Platelet Calcium Research Articles

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca 2+] i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca 2+] i which suggests that a small portion of the thrombin mediated increase in [Ca 2+] i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca 2+] i by stimulating store-operated Ca 2+ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca 2+] i however it inhibited the ability of thrombin to increase [Ca 2+] i and this effect was reversed by ODQ. The addition of low concentrations (2.0 – 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca 2+] i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca 2+] i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba 2+ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba 2+ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Diethylstilbestrol and Other Nonsteroidal Estrogens: Novel Class of Store-Operated Calcium Channel Modulators

Compounds with the stilbene pharmacophore and other nonsteroidal estrogens have previously been shown to inhibit thrombin-induced elevation of intracellular free calcium ([Ca]i) in human platelets. Thrombin elevates [Ca]i in platelets predominantly by activating a store-operated Ca entry (SOCE) mechanism, probably involving STIM1 and Orai1 although other components may be involved. Human platelets were loaded with the Ca sensitive indicator fura-2, various concentrations of stilbene compounds and other nonsteroidal estrogens were added to the platelets, and thrombin was added to elevate [Ca]i. The degree of inhibition by each compound was determined at the peak increase in [Ca]i induced by thrombin. The additional compounds that were examined in the present study were analogs of diethylstilbestrol (DES), namely trans-resveratrol, hexestrol, tetrahydrochrysene (THC), indenestrol, isoflavones, flavones, and flavanones. DES, indenestrols, and substituted THC diols had the highest inhibitory activity. Dietary polyphenols were less active, and isoflavones were more active than flavones. Glycosides of flavones, flavanones, and isoflavones were inactive. Equol (a product of isoflavone metabolism) had low activity. Among the compounds with a stilbene moiety the presence of unsubstituted phenyl hydroxyls in the para- position were required for activity. Esterification of hydroxyls and bulky substituents at a hydroxyl group diminished or abolished activity. Presence of the ethyl side chains enhanced activity, and shortening or removal of these side chains was detrimental to activity. Presence of the conjugated double bound was necessary for activity. Reduction of the double bond (in fused rings such as equol, dihydrogenistein, indanestrol, or in open chain stilbene derivatives) or repositioning of this double bond outside the stilbene moiety was detrimental to activity, because phenyl rings are not co-planar and have to be at a certain angle to each other. DES likely represents the pharmacophore of this group of nonsteroidal estrogens as an inhibitor of SOCE in platelets.

Role of platelet plasma membrane Ca2+-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis. Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders. On the other hand, hyperactive platelets lead to heart attack and stroke. Calcium is a major second messenger in platelet activation, and elevated intracellular calcium leads to hyperactive platelets. Elevated platelet calcium has been documented in hypertension and diabetes; both conditions increase the likelihood of heart attack and stroke. Thus, proper regulation of calcium metabolism in the platelet is extremely important. Plasma membrane Ca(2+)-ATPase (PMCA) is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux. In keeping with the important role of calcium in platelet function, PMCA is a highly regulated transporter. In human platelets, PMCA is activated by Ca(2+)/calmodulin, by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide. It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis. In addition, the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation. Rapid regulation by phosphorylation results in changes in the rate of platelet activation, whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction. In hypertension and diabetes, PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation. Clearly, a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.

Abstract 770Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt. Disclosures:No relevant conflicts of interest to declare.

Pathogen Inactivation of Platelet Concentrates by UVC-Light: Influence on Platelet Quality, Vitality, Membrane Integrity and Mitochondrial Function.

Introduction: Safety of blood products today is mainly based on strict donor selection, screening systems to detect markers for transfusion relevant viruses and bacterial culturing. Window period, pathogens not tested, emerging viruses and slow growing bacteria are remaining risks for the recipient and will cause a failure of all safety measures. Pathogen inactivation will overcome those problems, at least for platelet and plasma units. UVC light without additional substances has recently proven to inactivate a broad range of viruses (with the exception of HIV) and bacteria (Fokke et al., Transfusion 2008). We investigated the influence of UVC irradiation on platelet storage parameters, apoptosis rate and aggregation capacity. In addition we visualized membrane integrity, mitochondrial activity and intra-platelet calcium content in living platelets using live confocal microscopy.Method: 12 double dose platelet concentrates (6.0x1011 platelets, WBC content below 106) were prepared by means of apheresis (Trima, Gambro BCT, USA), automatically suspended in platelet additive solution (SSP+, Macopharma, BRD) in a 60:40 ratio (SSP+ vs. plasma) and divided into two single units after a two hours resting period. One unit was UVC irradiated immediately after resting at a dose of 0.3J/cm2 (group 2), one remained untreated and both were stored at 22° C for 7 days (group 1). Storage parameters (glucose, lactate, LDH, pH) were analyzed before irradiation and on day two, five and seven. Apoptosis rate was calculated as the percentage of Annexin V positive cells within CD-41 pos cells. Combining life confocal imaging with three fluorescent dyes, fluorescent wheat germ agglutinin (WGA; Molecular Probes, Eugene, OR, USA), tetramethylrhodamine methyl ester perchlorate (TMRM; Sigma-Aldrich) and Rhod-2 (Molecular Probes) we assessed platelet morphology (WGA), mitochondrial activity (TMRM) and intra-platelet calcium (Rhod-2). A students paired T-test was used for statistical analysis and a p value of < 0.05 was considered significant.Results: On day two, the only significant difference in the measured metabolic parameters could be seen in pH (7.28±0.04 vs. 7.25±0.05, group 1 vs. group2;p < 0.01) and differences remained significant over the whole observation period (7.37±0.06 vs. 7.18±0.07, group 1 vs. group2;p < 0.01). On day seven lactate values mg/dl (58.08±13.51 vs. 81.33±13.45, group 1 vs. group2; p < 0.01) and glucose consumption mg/dl (80.33±19.49 vs. 59.50±16,97, group 1 vs. group2; p < 0.01) reached statistical significance, only LDH (U/L) increase did not differ significantly between both groups even not on day seven (68.83±22.17 vs. 75.25±15.08 group 1 vs. group2; p= n.s.). Differences in aggregation capacity reach significance on day 5 (59.50%±10.45% vs. 55.92%±12.30%, group 1 vs. group2; p < 0.05) but not on day seven. Rate of apoptotic platelets was similar on day 2, reaches significance on day 5 (6.53%±3.02% vs. 8.52±3.53, group 1 vs. group2; p < 0.01) and increased on day seven (9.98%±7.34% vs. 18.89%±14.96%, group 1 vs. group2 ; p < 0.01). Image analysis of the confocal data comparing paired samples from untreated and UVC treated platelets revealed no significant differences for all time points. Neither mitochondrial activity, intra platelet calcium content, platelet morphology nor platelet membrane integrity differed between the compared samples.Conclusion: Influence of UVC irradiation of platelet concentrates as a pathogen inactivation method could be witnessed in metabolic parameters as well as in a decrease of aggregation capability and an increase in the rate of apoptotic cells after seven days in a paired comparison with untreated platelets. However differences are small and the total values of the measured parameters meet the quality requirements for platelets. Live confocal imaging of living platelets supports our conclusion but further recovery and survival studies with UVC treated platelets are necessary to compare in-vitro data with in-vivo data.

ATP, calcium and magnesium levels in platelets of patients with primary fibromyalgia

Objectives To evaluate the intracellular levels of the high energy adenosine triphosphate nucleotide ATP and essential divalent cations, calcium and magnesium, in platelets of patients affected by primary fibromyalgia syndrome (FMs). Design and method Platelet ATP and cation concentrations were measured in 25 patients affected by FMs and 25 healthy volunteers through a chemiluminescent and a fluorimetric assay, respectively. Results Significant lower ATP levels were observed inside platelets of FM patients (fmol ATP/plt: 0.0169 ± 0.0012 vs. healthy controls, fmol ATP/plt: 0.0306 ± 0.0023, mean ± SEM) (⁎⁎⁎ P < 0.0001). A trend towards higher calcium concentrations ( P = 0.06) together with significant increased magnesium levels were also reported in platelets of patients by comparison with controls ( P = 0.02). Conclusions This preliminary study suggests that disturbances in the homeostasis of platelet ATP metabolism-signaling and calcium-magnesium flows might have a relevance in the pathogenesis of FMs.

Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Platelet hyperactivity in type 2 diabetes: role of antiplatelet agents

Type 2 diabetes mellitus increases atherothrombotic risk. Platelets in individuals with diabetes show increased activity at baseline and in response to agonists, ultimately leading to increased aggregation. Increased expression of platelet surface adhesion molecules and receptors, enhanced production of thromboxane and thrombin and disturbances in platelet calcium homeostasis are well documented. As intra-arterial thrombi are initiated by platelets, strategies to limit acute thrombotic events have largely focused on antiplatelet agents. Aspirin remains the cornerstone of antiplatelet therapy but appears to have limited benefit in diabetes. Use of thienopyridines and platelet glycoprotein IIb/IIIa receptor inhibitors has been shown to benefit high-risk patient populations. This review summarises the different platelet abnormalities characterised in diabetes and the role of currently used antiplatelet agents.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization

Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

Diabetes mellitus as a prothrombotic condition

Diabetes mellitus (DM) is characterized by fasting hyperglycaemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral and peripheral arterial trees. The risk of myocardial infarction (MI) is 3-5 fold higher in Type 2 DM and a DM subject with no history of MI has the same risk as a non-DM subject with a past history of MI. In total around 70% of deaths are vascular with poorer outcomes to both acute events and cardiological interventions. It was proposed that clustering of vascular risk factors (hyperinsulinaemia, dysglycaemia, dyslipidaemia and hypertension) around insulin resistance (IR) accounted for the increase in risk with Type 2 DM. The importance of this became apparent with the recognition that risk clustering occurs in normoglycaemic and impaired glucose tolerance (IGT) subjects with IR, in total around 25% of the population in addition to long-standing Type 1 subjects with renal disease. Evidence indicates that thrombotic risk clustering also occurs in association with IR, suppression of fibrinolysis due to elevated concentrations of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1) is invariable with IR and there is evidence that this is regulated by the effects of triglyceride on the PAI-1 gene promoter. Other studies indicated that prothrombotic risk (coagulation factors VII, XII and fibrinogen) also associates with the IR syndrome. The development of endothelial cell dysfunction with suppression of nitric oxide and prostacyclin synthesis, combined with platelet resistance to the anti-aggregatory effects of these hormones leads to loss of control over platelet activation. In addition, hyperglycaemia and glycation have marked effects on fibrin structure function, generating a clot which has a denser structure, resistant to fibrinolysis. The combination of increased circulating coagulation zymogens, inhibition of fibrinolysis, changes in fibrin structure/function and alterations in platelet reactivity creates a thrombotic risk clustering which underpins the development of cardiovascular disease.

Molecular mechanism of tamoxifen‐induced calcium influx in human platelets

Tamoxifen, a non steroidal estrogen antagonist, is the most widely used drug for treatment and prevention of breast cancer. However, tamoxifen can cause thrombosis by an unknown mechanism. Using fura-2 to measure intracellular calcium in freshly isolated human platelets, we found that tamoxifen and its major metabolite 4-hydroxy tamoxifen at 5–10 uM promote calcium influx into platelets. Tamoxifen also potentiates action of platelet agonists (thrombin, vasopressin, ADP) to raise calcium. Tamoxifen effect was not attenuated by estrogen receptor antagonist ICI 182780 or COX inhibitor aspirin, but was blocked by the PLC inhibitor U-73122. Tamoxifen-induced calcium elevation was blocked by the lipid-soluble antioxidant vitamin E, but was unaffected by water-soluble vitamin C and quercetin. Tamoxifen effect was also blocked by the NADPH inhibitor apocynin and by wortmannin, an inhibitor of PI3 kinase, an upstream effector of NADPH oxidase. High molecular weight derivatives of 4-hydroxy tamoxifen conjugated to poly-methacrylic acid also promoted calcium elevation in platelets. An anti-estrogenic drug raloxifene, which is structurally related to tamoxifen, does not affect platelet calcium entry. Therefore tamoxifen may affect membrane-bound PLC, NADPH oxidase and PI3 kinase, leading to the activation of store-operated calcium channels, calcium entry into platelets, platelet activation and aggregation, which may be an underlying mechanism of tamoxifen-induced thrombosis. (Supported by grants from CHRB of Virginia and Cancer Research and Prevention Foundation to YD and Army Breast Cancer Research Program to RW).

Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood. 2006; 107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

Thrombin-induced caspases 3 and 9 translocation to the cytoskeleton is independent of changes in cytosolic calcium in human platelets

Apoptosis has been shown to be associated with changes in cytosolic free calcium concentration ([Ca 2+] c). Here we show that the agonist thrombin induces activation of caspases 9 and 3 and translocation of the caspase active forms and procaspases to the cytoskeleton in human platelets. Dimethyl-BAPTA loading did not affect thrombin-induced caspase 9 and 3 activation or translocation suggesting that these responses are independent of increases in [Ca 2+] c. Treatment with thapsigargin plus ionomycin, to induce extensive Ca 2+ store depletion and subsequent increase in [Ca 2+] c, stimulates caspase activation although it was unable to induce caspase translocation to the cytoskeleton. Similar results were observed in cells loaded with dimethyl-BAPTA, suggesting that activation of caspases 9 and 3 by thapsigargin plus ionomycin does not require rises in [Ca 2+] c. These findings suggest that thrombin-induced caspase 9 and 3 activation and translocation are independent on rises in [Ca 2+] c but might require store depletion in human platelets.

PI3-kinase is essential for ADP-stimulated integrin αIIbβ3-mediated platelet calcium oscillation, implications for P2Y receptor pathways in integrin αIIbβ3-initiated signaling cross-talks

Phosphatidylinositol 3-kinase (PI3K) pathway is important for platelet activation. Recent studies showed that PI3K and oscillative calcium could cross talk to each other and positively regulate integrin alpha (IIb)beta3-mediated outside-in signaling. However, the mechanism of this feedback regulation remains to be further characterized. Here we found that treatments of both PI3K inhibitor wortmannin and P2Y1 inhibitor A3P5P could inhibit granular secretion in platelets. Additionally, when RGD-substrate adherent platelets were treated with the ADP scavenger apyrase to deplete the granular-released ADP, their attachments in engaging with substrates became looser and the frequency of calcium oscillation decreased. Since it is known that ADP stimulates the PI3K and calcium signal primarily through P2Y12 and P2Y1 receptors respectively, our data indicated that integrin alpha(IIb)beta3 downstream PI3K and calcium activation might be not completely coupled to integrin associated signaling complex, but in part through feedback stimulation by granular released ADP. Our data indicates the important roles of PI3K and granular released ADP in coordinating the feedback regulations in integrin alpha(IIb)beta3-mediated platelet activation.

Paraformaldehyde induces elevation of intracellular calcium and phosphatidylserine externalization in platelets

Paraformaldehyde fixation of platelets stabilizes surface antigens while altering some that are associated with cellular activation. Present experiments show that the asymmetric distribution of phosphatidylserine in platelets was especially sensitive to paraformaldehyde treatment. It was found that this reagent induced a dose- and time-dependent translocation of phosphatidylserine to the membrane surface as measured by annexin V binding and flow cytometry. The percent phosphatidylserine-positive cells increased from about 5% to > 90%. Chelation of extracellular Ca 2+ with EGTA partially blocked this translocation. Spectrofluorimetric analysis of fluo-3 loaded platelets indicates that paraformaldehyde caused a concomitant elevation of intracellular Ca 2+ concentrations, [Ca 2+] i. ATP levels also declined in paraformaldehyde-treated cells, suggesting that the rise in [Ca 2+] i ensued in part from decreased activity of calcium pumps. Previous studies indicate that phosphatidylserine externalization arises from Ca 2+-activated randomization of membrane phospholipids and decreased transport of phosphatidylserine from the outer to the inner leaflet of the plasma membrane. In light of present results, paraformaldehyde fixation is best avoided particularly in studies involving platelet apoptosis or activation.

Quantitative Trait Loci Mapping for Intracellular Calcium in Spontaneously Hypertensive Rats

Increased intracellular calcium ([Ca2+]i) in platelets is also proposed as an intermediate phenotype for hypertension in spontaneously hypertensive rats (SHR). Increased [Ca2+]i in platelets is hypothesized to contribute to atherothrombotic events. Platelet hyperactivity is frequently associated with cardiovascular disease. In a genome scan, we performed the quantitative trait loci (QTL) mapping for [Ca2+]i in back-crossed rats derived from SHR and normotensive Fischer 344 rats, which demonstrated a single major QTL for hypertension on chromosome 1. Thrombin-stimulated [Ca2+]i in Ca2+-free and in Ca2+-containing buffers was measured in platelets using the Fura-2 method. Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2+]i were significantly greater in SHR than in Fischer 344 and F1 rats. The sarco(endo)plasmic reticulum Ca2+-dependent ATPase II gene locus (Serca2) between D12Mgh5 and D12Mgh6 showed the significant linkage for thrombin-stimulated [Ca2+]i in Ca2+-free and Ca2+-containing buffers. The peak logarithm of the odds scores were 3.6 and 3.3, respectively. These QTL explained 19.8% and 17.4% of the total variances, respectively. D3Mit13 and DXMgh1 showed suggestive linkage for thrombin-stimulated [Ca2+]i in Ca2+-free and in Ca2+-containing buffers, respectively. The peak logarithm of the odds scores were 2.6 and 2.1, respectively. A significant QTL for [Ca2+]i was mapped near Serca2 on chromosome 12, and suggestive QTL were identified near D3Mit13 and DXMgh1 in a genome scan. Genetic abnormalites in platelet [Ca2+]i may contribute to cardiovascular disease via platelet hyperactivity, independent of blood pressure elevation.

Cytoplasmic free calcium mobilization in platelets, expression of P-selectin, phosphatidylserine, and microparticle formation, measured by whole blood flow cytometry, in hypertensive patients. Effect of doxazosin GITS

The effects of doxazosin on expression of CD62 (P-selectin) and phosphatidylserine on platelet membrane and platelet calcium flux were studied in 50 uncomplicated essential hypertensive patients (World Health Organization stages 1–2) and 80 normotensive control subjects, matched for age, sex, and cardiovascular risk factors. Hypertensive patients showed greater in vivo platelet activation at baseline than control patients (percentage of CD62-positive platelets, 4.1 ± 2.2% versus 2.4 ± 1.5%, p < 0.001; percentage of phosphatidylserine-positive platelets, 0.8 ± 0.5% versus 0.5 ± 0.3%, p < 0.001). Increased platelet activation was associated with significant changes in the mobilization of free intraplatelet calcium, evaluated by a whole blood flow cytometric kinetic method. With this method, an arbitrary Ca 2+ mobilization index was defined as the ratio of cytoplasmic free calcium before activation with thrombin to the slope of the calcium removal rate following the action of the agonist. This index was significantly higher in untreated hypertensive patients than in normotensive controls (0.12 ± 0.06 versus 0.05 ± 0.08, p < 0.001). Treatment of hypertensive patients with doxazosin gastrointestinal therapeutic system (4 mg/day as a single dose) for 2 months normalized both platelet activation and Ca 2+ mobilization. Changes in the expression of CD62 and phosphatidylserine in the platelet membrane after treatment with doxazosin gastrointestinal therapeutic system may be related to normalization of the kinetics of cytoplasmic free Ca 2+. Normalization of platelet activation may represent an additional beneficial effect to the known antihypertensive action of doxazosin gastrointestinal therapeutic system.

Decreased Dense Granule Volume in Neonatal Platelets.

Two models of biogenesis have been proposed for the development of platelet granules. One model postulates that granule contents develop in the maturing megakaryocyte via a direct biosynthetic pathway from the golgi, whereas the second model is thought to involve the multivesicular body for sorting and targeting granular constituents via an endosomal pathway. While these models may appropriately apply to the mechanism of the development of platelet alpha granules, dense granule biosynthesis is not readily explained. In animal studies and in human cultured megakaryocyte experiments, molecular markers (CD63 and serotonin) have been reported to appear concomitant with alpha granule formation, yet the characteristic density of the delta granule is not present until later in the maturation of the megakaryocyte. This suggests that dense granule vesicles must be “loaded” for a fully functioning platelet. Our evaluations of platelets obtained from umbilical cord blood would suggest that circulating platelets may become fully functional after birth via a mechanism that loads specific vesicles. The neonate is known to have a transient platelet dysfunction at the time of birth which has been explained in the literature as being due to 1) a diminished response to physiologic agonists, 2) defective platelet dense granule secretion, and/or 3) ineffective mobilization of intracellular calcium in platelets. Studies to define the transient platelet dysfunction in the neonate have been limited. We and others have found that the adenine content of platelets obtained from umbilical cord blood is significantly less than seen in adults. We have found that platelets obtained from umbilical cord blood have an average of 0.79 ± 0.08 DG/PL (n=62), which is significantly decreased compared to the adult population and that the ATP:ADP ratio in these platelets is 1.95 (n=61), consistent with established adult normal range ratio values (1.4–1.95). The actual measured adenine content was found to be decreased in the neonatal platelets (ATP = 2.76 ± 0.53 mmol/1011 PL; ADP=0.96±0.19 mmol/1011 PL). The normal adenine nucleotide DG contents have been reported to be 3.5–7 mmol/1011 PL for ATP and 1.9–3.7 mmol/1011 PL for ADP in adults. Using the uranaffin reaction technique to classify the four types (I–IV) of dense granules present in neonatal platelets, we have found a significant difference in the percentage of each type than is found in adult controls. We have employed an image analysis technique (Blood , 90: 140a, 1997) to calculate the mean volume of dense granules in neonatal platelets. We report that not only do neonatal platelets have a deficiency of dense granules, but that these granules are also extremely small in size having an average volume of 6.45 X 105 nm3 (n =36) (normal adult = 8–12 X 106 nm3). These results would suggest that the neonatal platelet has an incomplete incorporation of constituents (i.e., calcium, ADP) into the dense granules at the time of birth and that an unknown mechanism exists that induces dense vesicle “loading” at some time after birth. These observations may prove useful in pursuit of understanding the mechanism of platelet dense granule biosynthesis.

Direct Real Time Visualization of Platelet Calclium Signaling In Vivo: Role of Platelet Activation and Thrombus Formation in a Living Mouse.

Intracellular calcium mobilization plays a critical role in platelet signaling. Upon platelet activation, an intracellular calcium mobilization leads to the activation of various intracellular and membrane proteins, including integrins involved in both platelet shape change and aggregation. The goal of the present study was to monitor platelet calcium mobilization in vivo in an intact animal and to determine which intracellular pathways are dominant in platelet accumulation into the developing thrombus. Platelets were isolated from mice, washed, loaded with a calcium-sensitive fluorochrome, Fura2-AM and then infused into a recipient mouse. We studied Fura2-AM loaded platelet incorporation during arterial thrombus development following laser injury of the vessel wall in the cremaster microcirculation of living mice using high speed intravital widefield digital microscopy. Fura-2 loaded platelets were monitored by excitation at 380 nm; this fluorescence reports the basal calcium levels in platelets. Calcium mobilization was monitored by excitation at 340 nm where the fluorescence intensity reflects Fura2-calcium complex formation. We observed that platelets bind to the growing thrombus independent of calcium mobilization. However, the stable incorporation of platelets into the thrombus correlated with a significant intracellular calcium increase. Once the thrombus reached maximal size at about 100 seconds, the calcium mobilization also reached maximal intensity. Subsequently, platelets that did not mobilize calcium dissociated from the thrombus. We confirmed these observations by using platelets treated with the calcium chelators, BAPTA-AM or EGTA-AM. We observed a significant inhibition of platelet accumulation into the thrombus, indicating that the intracellular calcium increase is necessary in vivo for the stable accumulation of the platelets into the thrombus. We also evaluated the involvement in vivo of two platelet agonists, ADP and thromboxane A2 (TxA2), on calcium mobilization and platelet incorporation into thrombi. When platelets were treated with aspirin or with the P2Y1 antagonist A3P5P (adenosine 3′-phosphate-5′-phosphate), we observed a partial decrease in both calcium mobilization and platelet accumulation into the thrombus. These results indicate that TxA2 and ADP via the P2Y1 receptor are involved in vivo in platelet activation upon vessel wall injury in this thrombosis model. When platelets were treated with both compounds, we completely inhibited the calcium increase and the incorporation of platelets into the thrombus. Altogether, our results directly show, for the first time in vivo, the importance of the calcium mobilization on platelet accumulation into the developing thrombus. The platelet agonists TxA2 and ADP both play an important and complementary role on platelet activation by acting on the mobilization of the intracellular calcium.