Platelet Suspensions Research Articles

Sedimentation and Phase Transitions of Colloidal Gibbsite Platelets

We study the competition between sedimentation, gelation, and liquid crystal formation in suspensions of colloidal gibbsite platelets of five different sizes at three ionic strengths. For large particles (with diameters of 350, 420, and 570 nm) sedimentation is initially the most important factor determining the macroscopic behavior. Only after the main part of the sample has sedimented in an amorphous phase, phase separation takes place. For the smallest particles (diameter 210 and 270 nm), it is the other way around: fast (within one week) phase separation or gelation takes place, after which sedimentation determines the final macroscopic appearance. We distinguish six different scenarios within this two-fold scheme and interpret these on the basis of the previously obtained phase diagram of colloidal gibbsite platelets (van der Beek, D.; Lekkerkerker, H. N. W. Langmuir 2004, 20, 8582).

Selective and Controlled Synthesis of α- and β-Cobalt Hydroxides in Highly Developed Hexagonal Platelets

We report on the controlled synthesis of single-crystal platelets of alpha- and beta-Co(OH)2 via homogeneous precipitation using hexamethylenetetramine as a hydrolysis agent. The alpha- and beta-Co(OH)2 hexagonal platelets of several micrometers in width and about 15 nm in thickness were reproducibly yielded in rather dilute CoCl2 solutions in the presence and absence of NaCl at 90 degrees C, respectively. The phase and size control of the products were achieved by varying both CoCl2 and NaCl concentrations. Polarized optical microscope observations revealed clear liquid crystallinity of colloidal suspensions of these high aspect-ratio platelets. The as-prepared alpha-Co(OH)2 containing interlayer chloride ions was intercalated with various inorganic or organic anions, keeping its high crystallinity and hexagonal platelike morphology.

Producing Accurate Platelet Counts for Platelet Rich Plasma: Validation of a Hematology Analyzer and Preparation Techniques for Counting

Platelet rich plasma (PRP) has been shown to clinically accelerate healing of both soft and hard tissues. As a result, it has gained increasing popularity. However, the clinical effectiveness of each type of PRP preparation method can vary in technique and efficiency, and current methods to evaluate the platelet concentration efficiency of PRP systems have several limitations. Therefore, the purpose of this study was to validate an automated hematology analyzer, the Cell-Dyn 3700, to accurately count platelets in concentration ranges of approximately 2,000,000-4,800,000 platelets/microL. PRP platelets were counted by way of a manual counting method and on the Cell-Dyn 3700, and the statistical evaluation indicated no difference between the groups (P > 0.05). Dilution of the PRP was not required, and accurate platelet counts could be achieved up to platelet concentrations of 4,800,000 platelets/microL. PRPs must be resuspended on a rocker for at least 5 minutes before platelet counts, and the entire PRP sample must be resuspended to allow for equal distribution of platelets before counting. With use of the validated Cell-Dyn 3700, a platelet concentrate system was used to prepare 153 PRPs. The baseline whole blood platelet concentration (328,000 platelets/microL +/- 69,000 platelets/microL) and the average PRP samples (2,645,000 platelets/microL +/- 680,000 platelets/microL) were compared, resulting in an eightfold increase in concentration and an average platelet percent recovery of approximately 76%. Automated hematology analyzers can be used to accurately count platelets in PRP given the system has been validated appropriately and the PRP samples are prepared properly to provide adequate platelet suspension.

NAD(P)H-dependent superoxide production in platelets: The role of angiotensin II and protein kinase C

Objectives: Vascular NAD(P)H oxidase represents a major source for excessive superoxide production in hypertension. Angiotensin II (AngII) can activate NAD(P)H oxidase via the angiotensin II type 1 (AT1) receptor and protein kinase C (PKC). Platelets possess AT1 receptors and all the components of the NAD(P)H oxidase system. We employed this tissue model to explore mechanisms involved in AngII-mediated superoxide production. Design and methods: Platelet suspensions from hypertensive patients' blood were activated with AngII or phorbol 12-myristate 13-acetate (PMA). Inhibitors of NAD(P)H oxidase, PKC, and the AT1 receptor were employed to study their effects on superoxide production. Results: Superoxide production was stimulated by AngII and PMA and attenuated by AT1 receptor antagonists (mean percentage reduction 80.2%, P < 0.01) and inhibitors of PKC (mean reduction 94.8%, P < 0.001) and NAD(P)H oxidase (mean reduction 100%, P < 0.001). Conclusions: AngII stimulates platelet superoxide production through activation of vascular NAD(P)H oxidase via the AT1 receptor and PKC.

Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V

Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.

Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase Cβ

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.

Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells

BackgroundThe growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes.ResultsHere we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1.ConclusionsThese studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease.

Plasminogen interaction with platelets: The importance of carboxyterminal lysines

Introduction Thrombin stimulation enhances plasminogen binding to platelets and promotes platelet-dependent plasmin generation. The objective of this study was to determine whether carboxyterminal lysines (C-lysines) are important for these processes, as they are in other cell types. Materials and methods 125I-plasminogen and varying concentrations of unlabeled plasminogen were added to washed platelets that were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. In some experiments the platelets were digested with carboxypeptidase B to remove C-lysines. Platelet-dependent plasmin generation was also studied by adding plasminogen and tissue plasminogen activator to platelet suspensions and monitoring the conversion of a plasmin specific chromogenic substrate. The cells were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. The effect of the thrombin inhibitor lepirudin and the plasmin inhibitor aprotinin on plasminogen binding and the appearance of C-lysines was also investigated. Results Thrombin, but not thrombin receptor activating peptide or ADP, stimulated high-affinity binding of plasminogen and greatly promoted platelet-dependent plasmin generation. Digestion with carboxypeptidase B eliminated thrombin-induced high-affinity binding and reduced thrombin-induced plasmin generation by increasing the Michaelis constant. Lepirudin, but not aprotinin, inhibited thrombin-stimulated plasminogen binding to platelets. Conclusion C-terminal lysines are necessary for high-affinity binding of plasminogen to platelets and for platelet-supported plasmin generation. The origin of the C-lysines is not clear, but they may result from a direct effect of thrombin, rather than an intermediate enzyme such as plasmin.

Structure and equation of state of interaction site models for disc-shaped lamellar colloids

We apply RISM (reference interaction site model) and PRISM (polymer-RISM) theories to calculate the site–site pair structure and the osmotic equation of state of suspensions of circular or hexagonal platelets (lamellar colloids) over a range of ratios of the particle diameter over thickness Dσ. Despite the neglect of edge effects, the simpler PRISM theory yields results in good agreement with the more elaborate RISM calculations, provided the correct form factor, characterizing the intramolecular structure of the platelets, is used. The RISM equation of state is sensitive to the number n of sites used to model the platelets, but saturates when the hard spheres, associated with the interaction sites, nearly touch; the limiting equation of state agrees reasonably well with available simulation data for all densities up to the isotropic–nematic transition. When properly scaled with the second virial coefficient, the equations of state of platelets with different aspect ratios Dσ nearly collapse on a single master curve.

Mechanisms Involved in the Antiplatelet Activity of Ketamine in Human Platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca²⁺ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A₂formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca²⁺ mobilization and TxA₂ formation, ultimately resulting in inhibition of platelet aggregation.

The β3 Subunit of the Integrin αIibβ3 Regulates αIib-Mediated Outside-In Signaling.

Bidirectional signaling is an essential feature of integrin function. The membrane distal residues of the αIIb subunit cytoplasmic domain regulate β3 subunit mediated inside-out signaling, but little is known about the regulation of αIIb subunit mediated outside-in signaling. We show that a mutation causing truncation of 39 membrane distal residues of the β3 cytoplasmic domain enables or enhances αIIb-mediated outside-in signaling in human platelets in suspension. Although the abnormal integrin complexes of these platelets (RM) are not constitutively active and cannot be activated by αIIbβ3-mediated inside-out signaling in response to thrombin and other agonists, they can be activated and induced to bind ligand by LIBS (ligand-induced binding site)-specific monoclonal antibodies. RM platelets fail to aggregate in response to physiological agonists because of the inability of the truncated β3 cytoplasmic domain to mediate the inside-out signaling required to activate αIIbβ3 in response to those agonists. Platelets lacking the β3 cytoplasmic domain residues were treated with the LIBS specific antibodies D3 and PT25-2, agents that cause αIIbβ3 activation, and facilitate aggregation in the presence of fibrinogen, but do not elicit TxA2 production or secretion by normal platelets. The rationale for the use of these platelets is that the absence of 39 membrane distal β3 cytoplasmic domain residues might result in abnormal regulation of αIIb-mediated outside-in signaling. Normal and RM platelets were treated independently with D3 and PT25-2 in the presence of fibrinogen with stirring. RM and normal platelets aggregated in response to both D3 and PT25-2 + fibrinogen. In contrast to normal platelets which neither underwent shape change nor produced TxA2 or secreted the contents of their storage granules, RM platelets changed shape, produced TxA2 and secreted ATP and PF4. Those responses by RM platelets required ligand crosslinking of the D3-treated αIIbβ3 demonstrating that receptor activation is insufficient to cause that signaling. This enablement or enhancement of outside-in signaling was not Fc receptor-dependent, but was eliminated by a palmitoylated peptide corresponding to part of the β3 cytoplasmic domain missing in RM platelets, but not by a palmitoylated, scrambled control peptide (palmitoylation of peptides facilitates their entry into the platelets). These results complement data showing that a palmitoylated peptide corresponding to a membrane distal region of the cytoplasmic domain of αIIb inhibits β3 inside-out signaling. It is plausible that the ability of the palmitoylated peptide to restore a wild type-like signaling phenotype to the RM platelets resulted from restoration of β3 negative regulation of αIIb outside-in signaling by binding of the β3 peptide to αIIb. Our data demonstrate negative regulation of αIIb outside-in signaling by the membrane distal cytoplasmic domain of β3 and provide new insight into the regulation of αIIb-mediated outside-in signaling in platelets in suspension

Direct Activation of Platelets by Tissue Factor Is Amplified by FVIIa and Potentiated by Serotonin: Influence of Shear Rate.

Although exposure of tissue factor (TF) at sites of disrupted atherosclerotic plaques plays a critical role in platelet-triggered ischemic complications, a direct interaction of TF with platelets has not been yet demonstrated. We have investigated the effects of TF on platelets under different shear rate conditions. Exposure of isolated washed platelets to TF derived from human placenta resulted in reversible aggregation (35-50% max at 1 min) using standard platelet aggregometry. This response was αIIb-β3 dependent and occurred in the presence of residual concentrations of calcium. TF-induced reversible aggregation was enhanced and made irreversible to 90–100% by the addition of FVIIa (5μg/ml). This second phase of activation was blocked by the presence of serine-protease inhibitors. A combination of ultrastructural and flow cytometry studies revealed that although TF induced shape change and moderate expression of P-selectin an ANV positive phospholipids, internal contraction and full platelet degranulation only occurred after addition of FVIIa. All these transformations occurred together with tyrosine phosphorylation of proteins. Platelets adhered, spread and aggregated onto synthetic membranes coated exclusively with TF that had been exposed to circulating blood. Addition of FVIIa caused a 50% enhancement on platelet interactions and a two-fold increase in fibrin deposition. Serotonin enhanced overall TF induced platelet interactions under the different experimental approaches. A selective serotonin reuptake inhibitor (citalopram) reduced the intensity and extent of the effects of TF in the two experimental settings used in our investigations. Our results are consistent with a direct role of TF on platelet-triggered ischemic complications. We demonstrate that a lipidated from of TF activates isolated platelets in suspension. In studies conducted with flowing whole blood we show that TF firmly attached to a surface acts itself as an adhesive substrate, and promotes formation of aggregates. These TF-platelet interactions are enhanced through serotoninergic mechanism and markedly potentiated in the presence of FVIIa. The possible synergism between FVIIa and serotoninergic mechanisms may well contribute to the development of severe ischemic complications triggered on ruptured atherosclerotic plaques.

Mechanisms involved in the antiplatelet activity of ketamine in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100-350 microM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca2+ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 microM) significantly inhibited thromboxane (Tx) A2 formation stimulated by collagen. Moreover, ketamine (200 and 350 microM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 microM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca2+ mobilization and TxA2 formation, ultimately resulting in inhibition of platelet aggregation.

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca 2+ mobilization in activated human platelets

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca 2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca 2+ mobilization is regulated by phospholipase C-2 (PLC-γ2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-γ2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca 2+ mobilization had no effects on tyrosine phosphorylation of PLC-γ2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca 2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca 2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca 2+ mobilization.

Sphingosine 1-Phosphate Breakdown in Platelets

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.

Inhibition of 5-lipoxygenase and Leukotriene C4 Synthase in Human Blood Cells by Thymoquinone

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 μM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 μM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 μM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.

Brownian dynamics simulations of Laponite colloid suspensions.

Colloidal suspensions of Laponite clay platelets are studied by means of Brownian dynamics simulations. The platelets carry discrete charged sites which interact via a Yukawa potential. As in the paper by S. Kutter et al. [J. Chem. Phys. 112, 311 (2000)], two models are considered. In the first one all surface sites are identically negative charged, whereas in the second one, rim charges of opposite sign are included. These models mimic the behavior of the Laponite particles in different media. They are employed in a series of simulations for different Laponite concentrations and for two values of the Debye length. For the equilibrium states, the system structure is studied by center-to-center and orientational pair distribution functions. Long-time translational and rotational self-diffusion coefficients are computed by two different methods, which yield very similar results.

Liquid crystal phases of charged colloidal platelets.

The liquid crystal phase behavior of a suspension of charged gibbsite [Al(OH)3] platelets is investigated. By variation of the ionic strength, we are able to tune the effective thickness-to-diameter ratio of the platelets in suspension. This enables us to experimentally test the liquid crystal phase transition scenario that was first predicted a decade ago by computer simulations for hard platelets (Veerman, J. A. C.; Frenkel, D. Phys. Rev. A 1992, 45, 5632), that is, the isotropic (I) to nematic (N) and isotropic to columnar (C) phase transitions in one colloidal suspension. In addition to the shape-dependent thermodynamic driving force, the effect of gravity is important. For example, a biphasic (I-N) suspension becomes triphasic (I-N-C) on prolonged standing. This effect is described by a simple osmotic compression model.

Techniques to examine platelet adhesive interactions under flow.

Platelet adhesion and aggregation at sites of vessel-wall injury are critical for the arrest of bleeding and for the development of vaso-occlusive thrombi at sites of atherosclerotic-plaque rupture. These adhesive interactions are critically dependent on multiple receptors on the platelet surface (GPIb/V/IX, GPVI, integrins αIIbβ3 and α2β1) and their specific ligands in the subendothelium (von Willebrand Factor, collagen) and plasma (von Willebrand Factor, fibrinogen) (,). In vivo, these receptor-ligand interactions are exposed to a broad range of shear stresses generated by blood flow, ranging from 20–200/s in veins to 800–10,000/s in arteries (). In stenotic vessels, shear rates can approach 40,000/s. The development of in vitro methodologies mimicking physiological and pathophysiological flow conditions has significantly improved our understanding of the role of shear in regulating platelet functional responses. In general, the effects of shear stress have been studied with platelets in suspension using rotational devices such as the Couette or cone-plate viscometer. Alternatively, the effects of shear on platelets have been evaluated in a laminar-flow device such as the tubular, annular, or parallel-plate flow chamber. Rotational viscometers are ideal for the examination of shear effects on platelet adhesive interactions in the absence of platelet-surface interactions (i.e., platelets in suspension). Such studies are important in determining the mechanisms of platelet activation occurring in areas of vascular stenosis where shear rates are elevated well above physiological levels. Thrombus formation, however, does not generally occur with platelets in suspension but rather involves the progressive accrual of platelets onto vascular subendothelium and subsequently onto immobilized platelets. As such, the in vitro investigation of platelet function under conditions of physiological and pathological shear has been greatly facilitated by laminar flow devices.

Measurement and manipulation of [Ca2+]i in suspensions of platelets and cell cultures.

Measurement and manipulation of [Ca2+]i in suspensions of platelets and cell cultures.