Influx In Platelets Research Articles

A Low-Fat/Sucrose Diet Rich in Complex Carbohydrates Reverses High-Fat/Sucrose Diet-Induced Corneal Dysregulation.

High-fat/sucrose diet feeding in mice causes loss of corneal nerve function and impairs corneal wound healing. While changing to a diet with a low fat/sugar composition and enrichments in complex carbohydrates mitigates the reduction in nerve function, it remains to be determined if it has an effect on corneal wound healing. In this study, 6-week-old C57BL/6 male mice were fed either a normal diet or a high-fat/sucrose diet for 20 weeks. A third group (diet reversal) was placed on a high-fat/sucrose diet for 10 weeks followed by a normal diet for an additional 10 weeks. A central corneal epithelial abrasion wound was created, and wound closure was monitored. Neutrophil and platelet recruitment was assessed by immunofluorescence microscopy. Mice fed the high-fat/sucrose diet-only had greater adiposity (p < 0.005) than normal diet-only fed mice; diet reversal markedly reduced adiposity. Following corneal abrasion, wound closure was delayed by ~6 h (p ≤ 0.01) and, at 30 h post-wounding, fewer neutrophils reached the wound center and fewer extravascular platelets were present at the limbus (p < 0.05). Diet restored normal wound closure and neutrophil and platelet influx in the injured cornea. These data suggest compositional changes to the diet may be an effective diet-based therapeutic strategy for maintaining or restoring corneal health.

BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation.

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Role of Protein Kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis.

Sepsis is characterized by an intense systemic inflammatory response activating a cascade of proinflammatory events resulting in leukocyte dysregulation and host tissue damage. The lung is particularly susceptible to systemic inflammation, leading to acute lung injury. Key to inflammation-induced lung damage is the excessive migration of neutrophils across the vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are not well defined but there is growing evidence that platelets are actively involved and play a key role in microvascular permeability and neutrophil-mediated organ damage. We previously identified PKC-delta (PKCδ) as a critical regulator of the inflammatory response in sepsis and demonstrated PKCδ inhibition was lung protective. However, the role of PKCδ in sepsis-induced platelet activation and platelet-leukocyte interactions is not known. In this study, rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Following surgeries, a PKCδ inhibitor (200μg/kg) or vehicle (PBS) was administered intra-tracheally. At 24 hours post-surgeries, lung tissue, BAL fluid, and blood samples were collected. While sepsis caused thrombocytopenia, the remaining circulating platelets were activated as demonstrated by increased p-selectin expression, elevated plasma PF4, and enhanced platelet-leukocyte aggregate formation compared to Sham animals. Platelet activation was associated with increased platelet PKCδ activity. Inhibition of PKCδ attenuated sepsis-induced platelet activation, secretion and aggregate formation. Sepsis-induced thrombocytopenia was also significantly reduced and circulating platelet numbers were similar to sham animals. In the lung, sepsis induced significant influx of platelets and neutrophils and the development of lung injury. Administration of the PKCδ inhibitor decreased platelet and neutrophil influx, and was lung protective. Thus, PKCδ inhibition modulated platelet activity both locally and systemically, decreased neutrophil influx into the lung, and was lung protective. We demonstrate for the first time that PKCδ plays an important role in platelet activation and platelet-neutrophil interaction during sepsis.

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.

PS 11-40 MENTHOL INHIBITS ENHANCED STORE-OPERATED CALCIUM ENTRY IN PLATELETS IS ASSOCIATED WITH PERIPHERAL ARTERY DISEASE FROM HYPERTENSION WITH TYPE 2 DIABETES

Objective: Menthol, a natural compound, is responsible for the minty flavor and smell of the mint plant and is widely used in food and drugs. A recent study showed that menthol modifies PLC signaling, which then leads to changes in cellular functions. Platelet dysfunction plays an important role in thrombosis in diabetes with peripheral artery disease (PAD). Store-operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) regulate platelet activity by modulating calcium influx. We hypothesized that enhanced SOCE in platelets is associated with diabetes with PAD. Design and Method: We studied the activity of platelets from healthy participants (NT), type 2 diabetic patients (DM) and hypertension with type 2 diabetes (HDM). Platelet calcium influx and protein expression of STIM1 and sarcoendoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were investigated. Results: Compared with that in platelets from DM, calcium influx was significantly increased in platelets from HDM in the absence of extracellular calcium after the discharge of intracellular Ca2+ stores by thapsigargin. Compared with platelets from DM without PAD, platelets from DM with PAD exhibited significantly increased SOCE. Menthol administration completely inhibited calcium influx in platelets from diabetic patients without PAD, but this effect was blunted in those from diabetic patients with PAD. Furthermore, the increased SOC calcium influx was correlated with the ankle brachial index (ABI) in diabetic patients. High glucose significantly up-regulated STIM1 and SERCA3 protein expression and induced the phosphorylation of phospholipase C (PLC) in platelets from healthy participants. This effect was attenuated in the presence of menthol or U73122, an inhibitor of PLC. Similarly, significant increases in STIM1 and SERCA3 protein expression were found in platelets from diabetic patients compared to those from healthy participants. Conclusions: Platelets from hypertension diabetic patients with PAD exhibited enhanced Store-operated calcium influx, which was associated with elevated STIM1/SERCA3 expression via a PLC-dependent pathway and was inhibited by menthol.

Enhanced Store-Operated Calcium Entry in Platelets is Associated with Peripheral Artery Disease in Type 2 Diabetes

Background/Aims: Platelet dysfunction plays an important role in thrombosis in diabetes with peripheral artery disease (PAD). Store-operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) regulate platelet activity by modulating calcium influx. We hypothesized that enhanced SOCE in platelets is associated with diabetes with PAD. Methods: We studied the activity of platelets from healthy participants and from type 2 diabetic patients. Platelet calcium influx and protein expression of STIM1 and sarcoendoplasmic reticulum Ca²⁺-ATPase 3 (SERCA3) were investigated. Results: Compared with platelets from diabetic patients without PAD, platelets from diabetic patients with PAD exhibited significantly increased SOCE . Menthol administration completely inhibited calcium influx in platelets from diabetic patients without PAD, but this effect was blunted in those from diabetic patients with PAD. Furthermore, the increase in SOCE was correlated with the ankle brachial index (ABI) in diabetic patients. High glucose significantly up-regulated STIM1 and SERCA3 protein expression and induced the phosphorylation of phospholipase C (PLC) in platelets from healthy participants. This effect was attenuated in the presence of menthol or U73122, an inhibitor of PLC. Similarly, significant increases in STIM1 and SERCA3 protein expression were found in platelets from diabetic patients compared to those from healthy participants. Conclusion: Platelets from diabetic patients with PAD exhibited enhanced Store-operated calcium influx, which was associated with elevated STIM1/SERCA3 expression via a PLC-dependent pathway and was inhibited by menthol.

Where do the platelets go? A simulation study of fully resolved blood flow through aneurysmal vessels

Despite the importance of platelets in the formation of a thrombus, their transport in complex flows has not yet been studied in detail. In this paper we simulated red blood cells and platelets to explore their transport behaviour in aneurysmal geometries. We considered two aneurysms with different aspect ratios (AR = 1.0, 2.0) in the presence of fast and slow blood flows (Re = 10, 100), and examined the distributions of the cells. Low velocities in the parent vessel resulted in a large stagnation zone inside the cavity, leaving the initial distribution almost unchanged. In fast flows, an influx of platelets into the aneurysm was observed, leading to an elevated concentration. The connection of the platelet-rich cell-free layer (CFL) with the outer regions of the recirculation zones leads to their increased platelet concentration. These platelet-enhanced recirculation zones produced a diverse distribution of cells inside the aneurysm, for the different aspect ratios. A thin red blood CFL that was occupied by platelets was observed on the top of the wide-necked aneurysm, whereas a high-haematocrit region very close to the vessel wall was present in the narrow-necked case. The simulations revealed that non-trivial distributions of red blood cells and platelets are possible inside aneurysmal geometries, giving rise to several hypotheses on the formation of a thrombus, as well as to the wall weakening and the possible rupture of an aneurysm.

Inhibition of thrombin-induced Ca2+ influx in platelets by R59949, an inhibitor of diacylglycerol kinase

The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²⁺ influx of platelets. Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²⁺ concentration ([Ca²⁺](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. Thrombin-induced increase in [Ca²⁺](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²⁺](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²⁺](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²⁺](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²⁺](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²⁺](i) , while thrombin-induced increase in [Ca²⁺](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²⁺ entry induced by thrombin, while store-operated Ca²⁺ entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²⁺ influx of platelets.

Research on the correlation between platelet gelsolin and blood-stasis syndrome of coronary heart disease

To study the distribution of gelsolin in human platelet and plasma, and the association with blood-stasis syndrome (BSS) of coronary heart disease (CHD). Sixty patients with CHD (30 in BSS group and 30 in non-BSS group) and 30 healthy subjects (control group) were included in this study. The classification of the syndrome was based on clinical symptoms and signs. Gelsolin concentration in platelet rich plasma (PRP), platelet poor plasma (PPP), filamentous actin (F-actin) and group-specific component globulin (Gc-globulin) of PPP were determined by enzyme-linked immunosorbent assay (ELISA). The fluorescence intensity of CD62p and cytoplasmic calcium ([Ca(2+)](i)) in human platelets of patients and healthy persons was measured with flow cytometry. Compared with the control group, gelsolin in PRP of the BSS group increased significantly (P<0.01), while that in PPP of the BSS and non-BSS groups decreased markedly (P<0.05), the CD62p, [Ca(2+)](i) of platelet, F-actin, and Gc-globulin of the BSS and non-BSS groups increased significantly (P<0.01). Compared with the non-BSS group, the gelsolin concentration in PRP of BSS group increased significantly (P<0.01), the [Ca(2+)](i) of platelet of the BSS group increased markedly (P<0.01), while the F-actin and Gc-globulin of the BSS group had no statistical defference (P>0.05). Gelsolin concentration in PRP was increased and accompanied by the elevated [Ca(2+)](i) of platelet in CHD with BSS, while gelsolin in PPP were lowered markedly. We speculate that plasma gelsolin may clear F-actin from circulation, thus resulting in depletion of plasma gelsolin significantly. This, in addition to the increased calcium influx of platelets, may lead to the gelsolin abnormal expression on platelets during the process of BSS in CHD. Therefore, platelet gelsolin may serve as a new potential biomarker and a therapeutic target of BSS in CHD.

CCL20, γδ T cells, and IL‐22 in corneal epithelial healing

After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6(+) IL-17(+) γδ T cells migrate into the epithelium. γδ T-cell-deficient (TCRδ(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mRNA increased 19-fold at 3 h, and protein increased ∼ 16-fold at 6 h after injury. Systemic or topical treatment of wild-type C57BL/6 mice with anti-CCL20 reduced γδ T-cell accumulation in the cornea by >50% with a concomitant decrease in epithelial healing and stromal inflammation. In addition to CCR6 and IL-17, corneal γδ T cells stained positively for RORγt, IL-23R, and IL-22. Anti-IL-22 reduced peak cell division of the healing epithelium by 52%. Treatment of TCRδ(-/-) mice with rIL-22 significantly promoted wound closure, with peak epithelial cell division increased >3-fold. In addition, rIL-22 restored neutrophil and platelet influx in the TCRδ(-/-) mice to wild-type levels and increased CXCL1 production by wounded corneal explants >2-fold. These results indicate that an important aspect of the healing response to corneal epithelial abrasion includes CCL20-dependent influx of CCR6(+) IL-17(+) IL-22(+) γδ T cells and that IL-22 contributes to the inflammatory response and promotes epithelial healing.

Store-operated Ca 2+ entry is sensitive to the extracellular Ca 2+ concentration through plasma membrane STIM1

Store-operated Ca 2+ entry (SOCE) is a major mechanism for Ca 2+ influx in platelets and other cells activated by a reduction in Ca 2+ concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca 2+, although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca 2+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn 2+ entry, which was inhibited by extracellular Ca 2+ in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25–139 of PM-STIM1 that contains the Ca 2+-binding domain, prevented the inactivation of Ca 2+ entry induced by extracellular Ca 2+. TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca 2+-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca 2+, an event that is likely to be mediated by interaction with Orai1.

Involvement of SNARE proteins in thrombin-induced platelet aggregation: Evidence for the relevance of Ca 2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca 2+ mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca 2+ entry, the main mechanism for Ca 2+ influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca 2+ entry for full activation. Depletion of the intracellular Ca 2+ stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca 2+ concentration reached a critical level (around 1.5 μM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca 2+ mobilization in human platelets.

A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets

AbstractStore-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in platelets and other cells. De novo conformational coupling between elements in the plasma membrane and Ca2+ stores, where the actin cytoskeleton plays an important regulatory role, has been proposed as the most likely mechanism to activate SOCE in platelets. Here we have examined for the first time changes in platelet F-actin levels on a subsecond time scale. Using stopped-flow fluorimetry and a quenched-flow approach, we provide evidence for the involvement of cofilin in actin filament reorganization and SOCE in platelets. Thrombin (0.1 U/mL) evoked an initial decrease in F-actin that commenced within 0.1 second and reached a minimum 0.9 second after stimulation, prior to the activation of SOCE. F-actin then increased, exceeding basal levels approximately 2.5 seconds after stimulation. Thrombin also induced cofilin dephosphorylation and activation, which paralleled the changes observed in F-actin, and rapid Btk activation. Inhibition of cofilin dephosphorylation by LFM-A13 resulted in the loss of net actin depolymerization and an increased delay in SOCE initiation. These results suggest that cofilin is important for the rapid actin remodeling necessary for the activation of SOCE in platelets through de novo conformational coupling.

Vasodilator-Stimulated Phosphoprotein–Deficient Mice Demonstrate Increased Platelet Activation but Improved Renal Endothelial Preservation and Regeneration in Passive Nephrotoxic Nephritis

Vasodilator-stimulated phosphoprotein (VASP), an actin cytoskeletal protein, is expressed in various cell types including renal cells. In vitro studies provide evidence for a role of VASP regarding platelet activation, cell adhesion, migration, and capillary formation. The in vivo role of VASP was investigated in experimental inflammatory renal disease. Kidneys of healthy VASP deficient (-/-) and wild-type (wt) mice were compared regarding morphology and functional parameters. Passive nephrotoxic nephritis was induced in 28 VASP -/- and 28 wt mice; kidneys were harvested; and tissues were analyzed by morphometric, immunohistochemical, and electron microscopic techniques on days 3, 7, 14, and 28. The time course of disease in VASP -/- mice differed substantially and biphasically from that in wt controls. Early on, VASP -/- mice demonstrated increased platelet influx associated with augmented glomerular and tubulointerstitial inflammation and sclerosis. Whereas renal disease continuously worsened up to day 28 in wt controls, renal disease in VASP -/- mice hardly progressed after day 3 as assessed by various injury indices. This long-term improvement of renal histology in VASP -/- compared with wt mice was associated with remarkable capillary preservation/regeneration up to day 28 mediated via an increased proliferative and a reduced apoptotic activity of VASP-negative peritubular endothelial cells. Despite an enhanced injury response early on, VASP -/- mice are protected from long-term progression of nephrotoxic nephritis, which is associated with improved renal endothelial cell preservation and regeneration.

Inhibition of l‐arginine transport in platelets by asymmetric dimethylarginine and NG‐monomethyl‐l‐arginine: Effects of arterial hypertension

Nitric oxide (NO) produced by human platelets plays an important role in all stages of platelet activation. l-Arginine, the precursor for NO synthesis, modulates NO production by platelets. The l-arginine analogues asymmetric dimethylarginine (ADMA) and N(G)-monomethyl-l-arginine (l-NMMA) are endogenous inhibitors of nitric oxide synthase (NOS), involved in the physiopathology of arterial hypertension. The aim of the present study was to investigate the inhibitory effects of endogenous and exogenous l-arginine analogues on l-arginine influx in platelets from healthy controls and hypertensive patients. Twelve patients with uncomplicated essential hypertension (stage I) and 15 age-matched normotensive controls participated in the present study. Platelets were isolated and incubated with l-[(3)H]-arginine and increasing concentrations of l-arginine analogues (5-2000 micromol/L). The influx of l-arginine was inhibited in a concentration-dependent manner by l-NMMA in platelets from controls (K(i) = 42 +/- 6 micromol/L) and this inhibitory effect was markedly higher in hypertensive platelets (K(i) = 23 +/- 4 micromol/L). Similarly, the K(i) for ADMA inhibition of l-arginine transport was significantly more pronounced in platelets from hypertensive patients (K(i) = 16 +/- 1 micromol/L) compared with controls (K(i) = 27 +/- 2 micromol/L). In contrast, N(G)-nitro-l-arginine methyl ester (l-NAME) was found to be a weak inhibitor of l-arginine influx in platelets from controls (K(i) = 1917 +/- 319 micromol/L) and hypertensive patients (K(i) = 2279 +/- 578 micromol/L). Aminoguanidine, a selective inhibitor of inducible NOS, failed to inhibit l-arginine transport. Our findings provide the first evidence that ADMA and l-NMMA markedly inhibit l-arginine transport in human platelets, an effect that is more pronounced in hypertensive patients. It is possible that endogenous l-arginine analogues, by inhibiting NO synthesis, are involved in the platelet activation present in hypertension.

Abnormal platelet function and calcium handling in Dahl salt-hypertensive rats.

The effect of dietary salt on platelet function and Ca(2+) homeostasis was studied in Dahl (DS) rats, a genetic model of salt-sensitive hypertension. DS rats were fed a high-salt (DSHS) or a low-salt diet (DSLS) for up to 4 weeks, and the effects of salt loading on systolic blood pressure, platelet P-selectin expression, and platelet Ca(2+) homeostasis were measured. The high-salt diet increased blood pressure and markedly increased the amount of ionomycin (IM)-releasable Ca(2+) in platelet intracellular stores (Ca(2+)/IM). The alteration in Ca(2+) stores was not prevented when the hypertension was prevented by treatment with hydralazine and reserpine. The Ca(2+) store filling during platelet exposure to 1 mmol/L Ca(2+) for 5 minutes and the rate of sarcoplasmic/endoplasmic Ca(2+) ATPase-dependent Ca(45) uptake were higher in DSHS compared with that in DSLS. There was a decrease in thrombin-induced Ca(2+) influx in platelets from DSHS; consistent with this, agonist-induced P-selectin expression was decreased. In DSLS, nitric oxide accelerated reloading of platelet Ca(2+) stores after their emptying by thrombin but failed to do so in DSHS. These results indicate that in DS rats, a high-salt diet increases sarcoplasmic/endoplasmic Ca(2+) ATPase activity and the Ca(2+)/IM but decreases the reuptake of Ca(2+) caused by nitric oxide. Decreases in Ca(2+) influx and platelet P-selectin expression might be explained by changes in intracellular Ca(2+) stores in DSHS rats, which apparently is a heritable response to a high-salt diet.

The actin cytoskeleton in store-mediated calcium entry.

Store-mediated Ca2+ entry is the main pathway for Ca2+ influx in platelets and many other cells. Several hypotheses have considered both direct and indirect coupling mechanisms between the endoplasmic reticulum and the plasma membrane. Here we pay particular attention to new insights into the regulation of store-mediated Ca2+ entry: the role of the cytoskeleton in a secretion-like coupling model. In this model, Ca2+ entry may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, that shows close parallels to the events mediating secretion. As with secretion, the actin cytoskeleton plays an inhibitory role in the activation of Ca2+ entry by preventing the approach and coupling of the endoplasmic reticulum with the plasma membrane, making cytoskeletal remodelling a key event in the activation of Ca2+ entry. We also review recent advances investigating the regulation of store-mediated Ca2+ entry by small GTPases and phosphoinositides, which might be involved in the store-mediated Ca2+ entry pathway through roles in the remodelling of the cytoskeleton.

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-plate activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.

Endogenous fibroblast growth factor-2 mediates cytotoxicity in experimental mesangioproliferative glomerulonephritis.

Fibroblast growth factor-2 (FGF-2) is released from mesangial cells in experimental mesangioproliferative glomerulonephritis induced with anti-Thy 1.1 antibody. To investigate the functional role of released FGF-2, rats received either neutralizing anti-FGF-2 IgG or a functional peptide antagonist of FGF-2 (FGF119-126) before or shortly after induction of anti-Thy 1.1 nephritis. In additional experiments, rats were treated with bolus injections of FGF-2 from 2 to 6 h after disease induction. The data showed that anti-FGF-2 therapy led to significant reductions of early mesangial cell injury (mesangiolysis, microaneurysm formation) and the subsequent mesangioproliferative changes (glomerular de novo expression of alpha-smooth muscle actin, mesangial cell proliferation, matrix accumulation, and platelet influx). Conversely, injections of FGF-2 augmented both mesangial injury and the subsequent mesangioproliferative changes. Studies on the mechanisms underlying the amplification of mesangial cell injury by FGF-2 showed that anti-FGF-2 therapy reduced cell death at 2 and 8 h after disease induction by 58 and 54%, respectively. This was associated with significant reductions in the number of glomerular H2O2- and OH -producing cells, as well as reduced glomerular production of nitric oxide. These data suggest that release of constitutively expressed FGF-2 after immune-mediated cell injury contributes to glomerular cell damage and thus identify FGF-2 as a novel mediator of cytotoxicity.

Molecular Basis for ADP-induced Platelet Activation: I. EVIDENCE FOR THREE DISTINCT ADP RECEPTORS ON HUMAN PLATELETS

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, alpha,beta-methylene adenosine 5'-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. alpha,beta-Methylene adenosine 5'-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.