model’s parameters were estimated automatically from experimental data in COPASI software. Further analysis was performed in Python 2.7. Contrary to our expectations, for a broad range of parameter values, the model had only one steady state of the stable type node, thus eliminating the initial assumption that the reversibility of the aggregation curve could be explained by the system’s being near a stable focus. Therefore, we conclude that during platelet aggregation, the system is outside of the influence area of the steady state. Further analysis of the model’s parameters demonstrated that the rate constants for the reaction of aggregate formation from existing aggregates determine the reversibility of the aggregation curve. The other parameters of the model influenced either the initial aggregation rate or the quasi-steady state aggregation values.
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Hubbard, William Brad, Meenakshi Banerjee, Hemendra Vekaria, et al. "Differential Leukocyte and Platelet Profiles in Distinct Models of Traumatic Brain Injury." Cells 10, no. 3 (2021): 500. http://dx.doi.org/10.3390/cells10030500.
Full textAbstract:
Traumatic brain injury (TBI) affects over 3 million individuals every year in the U.S. There is growing appreciation that TBI can produce systemic modifications, which are in part propagated through blood–brain barrier (BBB) dysfunction and blood–brain cell interactions. As such, platelets and leukocytes contribute to mechanisms of thromboinflammation after TBI. While these mechanisms have been investigated in experimental models of contusion brain injury, less is known regarding acute alterations following mild closed head injury. To investigate the role of platelet dynamics and bioenergetics after TBI, we employed two distinct, well-established models of TBI in mice: the controlled cortical impact (CCI) model of contusion brain injury and the closed head injury (CHI) model of mild diffuse brain injury. Hematology parameters, platelet-neutrophil aggregation, and platelet respirometry were assessed acutely after injury. CCI resulted in an early drop in blood leukocyte counts, while CHI increased blood leukocyte counts early after injury. Platelet-neutrophil aggregation was altered acutely after CCI compared to sham. Furthermore, platelet bioenergetic coupling efficiency was transiently reduced at 6 h and increased at 24 h post-CCI. After CHI, oxidative phosphorylation in intact platelets was reduced at 6 h and increased at 24 h compared to sham. Taken together, these data demonstrate that brain trauma initiates alterations in platelet-leukocyte dynamics and platelet metabolism, which may be time- and injury-dependent, providing evidence that platelets carry a peripheral signature of brain injury. The unique trend of platelet bioenergetics after two distinct types of TBI suggests the potential for utilization in prognosis.
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Stritt, Simon, Sarah Beck, Isabelle C. Becker, et al. "Twinfilin 2a regulates platelet reactivity and turnover in mice." Blood 130, no. 15 (2017): 1746–56. http://dx.doi.org/10.1182/blood-2017-02-770768.
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Key Points Deficiency in twinfilin 2a causes macrothrombocytopenia and hyperreactivity of platelets in mice. We provide the first in vivo evidence for an inhibitory function of twinfilin 2a in platelet actin dynamics.
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Vyshynska, M. B. "Changes in the functional state of platelets in patients with polytrauma." EMERGENCY MEDICINE 17, no. 1 (2021): 27–32. http://dx.doi.org/10.22141/2224-0586.17.1.2021.225716.
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Background. Polytrauma remains the leading cause of global morbidity and mortality and is the cause of more than 10 % of deaths. The purpose of this research was to study the literature data about changes in vascular platelet hemostasis, to investigate the dynamics of the morphofunctional state of platelets, to analyze changes in intravascular platelet activation in patients with polytrauma. Results. Normal blood clotting requires at least 4 components — blood vessels, platelets, the ability of blood to coagulate and fibrinolysis. Determination of components such as indicators of intravascular platelet activation can be an important step in assessing disorders of platelet hemostasis in patients with polytrauma. Vascular platelet hemostasis begins with primary reflex spasm of arterioles, followed by secondary spasm of arterioles, then primary platelet plug is formed (adhesion and aggregation), and, accordingly, the consolidation of the thrombus, resulting in the formation of the final platelet thrombus. Even before the contact of platelets with naked collagen, the primary activation of platelets occurs. Initially, the shape of intact platelets changes from discoid form to activated cells of discoechinocytes, spherocytes and, or, spheroechinocytes. We found that on day 3 after injury, with a normal number of platelets in the venous blood, the number of intact platelets, discocytes, decreases, the number of active forms of thrombocytes, discoechinocytes and spheroechinocytes, increases, and, accordingly, the total amount of active forms of thrombocytes increases. Normal platelet counts in patients with polytrauma may mask the severity of coagulopathy, and studies of intravascular platelet activation may be a diagnostic component of the vascular platelet hemostasis in patients with polytrauma. Conclusions. In patients with coagulopathy due to polytrauma, there are changes in intravascular platelet activation and platelet aggregation, induced by adrenaline and adenosine diphosphate, on day 3.
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Pula, Giordano, Kai Schuh, Keiko Nakayama, Keiichi I. Nakayama, Ulrich Walter та Alastair W. Poole. "PKCδ regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation". Blood 108, № 13 (2006): 4035–44. http://dx.doi.org/10.1182/blood-2006-05-023739.
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Abstract Protein kinase Cδ (PKCδ) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCδ–/– platelets, we show that the mechanism did not involve altered inside-out signaling to integrin αIIbβ3 and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCγ2 between wild-type and PKCδ–/– platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCδ–/– platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCδ is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCδ physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP–/– platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCδ is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.
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Tauber, Helmuth, Nicole Innerhofer, Daniel von Langen, et al. "Dynamics of Platelet Counts in Major Trauma: The Impact of Haemostatic Resuscitation and Effects of Platelet Transfusion—A Sub-Study of the Randomized Controlled RETIC Trial." Journal of Clinical Medicine 9, no. 8 (2020): 2420. http://dx.doi.org/10.3390/jcm9082420.
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Although platelets play a central role in haemostasis, the dynamics of platelet counts during haemostatic resuscitation, the response to platelet transfusion, and effects on clinical outcome are poorly described for trauma patients. As a sub-study of the already published randomized controlled RETIC Study “Reversal of Trauma-induced Coagulopathy using First-line Coagulation Factor Concentrates or Fresh-Frozen Plasma” trial, we here analysed whether the type of first-line haemostatic resuscitation influences the frequency of platelet transfusion and determined the effects of platelet transfusion in coagulopathic patients with major trauma. Patients randomly received first-line plasma (FFP) or coagulation factor concentrates (CFC), mainly fibrinogen concentrate. In both groups, platelets were transfused to maintain platelet counts between 50 and 100 × 109/L. Transfusion rates were significantly higher in the FFP (n = 44) vs. CFC (n = 50) group (FFP 47.7% vs. CFC 26%); p = 0.0335. Logistic regression analysis adjusted for the stratification variables injury severity score (ISS) and brain injury confirmed that first-line FFP therapy increases the odds for platelet transfusion (odds ratio (OR) 5.79 (1.89 to 20.62), p = 0.0036) and this effect was larger than a 16-point increase in ISS (OR 4.33 (2.17 to 9.74), p = 0.0001). In conclusion, early fibrinogen supplementation exerted a platelet-saving effect while platelet transfusions did not substantially improve platelet count and might contribute to poor clinical outcome.
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de Vrij, Edwin L., Hjalmar R. Bouma, Maaike Goris, et al. "Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids." Journal of Comparative Physiology B 191, no. 3 (2021): 603–15. http://dx.doi.org/10.1007/s00360-021-01351-3.
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AbstractImmobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals—who endure lengthy periods of immobility—do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.e., the hibernation phase with reduction of metabolic rate, low blood flow and immobility. It is unknown whether these platelet dynamics in hibernating hamsters originate from storage and release, as suggested for ground squirrel, or from breakdown and de novo synthesis. A reduction in detaching forces due to low blood flow can induce reversible adhesion of platelets to the vessel wall, which is called margination. Here, we hypothesized that storage-and-release by margination to the vessel wall induces reversible thrombocytopenia in torpor. Therefore, we transfused labeled platelets in hibernating Syrian hamster (Mesocricetus auratus) and platelets were analyzed using flow cytometry and electron microscopy. The half-life of labeled platelets was extended from 20 to 30 h in hibernating animals compared to non-hibernating control hamsters. More than 90% of labeled platelets were cleared from the circulation during torpor, followed by emergence during arousal which supports storage-and-release to govern thrombocytopenia in torpor. Furthermore, the low number of immature platelets, plasma level of interleukin-1α and normal numbers of megakaryocytes in bone marrow make platelet synthesis or megakaryocyte rupture via interleukin-1α unlikely to account for the recovery of platelet counts upon arousal. Finally, using large-scale electron microscopy we revealed platelets to accumulate in liver sinusoids, but not in spleen or lung, during torpor. These results thus demonstrate that platelet dynamics in hibernation are caused by storage and release of platelets, most likely by margination to the vessel wall in liver sinusoids. Translating the molecular mechanisms that govern platelet retention in the liver, may be of major relevance for hemostatic management in (accidental) hypothermia and for the development of novel anti-thrombotic strategies.
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Nikolaieva, I., T. Halenova, and O. Savchuk. "Modern concepts of the role of platelet receptors in the dynamics of thrombus formation." Bulletin of Taras Shevchenko National University of Kyiv. Series: Biology 70, no. 2 (2015): 5–11. http://dx.doi.org/10.17721/1728_2748.2015.70.5-11.
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The study of molecular and biological aspects of the platelets functioning with the use of biochemical methods, new technologies of cell and molecular biology became the basis for understanding signaling cascades regulating the activation, adhesion and aggregation of these cells. In this review, the general modern information of the role of platelet membrane receptors in physiological and pathological processes of thrombus formation was performed. The possible role of platelet receptors as target of antiagregatory agents was analyzed. Also, new promising areas of searching for effective and specific antithrombotic agents were identified.
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Stritt, Simon, Inga Birkholz, Sarah Beck, et al. "Profilin 1–mediated cytoskeletal rearrangements regulate integrin function in mouse platelets." Blood Advances 2, no. 9 (2018): 1040–45. http://dx.doi.org/10.1182/bloodadvances.2017014001.
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Key Points Profilin 1–mediated cytoskeletal dynamics regulate platelet β1- and β3-integrin function and turnover. Profilin 1 deficiency in platelets impairs hemostasis and results in a marked protection from arterial thrombosis.
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Stefanich, Eric, Tauri Senn, Ramon Widmer, Christine Fratino, Gilbert-André Keller, and Paul J. Fielder. "Metabolism of Thrombopoietin (TPO) In Vivo: Determination of the Binding Dynamics for TPO in Mice." Blood 89, no. 11 (1997): 4063–70. http://dx.doi.org/10.1182/blood.v89.11.4063.
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AbstractPrevious in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the “in vivo binding affinity” for rmTPO was approximately 6.4 μg/kg. These data indicate that after a dose of approximately 6.4 μg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.
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Babur, Özgün, Anh T. P. Ngo, Rachel A. Rigg, et al. "Platelet procoagulant phenotype is modulated by a p38-MK2 axis that regulates RTN4/Nogo proximal to the endoplasmic reticulum: utility of pathway analysis." American Journal of Physiology-Cell Physiology 314, no. 5 (2018): C603—C615. http://dx.doi.org/10.1152/ajpcell.00177.2017.
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Upon encountering physiological cues associated with damaged or inflamed endothelium, blood platelets set forth intracellular responses to ultimately support hemostatic plug formation and vascular repair. To gain insights into the molecular events underlying platelet function, we used a combination of interactome, pathway analysis, and other systems biology tools to analyze associations among proteins functionally modified by reversible phosphorylation upon platelet activation. While an interaction analysis mapped out a relative organization of intracellular mediators in platelet signaling, pathway analysis revealed directional signaling relations around protein kinase C (PKC) isoforms and mitogen-activated protein kinases (MAPKs) associated with platelet cytoskeletal dynamics, inflammatory responses, and hemostatic function. Pathway and causality analysis further suggested that platelets activate a specific p38-MK2 axis to phosphorylate RTN4 (reticulon-4, also known as Nogo), a Bcl-xl sequestration protein and critical regulator of endoplasmic reticulum (ER) physiology. In vitro, we find that platelets drive a p38-MK2-RTN4-Bcl-xl pathway associated with the regulation of the ER and platelet phosphatidylserine exposure. Together, our results support the use of pathway tools in the analysis of omics data sets as a means to help generate novel, mechanistic, and testable hypotheses for platelet studies while uncovering RTN4 as a putative regulator of platelet cell physiological responses.
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Dopheide, Sacha M., Mhairi J. Maxwell, and Shaun P. Jackson. "Shear-dependent tether formation during platelet translocation on von Willebrand factor." Blood 99, no. 1 (2002): 159–67. http://dx.doi.org/10.1182/blood.v99.1.159.
Full textAbstract:
The adhesion and aggregation of platelets at sites of vascular injury is dependent on the initial binding of the GP Ib/V/IX receptor complex to immobilized von Willebrand factor (VWF). Under flow conditions, this interaction supports platelet translocation that is characteristically stop-start in nature. High resolution imaging of platelets during surface translocation on immobilized VWF revealed that thin membrane tethers (length: 0.91 μm-47.90 μm) were pulled from the surface of these cells. Membrane tethers were dynamic structures that extended from small, localized adhesion contacts under the influence of flow. Perfusion of platelets in the presence of blocking antibodies against integrin αIIbβ3, or over isolated A1 domains, demonstrated that the VWF–GP Ib interaction was sufficient to induce membrane tether formation. The rate and extent of tether elongation was shear-dependent (shear range: 150 s−1-10 000 s−1), with mean tether length ranging from 3.23 μm to 16.55 μm, tether frequency from 2.67% to 97.33%, and tether growth rate from 0.04 μm/sec to 8.39 μm/sec. Tether formation and retraction did not require platelet activation; however, the growth rate, lifetime, and dimensions were significantly affected by the actin polymerization inhibitor, cytochalasin D, and by chelating intracellular calcium. Single-cell analysis revealed that formation of membrane tethers regulates the stop-start phases of platelet translocation on VWF, suggesting a potentially important role for this phenomenon in regulating the dynamics of the platelet-VWF interaction under flow.
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Kim, Dongjune A., Katrina J. Ashworth, Jorge Di Paola та David N. Ku. "Platelet α-granules are required for occlusive high-shear-rate thrombosis". Blood Advances 4, № 14 (2020): 3258–67. http://dx.doi.org/10.1182/bloodadvances.2020002117.
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Abstract von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.
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Sakurai, Yumiko, David R. Myers, Ashley Kita, and Wilbur Lam. "A Novel Assay That Integrates the Effects of Multiple Agonists At the Single-Platelet Level,." Blood 118, no. 21 (2011): 3258. http://dx.doi.org/10.1182/blood.v118.21.3258.3258.
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Abstract Abstract 3258 Background: At sites of vascular injury, platelets are exposed to multiple agonists that lead to overall activation and platelet plug formation. Signaling pathways induced by these agonists are known to interact with each other. For example, collagen binding to the platelet collagen receptors, α2β1 integrin, CD36, and glycoprotein VI, induces inside-out signaling that ultimately leads to the activation of the glycoprotein IIb/IIIa receptor for fibrinogen on the platelet surface (Nakamura et al, JCB, 1999). An assay capable of tracking the biological effects of multiple different agonists simultaneously within a single platelet would enhance our understanding of how platelets integrate these different signals at the single-platelet level. This assay would have advantages over current clinical assays in that platelet function tests such as platelet aggregometry or the PFA-100 assess only collective behavior of platelet populations without single platelet resolution. Flow cytometry achieves single platelet resolution but cannot monitor the dynamic changes induced by agonists over time. Platelet adhesion assays have the capability to track individual platelets over time via microscopy, but currently cannot simultaneously monitor the different effects of multiple agonists and their potential interactions. To that end, we developed a modified platelet adhesion assay by using microcontact printing to “stamp” distinct micro-to-nanoscale patterns of different platelet agonists and ligands on glass coverslips. Different parts of each platelet are then exposed to different agonists/ligands with our assay and how the different downstream biological signals interact, synergize, and potentially compete can be monitored overall time at the single platelet level via epifluorescence microscopy. Results: Using microfabrication techniques, patterned polydimethylsiloxane (PDMS) stamps were” inked” with two different known platelet agonists/ligands: Collagen type 1 conjugated with FITC (green) and fibrinogen conjugated to Alexa Fluor 594 (red). Using those stamps, protein micropatterns were then “microstamped” and transferred onto the glass surface, creating spatially distinct microprints with two different proteins (Fig. 1). The protein surface was then blocked with 1% BSA. Platelets were isolated and dyed with a fluorescent membrane marker. Platelets were suspended in Tyrode's buffer (20 million platelets per mL) and incubated with the double micro-patterned surface. After incubation, the surface was washed and imaged with epifluorescence microscopy using a 40x objective. For these initial proof-of-concept experiments, we observed that when individual platelets are simultaneously exposed to separate areas of fibrinogen as well as collagen, they exhibit a strong “preference” for the fibrinogen over collagen (Fig. 2). Indeed, 79.1% of platelets were observed to almost completely (>75% of the surface area) migrate to the fibrinogen micropattern from the collagen micropattern (Fig. 3). Few platelets were spread equally between the two protein micropatterns, which suggest that platelets eventually “choose” which agonist to settle before it get fully spread and immobilized. Interestingly, platelets with more than 75 % surface area on collagen (8.96 % of total platelets measured) tended to be smaller in size, have filopodia, and have intense granule staining compared to platelets on fibrinogen, which were more fully spread with broad lamellipodia. These data suggest that the platelets that migrated to the fibrinogen micropattern compared to those that preferred collagen may be physiologically distinct. Conclusions and Ongoing Efforts: These data establish the viability of our system to investigate the integrative effects of different agonists at the single platelet level. This assay will enable the further understanding of how different agonist-induced signaling pathways interact. Ongoing experiments will include other agonists/ligands such as von Willebrand factor and thrombin. Although the current data assesses morphologic changes of platelets, we are focusing our efforts on using high resolution fluorescence microscopy to measure and monitor the spatial dynamics of calcium signaling, protein phosphorylation of relevant signaling pathways, and cytoskeletal rearrangement when different parts of single platelets are exposed to different agonists. Disclosures: No relevant conflicts of interest to declare.
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Lorenz, Martin, Herbert Merk, Michael Buchanan, Wolfgang Eisert, and Joanne van Ryn. "Accumulation of radiolabelled platelets and fibrin on the carotid artery of rabbits after angioplasty: effects of heparin and dipyridamole." Thrombosis and Haemostasis 90, no. 12 (2003): 1179–86. http://dx.doi.org/10.1160/th03-05-0305.
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SummaryWe investigated the dynamic accumulation of platelets and fibrin after balloon injury of the carotid arteries in rabbits in vivo. In addition, effects of heparin and dipyridamole treatment were also tested. Autologous 99mTc-labelled platelet and 123I-labelled fibrin accumulation was measured at one minute intervals for 4 hours following balloon injury of the carotid artery. Platelet accumulation occurred rapidly, with a ~125% increase occurring within 30 min after injury. There was no further activity for up to 4 hours. This accumulation could be inhibited with an intravenous infusion of PGI2 (500 ng/kg/hr). Fibrin accumulation occurred slowly and continuously over the 4 hour measurement period. Injection of an anti-fibrin antibody inhibited fibrin accumulation. Heparin (25 U/kg/hr for 4 hrs) administration resulted in a significant 82 ± 19% and 68 ± 13% reduction in platelet and fibrin accumulation, respectively. This dose of heparin was associated with a 2-fold prolongation of the aPTT. Dipyridamole (0.45 mg/kg/hr for 4 hrs) resulted in a 46 ± 12% and 70 ± 25% reduction of platelet and fibrin accumulation, respectively. Thus, we demonstrated that the dynamics of platelet and fibrin accumulation following balloon injury in rabbits are very different. The vessel wall continues to be thrombogenic for fibrin up to 4 hours after injury even though platelet accumulation has ceased after one hour. We conclude that the local thrombotic events following balloon injury are complex and that not only platelets but also fibrin is important in regulating responses to injury.
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Wang, Yanfeng, Aae Suzuki, Lurong Lian та ін. "Platelets Lacking PIP5KIγ Have Impaired Cytoskeletal Dynamics and Adhesion, but No Defect in Integrin Activation." Blood 114, № 22 (2009): 772. http://dx.doi.org/10.1182/blood.v114.22.772.772.
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Abstract Abstract 772 Following thrombin stimulation, platelet PIP5KI synthesizes phosphatidylinositol 4,5-bisphosphate (PIP2), which can be hydrolyzed by phospholipase C to generate second messengers such as IP3. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins such as talin. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. We have generated and characterized murine lines lacking individual PIP5KI isoforms. While mice lacking PIP5KIα and PIP5KIα have absent second messenger formation and partially impaired integrin activation, they are viable. In contrast, mice lacking PIP5KIγ die in utero due to a cardiovascular developmental defect. Megakaryocytes derived from PIP5KIγ-null embryos bleb their membranes due to impaired anchoring of the cell membrane with the underlying cytoskeleton. Since platelets can not be obtained from these embryos, we employed a genetic approach. We used a MLC-2v Cre transgene that targets Cre expression to myocytes, and generated living mice lacking PIP5KIγ by a conditional rescue. PIP5KIγ-/- MLC-2v Cre+ mice expressed PIP5KIγ in the myocardium, but they had absent expression of PIP5KIγ in all other analyzed tissue. These mice had normal appearing hearts, brains, livers, and bone marrow morphology, as well as normal platelet counts. Since mice lacking PIP5KIα and PIP5KIβ have impaired platelet PIP2 production that causes absent IP3 formation, we analyzed platelets lacking PIP5KIγ for second messenger formation. Even though PIP5KIγ an abundant PIP5KI isoform in platelets, loss of PIP5KIγ does not affect IP3 formation or Akt phosphorylation. It has been previously demonstrated that PIP5KIγ can directly bind talin, a protein that regulates the function of integrins. An existing proposed model for integrin activation is that talin-associated PIP5KIγ synthesize PIP2. This newly synthesized PIP2 then binds a FERM domain within talin. The model suggests that this complex of PIP5KIγ-PIP2-talin is critical for the final step that stimulates integrins to bind their ligand. We found three lines of evidence that disprove this model of integrin activation. First, we found that PIP5KIγ-/- platelets had normal integrin-mediated aggregation in response to all analyzed doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). Second, we observed that PIP5KIγ-null platelets exhibited normal binding of Jon/A, an antibody that recognizes only the activated form of αIIb/β3. Third, we determined that platelets lacking PIP5KIγ spread normally upon adherent fibrinogen. Together, these results disprove the existing model that PIP5KIγ is a critical component of talin-mediated integrin activation. To determine the true function of PIP5KIγ within platelets, we extended our previous studies by analyzing the role PIP5KIγ plays in the regulation of the cytoskeleton. Therefore, we analyzed platelets lacking this enzyme for their ability to anchor the cytoskeleton to the cell membrane. We used optical tweezers to pull the cell membrane apart from the cytoskeleton. Wild type cells had rigid membranes that resisted stretching by trapped fibrinogen-coated beads that were pulled by the optical trap. In contrast, the PIP5KIγ-null platelets had flexible membranes that were easily stretched, and ultimately allowed membrane tethers to form. We further analyzed whether this defect in anchoring the cell membrane to the underlying cytoskeleton causes a defect in vivo using a carotid artery arterial injury model. Mice lacking platelet PIP5KIγ exhibited unstable adhesion in vivo suggesting that impaired cytoskeletal dynamics causes impaired platelet adhesion under flow. Together, our studies demonstrate that the abundant PIP5KI isoform, PIP5KIγ does not contribute to a pool of PIP2 required for second messenger formation or integrin activation. However it does synthesize the pool of PIP2 required to preserve the integrity of the membrane cytoskeleton, and support stable platelet adhesion under conditions of shear. Disclosures: No relevant conflicts of interest to declare.
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Emel’yanova, A. N., and Yu A. Vitkovskiy. "Lymphocyte-platelet adhesion and aggregation of platelets in patients with erysipelas." Kazan medical journal 93, no. 2 (2012): 216–20. http://dx.doi.org/10.17816/kmj2292.
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Aim. To study the lymphocyte-platelet adhesion in patients with various forms of erysipelas in the dynamics of treatment. Methods. 90 patients with erysipelas participated in the study: 20 with the erythematous-bullous form and 20 with the erythematous form. According to the recurrence of the disease the patients were divided into two groups: the main group (40 patients with the primary form of erysipelas) and the comparison group (50 people with recurrent erysipelas). The control group, which was comparable by sex and age, was comprised of 55 healthy people. The index of lymphocyte-platelet adhesion and hemostasis were studied in all patients. Results. The index of lymphocyte-platelet adhesion sharply declined already during the onset of the disease, despite the fact that no significant changes in the content of the absolute number of lymphocytes compared to healthy individuals during this period were identified. Severe course (erythematous-bullous form) was accompanied by significant shifts in the parameters of lymphocyte-platelet adhesion, and their dynamics made it possible to evaluate the effectiveness of treatment. Conclusion. The ability of lymphocytes to adhere to the surface of platelets is reduced during erysipelas; platelet hyperaggregation develops during the first days of onset of clinical manifestations, the nature and duration of which depends on the form of the disease.
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Savolainen, S., M. T. Syrjälä, K. Liewendahl, J. Gripenberg, and U. Nieminen. "Compartmental analysis of short-lived platelet dynamics." Scandinavian Journal of Clinical and Laboratory Investigation 50, no. 6 (1990): 679–86. http://dx.doi.org/10.3109/00365519009089187.
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Dragnea, Elena-Mihaela. "The dynamics of platelet volume in sepsis." Journal of Contemporary Clinical Practice 2, no. 2 (2016): 75–76. http://dx.doi.org/10.18683/jccp.2016.1016.
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Vignesh, Pandiarajan, and Surjit Singh. "Platelet Activation Dynamics in Kawasaki Disease– Revisited." Indian Journal of Pediatrics 86, no. 3 (2019): 216–17. http://dx.doi.org/10.1007/s12098-019-02890-y.
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Lee, Dooyoung, Karen P. Fong, Michael R. King, Lawrence F. Brass, and Daniel A. Hammer. "Differential Dynamics of Platelet Contact and Spreading." Biophysical Journal 102, no. 3 (2012): 472–82. http://dx.doi.org/10.1016/j.bpj.2011.10.056.
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Tesakov, I. P., A. A. Martyanov, A. E. Drui, and A. N. Sveshnikova. "Analysis of platelet RNA: a non-invasive method for studying the expression of tumor genes." Pediatric Hematology/Oncology and Immunopathology 20, no. 1 (2021): 207–17. http://dx.doi.org/10.24287/1726-1708-2021-20-1-207-217.
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Nowadays much attention is paid to non-invasive methods of cancer diagnostics and monitoring. One of the promising methods is the sequencing of platelet RNA (tumor-educated platelets), in which, as it was previously established, the mRNA repertoire changes in various oncological diseases. Thus, platelets can contain information about the molecular genetic characteristics of tumor. This review summarizes the current understanding of the mechanisms of interaction between tumor cells and platelets, and also discusses the possibilities of using platelet transcriptome analysis methods for diagnosing and assessing the dynamics of the tumor state, in particular, in response to therapy, as well as the current limitations for a wide range of introduction of this method into clinical practice.
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Horev, Melanie B., Yishaia Zabary, Revital Zarka, et al. "Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces." F1000Research 9 (May 27, 2020): 449. http://dx.doi.org/10.12688/f1000research.23598.1.
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Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
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Horev, Melanie B., Yishaia Zabary, Revital Zarka, et al. "Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces." F1000Research 9 (July 3, 2020): 449. http://dx.doi.org/10.12688/f1000research.23598.2.
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Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
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Tablin, Fern, and John H. Crowe. "Human Platelet Membrane Lipid Dynamics: Agonist Stimulation Results in the Aggregation of Lipid Rafts and the Lateral Phase Separation of like Lipids in Intact Human Platelets." Blood 104, no. 11 (2004): 3530. http://dx.doi.org/10.1182/blood.v104.11.3530.3530.
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Abstract Sphingomyelin and cholesterol rich platelet membrane domains are organized into lipid rafts, which are present in the liquid ordered state, and which have been suggested to be key membrane components both during cold-induced human platelet activation (Gousset et al. 2002 Evidence for a physiological role for membrane rafts in human platelets. J. Cellular Physiol. 190:117–128) as well as during agonist induced activation. We have previous demonstrated that platelets have two membrane phase transitions, a raft transition at 34–40°C (Gousset et al., 2002 ) and a phospholipid phase transition at 10–20°C (Tablin et al. 1996 The membrane phase transition of intact human platelets: correlation with cold-induced activation. J. Cellular Physiol. 168:305–313). Using Fourier transform infrared spectroscopy (FTIR) we are able to sample all the key phases adopted by intact platelet phospholipids and sphingolipids (gel, liquid crystalline, liquid ordered (raft<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>)</INS>, hexagonal). This method offers unique advantages for the study of phospholipid acyl chain structures and interactions, as it is non-invasive and can detect molecular vibrations produced by dipole moment oscillations at infrared frequencies. Equally important<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>,</INS> these vibrational frequencies are well characterized for individual phospholipids, permitting unambiguous assignment of the transitions to membranes in intact cells (Crowe et al. 1999. Are lipid phase transitions responsible for chilling damage in human platelets? Cryobiology 38:180-101). Most recently, using FTIR we have examined platelet lipid membrane dynamics associated with agonist induced human platelet activation. FTIR analysis of thrombin ( 1U/ml and 0.5U/ml) activated human platelets demonstrates that agonist stimulation results in the development of multiple (four or more) phase transitions, strongly suggestive of phase separation of lipids into like-lipid domains, also referred to as lateral phase separation. These like-lipid domains have phase transition temperatures very similar to that of isolated single lipid species, which, we have previously demonstrated combined to form the main phospholipids phase transition at between 10–20°C (Crowe et al. 1999). Furthermore, when human platelets are repeatedly scanned by FTIR - despite the temperature excursions which might normally result in the remixing of pure lipid populations, the thrombin treated platelets maintained their multiple phase transitions, suggesting that the lateral phase separation is an irreversible event during agonist induced activation. In addition to FTIR studies of human platelet populations, we have also examined the distribution of diIC18 in washed resting, and agonist stimulated human platelets. This dye, which preferentially partitions into liquid ordered or gel phases was uniformly distributed in resting platelets, but became aggregated when platelets were treated by either low concentrations of thrombin (0.01U/ml) or collagen (3.0ug/ml). In addition, platelets treated with higher concentrations of either agonist, demonstrated lipid rafts which further aggregated into larger liquid ordered domains. These results strongly suggest that platelet membrane lipid organization plays a key role in membrane function and further downstream signaling. Funded by NHLBI and DARPA.
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Yangfan Zhou, Mengjiao Hu, Xiaoyan Chen та ін. "Migfilin supports hemostasis and thrombosis through regulating platelet αIIbβ3 outside-in signaling." Haematologica 105, № 11 (2019): 2608–18. http://dx.doi.org/10.3324/haematol.2019.232488.
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Elucidating the regulation mechanism of integrin αIIbβ3 is key to understand platelet biology and thrombotic diseases. Previous in vitro studies have implicated a role of migfilin in the support of platelet αIIbβ3 activation, however, contribution of migfilin to thrombosis and hemostasis in vivo and a detailed mechanism of migfilin in platelets are not known. In this study, with migfilin deletion (migfilin-/-) mice, we report that migfilin is a pivotal positive regulator of hemostasis and thrombosis. Migfilin-/- mice showed a nearly doubled tail-bleeding time and a prolonged occlusion time in Fecl3-induced mesenteric arteriolar thrombosis. Migfilin deficiency impedes platelet thrombi formation on collagen surface and impairs platelet aggregation and dense-granule secretion. Supported by characteristic functional readings and phosphorylation status of distinctive signaling molecules in the bidirectional signaling processes of αIIbβ3, the functional defects of migfilin-/- platelets appear to be mechanistically associated with a compromised outside-in signaling, rather than inside-out signaling. A synthesized cell-permeable migfilin peptide harboring filamin A binding sequence rescued the defective function and phosphorylation of signaling molecules of migfilin-/- platelets. Finally, migfilin does not influence the binding of filamin A and β3 subunit of αIIbβ3 in resting platelets, but hampers the re-association of filamin A and β3 during the conduct of outside-in signaling, suggesting that migfilin functions through regulating the interaction dynamics of αIIbβ3 and filamin A in platelets. Our study enhances the current understanding of platelet integrin αIIbβ3-mediated outside-in signaling and proves that migfilin is an important regulator for platelet activation, hemostasis and thrombosis.
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Gill, J., C. S. Thompson, J. Y. Jeremy, and D. P. Mikhailidis. "Adrenoceptor-linked [45Ca2+] uptake in platelets from diabetic rats: a model for human platelets." Laboratory Animals 28, no. 2 (1994): 143–47. http://dx.doi.org/10.1258/002367794780745326.
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Adrenoceptor-linked 45calcium uptake was investigated in platelets from diabetic rats (hyperglycaemic, streptozotocin-induced, of 60 days duration). Basal uptake was markedly enhanced in platelets from diabetic rats compared with controls. However, whereas adrenaline-stimulated uptake was unchanged, isoprenaline-stimulated uptake was significantly reduced and noradrenaline-stimulated uptake significantly increased. These latter data indicate that there are differential alterations of adrenoceptor subtypes in diabetes (i.e. an increase in alpha- but decrease in beta-adrenoceptors). These changes in calcium uptake and in adrenoceptor activity may relate to altered platelet function known to occur in experimental diabetes. Furthermore, the similarity of the rat platelet with that of the human (in terms of absolute calcium uptake, responses to agonists and changes in diabetes), renders the rat platelet an appropriate model for studying calcium dynamics and linked adrenoceptors in diabetes.
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Diamond, Scott L. "Systems Biology to Predict Platelet Function." Blood 116, no. 21 (2010): SCI—38—SCI—38. http://dx.doi.org/10.1182/blood.v116.21.sci-38.sci-38.
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Abstract Abstract SCI-38 Systems Biology seeks to provide patient-specific prediction of dynamic cellular response to multiple stimuli, critical information toward predicting risk, disease progression, or response to therapy. We deployed two distinct approaches, bottom-up and top-down analyses, to gain insight into platelet signaling. The bottom-up approach required a definition of reaction network and kinetic equations (topology), kinetic parameters, and initial concentrations in order to simulate platelet signaling. We developed a computational platelet model – assembled from 24 peer-reviewed platelet studies to yield 132 measured kinetic rate constants – that accurately predicts resting levels of cytosolic calcium, IP3, diacylglycerol, phosphatidic acid, phosphoinositol, PIP, and PIP2. The model accurately predicts the full transient calcium dynamics in response to increasing levels of ADP. In the first full stochastic simulation of single platelet response to ADP, the model provides an extremely accurate prediction of the statistics of the asynchronous [Ca]i spikes observed in single platelets. Specifically, this is the first work to provide a quantitative molecular explanation of the asynchronous calcium spiking observed in ADP-activated human platelets. We show the asynchronous spiking is a result of the fundamentally stochastic nature of signal transduction in cells as small as human platelets. Specific testable predictions have emerged about the requirement of high SERCA/IP3R ratios in functional platelets, limits on the concentration of calcium in the DTS, and relative potencies of PAR peptides and ADP. For functional phenotyping platelets, a top-down approach linking multiple inputs to functional outputs was used to understand how human platelets integrate diverse signals encountered during thrombosis. We developed a high-throughput platform that measures the human platelet calcium mobilization in response to all pairwise combinations of six major agonists. Agonists tested in this study were: convulxin (CVX; GPVI activator), ADP, the thromboxane analog U46619, PAR1 agonist peptide (SFLLRN), PAR4 agonist peptide (AYPGKF), and PGE2 (activator of IP and EP receptor). The calcium responses to single agonists at 0.1, 1, 10′ EC50 and 135 pairwise combinations trained a neural network (NN) model to predict the entire 6-dimensional platelet response space. The NN model successfully predicted responses to sequential additions and 27 ternary combinations of [ADP], [convulxin], and [SFLLRN] (R=0.881). With 4077 NN simulations spanning the 6-dimensional agonist space, 45 combinations of 4–6 agonists (ranging from synergism to antagonism) were selected and confirmed experimentally (R=0.883), revealing a highly synergistic condition of high U46619/PGE2 ratio, consistent with the risk of COX-2 therapy. Furthermore, pairwise agonist scanning (PAS) provided a direct measurement of 135 synergy values, thus allowing a unique phenotypic scoring of 10 human donors. Patient-specific training of NNs represent a compact and robust approach for prediction of cellular integration of multiple signals in a complex disease milieu. Either bottom-up models or top-down NN models are ideal for incorporation into systems biology simulations of thrombotic pathways under flow conditions. Disclosures: No relevant conflicts of interest to declare.
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Lian, Lurong, Yanfeng Wang, Julia Draznin та ін. "The relative role of PLCβ and PI3Kγ in platelet activation". Blood 106, № 1 (2005): 110–17. http://dx.doi.org/10.1182/blood-2004-05-2005.
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Stimulation of platelet G protein–coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP2) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCβ). It also results in the phosphorylation of PIP2 by the γ isoform of phosphatidylinositol 3-kinase (PI3Kγ) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCβ (PLCβ2 and PLCβ3) or lacking the Gβγ-activated isoform of PI3K (PI3Kγ). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCβ2/β3–/– platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kγ disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCβ2/β3–/– and PI3Kγ–/– platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCβ2/β3 and PI3Kγ play vital roles in platelet cytoskeletal dynamics.
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Wiśniewski, Adam, Joanna Sikora, Aleksandra Karczmarska-Wódzka, Joanna Bugieda, Karolina Filipska, and Robert Ślusarz. "Unfavorable Dynamics of Platelet Reactivity during Clopidogrel Treatment Predict Severe Course and Poor Clinical Outcome of Ischemic Stroke." Brain Sciences 11, no. 2 (2021): 257. http://dx.doi.org/10.3390/brainsci11020257.
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Background: Previous studies have revealed that high platelet reactivity while on clopidogrel may affect the severe course and worse prognosis of ischemic stroke. However, the above findings were based on a single measurement of platelet function. We aimed to investigate whether the dynamics of platelet reactivity over time would more accurately determine its actual impact on clinical outcome. Methods: We enrolled 74 ischemic stroke subjects, taking a dose of 75 mg a day of clopidogrel to this prospective, single-center, and observational study. The determination of platelet function was based on the impedance aggregometry 6–12 h after the first dose of clopidogrel and 48 h later. We defined a favorable dynamics of platelet reactivity as a decrease in values at least equal to the median obtained in the entire study. The clinical condition was assessed by the National Institutes of Health Stroke Scale on the first, third, and ninetieth days and the functional status by modified Rankin Scale, respectively. Results: A favorable dynamics of platelet reactivity was associated with the mild clinical condition and favorable functional status, both early and late. Early neurological deterioration was related to unfavorable dynamics of platelet reactivity over time. In multivariate regression models, we found that unfavorable dynamics of platelet reactivity, alone and combined with a high baseline value of platelet reactivity, is an independent predictor of a severe clinical condition, the risk of deterioration, and poor early and late prognosis. Conclusion: We highlighted that dynamics of platelet reactivity over time predict the clinical course and prognosis of stroke better than a single value.
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Mody, Nipa A., and Michael R. King. "Platelet Adhesive Dynamics. Part I: Characterization of Platelet Hydrodynamic Collisions and Wall Effects." Biophysical Journal 95, no. 5 (2008): 2539–55. http://dx.doi.org/10.1529/biophysj.107.127670.
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