Intracellular Signaling Processes Research Articles

Responses of Normal Cells to Ionizing Radiation

Radiation-induced alterations in cellular tissue homeostasis triggered by various molecular responses at the level of inter- and intracellular signaling processes cause both acute and late effects in normal tissue after radiation therapy. Some of the underlying molecular and cellular response pathways leading to radiation-induced tissue remodeling will be discussed, with special emphasis on vascular and parenchymal tissues.

Expanding the Neuron's Calcium Signaling Repertoire: Intracellular Calcium Release via Voltage-Induced PLC and IP3R Activation

Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

Phospholipid Transfer Protein Augments Apoptosis in THP-1–Derived Macrophages Induced by Lipolyzed Hypertriglyceridemic Plasma

Lipolysis of triglyceride-rich lipoproteins (TGRLPs) generates phospholipid-rich surface remnants and induces cytotoxic effects in adjacent vascular cells. We hypothesized that by integrating surface remnants into HDL, phospholipid transfer protein (PLTP) alleviates cytotoxicity. To test this hypothesis and gain insight into cytotoxicity during the postprandial phase in vivo, we injected normo-TG and hyper-TG human volunteers after a standardized fat meal (postprandial sample) with heparin, thereby stimulating lipolysis (postprandial heparinized sample). Incubation of (primary) human macrophages and primary human endothelial cells with postprandial heparinized hyper-TG plasma induced pronounced cytotoxic effects that were dose dependent on the TG content of the sample. No such effects were seen with normo-TG and postprandial hyper-TG plasma. In vitro lipolysis of VLDL and chylomicrons indicated that both lipoprotein fractions can cause cytotoxicity. Interestingly, in experiments with THP-1-derived macrophages stably transfected with PLTP, PLTP substantially augmented both net phospholipid uptake and apoptotic cell death due to postprandial heparinized hyper-TG plasma. We observed that activation of caspase-3/7, poly-ADP-ribose polymerase, and enhanced bioactivity of acid sphingomyelinase may all contribute to this augmented apoptosis. Our data show that lipolysis of TGRLPs and their remodelling by PLTP interact to disturb cellular phospholipid flux and intracellular signaling processes, ultimately leading to apoptosis in human macrophages and endothelial cells.

Hyperosmotic stress response: comparison with other cellular stresses

Cellular responses induced by stress are essential for the survival of cells under adverse conditions. These responses, resulting in cell adaptation to the stress, are accomplished by a variety of processes at the molecular level. After an alteration in homeostatic conditions, intracellular signalling processes link the sensing mechanism to adaptive or compensatory changes in gene expression. The ability of cells to adapt to hyperosmotic stress involves early responses in which ions move across cell membranes and late responses characterized by increased synthesis of either membrane transporters essential for uptake of organic osmolytes or of enzymes involved in their synthesis. The goal of these responses is to return the cell to its normal size and maintain cellular homeostasis. The enhanced synthesis of molecular chaperones, such as heat shock proteins, is another important component of the adaptive process that contributes to cell survival. Some responses are common to different stresses, whereas others are specific. In the first part of the review, we illustrate the characteristic and specific features of adaptive response to hypertonicity; we then describe similarities to and differences from other cellular stresses, such as genotoxic agents, nutrient starvation and heat shock.

Cytosolic 2‐cys peroxiredoxins regulate TNF[alpha]‐induced apoptosis via controling caspase 8 at the mitochondria dependent manner.

Tumor necrosis factor α (TNFα) is a pleiotrophic cytokine which induces the activation of NF-κB, MAP kinases, and cell death. Therefore, intracellular signaling processes including the inhibition of NF-κB activation followed by the sustained JNK activation and robust increase of reactive oxygen species (ROS) are prerequisite for the induction of cell death programming. In this study, we targeted peroxiredoxin (Prx) II, one of the cytosolic 2-cys Prxs, in order to elucidate the molecular mechanisms underlying the involvement of ROS in TNF α-induced cell death. So we used the HeLa cervical cancer cells (DN) stably expressing a dominant negative mutant version of Prx II, in which two active site cysteine residues were substituted to serine residues. Besides overexpression DN form of 2-cys Prxs,, small interfering siRNA was also introduced to cells as a mean to deplete of endogenous PrxI and PrxII. The DN cells exhibited the dramatic increases in intracellular level of H2O2 and cell death induced by either TNF α in the presence of cycloheximide compared to control cells (CT). The cell death aggravated in DN cells was dependent on a typical FADD/caspase death pathway. The TNF α-treated DN cells showed the enhancement of caspase-8 activation, tBid translocation to mitochondria, and cytochrome c release compared to CT cells. Interestingly, CT and DN cells were equally sensitive to cell death induced by a mitochondrial damaging agent, staurosporine, suggesting that cytosolic 2-cys Prx regulates cell death before mitochondrial participation. Collectively, the results suggest that intracellular H2O2 regulated by cytosolic 2-cys Prx is involved in the TNF α-induced cell death via controlling caspase-8 activation at the upstream of mitochondria.

Feedback loops in intracellular signal processing and their potential for identifying novel signalling proteins

Mapping of intracellular signalling networks has attracted a lot of attention during recent years. Approaches which have been used to characterise functional and physical relationships within signalling systems and to identify novel components have included bioinformatics, genomics, proteomics and genetics. However, the importance of feedback loops in the regulation of signal processing systems has often been ignored. Here we report a novel focus for our previous screening platform, which evaluates the potential of autocatalytic feedback for functional screening of cDNA expression libraries. We confirm that the use of such systems is feasible. We propose that the use of feedback loops in functional screens may introduce a bias towards the detection of signalling molecules, which are likely to be of key regulatory importance due to the sensitivity of the system towards their expression levels.

Decreased NR1, NR2A, and SAP102 transcript expression in the hippocampus in bipolar disorder

Objectives: Schizophrenia is associated with dysfunction of glutamatergic neurotransmission, and several studies have suggested glutamatergic abnormalities in bipolar disorder. Recent data suggest involvement of the NMDA receptor signaling complex, which includes NMDA receptor subunits as well as associated intracellular interacting proteins critical for NMDA receptor assembly, trafficking, and activation; the most well-characterized being PSD93, PSD95, SAP102, and NF-L. Previously, studies from our laboratories have described changes in glutamate receptor subunit transcript and binding site expression in schizophrenia and changes in NMDA receptor binding site expression in bipolar disorder in postmortem brain tissue. In the present work, we focus on the expression of these molecules in hippocampus in schizophrenia and bipolar affective disorder I. Methods: We performed in situ hybridization to assess hippocampal expression of the transcripts encoding NMDA receptor subunits NR1, 2A, 2B, 2C and 2D, and the transcripts for the NMDA receptor associated PSD proteins PSD95, PSD93, NF-L, and SAP102 in subjects with schizophrenia, bipolar affective disorder I, and a comparison group. We also measured [ 3H]CGP39653 and [ 3H]MK-801 binding site expression in the hippocampus in schizophrenia. Results: There was a significant decrease in the expression of transcripts for NR1 and NR2A subunits and SAP102 in bipolar disorder. We did not detect any changes in these transcripts or in binding site expression in the hippocampus in schizophrenia. Conclusions: We propose that the NMDA receptor signaling complex, including the intracellular machinery that is coupled to the NMDA receptor subunits, is abnormal in the hippocampus in bipolar disorder. These data suggest that bipolar disorder might be associated with abnormalities of glutamate-linked intracellular signaling and trafficking processes.

Swelling-Activated K+ Efflux and Regulatory Volume Decrease Efficiency in Human Bronchial Epithelial Cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.

Design and synthesis of phenethyl benzo[1,4]oxazine-3-ones as potent inhibitors of PI3Kinaseγ

The Type 1 PI3Kinases comprise a family of enzymes, which primarily phosphorylate PIP2 to give the second messenger PIP3, a key player in many intracellular signaling processes [ Science, 2002, 296, 1655; Trends Pharmacol. Sci. 2003, 24, 366]. Of the four type 1 PI3Ks, the γ-isoform, which is expressed almost exclusively in leukocytes [ Curr. Biol., 1997, 7, R470], is of particular interest with respect to its role in inflammatory diseases such as rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD) [ Mol. Med. Today, 2000, 6, 347]. Investigation of a series of 4,6-disubstituted-4 H-benzo[1,4]oxazin-3-ones has led to the identification of single-digit nanomolar inhibitors of PI3Kγ, several of which had good cell based activity and were shown to be active in vivo in an aspectic peritonitis model of inflammatory cell migration.

Selective effects of external noise on Ca 2+ signal in mesoscopic scale biochemical cell systems

We have investigated how the external noise would influence the intracellular calcium signaling processes due to the existence of internal noise in small systems. Using chemical Langevin equations, we demonstrate numerically that the behavior of ‘system size bi-resonance’ induced by internal noise will change with the external noise intensity in different regions, indicating the occurrence of selective effects of external noise. Meanwhile, cell system may also automatically select an optimal cell size to obtain the best performance in the presence of external noise. These results may imply that such selective-effect phenomenon should have some inherent relevance with the distinct deterministic bifurcation features of the system.

Activation dependent expression of MMPs in peripheral blood mononuclear cells involves protein kinase A

Monocyte/Macrophages are integral cellular components of inflammation. Matrix metalloproteinases (MMPs) produced by these cells play a crucial role in every aspect of inflammation. Results of the investigations on activation dependent upregulation of MMPs in human peripheral blood mononuclear cells in culture using different lectins as an in vitro model system to mimic inflammatory monocytes are presented. Under normal physiological conditions the monocytes produced only very low amount of MMPs in an indomethacin insensitive PG/cAMP independent manner. Zymographic analysis and ELISA showed that treatment of monocyte with lectins like concanavalin A (ConA), wheat germ agglutinin (WGA) and Artocarpus lakoocha agglutinin (ALA) caused upregulation of MMPs and the maximum effect was produced by ALA. ALA significantly upregulated MMP-9 in a concentration and time dependent manner. Immunoblot analysis and RT-PCR confirmed ALA mediated upregulation of MMP-9 production. Inhibition of ALA effect by indomethacin and reversal of the indomethacin effect by Bt(2)cAMP indicated involvement of cAMP dependent signaling pathway. Further support for the prostaglandin mediated effect was obtained by the upregulation of cyclooxygenase by ALA. H-89, an inhibitor of protein kinase A (PKA), inhibited the expression of MMP-9 indicating that ALA mediated upregulation of MMP-9 is mediated through PKA pathway. Increase in MMP production and increase in cyclooxygenase activity and inhibition of the effect of ALA on MMP production by indomethacin suggested that the ALA activated monocytes in culture can be used as an in vitro model system to study the intracellular signaling process involved in the mediation of inflammatory response.

Regulation of Peripheral Inflammation by Spinal p38 MAP Kinase in Rats

BackgroundSomatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.Methods and FindingsSelective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and −6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor α (TNFα) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.ConclusionsThese data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFα-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.

Cannabinoids attenuate norepinephrine‐induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N‐acetyltransferase activity without involvement of cannabinoid receptors

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.

Coincident signaling in mesolimbic structures underlying alcohol reinforcement

The medium spiny neurons (MSNs) of the nucleus accumbens function in a critical regard to examine and integrate information in the processing of rewarding behaviors. These neurons are aberrantly affected by drugs of abuse, including alcohol. However, ethanol is unlike any other common drug of abuse, due to its pleiotropic actions on intracellular and intercellular signaling processes. Intracellular biochemical pathways appear to critically contribute to long-term changes in the level of synaptic activation of these neurons, which have been implicated in ethanol dependence. Additionally, these neurons also display a fascinating pattern of up/down activity, which appears to be, at least in part, regulated by convergent activation of dopaminergic and glutamatergic (NMDA) inputs. Thus, dopaminergic and NMDA receptor-mediated synaptic transmission onto these neurons may constitute a critical site of ethanol action in mesolimbic structures. For instance, dopaminergic inputs alter the ability of ethanol to regulate NMDA receptor-mediated synaptic transmission onto accumbal MSNs. Prior activation of D1-signaling cascade through the cAMP-regulated phosphoprotein-32 kD (DARPP-32) and protein phosphatase-1 (PP-1) pathway significantly attenuates ethanol inhibition of NMDA receptor function. Therefore, the interaction of D1-signaling and NMDA receptor signaling may alter NMDA receptor-dependent long-term synaptic plasticity, contributing to the development of ethanol-induced neuroadaptation of the reward pathway.

Reduced Apoptosis Rates in Human Schwannomas

Schwannomas, tumors originating from Schwann cells, represent a frequent neurological tumor and can occur both in a genetic disorder called neurofibromatosis type 2 (NF2) and sporadically. In both cases the genetic background is identical as all schwannomas are caused by biallelic mutations in the tumor suppressor gene NF2 coding for merlin. Mutations in this gene have also been found to be responsible for 50% to 60% of spontaneous and 100% of the NF2 associated meningiomas. The NF2 gene product, merlin, links transmembrane proteins to the cytoskeleton and is involved in intracellular signaling processes. It has previously been shown that reexpression of wild-type merlin in primary human schwannoma cells leads to an increase in the number of apoptotic cells. Here, we report in vivo and in vitro evidence that the basal apoptosis rate of primary human schwannoma cells is reduced in comparison to that of normal Schwann cells, supporting the idea that in this benign tumor type, apoptosis has a role in tumorigenesis.

Swelling‐activated K efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o−1. Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (Tc), as an index of cell volume, whereas 86Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H2O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral 86Rb efflux markedly increased during the hyposmotic shock, from 0.50 ± 0.03 min−1 to a peak value of 6.32 ± 0.07 min−1, while apical 86Rb efflux was negligible. Channel blockers, such as GdCl3 (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 μM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 μM) and genistein (150 μM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in 86Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K+ and Cl−1 efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral 86Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl−1 from 16HBE14o−1 cells in response to cell swelling determines RVD efficiency.

Immunochemical characterization of human estrogen sulfotransferase SULT1E1 in human embryonic kidney 293 cells

Human sulfotransferase SULT1E1 is the major cytosolic sulfotransferase isoform that catalyzes the sulfation of estrogens. This reaction increases the water solubility of the estrogen molecule, which may lead to altered cellular estrogen distribution and, as a result, may affect the cellular response to estrogenic signaling. To date, little is known about the role(s) of SULT1E1-catalyzed estrogen sulfation in the regulation of intracellular estrogen-activated signaling processes. In the current studies, the expression of SULT1E1 in human embryonic kidney 293 (HEK293) cell line was determined using immunocytochemistry and green fluorescent protein (GFP)-tagging methods. The results showed that immunoreactivity to anti-SULT1E1 antibody was highly detectable in the cytoplasm of HEK293 cells. The GFP-SULT1E1 fusion protein also had the same cytosolic location as the native protein. Additional studies using immunoblot analysis were not able to detect the expression levels of human nuclear estrogen receptors α and β in HEK293 cells. This is consistent with previous results (Thomas et. al., 2005) and suggests that the physiological role(s) of SULT1E1-catalyzed sulfation reactions in HEK293 cells are not associated with estrogen receptors α and β coupled cellular signaling pathways.

A 3D Monte Carlo Analysis of the Role of Dyadic Space Geometry in Spark Generation

In multiple biological systems, vital intracellular signaling processes occur locally in minute periplasmic subspaces often referred to as signaling microdomains. The number of signaling molecules in these microdomains is small enough to render the notion of continuous concentration changes invalid, such that signaling events are better described using stochastic rather than deterministic methods. Of particular interest is the dyadic cleft in the cardiac myocyte, where short-lived, local increases in intracellular Ca 2+ known as Ca 2+ sparks regulate excitation-contraction coupling. The geometry of dyadic spaces can alter in disease and development and display significant interspecies variability. We created and studied a 3D Monte Carlo model of the dyadic cleft, specifying the spatial localization of L-type Ca 2+ channels and ryanodine receptors. Our analysis revealed how reaction specificity and efficiency are regulated by microdomain geometry as well as the physical separation of signaling molecules into functional complexes. The spark amplitude and rise time were found to be highly dependent on the concentration of activated channels per dyadic cleft and on the intermembrane separation, but not very sensitive to other cleft dimensions. The role of L-type Ca 2+ channel and ryanodine receptor phosphorylation was also examined. We anticipate that this modeling approach may be applied to other systems (e.g., neuronal growth cones and chemotactic cells) to create a general description of stochastic events in Ca 2+ signaling.

Regulation of Sodium and Calcium Channels by Signaling Complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.

Shear Induces a Unique Series of Morphological Changes in Translocating Platelets

The platelet glycoprotein (GP) Ib/V/IX complex plays an important role in regulating the morphology of resting platelets and can induce shape change during adhesion to immobilized von Willebrand factor (vWf). In this study we have examined the effects of fluid shear stress on GPIb-dependent changes in platelet morphology during translocation on vWf. We demonstrate that translocating platelets undergo a unique series of morphological changes in response to increasing fluid shear stress. Under moderately low shear conditions (600 s(-1)), initial shape change involved extension of membrane tethers and/or filopodia from the platelet surface. With increasing shear rate, platelets adopted a spherical morphology with numerous surface projections (1800 to 5000 s(-1)). At high wall shear rates (10000 to 20,000 s(-1)), translocating platelets retracted filopodia, developing a smooth ball-like appearance. These changes in morphology were dependent on reorganization of the actin and microtubule components of the cytoskeleton and were regulated by intracellular signaling processes linked to Src kinases. Functionally, alterations in platelet shape had a major effect on translocation dynamics in that conversion from discs to spheres resulted in a 3- to 8-fold increase in rolling velocity. These studies demonstrate that platelets undergo shear-specific morphological changes during surface translocation on vWf that may serve to regulate translocation dynamics under flow.