Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.     

Low-density lipoprotein and its effect on human blood platelets

Events leading to hyperactivity of human blood platelets are accompanied by an enhanced risk of atherosclerosis and arterial thrombosis. Lipoprotein disorders affect platelet functions, and hypersensitive platelets are observed in various stages of hyperlipidemia. Low-density lipoprotein (LDL), a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and increases sensitivity of platelets to several naturally occurring agonists. LDL sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane. The receptor that mediates binding of LDL to the platelet and initiates subsequent intracellular signaling cascades has not yet been identified. Modification of native LDL generates a platelet-activating particle, and this interaction might contribute to the development of the atherosclerotic plaque. Lysophosphatidic acid is formed upon mild oxidation of LDL and is responsible for subsequent platelet activation induced by the modified LDL particle. Thus, LDL changes the functions of platelets via a broad spectrum of interactions.     

Anandamide extends platelets survival through CB1-dependent Akt signaling

Platelets are stored at 22°C, since incubation at 37°C results in loss of viability. Nonetheless, in our body (37°C), platelets survive for 8–10 days. This discrepancy has been explained in terms of deprivation of viability factors or accumulation of apoptotic factors during storage. We report that the endocannabinoid anandamide (AEA) may be one of the agents allowing platelet survival. In fact, at 37°C, human platelets enhance the expression of pro-apoptotic proteins (caspases, Bax, Bak) and decrease the expression of Bcl-xL, thus changing the Bcl-xL/Bak ratio, a key platelet biological clock. AEA or its non-hydrolyzable analogue, methanandamide, extend platelet life span, without reversing the changes in Bcl-xL/Bak ratio induced by heat stress. Instead, AEA binding to type-1 cannabinoid receptor activates Akt, which regulates, through phosphorylation of Bad, the interactions among different Bcl-2 family members. These findings could have implications for platelet collection and, potentially, for their clinical use.     

Human endoglin as a potential new partner involved in platelet–endothelium interactions

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng ^+/?) mice compared to Eng ^+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.     

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor α _IIb  β ₃ , has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of α _IIb  β ₃ inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.     

Platelet–bacterial interactions

Many bacteria are capable of interacting with platelets and inducing platelet aggregation. This interaction may be a direct interaction between a bacterial surface protein and a platelet receptor or may be an indirect interaction where plasma proteins bind to the bacterial surface and subsequently bind to a platelet receptor. However, these interactions usually do not trigger platelet activation as a secondary co-signal is also required. This is usually due to specific antibody bound to the bacteria interacting with FcγRIIa on the platelet surface. Secreted bacterial products such as gingipains and lipopolysaccharide may also be capable of triggering platelet activation.     

Human platelet 5-hydroxytryptamine receptors: binding of [3H]-lysergic acid diethylamide (LSD). Effects of chronic neuroleptic and antidepressant drug administration

Chronic treatment with phenothiazines and thioxanthenes has been found to enhance 5-HT-induced aggregation of human platelets. A method has been developed to study 5-HT2 receptor binding sites on platelets utilising [3H]-LSD and more recently 125I/LSD. Results are presented which suggest that the LSD binding site is indeed the 5-HT2 binding site and that the LSD binding characterises the specific receptor responsible for 5-HT-induced shape change and aggregation. In a group of patients receiving phenothiazines or thioxanthenes, the Bmax of LSD binding was increased. The mean binding affinity was decreased possibly due to a persistence of neuroleptic in the platelet membrane preparation. Analysis showed that this was not the reason why the mean binding capacity was increased. The results show that chronic phenothiazine and thioxanthene delta treatment 'up-regulates' platelet 5-HT2 binding sites and that this may be accompanied by increased sensitivity to platelet aggregation by 5-HT. In normal subjects desipramine treatment increased the Bmax of platelet LSD binding and this was accompanied by an increased prolactin response to tryptophan which is thought to be mediated by central 5-HT function.     

Immunochemical study of protein anomalies in platelets of patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome]

Crossed immunoelectrophoresis of "Triton X-100" solubilized proteins from (125I) iodinated normal and abnormal (Bernard-Soulier and thrombasthenic) platelets was performed using rabbit antibodies raised against whole, normal platelets. In Bernard-Soulier platelets neither of the two surface oriented forms of GP I, glycocalicin and glycoprotein Ib (band 13) were detected. In two patients with type I thrombasthenia, the most prominent precipitate (No. 16) and platelet fibrinogen (No 24) were not detected whereas in one patient with type II thromblasthenia, fibrinogen was normally detected and the amount of protein 16 was around 15% of the normal level. The alloantibody IgG L..., able to induce a "thrombasthenic-like" reactivity in normal human platelets, has been shown to be directed against protein 16.     

Cytosolic calcium in platelet activation

Experiments with permeabilised platelets, and with intact platelets loaded with fluorescent Ca2+-indicators, over the past several years have greatly extended our knowledge and understanding of cytosolic Ca2+ as a platelet activator and its interactions with other cytosolic regulators. This article outlines insights, gained from the use of the fluorescent dyes, into maintenance and restoration of basal [Ca2+]i, mechanisms of receptor-mediated Ca2+-mobilisation and quantitation of [Ca2+]i/response relations in intact human platelets.     

Cytosolic calcium in platelet activation

Experiments with permeabilised platelets, and with intact platelets loaded with fluorescent Ca²⁺-indicators, over the past several years have greatly extended our knowledge and understanding of cytosolic Ca²⁺ as a platelet activator and its interactions with other cytosolic regulators. This article outlines insights, gained from the use of the fluorescent dyes, into maintenance and restoration of basal [Ca²⁺]_i, mechanisms of receptor-mediated Ca²⁺-mobilisation and quantitation of [Ca²⁺]_i/response relations in intact human platelets.     

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca²⁺-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.Received 8 March 2005; received after revision 19 April 2005; accepted 13 May 2005     

Platelets in defense against bacterial pathogens

Platelets interact with bacterial pathogens through a wide array of cellular and molecular mechanisms. The consequences of this interaction may significantly influence the balance between infection and immunity. On the one hand, recent data indicate that certain bacteria may be capable of exploiting these interactions to gain a virulence advantage. Indeed, certain bacterial pathogens appear to have evolved specific ways in which to subvert activated platelets. Hence, it is conceivable that some bacterial pathogens exploit platelet responses. On the other hand, platelets are now known to possess unambiguous structures and functions of host defense effector cells. Recent discoveries emphasize critical features enabling such functions, including expression of toll-like receptors that detect hallmark signals of bacterial infection, an array of microbicidal peptides, as well as other host defense molecules and functions. These concepts are consistent with increased risk and severity of bacterial infection as correlates of clinical abnormalities in platelet quantity and quality. In these respects, the molecular and cellular roles of platelets in host defense against bacterial pathogens are explored with attention on advances in platelet immunobiology.     

Lactoferrin

Mammalian lactoferrin (Lf) receptors are suggested to have pivotal roles for mediating multiple functions of Lf. In this review, we focus on current knowledge of the structure and function of mammalian Lf receptors, mainly the first cloned Lf receptor that has been shown to be expressed in the infant small intestine at high levels but also in virtually all other tissues. The small intestinal Lf receptor takes up iron from Lf into cells and presumably exerts other physiological functions. Other Lf receptors in various tissues have also been reported to mediate some functions of Lf, such as modulating immune function, inhibiting platelet aggregation and enhancing collagen gel contractile strength. The detailed mechanisms behind the receptor-Lf interactions still need to be elucidated.     

Platelets and innate immunity

Although platelets are best known as primary mediators of hemostasis, this function intimately associates them with inflammatory processes, and it has been increasingly recognized that platelets play an active role in both innate and adaptive immunity. For example, platelet adhesive interactions with leukocytes and endothelial cells via P-selectin can lead to several pro-inflammatory events, including leukocyte rolling and activation, production of cytokine cascades, and recruitment of the leukocytes to sites of tissue damage. Superimposed on this, platelets express immunologically-related molecules such as CD40L and Toll-like receptors that have been shown to functionally modulate innate immunity. Furthermore, platelets themselves can interact with microorganisms, and several viruses have been shown to cross-react immunologically with platelet antigens. This review discusses the central role that platelets play in inflammation, linking them with varied pathological conditions, such as atherosclerosis, sepsis, and immune thrombocytopenic purpura, and suggests that platelets also act as primary mediators of our innate defences.     

Glycoprotein IIb/IIIa blockade inhibits platelet aminophospholipid exposure by potentiating translocase and attenuating scramblase activity

The present study investigated the mechanisms underlying the inhibition of platelet phosphatidylserine (PS) exposure by GPIIb/IIIa blockade. Platelet PS exposure induced by thrombin stimulation was cell-cell contact dependent. GPIIb/IIIa blockade by c7E3 or SR121566 inhibited thrombin-induced platelet PS exposure. Thrombin stimulation induced mild, while A23187 induced extensive platelet-derived microparticle (PDMP) generation. Thrombin-induced PDMP generation was not inhibited by GPIIb/IIIa blockade. Aminophospholipid translocase activity was reduced upon platelet activation by thrombin. The reduction of non-PS-exposing platelets was attenuated by GPIIb/IIIa blockade, while little translocase activity was seen in PS-exposing platelets. Thrombin increased scramblase activity slightly in non-PS-exposing platelets, which was inhibited by GPIIb/IIIa blockade, and markedly enhanced scramblase activity in PS-exposing platelets. Activation of platelet calpain and caspase-3 or cytosolic calcium mobilization were not altered by GPIIb/IIIa inhibition. Thus, GPIIb/IIIa blockade inhibits platelet PS exposure by enhancing translocase activity and attenuating scramblase activity, but does not inhibit PDMP generation. Received 13 December 2006; received after revision 5 February 2007; accepted 9 March 2007     

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with β,γ-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 μM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36. Received 18 July 2002; received after revision 29 August 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Components of biological,including seasonal,variation in hematological measurements and plasma fibrinogen concentrations in normal humans

This study has been carried out in order to examine the components of biologicalaand, in particular, seasonal variation in hematologic measurements in normal humans. Toward this end, 26 normal volunteers had monthly blood samplings during one calendar year for determination of number of red blood cells (RBC) and platelets, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), MC Hb (MCH), MC Hb concentration (MCHC), RBC distribution width (RDW), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), and plasma fibrinogen concentrations. The data were analyzed by means of spectral analyses of a group of time series or a single time series, and by means of repeated measures analyses of variance. Most of the hematologic variables show seasonal rhythms, such as annual rhythms or harmonics, which are expressed as a group phenomenon. An important part of the variance (>15%) in Ht, MCV, MCH, MCHC, RDW, number of platelets, MPV and plasma fibrinogen was explained by a yearly variation. The peak-trough differences (expressed as a percentage of the mean) in the yearly variations in number of RBC, Ht, MCV, MCH, MCHC and RDW were very low (all<8.5%). Number of platelets (14.4%) and plasma fibrinogen values (28%) showed a high-amplitude yearly variation. All hematological variables, except MCHC, show a high interindividual variability which exceeds by far the intraindividual variability.     

LDL receptor relatives at the crossroad of endocytosis and signaling

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.