Search

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin α IIb β 3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases.

Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride-induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo., (© 2019 by The American Society of Hematology.)

The present study was designed to target fish for potential bioactive components contained in a Huang Lian Jie Du decoction (HLJDD) and identify the underlying mechanisms of action for the treatment of sepsis at the molecular level. he bioactive components database of HLJDD was constructed and the sepsis-associated targets were comprehensively investigated. The 3D structures of the PAFR and TXA2R proteins were established using the homology modelling (HM) method, and the molecular effects for sepsis treatment were analysed by comparing the bioactive components database and the sepsis targets using computational biology methods. The results of the screening were validated with biological testing against the human oral epidermal carcinoma cell line KB in vitro. We found that multiple bioactive compounds contained in the HLJDD interacted with multiple targets. We also predicted the promising compound leads for sepsis treatment, and the first 28 compounds were characterized. Several compounds, such as berberine, berberrubine and epiberberine, dose-dependently inhibited PGE2 production in human KB cells, and the effects were similar in the presence or absence of TPA. This study demonstrates a novel approach to identifying natural chemical compounds as new leads for the treatment of sepsis., (Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Background: We recently published that platelet-activating factor receptor (PAFr) is upregulated on the epithelium of the proximal airways of current smokers and also in bronchial epithelial cells exposed to cigarette smoke extract. These treated cells also showed upregulation of Streptococcus pneumoniae adhesion. Bacterial wall phosphorylcholine specifically binds to PAFr expressed on airway epithelium, thus facilitating adherence and tissue invasion, which may be relevant to chronic obstructive pulmonary disease (COPD). Moreover, the use of inhaled corticosteroids (ICS) in COPD patients is associated with an increased risk of invasive respiratory pneumococcal infections., Objective: In this study, we have investigated whether PAFr expression is especially upregulated in airway epithelium in COPD patients and whether this expression may be modulated by ICS therapy., Methods: We cross-sectionally evaluated PAFr expression in bronchial biopsies from 15 COPD patients who were current smokers (COPD-smokers) and 12 COPD-ex-smokers, and we compared these to biopsies from 16 smokers with normal lung function. We assessed immunostaining with anti-PAFr monoclonal antibody. We also used material from a previous double-blinded randomized placebo-controlled 6-month ICS intervention study in COPD patients to explore the effect of ICS on PAFr expression. We employed computer-aided image analysis to quantify the percentage of epithelium stained for PAFr., Results: Markedly enhanced expression of PAFr was found in both COPD-smokers (P<0.005) and COPD-ex-smokers (P<0.002) compared to smokers with normal lung function. There was little evidence that PAFr expression was affected by ICS therapy over 6 months., Conclusion: Epithelial PAFr expression is upregulated in smokers, especially in those with COPD, and is not obviously affected by ICS therapy.

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided., (© 2012 International Society on Thrombosis and Haemostasis.)

Phytochemical investigation on the bark of Goniothalamus tapis Miq. and G. uvaroides King has resulted in the isolation of eight styryl-lactones, (-)-cryptomeridiol, liriodenine, 3-methyl-1H-benz[f]indole-4,9-dione, (-)-stigmasterol and dimethyl terephthalate. The structures of the compounds were elucidated by spectroscopic techniques. The compounds were evaluated for their effect on platelet-activating factor (PAF) receptor binding on rabbit platelets using (3) H-PAF as a ligand. Among the compounds tested, (-)-cryptomeridiol, (+)-goniothalamin and (+)-isoaltholactone exhibited a significant and concentration-dependent inhibitory effect on PAF receptor binding, with inhibitory concentration (IC)(50) values of 17.5, 19.7 and 46.5 µm, respectively. The inhibitory effects of the first two compounds were comparable to that obtained from the positive control, cedrol. The results indicated that these compounds were strong PAF receptor binding inhibitors., (Copyright © 2011 John Wiley & Sons, Ltd.)

Streptococcus pneumoniae is a major pathogen of respiratory infections that utilizes platelet-activating factor receptor (PAFR) for firm adherence to host cells. The mucolytic agent S-carboxymethylcysteine (S-CMC) has been shown to exert inhibitory effects against infection by several respiratory pathogens including S. pneumoniae in vitro and in vivo. Moreover, clinical studies have implicated the benefits of S-CMC in preventing exacerbation of chronic obstructive pulmonary disease, which is considered to be related to respiratory infections. In this study, to assess whether the potency of S-CMC is attributable to inhibition of pneumococcal adherence to host cells, an alveolar epithelial cell line stimulated with interleukin-1α was used as a model of inflamed epithelial cells. Despite upregulation of PAFR by inflammatory activation, treatment with S-CMC efficiently inhibited pneumococcal adherence to host epithelial cells. In order to gain insight into the inhibitory mechanism, the effects of S-CMC on PAFR expression were also investigated. Following treatment with S-CMC, PAFR expression was reduced at both mRNA and post-transcriptional levels. Interestingly, S-CMC was also effective in inhibiting pneumococcal adherence to cells transfected with PAFR small interfering RNAs. These results indicate S-CMC as a probable inhibitor targeting numerous epithelial receptors that interact with S. pneumoniae.

The concept of "pharmacogenetics" addresses genetically determined variation in how individuals respond to drugs. Accordingly, specific genetic variants have been suggested as contributors to a reduced response to various antiplatelet drugs. Aspirin is a cornerstone in secondary cardiovascular prevention and has been thoroughly investigated. The efficacy of aspirin is well documented, although with considerable interindividual variation. According to meta-analyses, a reduced antiplatelet effect of aspirin confers an increased risk of cardiovascular events. The platelet response to aspirin is assessed by in vitro evaluation of thromboxane-dependent platelet function. A reduced antiplatelet effect of aspirin can be explained by several mechanisms, which are largely determined by clinical, pharmacodynamic, biological and genetic factors. During the past decade, numerous studies have identified genetic polymorphisms significantly associated with cardiovascular events and modulating the antiplatelet effect of aspirin. However, results have been contradictory allowing only few firm conclusions to be drawn. Polymorphisms in genes encoding glycoproteins (IIb/IIIa, Ia/IIa, VI and Ibα), cyclooxygenases (1 and 2), adenosine diphosphate receptors (P2Y1 and P2Y12) and proteins of importance for haemostasis (thromboxane A2 receptor, coagulation factor XIII, etc.) have been investigated. In particular, a polymorphism in the gene encoding glycoprotein IIb/IIIa has been associated with a reduced antiplatelet effect of aspirin. The additive value of an individual's genetic makeup in predicting the antiplatelet effect of aspirin and the risk of cardiovascular events remains controversial. The present review outlines the pharmacology of aspirin and provides an overview of specific genetic variations considered to influence the antiplatelet effect of aspirin.

Sodium-hydrogen exchanger (NHE), the principal sarcolemmal acid extruder in ventricular myocytes, is stimulated by a variety of autocrine/paracrine factors and contributes to myocardial injury and arrhythmias during ischemia-reperfusion. Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory phospholipid that is released in the heart in response to oxidative stress and promotes myocardial ischemia-reperfusion injury. PAF stimulates NHE in neutrophils and platelets, but its effect on cardiac NHE (NHE1) is unresolved. We utilized quiescent guinea pig ventricular myocytes bathed in bicarbonate-free solutions and epifluorescence to measure intracellular pH (pH(i)). Methylcarbamyl-PAF (C-PAF; 200 nM), a metabolically stable analog of PAF, significantly increased steady-state pH(i). The alkalosis was completely blocked by the NHE inhibitor, cariporide, and by sodium-free bathing solutions, indicating it was mediated by NHE activation. C-PAF also significantly increased the rate of acid extrusion induced by intracellular acidosis. The ability of C-PAF to increase steady-state pH(i) was completely blocked by the PAF receptor inhibitor WEB 2086 (10 μM), indicating the PAF receptor is required. A MEK inhibitor (PD98059; 25 μM) also completely blocked the rise in pH(i) induced by C-PAF, suggesting participation of the MAP kinase signaling cascade downstream of the PAF receptor. Inhibition of PKC with GF109203X (1 μM) and chelerythrine (2 μM) did not significantly affect the alkalosis induced by C-PAF. In summary, these results provide evidence that PAF stimulates cardiac NHE1, the effect occurs via the PAF receptor, and signal relay requires participation of the MAP kinase cascade.

Understanding genetic contributions to platelet function could have profound clinical ramifications for personalizing platelet-directed pharmacotherapy, by providing insight into the risks and possible benefits associated with specific genotypes. This article represents an integrated summary of presentations related to genetic regulation of platelet receptor expression and function given at the Fifth Annual Platelet Colloquium in January 2010. It is supplemented with additional highlights from the literature covering (1) approaches to determining and evidence for the associations of genetic variants with platelet hypo- and hyperresponsive phenotypes, (2) the ramifications of these polymorphisms with regard to clinical responses to antiplatelet therapies, and (3) the role of platelet function/genetic testing in guiding antiplatelet therapy.

Background: Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization., Objectives: Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation., Methods: Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells., Results: Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium., Conclusions: Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.

Sickle cell disease (SCD) is characterized by intravascular hemolysis and inflammation coupled to a 400-fold greater incidence of invasive pneumococcal infection resulting in fulminant, lethal pneumococcal sepsis. Mechanistically, invasive infection is facilitated by a proinflammatory state that enhances receptor-mediated endocytosis of pneumococci into epithelial and endothelial cells. As statins reduce chronic inflammation, in addition to their serum cholesterol-lowering effects, we hypothesized that statin therapy might improve the outcome of pneumococcal infection in SCD. In this study, we tested this hypothesis in an experimental SCD mouse model and found that statin therapy prolonged survival following pneumococcal challenge. The protective effect resulted in part from decreased platelet-activating factor receptor expression on endothelia and epithelia, which led to reduced bacterial invasion. An additional protective effect resulted from inhibition of host cell lysis by pneumococcal cholesterol-dependent cytotoxins (CDCs), including pneumolysin. We conclude therefore that statins may be of prophylactic benefit against invasive pneumococcal disease in patients with SCD and, more broadly, in settings of bacterial pathogenesis driven by receptor-mediated endocytosis and the CDC class of toxins produced by Gram-positive invasive bacteria.

MicroRNAs (miRs) are 21- to 23-nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs of proteins involved in cell growth and apoptosis. miR-92 is part of the mir-17-92 cluster, which comprises members with an effect on cell proliferation. However, the role of miR-92 is unknown, and its targets have not been identified. Here, we describe a mechanism through which miR-92 contributes to regulate cell proliferation. Using a miR-92 synthetic double-strand oligonucleotide, we demonstrate that miR-92 increases 32D myeloid cell proliferation and 5-bromo-2-deoxyuridine (BrdU) incorporation and inhibits cell death. The effect is miR-92 specific since the miR-92 antagomir inhibits cell proliferation. Moreover, we show that miR-92 acts by modulating p63-isoform abundance through down-regulatation of endogenous DeltaNp63beta. Using luciferase reporters containing p63 3'UTR fragments with wild-type or mutant miR-92 complementary sites, we demonstrate that the wild-type 3'UTR is a direct target of miR-92. Finally, we observed that a miR-92-resistant DeltaNp63beta isoform (without 3'UTR) inhibits cell proliferation and parallels the effect of the antagomir. We conclude that one of the molecular mechanisms through which miR-92 increases cell proliferation is by negative regulation of an isoform of the cell-cycle regulator p63.

Objective: Mitochondrial depolarization aids platelet activation. Oxidized LDL (oxLDL) contains the medium length oxidatively truncated phospholipid hexadecyl azelaoyl-lysoPAF (HAz-LPAF) that disrupts mitochondrial function in nucleated cells, so oxLDL may augment platelet activation., Methods and Results: Flow cytometry showed intact oxLDL particles synergized with subthreshold amounts of soluble agonists to increase intracellular Ca2+, and initiate platelet aggregation and surface expression of activated gpIIb/IIIa and P-selectin. oxLDL also induced aggregation and increased intracellular Ca2+ in FURA2-labeled cells by itself at low, although not higher, concentrations. HAz-LPAF, alone and in combination with substimulatory amounts of thrombin, rapidly increased cytoplasmic Ca2+ and initiated aggregation. HAz-LPAF depolarized mitochondria in intact platelets, but this required concentrations beyond those that directly activated platelets. An unexpectedly large series of chemically pure truncated phospholipids generated by oxidative fragmentation of arachidonoyl-, docosahexaneoyl-, or linoleoyl alkyl phospholipids were platelet agonists. The PAF receptor, thought to effectively recognize only phospholipids with very short sn-2 residues, was essential for platelet activation because PAF receptor agonists blocked signaling by all these medium length phospholipids and oxLDL., Conclusions: Intact oxLDL particles activate platelets through the PAF receptor, and the PAF receptor responds to a far wider range of oxidized phospholipids in oxLDL than anticipated.

During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.

Glycoprotein (GP)VI, that binds collagen, together with GPIb-IX-V which binds vonWillebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases. Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus ( approximately 60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, approximately 0.3 microg/ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming crotarhagin targeted GPVI. Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation. Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.

We investigated the vascular effects of a crude aqueous extract (AEEG) of Echinodorus grandiflorus (Alismataceae) using the in vitro experimental models of the rabbit isolated aorta and perfused kidney. Echinodorus grandiflorus, a native semi-aquatic plant widely distributed in Brazil, has been extensively used in Brazilian folk medicine for the treatment of high blood pressure and inflammatory diseases. The bolus injection of AEEG (0.1-10 mg) into the rabbit renal circulation pre-contracted with norepinephrine induced marked and dose-dependent vasodilator responses (maximum of 37+/-4%; n=6; P<0.001), which was similar to that induced by injection of 10 mmol acetylcholine (41+/-3%). Moreover, AEEG elicited a significant and concentration-dependent relaxation in the endothelium-intact, but not endothelium-denuded aortic rings, reaching the maximum of 81+/-5% (n=7, P<0.001). Inhibition of the nitric oxide-cGMP pathway with L-NAME (100 microM) or Methylene Blue (20 microM) reduced maximum relaxation induced by AEEG from 81+/-5% to 46+/-3 and 45+/-3%, respectively (n=7, P<0.001). A similar reduction was obtained with the incubation of the aortic rings with the selective PAF receptor antagonist WEB 2086 (10 microM) (from 81+/-5% to 55+/-3%; n=7; P<0.01). Conversely, blockade of muscarinic receptors with atropine (10 microM) did not affect the vasodilator effects of AEEG, while inhibition of the enzyme cyclooxigenase not only did not block, but rather potentiated vasodilation induced by AEEG (n=7, P<0.001). Finally, blockade of Ca(2+)- and ATP-activated K(+) channels using the specific blockers charydbotoxin (100 nM) and glibenclamide (3 microM), respectively, did not modify aortic relaxation induced by AEEG. We conclude that water-soluble extracts from leaves of Echinodorus grandiflorus elicit an endothelium-dependent, nitric oxide and PAF receptor-mediated vasodilation in rabbit aortic rings, which does not appear to involve the generation of vasodilating prostaglandins or the activation of K(+) channels. This potent vasodilator effect of the extracts was confirmed in the isolated rabbit renal circulation.

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.

Previous studies have shown that the extracts obtained from Phyllanthus amarus, and some of the lignans isolated from it, exhibit pronounced antiinflammatory properties. In the present study, we have assessed whether the antiinflammatory actions of these lignans can be mediated by interaction with platelet activating factor (PAF) receptor or interference with the action of this lipid. The local administration of nirtetralin, phyltetralin or niranthin (30 nmol/paw), similar to WEB2170 (a PAF receptor antagonist, 30 nmol/paw), significantly inhibited PAF-induced paw oedema formation in mice. The extracts of P. amarus (100 microg/ml) and niranthin (30 microM), but not nirtetralin or phyltetralin (30 microM), decreased the specific binding of [(3)H]-PAF in mouse cerebral cortex membranes. Furthermore, both niranthin and WEB2170 displaced, in a concentration-dependent manner, the [(3)H]-PAF binding sites. The mean IC(50) values from these effects were 6.5 microM and 0.3 microM, respectively. Additionally, both niranthin and WEB2170 (30 nmol/paw) inhibited the increase of myeloperoxidase activity induced by PAF injection in the mouse paw. When assessed the mouse model of pleurisy induced by PAF, pretreatment with niranthin (100 micromol/kg, p.o.) or WEB2170 (1.7 micromol/kg, i.p.) significantly inhibited PAF-induced protein extravasations. Moreover, in the rat model of PAF-induced allodynia, both niranthin (30 nmol/paw) and WEB2170 (30 nmol/paw) treatment significantly inhibited PAF-induced allodynia. In addition, niranthin had a rapid onset and long-lasting antiallodynic action when compared with WEB2170. Collectively, the present findings suggest that niranthin exhibits antiinflammatory and antiallodynic actions which are probably mediated through its direct antagonistic action on the PAF receptor binding sites.

Oxidation-sensitive signals are important in platelet activation. The novel, phenolic, intracellular and extra-cellular antioxidant AGI-1067 inhibits the expression of a number of proinflammatory genes involved in atherosclerosis. The effect of AGI-1067 on human platelets was evaluated. Blood obtained from 20 aspirin-naïve volunteers with multiple risk factors for vascular disease was preincubated with escalating concentrations of AGI-1067 for the assessment of its ex vivo effects on platelet aggregation and expression of major surface receptors flow cytometry, evaluated by flow cytometry. AGI-1067 resulted in significant inhibition of a variety of activation-dependent platelet biomarkers in healthy volunteers, including adenosine diphosphate-induced platelet aggregation and decreased surface platelet expression of glycoprotein IIb/IIIa antigen, activity with PAC-1 antibody, and glycoprotein Ib (CD42b). The effect of AGI-1067 differs from other known antiplatelet agents, suggesting opportunities for therapeutic combination. These data need to be confirmed in subjects receiving orally dosed AGI-1067 to be clinically relevant.

Platelets have traditionally been understood within the context of hemostasis and hemorrhagic disorders. However, with increasing procedures being performed in smaller vessels and with an increasing incidence of atherosclerosis, the often critical role platelets play is more evident. Platelets are no longer viewed as "scaffolding" for the events of the coagulation cascade but rather as important catalysts in hemostasis, thrombosis, and fibrinolysis. Improved understanding of platelet physiology has led to developments of pharmacologic adjuncts resulting in improved patency rates and improved patient outcomes. This review addresses the physiology of platelet function and the impact of new pharmacologic agents in percutaneous intervention.

Objectives: We sought to describe the responses of patients to clopidogrel using ex vivo measures of platelet aggregation and activation in a large, heterogeneous population., Background: Recently, a number of reports, using various definitions, have dichotomized patients who are treated with clopidogrel into a minority of "non-responders" and a majority of "responders." Such classifications imply that treatment leads to an all-or-none response, with potentially important clinical implications., Methods: We conducted secondary post-hoc analyses of a dataset consisting of volunteers (n = 94) and patients after coronary stenting (n = 405), with heart failure (n = 25), and after stroke (n = 20)., Results: The response of subjects to clopidogrel followed a normal, bell-shaped distribution, with a mean and standard deviation of 41.9 +/- 20.8% when aggregation was induced by 5 mumol/l of adenosine diphosphate. When hyporesponsiveness and hyper-responsiveness to clopidogrel were considered to be two standard deviations less than and greater than the mean, respectively, the prevalence of hyporesponsiveness and hyper-responsiveness in these patients was 4.2% and 4.8%, respectively. Pretreatment platelet activity and clinical characteristics were not associated with responsiveness to clopidogrel., Conclusions: Individuals receiving clopidogrel exhibit a wide variability in response that follows a normal distribution. The clinical implications of this variability are unknown but potentially are important. Clinical trials are needed to define whether hyporesponders to clopidogrel are at increased risk for thrombotic events and whether hyper-responders are at increased risk for bleeding. If so, the individualization of antiplatelet therapy, including clopidogrel dosing, may be possible in the future but will require the ability to easily and reproducibly measure responsiveness by a method that has been proven to be predictive of clinical events.

Platelets play a central role in the pathogenesis of atherosclerosis and thrombosis. Platelet adhesion and aggregate formation are critical events that occur in unstable coronary syndromes. Platelet activation precedes the formation of homotypic and heterotypic aggregates. In the last 10 years, researchers have described the presence of activated platelets in the systemic circulation in various cardiovascular disease states, particularly acute coronary syndromes. This review describes the evidence for platelet activation in acute myocardial ischemic syndromes, describes the pathophysiology responsible for its occurrence, and discusses how platelet activation and reactivity may affect the use of concomitant drug therapies and patient prognosis.

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.

The leflunomide metabolite analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRgamma-coupled collagen receptor signaling pathway. At low micromolar concentrations, LFM-A13 inhibited collagen-induced ultrastructural changes indicative of activation. LFM-A13 inhibited collagen (but not thrombin, TRAP-6, or ADP)-induced platelet aggregation in a concentration-dependent fashion with an IC50 value of 2.8 microM. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100 mg/kg. At nontoxic dose levels, LFM-A13 prolonged the tail bleeding times of mice and improved event-free survival in two mouse models of agonist-induced invariably fatal pulmonary thromboembolism. To our knowledge, LFM-A13 is the first anti-thrombotic agent which prevents platelet aggregation by inhibiting BTK.

Benzodiazepine (diazepam), triazolobenzodiazepines (brotizolam, triazolam) and platelet activating factor (PAF) antagonist (BN 52021) are administered to mice before acquisition and retrieval trials conducted using Morris water maze. Benzodiazepine has produced only anterograde amnesia and it has not produced retrograde amnesia. Triazolobenzodiazepines have produced both anterograde and retrograde amnesia. PAF antagonist (BN 52021) has only produced retrograde amnesia and it has not produced anterograde amnesia. The anterograde amnesia produced by benzodiazepine and triazolobenzodiazepines, has been prevented by benzodiazepine receptor antagonist (flumazenil). It suggests that benzodiazepine- and triazolobenzodiazepines-induced anterograde amnesia may be mediated through benzodiazepine receptors. On the other hand, retrograde amnesia produced by PAF antagonist (BN 52021) and triazolobenzodiazepines has been attenuated by PAF and PAF acetyl hydrolase inhibitors such as cigarette smoke extract (CSE) and phenylmethanesulfonylflouride. It suggests that triazolobenzodiazepine-induced retrograde amnesia may be mediated through blockade of PAF receptors.

Platelet adhesion to a damaged blood vessel is the initial trigger for arterial hemostasis and thrombosis. Platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib/V/IX (GPIb), an interaction especially important under high shear conditions[1]. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (alpha(2)beta(1), GPVI, ) in addition[2] resulting in platelet activation. This leads to the conformational activation of the platelet alpha(IIb)beta3 receptor, fibrinogen binding and finally to platelet aggregation. Over the past decades, modulation of platelet function has been a strategy for the control of cardiovascular disease. Lately, drugs have been developed that target the fibrinogen receptor alphaIIbbeta3 or the ADP receptor and many of these promising compounds have been tested in clinical trials. However the development of products that interfere with the first step of hemostasis, i.e. the platelet adhesion, has lagged behind. In this review we want to discuss (i) the in vivo studies that were performed with compounds that target proteins involved in different adhesion steps i.e. the VWF-GPIb-axis, the collagen-VWF axis and the collagen-collagen receptor axis and (ii) the possible advantages these putative new drugs could have over the current antiplatelet agents.

Most chemotherapeutic agents exert their cytotoxic effects in part through the induction of apoptosis. In addition, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors, it is not known whether PAF is involved in chemotherapeutic agent-induced apoptosis. These studies examined the role of the PAF system in chemotherapy-mediated cytotoxicity using model systems created by retroviral mediated transduction of the PAF receptor-negative human epidermal carcinoma cell line KB with the human PAF receptor (PAF-R) and ablation of the endogenous PAF-R in the carcinoma cell line HaCaT with a retroviral mediated inducible antisense PAF-R vector. The presence of the PAF-R in these models resulted in an augmentation of apoptosis induced by chemotherapeutic agents etoposide and mitomycin C but not by tumor necrosis factor-related apoptosis-inducing ligand or by C(2) ceramide. Oxidative stress and the transcription factor nuclear factor kappaB (NF-kappaB) are found to be involved in this augmentative effect because it was blocked by antioxidants and inhibition of the NF-kappaB pathway using a super-repressor form of inhibitor B. These studies provide evidence for a novel pathway whereby the epidermal PAF-R can augment chemotherapy-induced apoptotic effects through an NF-kappaB-dependent process.

Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphate-buffered saline-diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit-CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P-selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule-1 (PECAM-1)], CD107a [lysosomal associated membrane protein (LAMP)-1], CD107b (LAMP-2), CD63 (LIMP or LAMP-3), and CD151 (PETA-3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose-dependent inhibition of GPIIb/IIIa, P-selectin, CD63, and CD107a receptor expression was observed in the aspirin-treated whole-blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM-1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase-1, thereby blocking thromboxane A(2) synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors.

We sought to assess how one tablet of non-enteric coated aspirin (325 mg) affects human platelets in subjects with risk factors for coronary artery disease. Data from 63 individuals with multiple cardiac risk factors were analyzed. Platelets were assessed twice at baseline (pre-aspirin), and after 3-4 h (post-aspirin). We employed 5 microM epinephrine-induced conventional aggregometry, closure time with epinephrine/collagen cartridge by PFA-100(R) (Dade-Behring), and aspirin response units (ARU) stimulated by propyl gallat with Ultegra (Accumetrics, San Diego, CA, USA) for measuring platelet function. In addition, the expression of platelet receptors was determined by using the following monoclonal antibodies: anti-CD31, CD41, CD42b, CD51/CD61, CD62p, CD63, CD107a, and CD151. Platelet-leukocyte formation was detected utilizing dual antibodies for a pan-platelet marker CD151, and CD14, a monocyte/macrophage marker. PAC-1 was used to measure fibrinogen-platelet binding. One pill of aspirin significantly decreased platelet-rich plasma (PRP) aggregation (74.18+/-16.75% vs. 24.92+/-8.64%; p<0.0001) and resulted in reduction of the aspirin response units (ARU) (662.24+/-65.65 vs. 451.05+/-69.31; p<0.0001). There was also prolongation of the closure time (194.4+/-25.3 vs. 258.63+/-55.61 s; p<0.0001). High correlation (r(2)=0.73-0.86) between platelet analyzer readings and aggregation was observed. One tablet of aspirin moderately inhibited expression of most surface platelet receptors measured, and such inhibition reached significance (p<0.05) for PAC-1, CD31, CD41, CD42, CD62p, and CD151. We conclude that a single dose of aspirin affects major platelet receptors, presumably directly or indirectly through the inhibition of prostanoids via platelet cyclooxygenase-1 blockade. The Ultegra Analyzer with a novel cartridge seems to be reliable in reflecting aspirins' effects on platelets and could be used in the future in clinical practice for monitoring aspirin therapy.

Objective: Chemically modified hemoglobins are being developed as potential oxygen-carrying blood substitutes (HBOCs). Clinical and preclinical data demonstrate the vasoactive properties of HBOCs by trapping of nitric oxide, which is also known to have platelet inhibitory activities properties. This study evaluated the effects of three structurally different HBOCs (Hb-Dex-BTC, alphaalpha-Hb, and o-raffinose-poly-Hb) on platelet functions in vitro to compare to those elicited by plasma substitutes, such as hydroxyethylstarch., Design: Platelet activation state was assessed using platelet-rich plasma diluted to 20% (v/v) with the different solutions, by main measuring glycoproteins (GPIb, GPIIb/IIIa, and P-selectin) using flow cytometry. Aggregation was assessed by impedance aggregometry on whole blood hemodiluted to 20% (v/v) with the solutions., Setting: Biological hematology department of the university hospital of Nancy-Brabois., Patients and Participants: Ten healthy volunteers consent and informed of the study who denied taking any drugs at the time of the experiment., Measurements and Results: None of these solutions induced activation nor modified reactivity of platelets as measured by the surface expression of glycoproteins GPIb, GPIIb/IIIa, and P-selectin. Moreover, none of these solutions induced platelet aggregation when added alone, nor modified the aggregation patterns of platelets induced by collagen (0.5 microg/ml) and thrombin receptor agonist peptide (12.5 microM)., Conclusions: The three tested structurally different HBOCs, as with hydroxyethylstarch, did not alter platelet functions in vitro.

Objective: To evaluate the relationship between aspirin ingestion and postoperative bleeding complications, and to test the hypothesis that there is a subset of patients who are aspirin hyperresponders with a proclivity toward platelet dysfunction., Summary Background Data: Despite numerous retrospective and prospective analyses, it is still controversial as to whether aspirin ingestion before coronary artery bypass grafting (CABG) is associated with significant postoperative bleeding., Methods: Between January 1995 and December 1999, the records of 2,606 consecutive patients undergoing CABG were reviewed to identify patients with a history of aspirin ingestion up until the time of surgery. Aspirin ingestion was correlated with postoperative blood transfusion using multivariate analysis. In a subset of preoperative aspirin users (n = 40), bleeding times were measured before and after aspirin use. Flow cytometry was performed in another cohort of patients with known heart disease (n = 30) to determine the effect of aspirin on platelet surface receptors., Results: During the 5-year study period, 63% of the CABG patients were identified as aspirin users. Among these, 23.1% required blood transfusions compared with 19% for the nonusers. Non-red blood cell transfusions were more common in aspirin users, as was reexploration for bleeding. Stratification of these results according to the frequency of aspirin use showed that aspirin is an independent multivariate predictor of postoperative blood transfusion only in high-risk patients. In the prospective studies, aspirin treatment resulted in a significant increase in the template bleeding time, an increase in platelet PAR-1 thrombin receptor activity, and a decrease in the binding of platelets to monocytes., Conclusions: The findings support the hypothesis that aspirin is associated with a greater likelihood of postoperative bleeding. A platelet function testing algorithm that combines preoperative risk factor assessment, template bleeding times, and flow cytometry may allow the identification of aspirin hyperresponders who are at increased risk for bleeding.

Alboluxin, a potent platelet activator, was purified from Trimeresurus albolabris venom with a mass of 120 kDa non-reduced and, after reduction, subunits of 17 and 24 kDa. Alboluxin induced a tyrosine phosphorylation profile in platelets that resembles those produced by collagen and convulxin, involving the time dependent tyrosine phosphorylation of Fc receptor gamma chain (Fc gamma), phospholipase Cgamma2 (PLCgamma2), LAT and p72SYK. Antibodies against both GPIb and GPVI inhibited platelet aggregation induced by alboluxin, whereas antibodies against alpha2beta1 had no effect. Inhibition of alphaIIb beta3 reduced the aggregation response to alboluxin, as well as tyrosine phosphorylation of platelet proteins, showing that activation of alphaIIb beta3 and binding of fibrinogen are involved in alboluxin-induced platelet aggregation and it is not simply agglutination. N-terminal sequence data from the beta-subunit of alboluxin indicates that it belongs to the snake C-type lectin family. The C-type lectin subunits are larger than usual possibly due to post-translational modifications such as glycosylation. Alboluxin is a hexameric (alphabeta)3 snake C-type lectin which activates platelets via both GPIb and GPVI.

The components of inflammation, including macrophages, cytokines and lipid inflammatory mediators, have a role in atherosclerosis. A key lipid mediator in regulated, physiologic inflammation is platelet-activating factor (PAF). PAF activates cells, including monocytes, through a single molecularly characterized receptor, the PAF receptor (PAFR), at exceedingly low concentrations. The PAFR recognizes the short residue, an acetate residue, at the 2-position of the phospholipid, and this sharp specificity precludes receptor activation by other related phosphatidylcholines. Oxidation of low-density lipoproteins (LDLs) is an early and causal step in atherosclerosis that generates inflammatory compounds leading to foam cell formation. One class of oxidatively generated inflammatory compounds are phospholipids that structurally mimic PAF, the PAF-like lipids. Oxidation of LDLs fragments and derivatizes the fatty acid residues at the 2-position of the phosphatidylcholines that comprise the shell of LDLs, an event that allows certain oxidized phospholipids to interact with and activate the PAFR. We know that these products activate polymorphonuclear leukocytes, but because the function of the PAFR differs among cells, we do not know if monocytes or platelets themselves respond to PAF-like lipids. Here, we show that PAF-like lipids from oxidized LDLs are potent and serve as specific agonists for all cells that express the PAFR.

Platelet activating factor (PAF) is a potent mediator potentially involved in the pathogenesis of inflammatory disorders, including bronchial asthma. Recently, transgenic mice overexpressing the PAF receptor (PAFR) gene have been established, and exhibit bronchial hyperresponsiveness, one of the cardinal features of asthma. To elucidate the molecular and pathophysiologic mechanisms underlying PAF-associated bronchial hyperreactivity, we studied airway responsiveness to methacholine (MCh) and serotonin (5-hydroxytryptamine; 5-HT) in PAFR-transgenic mice. In addition, we examined the role of the muscarinic receptor in PAF-induced responses and the binding activities of the muscarinic receptor. The PAFR-transgenic mice exhibited hyperresponsiveness to MCh and PAF; however, no significant differences in 5-HT responsiveness were observed between the control and PAFR-transgenic mice. The administration of atropine significantly blocked PAF-induced responses in PAFR-transgenic mice. There were no differences between the two phenotypes in the binding activities of muscarinic receptor. Morphometric analyses demonstrated that PAFR overexpression did not affect airway structure. These findings suggest that the muscarinic pathway may have a key role in airway hyperresponsiveness associated with PAFR gene overexpression. More generally, PAFR-transgenic mice may provide appropriate models for study of the molecular mechanisms underlying PAF-associated diseases.

Background: Plaque disruption with resultant platelet activation and leukocyte-platelet aggregation is a pathophysiologic process common to both acute coronary syndromes and percutaneous coronary interventions. Unfractionated heparin is a standard antithrombotic therapy in patients with both acute coronary syndromes and in those undergoing percutaneous coronary interventions. Low-molecular-weight heparins have been reported to cause less platelet activation than unfractionated heparin., Methods: Monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet surface P-selectin, and platelet surface glycoprotein (GP) IIIa were measured serially by whole blood flow cytometry in 40 patients with unstable angina (randomly assigned to either unfractionated heparin 70 U/kg or the low-molecular-weight heparin dalteparin 60 IU/kg) undergoing coronary intervention with planned abciximab administration (in 2, one-half-dose boluses). Assays were performed at baseline, 5 minutes after administration of either type of heparin, 10 minutes after the first bolus of abciximab, 10 minutes after second bolus of abciximab, and 8 to 10 and 16 to 24 hours after administration of either heparin., Results: No significant differences in clinical outcomes were observed between patients receiving either unfractionated heparin or dalteparin. The number of circulating P-selectin-positive platelets was increased by unfractionated heparin but not dalteparin, and abciximab reversed this increase. The number of circulating P-selectin-positive platelets was reduced below baseline levels in both treatment groups 8 to 10 and 16 to 24 hours after study drug administration. At 8 to 10 and 16 to 24 hours after administration of study drug, platelet degranulation in response to iso-thrombin receptor agonist peptide 1.5 mmol/L was significantly reduced by almost 50% (compared with immediately after study drug administration). Both unfractionated heparin and dalteparin significantly increased the numbers of circulating monocyte-platelet and neutrophil-platelet aggregates, which were subsequently reduced to baseline levels after administration of the second abciximab bolus and to below baseline at both 8 to 10 and 16 to 24 hours in all patients. After both unfractionated heparin and dalteparin administration, platelet surface GP IIIa expression was significantly increased compared with baseline at both 8 to 10 and 16 to 24 hours., Conclusions: Dalteparin in combination with abciximab in patients with unstable angina undergoing coronary intervention appears to be safe. Unfractionated heparin, but not dalteparin, degranulates platelets in patients with unstable angina. Both heparins increase the number of circulating monocyte-platelet and neutrophil-platelet aggregates. Abciximab therapy during coronary interventions rapidly reduces the number of degranulated platelets and leukocyte-platelet aggregates.

Rapid proliferation of atypical megakaryoblasts is a characteristic of megakaryoblastic leukemia. Cells from patients with this disorder and cell lines established from this type of leukemia showed the presence of gelsolin but the absence of scinderin expression, 2 filamentous actin-severing proteins present in normal megakaryocytes and platelets. Vector-mediated expression of scinderin in the megakaryoblastic cell line MEG-01 induced a decrease in both F-actin and gelsolin. This was accompanied by increased Rac2 expression and by activation of the PAK/MEKK.SEK/JNK/c-jun, c-fos transduction pathway. The Raf/MEK/ERK pathway was also activated in these cells. Transduction pathway activation was followed by cell differentiation, polyploidization, maturation, and apoptosis with release of platelet-like particles. Particles expressed surface CD41a antigen (glycoprotein IIb/IIIa or fibrinogen receptor), had dense bodies, high-affinity serotonin transport, and circular array of microtubules. Treatment of particles with thrombin induced serotonin release and aggregation that was blocked by CD41a antibodies. PAC-1 antibodies also blocked aggregation. Exposure of cells to PD98059, a blocker of MEK, inhibited antigen CD41a expression, increases in cell volume, and number of protoplasmic extensions. Cell proliferation and cell ability to form tumors in nude mice were also inhibited by the expression of scinderin. MEG-01 cells expressing scinderin had the same fate in vivo as in culture. Thus, when injected into nude mice, they entered apoptosis and released platelet-like particles. The lack of scinderin expression in megakaryoblastic leukemia cells seems to be responsible for their inability to enter into differentiation and maturation pathways characteristic of their normal counterparts.

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist. We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.

A series of oligomaltose surfactant polymers were prepared by the simultaneous coupling of hydrophilic maltolactone [of 2(M2), 7(M7), or 15(M15) glucose units] and hydrophobic N-(hexanoyloxy)succinimide (Hex) groups to the amino groups of a poly(vinyl amine) backbone. The surfactants were characterized by FTIR and 1H-NMR spectroscopies for purity and composition. Contact-angle and AFM measurements confirmed full monolayer adsorption for all surfactants on a model surface, highly oriented pyrolitic graphite, while full coverage was observed on polyethylene only for PVAm (M7:Hex) due to the optimal M7:Hex ratio and Hex chain density. On graphite, protein resistance increased with increasing coating thickness from 81.4 to 85.8 to 95.8% for the M2, M7, and M15 surfactants, respectively. Additionally, static platelet adhesion on all three surfactants dropped substantially to 15% (M2), 17% (M7), and 16% (M15)compared to glass (adhesion normalized to 100%) and a polyurethane (24%) surface. Protein- and platelet-resistant properties of the controlled oligomaltose layers are discussed by analysis of molecular modeling, oligomaltose and hexanoyl chain densities, and surfactant stability.

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet-activating-factor (PAF). We investigated the effect of oxidized low-density lipoprotein (oxLDL) on lipopolysaccharide (LPS)-induced PAF receptor (PAF-R) expression in human macrophages and the implication of the nuclear factor (NF)-kappaB in this regulation. LPS-treatment (1 microg.mL(-1)) of macrophages increased PAF binding and PAF-R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low-density lipoprotein [ox24hLDL; 100 microg.mL(-1); thiobarbituric acid reacting substances: 31 +/- 3 nmol equiv. malondialdehyde (MDA).mg protein LDL-1] diminished PAF-R expression (-69%; P < 0.05) and mRNA level (- 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF-kappaB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS-induced binding of NF-kappaB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 microM), a major component of oxLDL, inhibited the LPS-induced NF-kappaB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF-R expression in human macrophages by inhibiting LPS-induced NF-kappaB binding to DNA.

A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for beta-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbbeta and GPIX but also a significantly lower expression of the nondirectly GATA1-regulated Gsalpha gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbalpha, GPIbbeta, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion, GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.

The glycoprotein (GP)-Ib-IX-V receptor complex has recently been reported to signal through a pathway similar to that used by the collagen receptor GPVI, with a critical role described for the Fc receptor gamma-chain. The evidence for this was based in part on studies with the GPIbalpha-selective snake venom toxin, alboaggregin-A. In the present study, it is reported that alboaggregin-A has activity at the collagen receptor GPVI in addition to GPIbalpha, and evidence is provided that this contributes to protein tyrosine phosphorylation, shape change, and GPIIb-IIIa-dependent aggregation. This may explain why responses to alboaggregin-A are distinct from those to von Willebrand factor-ristocetin. (Blood. 2001;97:3989-3991)

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC(50) values of 200 and 300 nM respectively, similar to that of the wild-type RGD motif (170 nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10-30-fold less potent at inhibiting K562 cell (containing alpha(5)beta(1) integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC(50) value to the RGD motif at inhibiting A375-SM cell (beta(3) integrin) adhesion to collagen, whereas the RCD motif was approx. 2-6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to beta(1) and beta(3) integrins can be modulated by the introduction of alternative cell recognition sequences.

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fcepsilon receptor gamma-chain (Fc Rgamma), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagen-related peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc Rgamma partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc Rgamma and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the alpha(2)beta(1) integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

Background: Trauma modulates polymorphonuclear neutrophil (PMN) function, predisposing to organ failure and infection. Many chemoattractants released by injury activate PMNs via G-protein-coupled (GPC) receptors, which elevate PMN cytosolic calcium ([Ca2+]i). Nonetheless, PMN GPC receptor function after injury is unstudied., Methods: PMNs from 11 major trauma patients (Injury Severity Score = 31 +/- 3, eight men and three women, age = 38 +/- 3) were obtained on days 1, 3, and 7 after injury. Nine developed organ failure and one died. PMNs were exposed to interleukin-8 (IL-8), growth regulated oncogene-alpha (GRO-alpha), and platelet-activating factor (PAF) to stimulate the CXCR1, CXCR2, and PAF receptors. [Ca2+]i flux measurements were used to quantify receptor responses. Receptor responses to individual as well as serial GPC agonists were studied over the week after injury and compared with the responses of PMNs from healthy volunteers (n = 10-23). Results were evaluated by one-way analysis of variance, and paired and unpaired t tests., Results: Responses to GRO-alpha and PAF were significantly depressed early after injury (p < 0.01). Responses to all agonists tested tended to be lowest on day 1, to peak on day 3, and to decrease again by day 7, but variations in response to GRO-alpha were the most marked (p < 0.03, analysis of variance). Whereas GRO-alpha primed IL-8 and IL-8 primed PAF in normal PMNs, GRO-alpha paradoxically suppressed IL-8 responses and IL-8 suppressed PAF responses in trauma PMNs. PAF priming of IL-8 responses was unaffected by injury., Conclusion: Receptor responses to individual GPC agonists are suppressed early after trauma, but increase by day 3. Normal chemokine priming of PMN calcium mobilization is reversed by injury; priming by PAF is intact. PMN GPC responses depend on the sequence in which agonists are encountered. Injury appears to alter these interactions, thus priming some aspects of PMN function while simultaneously suppressing others.

Abnormal platelet reactivity has been linked to unstable angina, myocardial infarction, post angioplasty stenosis, cerebral ischemia, thrombotic stroke and a variety of inflammatory vascular disorders associated with transplantation. Drugs that inhibit blood coagulation, promote fibrinolysis or block platelet activation are important therapeutic agents in cardiovascular medicine. However, many of the current antiplatelet modalities are nonspecific, ineffective or associated with severe side effects that limit their usefulness. In this article, we discuss some basic aspects of platelet pathophysiology to illustrate the importance of ADP stimulation and signaling in platelet activation. CD39, the ATP diphosphohydrolase (ATPDase) expressed on quiescent vascular endothelium, modulates platelet purinoreceptor activity by the sequential hydrolysis of extracellular ATP or ADP directly to AMP. This thromboregulatory potential of CD39 has been recently demonstrated by the generation of mutant mice with disruption of the gene, and by a series of experiments where high level ATPDase expression has been attained by adenoviral vectors in the injured vasculature. Systemic administration of soluble derivatives of CD39 or targeted expression of the native protein to sites of vascular injury may have future therapeutic application.