The Impact of Levetiracetam and Valproate on Platelet Functions-A Double-Blind, Placebo-Controlled Crossover Study.

It is known that valproate inhibits platelet functions; however, the exact mechanisms are not clearly identified. We studied 12 healthy adult volunteers (1 female, 11 male; age range 31.7 ± 7.8 years) before and after valproate 500 mg and compared the results to levetiracetam 1000 mg as a control substance and placebo. The study had a crossover and double-blind design. A blood sample was taken before and 90 min after medication intake, because the times to maximum serum concentration (Tmax) are 1.5 h for levetiracetam and 1 to 3 h for valproate. We analysed changes in platelet, erythrocyte, and leukocyte cell count and in platelet functions (CD62 expression (P selectin), thrombin binding, and fibrinogen binding). We found no significant differences in all cell counts before and after different study drugs. After valproate intake, but not after placebo or levetiracetam intake, the fibrinogen binding significantly decreased and the CD62 expression significantly increased resulting in decreased platelet aggregation. Our data suggest that the platelet dysfunctions reported for valproate result from decreased fibrinogen binding and from increased CD62 expression. This phenomenon might be one reason for the increased bleeding risk under valproate and cannot be observed for levetiracetam.

Plasmin-Induced Activation of Human Platelets Is Modulated by Thrombospondin-1, Bona Fide Misfolded Proteins and Thiol Isomerases.

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-ß-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN--denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.

Panton-Valentine Leukocidin associated with S. aureus osteomyelitis activates platelets via neutrophil secretion products.

Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4).

BACKGROUND: Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. METHODS: Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. RESULTS: Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. CONCLUSION: Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress.

Neutrophils Turn Plasma Proteins into Weapons against HIV-1.

As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases.

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Native high-density lipoproteins inhibit platelet activation via scavenger receptor BI: role of negatively charged phospholipids.

OBJECTIVES: HIGH-density lipoproteins (HDL) are a negative predictor of platelet-dependent thrombus formation and reduced platelet activation has been observed in vitro in the presence of HDL3, a major HDL fraction. However, mechanisms underlying the anti-thrombotic effects of HDL3 are poorly understood. Scavenger receptors class B represent possible HDL3 binding partners on platelets. We here investigated the role of scavenger receptor class B type I (SR-BI) and CD36 in mediating inhibitory effects of native HDL3 on thrombin-induced platelet activation. METHODS AND RESULTS: Rhodamine isothiocyanate-labeled HDL3 bound specifically to platelets and HDL3 binding was inhibited by scavenger receptor class B ligands such as phosphatidylserine (PS)- or phosphatidylinositol (PI)-containing liposomes or maleylated albumin (mBSA). By contrast, scavenger receptor class A ligands failed to displace HDL3 from platelets. HDL3, PS- and PI-liposomes, and mBSA inhibited thrombin-induced platelet aggregation, fibrinogen binding, P-selectin expression and mobilization of intracellular Ca(2+). In addition, PS- and PI-liposomes emulated HDL3-induced intracellular signaling cascades including diacylglycerol production and protein kinase C activation. The reduction of platelet activation by liposomes was related to their PS or PI content. Moreover, inhibitory effects of native HDL3 were enhanced after enriching lipoproteins with PS, while PS- and PI-poor HDL2 failed to inhibit platelet aggregation and Ca(2+) mobilization. Both, HDL3 and PS-containing liposomes failed to inhibit thrombin-induced activation of platelets obtained from SR-BI-deficient mice but not CD36-deficient mice. CONCLUSION: We suggest that SR-BI is a functional receptor for native HDL3 on platelets that generates an inhibitory signal for platelet activation. The content of negatively charged phospholipids (PS, PI) in HDL may be an important determinant of their anti-thrombotic potential.

Markers of Blood Cell Activation and Complement Activation in Factor VIII and von Willebrand Factor Concentrates.

BACKGROUND: Preparations of commercially available clotting factor VIII are complex protein mixtures. Most of them contain either von Willebrand factor or human serum albumin as stabilizers. The aim of the study was to quantify further proteins in twelve concentrates either of recombinant origin or derived from human plasma. METHODS: Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Some proteins were quantified by ELISA. RESULTS: Recombinant clotting factor preparations showed fewer protein spots in the 2D-PAGE, than plasma-derived preparations. Proteins identified in some of the plasma-derived concentrates included up to 90 ng/IU of the anaphylatoxin C3a, up to 40 ng/IU of the platelet a-granule protein thrombospondin-1, up to 0.85 ng/IU of the platelet a-granule protein platelet factor 4, 3.5 ng/IU myeloperoxidase secreted by leukocytes and up to 0.05 ng/IU of the leukocyte-secreted protein a-defensin. The protein content differed between concentrates from different manufacturers. CONCLUSIONS: The origin of the plasma used to prepare the factor concentrates might influence the protein impurities in these products. It is unknown whether the impurities observed have long-term consequences for chronic inflammatory conditions.

High-density lipoproteins, platelets and the pathogenesis of atherosclerosis.

1. Prospective and interventional studies demonstrate an inverse relationship between plasma high-density lipoprotein (HDL)-cholesterol and the incidence of coronary artery disease. Although the atheroprotective effects of HDL are usually attributed to the reverse cholesterol transport, in which HDL shuttles cholesterol from cells in the arterial wall to the liver, other mechanisms are also under investigation. 2. Platelets are involved in both the initiation and progression of atherosclerotic lesions. In addition, the formation of thrombi over ruptured atherosclerotic plaques results in the narrowing or complete occlusion of coronary arteries. Current experimental evidence suggests that HDL may exert antiplatelet effects and thereby counteract the development of atherothrombotic vascular disease. 3. In vitro studies show that HDL inhibits agonist-stimulated platelet aggregation, fibrinogen binding, granule secretion and liberation of thromboxane A(2). Inhibitory effects of HDL are mediated, in part, by scavenger receptor type B1 and/or the apolipoprotein E receptor apoER2/LRP8 and are linked to the induction of intracellular signalling cascades encompassing stimulation of protein kinase C, cytoplasmatic alkalization and generation of nitric oxide. 4. Populational studies demonstrate that there is an inverse association between plasma HDL levels and recurrent venous thromboembolism. In addition, HDL-cholesterol has been identified as an independent predictor of acute platelet thrombus formation. The administration of reconstituted HDL particles in humans attenuates ex vivo platelet activation. 5. The present review summarizes recent advances in understanding HDL-platelet interactions and discusses the potential use of HDL-like particles in the therapy of thrombosis.

Efficient non-viral transfection of THP-1 cells.

Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies in vitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.

Hydroxyethyl starch normalizes platelet and leukocyte adhesion within pulmonary microcirculation during LPS-induced endotoxemia.

Growing evidence supports substantial pathophysiological impact of platelets and their interactions on the development of septic lung failure. We developed a rat model of endotoxemia for direct in situ visualization of pulmonary microcirculation by in vivo fluorescence videomicroscopy. Male Sprague-Dawley rats were assigned to control, endotoxemia (Escherichia coli LPS, 15 mg/kg, i.v.), and fluid management for treatment of LPS-induced hypovolemia (Ringer lactate, hydroxyethyl starch [HES] 6%) groups (n = 7 each). Leukocytes were labeled in vivo by rhodamine, and 5 x 10(6) Calcein-AM-labeled nonactivated platelets were injected. Microcirculatory parameters (vessel diameter, ventilation-perfusion ratio) and adhesive characteristics of platelets and leukocytes (velocity, rolling, sticking) within the pulmonary microcirculation were quantified after endotoxin application under various regimens of fluid substitution for 60 min. A reduction of cell velocity and enhanced cell adhesion was seen in leukocytes and platelets (P < 0.05) after LPS injection. Fluid treatment with HES 6% resulted in a significant increase of platelet's velocity compared with the LPS group (442.86 +/- 20.60 vs. 343.93 +/- 11.17; P < 0.05), whereas Ringer lactate showed no beneficial effects. Similarly, HES 6% normalized LPS-induced platelet rolling and sticking as well as alterations in ventilation-perfusion ratio. Using direct visualization of the pulmonary microcirculation, we observed that platelet and leukocyte interactions are enhanced in the lung during LPS endotoxemia. Fluid therapy with HES 6% seems to have restorative effects on these cellular functions within the pulmonary microcirculation.

Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor.

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.