Platelet Membrane Phospholipids Research Articles

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). Client-owned dogs with platelet counts <50 000/μL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Incidence of antiphospholipid antibodies in new patients with non-Hodgkin lymphoma

Non-Hodgkin’s lymphoma (NHL) presents a group of histologically and biologically inhomogeneous B and T cell neoplasms of lymphoid tissue with a completely unidentified etiology. Antiphospholipid antibodies (aPL) are antibodies produced as a result of misinterpretation of platelet membrane phospholipids. It is well known that antiphospholipid antibodies are general risk factors that induce the disorder of the physiological process of hemostasis. Respectively, it is interesting to appreciate the incidence of antiphospholipid antibodies in new non-Hodgkin lymphomas patients depending on age, sex, type of non-Hodgkin’s lymphoma, the peculiarities of the onset of the disease, the degree of disease spread. According to the results of our study, we found a 14.8% incidence of aPL in primary patients with non-Hodgkin’s lymphoma, more frequently in men and people older than 50 years. The positivity of aPL antibodies depended on the immunohistochemical type of malignant lymphoma, the degree of dissemination of the tumor process and independent of the location of the tumor focus (nodal or extranodal) of NHL. The incidence of aPL antibody types was uneven with the obvious predominance of lupus anticoagulant. Th is study allowed the evaluation of the incidence of antiphospholipid antibodies in primary patients with non-Hodgkin’s lymphoma.

A SYSTEMATIC LITERATURE REVIEW OF BUDD-CHIARI SYNDROME IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Antiphospholipid syndrome is associated with multiple and recurrent episodes of arterial and venous thrombosis, thrombocytopenia, spontaneous fetal losses, and high levels of antiphospholipid antibodies. These antibodies can also be present in systemic lupus erythematosus (SLE), and predispose patients with Lupus to a hypercoagulable state. Budd-Chiari syndrome (BCS) is characterized by hepatic venous outflow obstruction that can lead to hepatic congestion and it is deeply associated with acquired or inherited hypercoagulable states. Therefore, the aim of this study was to conduct a systematic literature review to analyze the association between Budd-Chiari syndrome and SLE. A systematic literature review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Articles were selected from PubMed/Medline, SciELO and LILACS databases in August 2021 using the search terms [Lupus] OR [SLE] AND [Budd-Chiari syndrome]. The inclusion criterion was limited to observational studies, case reports or case series that reported an association between Budd-Chiari syndrome and SLE. Language was restricted to English, Spanish or Portuguese. Among 76 articles initially identified, 31 were eligible for this review after full texts were read. The vast majority of the studies have linked the antibodies lupus anticoagulant and anticardiolipin as etiological factors of BCS in patients with lupus. One study has reported six cases in which factor V Leiden was strongly related to SLE, suggesting that this mutation should also be investigated in patients with lupus and BCS. Few studies have mentioned isolated deficiency of protein C and/or protein S as common etiologies of BCS in patients with lupus. Studies suggest that Lupus anticoagulant can be sought in patients with idiopathic hepatic venous thrombosis. Possible mechanisms by which antiphospholipid antibodies induce thrombotic events in patients with SLE have been suggested: it can bind to platelet membrane phospholipids and accentuate their adhesion and aggregation ability, it can interfere in the interactions of proteins C and S and impair the formation of the coagulation control complex (activated protein C, protein S and factor V). Budd-Chiari syndrome can be a rare complication in patients with SLE. A methodical search for hepatic vein occlusion in patients with SLE who develop unexplained hepatomegaly should be performed. In addition, screening for lupus anticoagulant and anticardiolipin antibodies should take place to reduce the percentage of apparently idiopathic BCS cases.

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis in mice and humans

AbstractHyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. In this study, human medical examination showed that elevated human plasma Hcy levels correlated positively with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation in apolipoprotein E-deficient (ApoE−/−) mice with acute or chronic HHcy. Upon homocysteine (Hcy) administration in C57BL/6J mice, platelet aggregation, spreading and clot retraction were markedly induced. More important, Hcy increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE−/− mice. In this study, we identified a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently exacerbating thrombosis and the results revealed an innovative approach to treating HHcy-related thrombotic diseases.

EFFICACY OF TELMISARTAN GROUP IN PATIENTS ISCHEMIC HEART DISEASE WITH ARTERIAL HYPERTENSION

In the studyphospholipids, cholesterol and its metabolites play a special role in of biological membranes. Disruption of the phospholipid content of biological membranes is one of the main factors of pathological processes, because phospholipids make up the largest part of the lipids of biological membranes, as well as platelet membranes. The study examined the effect of telmisartan on the clinical hemodynamics and platelet membrane phospholipids, cholesterol metabolism in patients with ischemic heart diseaseandarterial hypertension during 8 weeks of therapy.Keywords:ischemic heart disease,arterial hypertension, telmisartan, enalapril, phospholipids, lysophosphotidylcholine, phosphotidylcholine, syphingomyelin, phosphotidylserine, phosphatidylethanolamine

Abstract P335: Greater Frequency of Olive Oil Consumption is Associated with Lower Platelet Activation in Obesity

Healthy lifestyle behaviors, including maintenance of normal weight, are associated with lower risk of atherothrombotic events. In the general population, consumption of olive oil and a Mediterranean diet are associated with reduced risk of cardiovascular disease (CVD). However, less is known about how this affects individuals with obesity. We investigated whether olive oil intake is associated with reduced platelet activation in a cohort of obese, non-smoking, non-diabetic subjects without known CVD. As part of a larger prospective study of platelet function in obesity, we assessed platelet activation (surface P-selectin and PAC1 MFI) with and without agonist (0.025U thrombin, 0.1uM ADP, 0.4uM epinephrine) exposure via flow cytometry. Food frequency surveys were used to estimate diet composition. Subjects were stratified by food intake frequency and platelet activation was compared using ANOVA, with post-hoc independent samples t-testing. Among 63 subjects, the mean age was 32.2 ± 8.0 years and BMI 44.1 ± 8.5 kg/m 2 . Olive oil intake frequency was stratified into 3 groups: ≤ 1 time/wk, n=21; 1 - 3 times/wk, n=18; ≥ 4 times/wk, n=24. The groups did not differ by age, weight, BMI, waist and hip circumferences, platelet count or mean platelet volume. Subjects with more frequent olive oil intake showed lower platelet activation to agonists than those consuming olive oil ≤ 1 time/wk (Figure). Non-agonized platelet activity did not differ significantly by olive oil intake frequency. Notably, we did not observe associations between platelet activity and consumption of red meat, eggs, butter, or margarine. In metabolically healthy obese individuals, more frequent olive oil intake is associated with suppressed platelet activation. We hypothesize that phenolic compounds in olive oil may alter platelet membrane phospholipid content, making platelets less susceptible to activation. For obese individuals, olive oil intake may lower risk of cardiovascular events.

Phospholipid-Dependent Mechanisms of Platelet Dysfunction in Rabbits with Hemorrhagic Shock.

The time course of phospholipid composition of platelet plasma membranes at the peak of hemorrhagic shock were studied in rabbit experiments. The results indicate the key role of impairment of the structure of platelet membrane phospholipid bilayer in the pathogenesis of shockogenic thrombocytopathy.

Apoptosis Signal-Regulating Kinase (ASK1) Regulates Thrombosis in Part By Regulating cPLA2 phosphorylation-Dependent TxA2 Generation

When vascular endothelium is injured, circulating platelets are activated by primary agonists. Activation causes platelets to change shape, aggregate, and release secondary agonists which reinforce initial platelet activation as well as help recruit additional platelets to the site of vascular injury. MAP kinases have been shown to be important regulators of platelet function and secondary agonist production. One important secondary agonist released by activated platelets is TxA2. TxA2 is generated by metabolism of Arachidonic acid (AA). AA is released from platelet membrane phospholipids via the activity of PLAs. In platelets cPLA2 activity has been shown to be regulated by MAP kinases, however, the mechanisms which regulate platelet MAP kinase activity are not well understood. Our laboratory has identified that ASK1 (a Ser/Thr kinase of the MAP3K family) is present in both human and murine platelets and is activated by physiological agonists. ASK1 is known to be activated by a number of cellular stress response pathways. When challenged by cellular stress, ASK1 auto phosphorylates Thr845 on its activation loop, which is required for its ability to phosphorylate its substrates. Here we show that ASK1 regulates platelet function in part by regulating agonist-induced TxA2 generation. To determine the role of Ask1 in hemostasis and thrombosis, we evaluated in vivo thrombosis using carotid artery injury induced by 10% FeCl3 or pulmonary thromboembolism induced by injecting mixture of collagen/epinephrine. We found that genetic ablation of Ask1 renders mice significant protection from thrombosis. To determine the mechanism by which Ask1 regulates platelet activation leading to thrombosis, we evaluated the MAP kinase cascade using Ask1 null platelets. We found that genetic ablation of Ask1 blocked agonist-induced activation of the MAP2Ks (MKK3 and MKK4) in murine platelets. Since MKK3 can activate p38 and MKK4 can activate both p38 and JNK, we assessed MAPKs activation in murine platelets. When stimulated by various agonists, activation of p38 was entirely lost in Ask1 null platelets while activation of ERK1/2 and JNK remained unaffected indicating that Ask1 solely regulates p38 activity in platelets. Activity of p38 has been linked to agonist-induced generation of TxA2, an important contributing factor to thrombosis. We therefore evaluated agonist-induced production of TxA2 by measuring TxB2 (a stable metabolite of TxA2). We saw a substantial reduction (~50% in thrombin- and ~70% in convulxin-induced) production of TxA2 in Ask1 null platelets suggesting a separate Ask1 independent mechanism for TxA2 generation. Since TxA2 is a metabolite of AA, whose production in platelets is caused by cPLA2 enzymatic activity and cPLA2 activity is regulated by phosphorylation of its Ser505 residue by p38, we evaluated phosphorylation of cPLA2 (p-Ser505). We found that agonist-induced phosphorylation of cPLA2 (Ser505) was completely lost in Ask1 null platelets. Although in Ask1 null platelets cPLA2 phosphorylation (Ser505) is completely abolished, substantial amount (~50%) of TxA2 was generated in response to thrombin suggesting that there exists an Ask1 independent mechanism of activation of cPLA2. To rule out the possibility that an alternative PLA2 is responsible for the residual TxA2 production found in Ask1 null platelets, we evaluated agonist-induced TxA2 production in the presence of pyrrophenone, a cPLA2 specific inhibitor. Pretreatment with pyrrophenone completely abolished agonist-induced TxA2 production in murine as well as human platelets, suggesting that cPLA2 is solely responsible for the majority of agonist induced AA/TxA2 in platelets. In addition to its phosphorylation, it is documented that cPLA2 activity is also dependent on intracellular Ca2+, which facilitates translocation of cPLA2 to AA containing membranes. It is therefore possible that the remainder of TxA2 formed is dependent on Ca2+-dependent activity of cPLA2. Taken together these in vivo and in vitro results strongly suggest that ASK1 plays a key role in regulating thrombosis, in part, by regulating the signaling mechanisms involved in agonist-induced production of TxA2. DisclosuresNo relevant conflicts of interest to declare.

Expression of anoctamins in retinal pigment epithelium (RPE).

The anoctamin (ANO, TMEM16) family of Ca2+-activated Cl- channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca2+-activated Cl- channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca2+-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca2+ signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca2+ signals. Anoctamins may also form the basolateral Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca2+-activated Cl- secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca2+-dependent Cl- secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca2+ signals.

TMEM16F-Mediated Platelet Membrane Phospholipid Scrambling Is Critical for Hemostasis and Thrombosis but not Thromboinflammation in Mice-Brief Report.

It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Порівняння ефективності антиагрегантної терапії у хворих на уперше виниклу і прогресуючу стенокардію за впливом на жирнокислотний спектр фосфоліпідів мембран тромбоцитів

Порівняння ефективності антиагрегантної терапії у хворих на уперше виниклу і прогресуючу стенокардію за впливом на жирнокислотний спектр фосфоліпідів мембран тромбоцитів

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca(2+)-dependent Cl(-) ion transport, and TMEM16F is responsible for Ca(2+)-dependent phospholipid scrambling. Here we established assay systems for the Ca(2+)-dependent Cl(-) channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F(-/-) immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl(-) channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl(-) channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

Class I Phosphatidylinositol Transfer Proteins (PITPs) Promote PIP2 Synthesis and IP3 Production in Mouse Platelets

Abstract 363Phosphoinositides are the phosphorylated forms of phosphatidylinositol. Although they constitute only 1% of platelet membrane phospholipids, phosphoinositides are important contributors to platelet signaling. As an example the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PIP2) has been shown to regulate integrin activation, actin assembly, and secretion in platelets. PtdIns(4,5)P2 contributes to these events by directly binding to signaling proteins, and also by serving as a substrate for PLC and PI3K to generate the second messengers Inositol(3,4,5)P3 (Ins(3,4,5)P3) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The phosphoinositide concentration in different cellular compartments is tightly regulated both in time and space, and their relative amounts of various phosphoinositides change within seconds of agonist stimulation. Given their importance in platelet biology, we sought to better understand the mechanism of phosphoinositide synthesis and function. Class I PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that bind to and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner. Although there are no studies on the function of PITPs in hematopoietic cells, the proteins are essential in yeast cells for the biosynthesis and metabolism of phosphoinositides. This suggests that PITPs may also be essential for phosphoinositide metabolism in platelets. There are two dominant Class I PITP family members, PITPα and PITPβ in murine platelets. We introduced conditional loss-of-function mutations into the murine PITPα and PITPβ genes These mice were crossed with transgenic mice expressing Cre recombinase under the control of the platelet specific promoter PF4, thereby generating mice lacking individual PITP isoforms only in platelets and megakaryocytes. Mice lacking either platelet PITP isoform have approximately one-third lower platelet counts than littermate controls, but otherwise have normal blood counts and appear phenotypically normal. We have also generated a few viable double knockout (PITPαfl/fl PITPβfl/fl PF4Cre+) mice and found that these mice produce some platelets even in the absence of both PITP isoforms. Next, we analyzed the concentration of individual phosphoinositides in platelets of the knockout mice by thin layer chromatography. Although loss of either PITP isoform did not affect PtdIns(4)P synthesis in resting or thrombin stimulated platelets, PtdIns(4,5)P2 synthesis was significantly inhibited in platelets lacking either PITPα (∼40% of normal) or PITPβ (∼50% of normal). This suggests that both PITPα and PITPβ are required for normal PtdIns(4,5)P2 synthesis in platelets. To test whether PITP isoforms are critical for second messenger formation, we analyzed the formation of Ins(3,4,5)P3 in response to thrombin stimulation. We found that the PITPα-null mutation causes an almost absence of Ins(3,4,5)P3 formation. The PITPβ-null mutation also caused a profound defect in Ins(3,4,5)P3 formation, albeit less than that seen following PITPα deletion. Nonetheless, both PITPα and PITPβ knockout platelets were able to increase their intracellular calcium concentration following thrombin stimulation to approximately 50% of normal. These data imply that a significant portion of the calcium response in platelets results from the influx of extracellular calcium and not by the Ins(3,4,5)P3–dependent release of calcium from intracellular stores. Further, platelets lacking either PITP isoform exhibited only a moderate (∼50%) defect in Akt phosphorylation – a PtdIns(3,4,5)P3 synthesis-dependent event. Although both Ins(3,4,5)P3 and PtdIns(3,4,5)P3 are derived from PtdIns(4,5)P2, it is remarkable that PITP enzymes are more critical for Ins(3,4,5)P3-synthesis than for PtdIns(3,4,5)P3-synthesis (as suggested by Akt phosphorylation). Finally, we performed pilot experiments of the ex vivo function of platelets lacking both PITP isoforms and found that double knockout platelets had a profound loss of agonist-induced platelet aggregation. Together, our data suggest that PITPα and PITPβ are both critical for the synthesis of essential platelet phosphoinositides and second messengers. Thus, these enzymes may represent novel therapeutic targets for the prevention and treatment of arterial occlusion. Disclosures:No relevant conflicts of interest to declare.

Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome.

Like all eukaryotic cells, platelets maintain plasma membrane phospholipid asymmetry in normal blood circulation via lipid transporters, which control transbilayer movement. Upon platelet activation, the asymmetric orientation of membrane phospholipids is rapidly disrupted, resulting in a calcium-dependent exposure of the anionic phospholipid, phosphatidylserine (PS), at the outer platelet surface. This newly-exposed PS surface is a major component of normal hemostasis because it supports platelet procoagulant function. Binding of blood clotting enzyme complexes to this negatively-charged membrane surface allows a dramatic increase in the rate of conversion of zymogens to active serine proteases, which in turn produce a burst of thrombin leading to the formation of a fibrin clot and further platelet activation. Cells have the capacity to catalyze transbilayer phospholipid exchange via ATP-requiring translocase enzymes (flippases and floppases), which control unidirectional phospholipid transport against a concentration gradient. They also use an energy-independent, calcium-dependent scramblase activity to govern the bidirectional exchange of phospholipids between the two leaflets of the bilayer; this activity is essential for PS exposure during platelet activation. Scramblase activity, biochemically characterized in the 1980s, is deficient in patients with Scott syndrome, a rare inherited bleeding disorder with defective platelet procoagulant activity. Despite considerable efforts, the platelet scramblase protein remained elusive for years but a significant advance has recently been made with the identification of TMEM16F, a membrane protein essential for calcium-dependent PS exposure whose loss of function mutations are found in Scott syndrome. This review recalls historical aspects of platelet membrane asymmetry characterization, summarizes the mechanisms and roles of PS exposure following platelet activation and discusses the recent identification of TMEM16F and its significance in the scrambling process.

Recombinant factor VIIa (rFVIIa): its potential role as a hemostatic agent

Recombinant activated coagulation factor VII (rFVIIa) was developed for the treatment of patients with hemophilia who have developed inhibitors against the factor they are missing. Hemophilia is a serious bleeding disorder and patients with hemophilia develop repeated spontaneous CNS, joint and muscle bleeding. Any trauma, even mild events, may cause life-threatening bleeding, and without treatment, these patients have a life expectancy of about 16 years. Thus, hemophilia can be regarded as a model of severe bleeding, and an agent capable of inducing hemostasis in severe hemophilia independent of the hemophilia proteins (FVIII or FIX) may also be effective in patients without hemophilia who experience serious bleeds. The availability of rFVIIa stimulated research on the role of FVII and tissue factor (TF) in the hemostatic process. As a result, a picture partly different from the one suggested by previous models has emerged. These previous models basically neglected the role of cells and cell membranes. The importance of platelets and platelet membrane phospholipids in hemostasis has been demonstrated, and the new concept of the hemostatic process, focusing on cell surfaces, has been outlined.

Advanced glycation end products elicit externalization of phosphatidylserine in a subpopulation of platelets via 5-HT2A/2C receptors

Advanced glycation end products (AGE) are substantially elevated in individuals with diabetes and/or chronic kidney disease (CKD). These patients are at greatly increased risk of cardiovascular events. The purpose of this study was to investigate the novel hypothesis that AGE elicit externalization of the platelet membrane phospholipid phosphatidylserine (PS). This contributes to hemostasis through propagation of the coagulation cascade leading to thrombus formation. Platelet-rich plasma (PRP) was prepared by differential centrifugation, and PS externalization was quantified by a fluorescence-activated cell sorter using annexin V-FITC. Human serum albumin (HSA)-AGE was generated by incubating HSA with glucose for 2, 4, or 6 wk, and total HSA-AGE was assessed by fluorescence intensity. The 2-wk HSA-AGE preparation (0-2 mg/ml) stimulated a concentration-dependent increase in PS externalization in a subpopulation of platelets that was threefold at 2 mg/ml. In contrast, the 4- and 6-wk preparations were maximal at 0.5 mg/ml and fivefold in magnitude. These effects mirrored the change in total HSA-AGE content of the preparations. The PS response was maximal at 10 min and inhibited by the PKC-delta inhibitor rottlerin and the serotonin [5-hydroxytryptamine (5-HT)](2A/2C) receptor antagonist ritanserin in a dose-dependent manner. Moreover, the 5-HT(2A/2C) receptor agonist 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane mimicked the effect of HSA-AGE on PS externalization. These data demonstrate, for the first time, that HSA-AGE stimulates PS externalization in a subpopulation of platelets via the 5-HT(2A/2C) receptor. This may have important consequences for platelet involvement in inflammatory responses and the increased cardiovascular risk observed in individuals with diabetes and/or CKD.

Intravenous fish oil blunts the physiological response to endotoxin in healthy subjects

To assess the effects of intravenous fish oil fat emulsion on the metabolic alterations induced by lipopolysaccharide (LPS) challenge in healthy volunteers. Two groups of eight healthy subjects were randomized to receive either two pharmacological doses of intravenous FO fat emulsion or no treatment. The FO group received twice 0.5 g/kg 10% emulsion (Omegaven) 48 and 24h before investigation. LPS (2 ng/kg) was injected as a bolus on the investigation day. Systemic parameters, indirect calorimetry, heart rate variability, and platelet membrane phospholipid composition were measured. Basal EPA and DHA content in platelet phospholipids was low (0.28% and 2.54%, respectively) and increased significantly after FO to 1.68% and 3.32%. LPS induced reproducible effects in all subjects. Fever was higher in the control [corrected] group than in FO group [corrected] the difference was significant from t (120) until t (360). FO blunted the neuroendocrine response: the rise in plasma norepinephrine was sevenfold lower at t (120) while the ACTH peak was fourfold lower. Tumor necrosis factor alpha was significantly lower between t (360) and t (180) in the FO group. Two doses of intravenous FO fat emulsion modified the phospholipid composition of platelets in healthy subjects. FO blunted fever and increased the neuroendocrine and the inflammatory responses to LPS.

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

In the present study, phospholipase A2 (PLA2)-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA2 activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanied by increased arachidonic acid and thromboxane B2 release and increased lysoplasmenylcholine production. Thrombin stimulation caused an increase in PLA2 activity measured in the cytosolic fraction with plasmenylcholine only; no increase in activity was measured with phosphatidylcholine. No change in membrane-associated PLA2 activity was observed with either substrate tested. Pretreatment with the Ca2+-independent PLA2-selective inhibitor, bromoenol lactone, inhibited completely any thrombin-stimulated phospholipid hydrolysis. Thus, thrombin stimulation of human platelets activates a cytosolic PLA2 that selectively hydrolyzes arachidonylated plasmalogen phospholipids.