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Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10–1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Objective Activation of thromboxane A2 synthase (TXAS)/thromboxane A2 (TXA2)/thromboxane prostanoid (TP) receptor leads to arterial constriction, platelet aggregation and vascular injury. We attempted to characterize the microvascular dysfunction in ischaemia/reperfusion injury using genetically modified TXAS−/−, TP−/− and TXAS−/−TP−/− mice. Approach and Results The cardiac micro-circulation and electrocardiograms were evaluated from B6, TXAS−/−, TP−/− and TXAS−/−TP−/− mice in response to intravenous saline, endothelin-1, U46619 (a TXA2 agonist) and myocardial ischaemia/reperfusion injury. Cardiac function was investigated with myocardial permeability, the troponin I concentration and the infarct size. Myocardial TXAS, TP, endothelial nitric oxide (NO) synthase (eNOS), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOx4), 4-hydroxynonenal, interleukin (IL)-1β, cell apoptosis, coronary effluent thromboxane B2 (TXB2) and superoxide anions (O2 −) and NO concentrations were measured. Mice mesenteric reactivity in response to various drugs was assessed by wire myography. In vivo fluorescent platelet adhesiveness to the mesenteric arterial endothelium after FeCl3 stimulation was examined. In B6 mice, ischaemia/reperfusion significantly increased levels of ST-segment elevation, myocardial TXAS, TP, NOx4, IL-1β, apoptosis, coronary endothelin-1, TXB2, O2 − release and the infarct size, with concomitant decreases in eNOS, NO concentrations and cardiac micro-circulation. These effects were remarkably depressed in TXAS−/−, TP−/− and TXAS−/−TP−/− mice. Aspirin treatment or depletion of the TXAS, TP or TXAS/TP gene significantly attenuated the exaggerated vascular reactivity by vasoconstrictors and vasodilators and efficiently reduced platelet adhesion to the mesenteric endothelium under FeCl3 stimulation. Conclusion Inhibiting TXAS/TXA2/TP signalling confers microvascular protection against oxidative injury in both cardiac and mesenteric arteries.

Aim Enhancement of thromboxane A2 (TXA2) synthase (TXAS) activity, TXA2 release, and thromboxane prostanoid (TP) receptor activation leads to vasoconstriction and oxidative injury. We explored whether genetic deletion of TXAS/TXA2/TP signalling may reduce renal ischaemia/reperfusion (I/R) injury in mice. Materials and Methods Renal haemodynamics and function were evaluated in TXAS+/+TP+/+ (wild-type, WT), TXAS−/− (TXS−/−), TP−/− and TXAS−/−TP−/− (double knockout, dKO) mice in response to intravenous TXA2 mimetic-U46619 and 45-minute renal ischaemia and 4-hour reperfusion injury. We examined renal TXAS and TP expression, blood urea nitrogen (BUN) and creatinine, reactive oxygen species (ROS) amount, pro-inflammatory cytokines and pathophysiologic mechanisms, including apoptosis, autophagy and pyroptosis under I/R injury. Results Renal I/R enhanced the levels of TXAS, TP, nuclear factor-κB, nicotinamide adenine dinucleotide phosphate oxidase gp91, Bax/Bcl-2/caspase-3/apoptosis, Beclin-1/LC3-II/autophagy, caspase-1/gasdermin D/interleukin-1β/pyroptosis, renal thromboxane B2 (TXB2) concentration, ROS amount, plasma BUN, creatinine and TXB2 and decreased renal endothelial nitric oxide synthase expression in WT mice. All these enhanced parameters were significantly decreased in three KO mice. Intravenous U46619 significantly decreased renal microcirculation and enhanced gp91 and Bax/Bcl-2 in WT and TXS−/− but not TP−/− in dKO mice. I/R significantly decreased renal microcirculation in all mice; however, the time for recovery to baseline renal blood flow level was significantly shortened in TXS−/−, TP−/−and dKO mice versus WT mice. Blockade of TXAS/TP signalling attenuated I/R-enhanced pro-inflammatory cytokine profile. Conclusion Blockade of TXAS/TXA2/TP signalling confers renal protection against I/R injury through the actions of anti-oxidation, anti-inflammation, anti-apoptosis, anti-autophagy and anti-pyroptosis.

Platelets play an important role in both physiological hemostasis and pathological thrombosis. Thromboxane (TX) A2 and prostaglandin (PG) I2 are well known as a potent stimulator and an inhibitor of platelet function, respectively. Recently, PGE2 has also been reported to regulate platelet function via PGE2 receptor subtypes. However, the effect of PGF2α on platelet function remains to be determined. The aim of the present study was to clarify the effect of PGF2α on murine platelet function both in vitro and in vivo. Platelets prepared from wild-type mice (WT platelets) expressed several types of prostanoid receptors, including the PGE2 receptor subtype EP3 and the TXA2 receptor TP, while expression of the PGF2α receptor FP was not detected. In WT platelets, PGF2α potentiated adenosine diphosphate-induced aggregation in a concentration-dependent manner, while PGF2α alone did not induce aggregation. In platelets prepared from mice lacking FP, however, PGF2α-induced potentiation was not significantly different from that in WT platelets. Interestingly, the potentiation was significantly blunted in platelets lacking EP3 or TP and disappeared completely in platelets lacking both EP3 and TP. Accordingly, PGF2α decreased the cyclic adenosine monophosphate level via EP3 and increased the inositol triphosphate level via TP in WT platelets. Intravenously administered PGF2α significantly shortened the bleeding time and aggravated arachidonic acid-induced acute thromboembolism in WT mice, suggesting that PGF2α works as a platelet stimulator also in vivo. In conclusion, PGF2α potentiates platelet aggregation in vitro via EP3 and TP but not FP. Accordingly, PGF2α facilitates hemostasis and thromboembolism in vivo.

SummaryA small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function. We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S–expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S–expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

SummaryDifferent aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel’s active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p

SummaryThe effect of lipopolysaccharide (LPS) on platelet aggregation is still controversial. We performed in vitro and ex vivo studies in controls and in patients with community-acquired pneumonia (CAP) to assess the effect of LPS on platelet activation (PA). LPS (15–100 pg/ml) significantly increased PA only if combined with sub-threshold concentrations (STC) of collagen or ADP; this effect was associated with increased platelet H2O2 production, Nox2 activation, PLA2 phosphorylation, thromboxane (Tx)A2 and 8-iso-PGF2α-III, and was inhibited by aspirin, TxA2 receptor antagonist or by Toll-like receptor 4 blocking peptide (TLR4bp). Analysis of up-stream signalling potentially responsible for Nox2 and PLA2 activation demonstrated that LPS-mediated PA was associated with phosphorylation of AKT, p38 and p47phox translocation. In 10 consecutive CAP patients serum endotoxins were significantly higher compared to 10 controls (145 [115–187] vs 18 [6–21] pg/ml; pSupplementary Material to this article is available online at www.thrombosis-online.com

SummaryLittle is known about the onset of action after intravenous or oral administration of acetylsalicylic acid (ASA) in patients with acute coronary syndromes (ACS). The aim of the study was to compare intravenous 250 or 500 mg acetylsalicylic acid (ASA) with oral 300 mg in ASA naïve patients with ACS concerning the onset of antiplatelet effects measured by time dependent thromboxane inhibition. A total of 270 patients with ACS < 24 hours were randomised into one of three treatment arms comprising administration of a single dose of ASA as soon as possible after admission. The primary endpoint was platelet inhibition assessed by measurement of arachidonic acid (AA)-induced platelet thromboxane release (TXB2) 5 minutes (min) after study drug administration. Both 250 mg and 500 mg ASA i. v. inhibited TXB2 formation nearly completely (geometric means: from 581.7 and 573.9 ng/ml at baseline to 3.9 and 3.1 ng/ml at 5 min, respectively) compared to 300 mg oral ASA (geometric means: from 652.0 to 223.7 ng/ml) (p-value, ANCOVA: < 0.0001). Similar results were obtained for inhibition of AA-induced platelet aggregation (Multiplate ASPItest; from means 86.41 and 85.72 U to 23.04 and 20.57 U at 5 min, respectively) compared to 300 mg oral ASA from mean 87.18 to 75.56 U (p-value, ANCOVA: Supplementary Material including a list of institutions where work was performed is available online at www.thrombosis-online.com.

SummaryPlatelets critically contribute to atherothrombosis and worsening ischaemia in patients with peripheral arterial disease (PAD), eventually leading to critical limb ischaemia (CLI). Furthermore, persistent platelet activation despite antiplatelet therapy has been reported in this setting. The prostacyclin analogue iloprost is currently recommended in CLI patients for its effects in relieving symptoms by promoting local perfusion. In this study, we investigated the effects of iloprost infusion on urinary 11-dehydro-TXB2 and 8-iso-PGF2α excretion rate, as in vivoindexes of thromboxane-dependent platelet activation and lipid peroxidation, respectively, and on platelet-derived proinflammatory sCD40L and nitric oxide bioavailability in 44 patients with CLI while on chronic treatment with low-dose aspirin. Daily iloprost infusion for one-week significantly decreased urinary 11-dehydro-TXB2 [499 (277 – 807) vs. 380 (189 – 560) pg/mg creatinine, p < 0.0001] and 8-iso-PGF2α [533 (316 – 842) vs. 334 (196 – 540) pg/mg creatinine, p < 0.0001] as well as plasma sCD40L [1540 (1005 – 3015) vs. 948 (845 – 2030) pg/ml, p < 0.0001]. Furthermore, a significant increase in plasma nitrate plus nitrite levels has been observed [26.8 (18.8 – 35.9) vs. 43.7 (33.0 – 75.5) μM, p < 0.0001]. A significant direct correlation was also found between urinary 8-iso-PGF2α and 11-dehydro-TXB2 before and after iloprost treatment (Rho = 0.695, p < 0.0001). In conclusion, we report that a short-term course of iloprost is able to significantly reduce residual thromboxane biosynthesis, oxidative stress, endothelial dysfunction and platelet-derived inflammation in low-dose aspirin treated patients with CLI.

SummaryEarly post-operative aspirin improves survival in patients undergoing coronary artery bypass graft (CABG). However, most patients do not benefit of aspirin after CABG, still remaining at risk of thrombotic events due to insufficient platelet inhibition, specifically via the thromboxane (TX) pathway. We evaluated the effect of two aspirin doses (100 or 325 mg daily, enteric coated formulations) on platelet function and TX biosynthesis in patients after CABG and assessed whether the incidence of residual platelet reactivity could be reduced by the higher dose. Fifty-six patients undergoing CABG were randomly assigned to 100 or 325 mg aspirin daily for five days in a prospective single-centre study. Treatment effect was assessed by measuring either platelet function (light-transmission aggregometry and point-of-care PFA-100®) or TX biosynthesis in collagen-stimulated platelets, serum, urine, and in lipopolysaccharide (LPS)-cultured whole blood (WB). An insufficient TX inhibition was observed with 100 mg aspirin but not with the higher dose. The different effect of the two doses was, however, highlighted by either TX (platelet- or serum-derived) or by PFA-100® but not by the other assays. In conclusion, early after CABG, the incidence of residual platelet activity was lower in patients who received 325 mg aspirin. Moreover, evidence was provided that different methods yield different results in the detection of aspirin resistance, rendering them not interchangeable.

SummaryCOX-1 polymorphism C50T, in complete linkage disequilibrium with the other polymorphism A-842G, has been depicted as a determinant of pharmacological response to aspirin treatment. Whether these polymorphisms exert an effect on response to aspirin both in vitro and ex vivo is still controversial. We geno-typed a population of 148 healthy individuals for the C50T/A-842G haplotype. Thirty of them underwent low-dose aspirin (100 mg daily) treatment for four weeks and were followed up for seven days after withdrawal. In this subgroup, we evaluated the thromboxane-dependence of biochemical and functional indexes used to monitor the antiplatelet effect of low-dose aspirin. Among the 148 subjects studied, 10 were heterozygous for the C50T/A-842G haplotype (6.7%) and only one was homozygous for the 50T/-842G haplotype (0.67%). In the group on low-dose aspirin, serum thromboxane (TX) B2 as well as urinary 11-dehydro-TXB2 and arachidonic acid (AA)-induced aggregation were similarly suppressed in carriers and non-carriers of the 50T/-842G haplotype, with an increase until basal levels of all the parameters within seven days after withdrawal. We found no relationship between the 50T/-842G haplotype and the so-called phenomenon of aspirin resistance. Platelet cyclooxygenase activity, as reflected by serum TXB2, was uniformly and persistently suppressed by low-dose aspirin in both carriers and non carriers of these polymorphisms.

Variable biological effect of aspirin is suggested to be related to pharmacological resistance. The incidence of this so-called "resistant" state varies with the study population and the assay used. We determined performance features of five assays used to assess aspirin effects in non-smoking healthy volunteers not taking any drug known to interfere with platelet function. Blood and urine samples were obtained immediately before and after 8-10 days of aspirin 80 mg intake. Forty-five participants 19-59 years old were enrolled. The sensitivity (SE), specificity (SP), and optimal cut-off (CO) value to detect the effect of aspirin were: light transmission aggregometry (LTA) with 1.6 mM arachidonic acid (AA) - SE 100%, SP 95.9%, CO 20%; LTA with adenosine diphosphate (ADP) 10 microM - SE 84.4%, SP 77.8%, CO 70%; VerifyNow Aspirin - SE 100%, SP 95.6%, CO 550 ARU; platelet count drop - SE 82.2%, SP 86,7%, CO 55%; TEG((R)) - SE 82,9%, SP 75,8%, CO 90%; and urinary 11-dehydrothromboxane B(2) levels (11-dHTB(2)) - SE 62.2%, SP 82.2%, CO 60 pg/ml. AA-induced LTA and the VerifyNow assay reliably detected aspirin intake in all subjects; there was wide overlap in pre- and post- aspirin results with ADP-induced LTA, platelet count drop, TEG((R)) and urinary 11-dHTB(2) assays. These results suggest that some of the variability in the reported incidence of "aspirin resistance" is unrelated to aspirin intake but related to inherent limitations of some assays to detect aspirin mediated effects or to underlying platelet reactivity variability independent of aspirin-mediated cyclooxygenase-1 inhibition.

SummaryRecently, we cloned from platelet mRNA a novel cyclooxygenase (COX)-2 splice variant, designated COX-2a, which is characterized by a partial deletion of exon 5. Preliminary studies of mRNA distribution of COX-2 isoforms in platelets from coronary artery bypass grafting (CABG) patients showed a variable increase in COX-2a mRNA expression after cardiac surgery. Thus, we assessed whether this variant may play a functional role in these patients. We report a marked (about 20346-fold) increase in the expression of COX-2a mRNA after CABG. Evidence is presented that ribosomal frame-shifting may correct the coding sequence resulting in the expression of a full-length COX-2a pro-tein. In addition, a reading frame-corrected COX-2a mutant (COX-2aΔG) was generated by site-directed mutagenesis and expressed in COS-7 cells using an adenoviral expression system. However, COX-2a protein was not active in terms of prostaglandin formation. Thus, alternative mRNA splicing might represent an intriguing posttranscriptional mechanism to oppose a transcriptional activation of the COX-2 gene. Evolutionary, this mechanism may prevent COX-2-dependent thromboxane synthesis in the platelet, which would potentiate the likelihood of thrombosis; pharmacologically, this mechanism would prevent an aspirin-insensitive pathway of thromboxane formation.

SummaryAcute thrombosis is a threat in patients undergoing percutaneous coronary intervention with stent implantation. Our objective was to determine if stent-induced thrombus formation could be inhibited by oral treatment with a thromboxane A2/prostaglandin H2 receptor antagonist (TPr; S18886) as an alternative to standard therapy. Pigs were allocated in the following treatment (p.o) groups: I) clopidogrel (CLOP); II) ASA; III) S18886; IV) ASA+CLOP; and V) placebo-control. Damaged vessel was placed in the Badimon chamber containing a stent and perfused at 212/s.Antithrombotic effects were assessed as 111In-platelet deposition (PD) in two series (60 and 180 min after drug intake). Fibrin(ogen) deposition, light transmittance aggregometry (LTA; collagen, U46619, and ADP), and bleeding time (BT) were also evaluated. After 60 min S18886 reduced PD ≤48%, 40%, and 35% vs placebo, CLOP-, and ASA-treated ani mals, respectively (P0.05), and ASA showed no inhibition versus placebo. Similar effects were found in S18886– and ASA+CLOP-treated animals at both times. Fibrin(ogen) deposition followed the same pattern. Collagen-induced LTA was significantly reduced by ASA, ASA+CLOP, and S18886; S18886 abolished U46619-induced LTA; and, CLOP±ASA reduced ADP-induced LTA in a time-dependent manner. TPr blockade did not prolong BT, whereas CLOP±ASA significantly did (P

Platelets stably interact with collagen via glycoprotein (GP)VI and α2β1integrin.With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects ,while the thromboxaneA2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice.We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.. See also the following videos: Video 1 Video 2 Video 3 Video 4

SummaryLeptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of αIIbβ3, as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p

SummaryVascular injury in acute coronary syndromes (ACS) involves a complex cross-talk between inflammatory mediators, platelets and thrombosis, where the interaction between platelets and coagulation factors (e. g. thrombin) is a central link between thrombosis and inflammation. In ACS, aspirin at antiplatelet doses exhibits anti-inflammatory effects as seen from the decrease in inflammation markers such as CRP, M-CSF, MCP-1 and others. These actions probably occur subsequent to inhibition of platelet COX-1-dependent thromboxane formation and its action as a multipotent autocrine and paracrine agent. This likely involves inhibition of thrombin formation as well as inhibition of secondary pro-inflammatory mediators, such as sphingosine-1-phosphate. Experimental and limited clinical data additionally suggest antiinflammatory effects of aspirin independent of its antiplatelet action. For example, aspirin at antiplatelet doses might acetylate COX-2 in vascular cells, directing the activity of the enzyme into a 15-lipoxygenase which by transcellular metabolism results in the formation of 15-epi-lipoxin (‘aspirin-triggered lipoxin’), an antiinflammatory mediator. Furthermore, aspirin stimulates eNOS via lysine-acetylation, eventually resulting in induction of heme oxygenase (HO-1), which improves the antioxidative potential of vascular cells. All of these effects have been seen at antiplatelet doses of 100–300 mg/day, equivalent to peak plasma levels of 10–30 μM. Many more potentially antiinflammatory mechanisms of aspirin have been described, mostly salicy-late-related, at low to medium millimolar concentrations and, therefore, are of minor clinical interest. Altogether, there is a wealth of data supporting antiiflammatory effects of aspirin in ACS, but studies generating direct evidence for antiinflammatory effects in ACS remain to be done.

SummaryWe have recently shown that dipyrone (metamizole), a non-opioid analgesic, can nullify aspirin (acetylsalicylic acid; ASA) antiplatelet effects in patients with coronary artery disease (CAD). In this study, we analysed the aspirin and dipyrone drug-drug interaction in order to identify strategies to prevent the dipyrone induced inhibition of asprin antiplatelet effects. Platelet function was measured by arachidonic acid-induced light-transmission aggregometry, thromboxane (TX) B2–formation by immunoassay. Dipyrone metabolite plasma levels were determined by high-performance-liquid-chromatography (HPLC). In seven healthy individuals, in vitro ASA (30 μM/ 100 μM/ 300 μM/ 1,000 μM) and dipyrone (10 μM) coincubation revealed, that the aspirin and dipyrone interaction can be overcome by increasing doses of aspirin. In 36 aspirin and dipyrone comedicated CAD patients, addition of ASA (30 μM/ 100 μM) in vitro inhibited, but did not completely overcome the dipyrone induced reduction of aspirin antiplatelet effects. Notably, the inhibition of thromboxane formation in aspirin and dipyrone comedicated CAD patients coincided with dipyrone plasma levels. In a cross-over designed study in four healthy individuals, we were able to prove that inhibition of aspirin (100 mg/ day) effects by dipyrone (750 mg/ day) was reversible. Furthermore, aspirin (100 mg/ day) medication prior to dipyrone (750 mg/ day) intake prevented the inhibition of antiplatelet effects by dipyrone in 12 healthy individuals. In conclusion, aspirin medication prior to dipyrone intake preserves antiplatelet effects, circumventing the pharmacodynamic drug-drug interaction at the level of cyclooxygenase-1.Clinical Trial Registration: NCT01402804; http://clinicaltrials.gov/ct2/show/NCT01402804.

SummaryADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

SummaryArachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro- TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wildtype homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

SummaryAspirin and statin therapy are mainstay treatments in patients with coronary artery disease (CAD). The relation between statin therapy, in vivo thromboxane (Tx) generation; a marker of inflammation, and blood thrombogenicity has never been explored. Urinary 11-dehydro (dh) TxB2 was determined in patients with suspected CAD on 325 mg daily aspirin therapy prior to undergoing cardiac catheterisation (n=281). Thrombogenicity was estimated by thrombelastographic measurement of thrombin-induced platelet-fibrin clot strength (TIP-FCS) and lipids/lipoproteins were determined by vertical density gradient ultracentrifugation/ELISA. The influence of statin therapy and dose was analysed by the atorvastatin equivalent dose (5–10 mg, 20–40 mg, or 80 mg daily). Statin therapy (n=186) was associated with a dose-dependent reduction in urinary 11-dh TxB2 (p=0.046) that was independent of LDL and apo B100 levels but was strongly related to TIP-FCS (p=0.006). By multivariate analysis, no statin therapy (n=95) and female gender were independently associated with high urinary 11-dh TxB2 [OR=2.95 (0.1.57–5.50, p=0.0007); OR=2.25 (1.24–4.05, p=0.007)], respectively. In aspirin-treated patients, statin therapy was independently and inversely associated with inflammation in a dose-dependent manner. Elevated 11-dh TxB2 was associated with a prothrombotic state indicated by high TIP-FCS. Our data suggest that measurement of urinary 11-dTxB2 may be a useful method to optimise statin dosing in order to reduce thrombotic risk.

Summary Background: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. Study design and methods: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in alpha2 integrin polymorphism and density. Results: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the alpha2-integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. Conclusion: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation. Abbreviations: 807 CC/CT/TT polymorphisms of the alpha2-integrin gene, CD36… GPIIIb, an alternative collagen receptor, CD42b… GpIb, the von Willebrand receptor, CD49b… alpha2-integrin, subunit of the main collagen receptor, CADP-CT… collagen adenosine diphosphate induced closure time, CEPI-CT… collagen/epinephrine induced closure time, PFA-100… platelet function analyzer, vWF-Ag… von Willebrand factor antigen, TXB2… thromboxane B2

SummaryPlatelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3’phosphate-5’-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T AC receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T AC receptor or the α2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2TAC receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.

SummaryThe release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

SummaryAmong the nine ellagitannins, rugosin E was the most potent platelet aggregating agent with an EC50 of 1.5 ± 0.1 µM in rabbit platelets and 3.2 ±0.1 µM in human platelets. The aggregations caused by rugosin E and ADP were inhibited by EGTA, PGE1, mepacrine, sodium nitroprusside and neomycin, but not by indomethacin, verapamil, TMB-8, BN52021 and GR32191B. Rugosin E-induced thromboxane formation was suppressed by indomethacin, EGTA, PGE,, verapamil, mepacrine, TMB-8 and neomycin. ADP-scavenging agents, such as CP/CPK and apyrase inhibited concentration-dependently ADP (20 εM)-, but not rugosin E (5 εM)-induced platelet aggregation. In thrombin (0.1 U/ml)-treated and degranulated platelets, rugosin E and ADP still caused 63.5 ± 3.0% and 61.2 ± 3.5% of platelet aggregation, respectively. Selective ADP receptor antagonists, ATP and FSBA inhibited rugosin E- and ADP-induced platelet aggregations in a concentration-dependent manner. Both rugosin E and ADP did not induce platelet aggregation in ADP (1 mM)-desensitized platelets. In contrast to ADP, rugosin E did not decrease cAMP formation in washed rabbit platelets. Both rugosin E and ADP did not cause phosphoinositide breakdown in [3H]myo-inositol-labeled rabbit platelets. In fura-2/AM- load platelets, both rugosin E and ADP induced increase in intracellular calcium concentration and these responses were inhibited by ATP and PGEj. All these data suggest that rugosin E may be an ADP receptor agonist in rabbit platelets.

SummaryEffects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37° C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2C1 (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

SummaryCirculating microparticles play a pro-inflammatory and procoagulant detrimental role in the vascular dysfunction of septic shock. It was the objective of this study to investigate mechanisms by which a pharmacological modulation of microparticles could affect vascular dysfunction in a rat model of septic shock. Septic or sham rats were treated by activated protein C (aPC) and resuscitated during 4 hours. Their microparticles were harvested and inoculated to another set of healthy recipient rats. Haemodynamic parameters were monitored, circulating total procoagulant microparticles assessed by prothrombinase assay, and their cell origin characterised. Mesenteric resistance arteries, aorta and heart were harvested for western blotting analysis. We found that a) the amount and phenotype of circulating microparticles were altered in septic rats with an enhanced endothelial, leucocyte and platelet contribution; b) aPC treatment significantly reduced the generation of leucocyte microparticles and norepinephrine requirements to reach the mean arterial pressure target in septic rats; c) Microparticles from untreated septic rats, but not from aPC-treated ones, significantly reduced the healthy recipients’ mean arterial pressure; d) Microparticle thromboxane content and aPC activity were significantly increased in aPC-treated septic rats. In inoculated naïve recipients, microparticles from aPC-treated septic rats prompted reduced NF-κB and cyclooxygenase-2 arterial activation, blunted the generation of pro-inflammatory iNOS and secondarily increased platelet and endothelial microparticles. In conclusion, in this septic shock model, increased circulating levels of procoagulant microparticles led to negative haemodynamic outcomes. Pharmacological treatment by aPC modified the cell origin and levels of circulating microparticles, thereby limiting vascular inflammation and favouring haemodynamic improvement.

SummaryPhysical activity is associated with cardiovascular risk reduction, but the effects of exercise on platelet activation remain controversial. We investigated the effects of regular high-amount, high intensity aerobic exercise on in vivo thromboxane (TX)-dependent platelet activation and plasma levels of platelet-derived proteins, CD40L and P-selectin, and whether platelet variables changes may be related to changes in high-density lipoprotein (HDL) and in the extent of oxidative stress and oxidative stress-related inflammation, as reflected by urinary isoprostane excretion and endogenous soluble receptor for advanced glycation end-products (esRAGE), respectively. Urinary excretion of 11-dehydro-TXB2 and 8-iso-prostaglandin (PG)F2α and plasma levels of P-selectin, CD40L and esRAGE were measured before and after a eight-week standardised aerobic high-amount–high-intensity training program in 22 sedentary subjects with low-to-intermediate risk. Exercise training had a clear beneficial effect on HDL cholesterol (+10%, p=0.027) and triglyceride (-27%, p=0.008) concentration. In addition, a significant (p

SummaryWe have previously shown that tight metabolic control by insulin therapy reduced thromboxane-dependent platelet activation in noninsulin-dependent diabetes mellitus (NIDDM) patients. The present study was undertaken to determine whether a similar effect could be obtained without switching diabetics in secondary failure to insulin treatment. For this purpose, we gave strict diet and exercise advise program and adjusted on a weekly basis the oral antidiabetic therapy (glipizide) that 26 patients with NIDDM had been given over the previous months.Basal measurements of urinary ll-dehydro-TXB2 and PAI-1 confirmed previous findings of enhanced levels of these parameters in NIDDM patients with macrovascular disease in comparison to age-and sex-matched controls. After 2-6 weeks, 16 patients achieved tight metabolic control associated with significant reduction of both thromboxane biosynthesis and PAI-1 levels; 10 patients remained in poor control and no significant decrease of both parameters was observed.We conclude that reduction of in-vivo platelet activation and PAI-1 antigen levels after metabolic improvement obtained by frequent reassessment of sulphonylurea therapy together with strict diet and exercise programs may have beneficial effects on the progression of diabetic micro- and macrovascular disease.

SummaryThere is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Previous in vitro studies suggest that platelets from pregnant women show increased sensitivity to agonists, the response to which has a thromboxane dependent component. The aim of this study was to determine whether this is due to increased activity of the thromboxane biosynthetic pathway or to increased platelet sensitivity to the effects of thromboxane. During pregnancy, platelets were more sensitive to the pro-aggregatory effects in vitro of the thromboxane mimetic U46619, in whole blood and in platelet rich plasma, compared to those from non-pregnant controls. The difference in extent of U46619-induced platelet aggregation between groups was abolished in the presence of a high concentration of the specific thromboxane antagonist ICI 192605, but not by prior incubation of blood with aspirin. Platelets from pregnant women were significantly less sensitive to inhibition of arachidonic acid induced activation by the thromboxane synthetase inhibitor dazmegrel, but there was no change in platelet cyclic AMP accumulation under these conditions. Arachidonic acid induced platelet thromboxane B2 production was similar in pregnant and non-pregnant subjects. In conclusion, platelets are more sensitive to the activating effects of thromboxane during pregnancy, but there is no change in the intrinsic reactivity of the thromboxane biosynthetic pathway.

SummaryCytochalasin D was used to investigate the role of intracellular cyto-skeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen y-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein Ilb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.

SummaryThe effects of Ro 44-9883, a new specific antagonist of platelet glycoprotein IIb-IIIa receptor, on thrombus formation and reocclusion after thrombolysis induced by tissue-type plasminogen activator (t-PA) were compared with those of vapiprost, a thromboxane (TX) A2 receptor antagonist, using a photochemically-induced thrombosis model in the guinea-pig femoral artery. Pretreatment with Ro 44-9883 (5,10 and 20 μg/kg/min, i. v.) prolonged the time required to occlude the artery in a dose-dependent manner. Ro 44-9883 at 10 and 20 μg/kg/min significantly inhibited ex vivo platelet aggregation in whole blood induced by collagen, ADP or U46619. Vapiprost 0.3 mg/kg inhibited thrombus formation and platelet aggregation induced by collagen or U46619, to the same extent as Ro 44-9883 at the higher doses. In the thrombolysis study, Ro 44-9883 at the higher doses given as comedication with t-PA reduced the time to achieve reperfusion and increased the vascular patency after successful reperfusion. Vapiprost also significantly reduced the time to reperfusion and prevented reocclusion. However, the vascular patency after thrombolysis by t-PA with vapiprost was significantly increased compared with Ro 44-9883. Ro 44-9883 inhibited platelet aggregation, but did not prevent TXA2 formation in platelets. Thus, vascular contraction mediated by platelet-derived TXA2 may be responsible for lower efficacy of Ro 44-9883 against reocclusion compared with vapiprost. These results indicate that not only platelet aggregation but also vasoconstriction may contribute to reocclusion after t-PA-induced thrombolysis in the guinea-pig.

SummaryThe effects of unfractionated heparin (UH) and recombinant hirudin (rH) on prothrombin activation, free thrombin generation, and platelet aggregation induced by endogenously generated thrombin after intrinsic activation of platelet rich plasma were compared. Free thrombin generation and platelet aggregation were assessed simultaneously by delaying fibrinogen polymerisation with GPRP.UH more effectively inhibited prothrombin activation and free thrombin generation than rH. Increasing concentrations of rH had hardly any effect on the peak amount of free thrombin, while in the presence of 400 nM UH only traces of free thrombin were detected. Comparison of TAT and THC (thrombin-hirudin complex) generated until the onset of platelet aggregation on a molar basis showed that much more thrombin was inactivated in the presence of rH than in plasma containing UH. The explosive generation of free thrombin in hirudinized plasmas was accompanied by a markedly steeper aggregation curve as compared to heparinized plasmas. The generation of thromboxane B2 was markedly delayed in the presence of UH but not influenced in the presence of rH.Our results suggest that UH is more effective than rH in inhibiting prothrombin activation after intrinsic activation of platelet rich plasma, while rH prevents clotting more by direct inactivation of already generated thrombin. The inability of even high concentrations of rH to prevent the explosive generation of free thrombin might contribute to the observed inefficiency of rH to inhibit platelet aggregation.

SummaryTiclopidine is thought to be a selective inhibitor of ADP-induced platelet function. Here we have investigated the effects of ticlopidine on platelet function in whole blood induced by ADP and by other platelet agonists. Whole blood was used because it was considered that ADP derived from red cells might act synergistically with other platelet agonists to enhance platelet responses, and that ticlopidine might interfere with this process. Measurements were performed using blood from 16 healthy volunteers before ticlopidine administration, after taking ticlopidine 250 mg daily for 10 days, after taking ticlopidine 250 mg twice daily for a further 10 days, and after 14 days off treatment.Ticlopidine proved to be a very effective inhibitor of the platelet aggregation induced by ADP; it was most effective in enhancing the reversibility of the aggregation response. The drug modestly but significantly reduced streptokinase, adrenaline, collagen, sodium arachidonate, PAF and U46619 – induced platelet aggregation. The drug significantly reduced the extent of the release reaction (14C-5HT release) induced by ADP, streptokinase, PAF, ristocetin and sodium arachidonate, and also reduced the extent of the synergistic 14C-5HT release induced by combinations of ADP and PAF, ADP and adrenaline and PAF and adrenaline.The various inhibitory effects of ticlopidine were evident after treatment with 250 mg daily but were more pronounced after 250 mg twice daily. All values had returned to normal after 14 days off treatment. Ticlopidine had no effect on serum thromboxane B2 production nor on several parameters of coagulation and fibrinolysis.We conclude that ticlopidine is an effective inhibitor of ADP-induced platelet aggregation and also the platelet aggregation and 14C-5HT release induced in whole blood by a number of platelet agonists and combinations of agonists. These latter effects are probably mainly via a selective effect on ADP. The inhibitory effects of the drug are dose-related.

SummaryKRDS, a tetrapeptide from human lactotransferrin, inhibits thrombin-induced platelet aggregation, secretion and thromboxane (TX) synthesis without interfering with phospholipase C (PLC) β activation, since in previous work we have shown that Ca2+ mobilization and phosphorylation of the myosin light chain kinase (20 kDa) and pleckstrin (47 kDa) were normal. However, the inhibition of arachido- nic acid-induced aggregation in the presence of KRDS is accompanied by normal TX synthesis suggesting that it does not interfere with the cyclooxygenase activity. To elucidate further the mechanisms of action of this peptide we tested its effect on U46619-induced platelet activation. KRDS inhibits U46619-induced platelet aggregation time- and dose-dependently without inhibiting the phosphorylation of pleckstrin. This suggests that the PLC pathway is not affected and that the inhibitory effect of KRDS is not due to an uncoupling of TXA2 from its receptor. In addition to the PLC pathway, protein tyrosine kinases play a major role in platelet signal transduction mechanisms. At least 7 tyrosine-phosphorylated proteins are detected upon stimulation of platelets by thrombin. KRDS strongly inhibits the tyrosine- phosphorylated substrates, in particular two 100-105 kDa substrates which are related to GP Ilb/IIIa activation and platelet aggregation. The absence of TX synthesis observed in the presence of KRDS could be due to the inactivation of cPLA2 since the latter needs tyrosine phosphorylation to be activated, thus explaining the inhibitory action of KRDS on platelet functions.

SummaryIn a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

SummaryPreviously we demonstrated that heparin administration during carotid endarterectomy (CEA) caused a marked, but transient increase in platelet aggregation to arachidonic acid (AA) and adenosine diphosphate (ADP), despite effective platelet cyclo-oxygenase-1 (COX-1) inhibition with aspirin. Here we investigated the metabolism of AA via platelet 12-lipoxygenase (12-LOX) as a possible mediator of the observed transient aspirin resistance, and compared the effects of unfractionated (UFH) and low-molecular-weight (LMWH) heparin. A total of 43 aspirinated patients undergoing CEA were randomised in the trial to 5,000 IU UFH (n=22) or 2,500 IU LMWH (dalteparin, n=21). Platelet aggregation to AA (4×10–3) and ADP (3×10–6) was determined, and the products of the COX-1 and 12-LOX pathways; thromboxane B2 (TXB2) and 12-hydroxyeicosatretraenoic acid (12-HETE) were measured in plasma, and in material released from aggregating platelets. Aggregation to AA increased significantly (∼10-fold) following heparinisation (p

SummaryVenous stasis occurs when people are at bedrest, because of altered venous flow characteristics. This is commonly believed to be one etiology behind the development of deep venous thrombosis (DVT). The hemostatic effects of bedrest and their possible role in DVT development have not been fully examined. We hypothesized that bedrest would lead to increases in hemostatic function and that these increases could be important in the development of DVT.Twelve non-smoking volunteers were studied during supine positioning for 36 hours. Platelet reactivity and plasma concentrations of fibrinogen, a2-antiplasmin, plasminogen, thromboxane (32, plasminogen activator inhibitor-1, tissue plasminogen activator and neuroendocrine hormones (cortisol, epinephrine and norepinephrine) were measured at 8:00 a.m., 10:00 a.m., 4:00 p.m. and 8:00 a.m.Cortisol demonstrated an early morning increase while catecholamines were unchanged throughout. Fibrinogen, a2-antiplasmin, plasminogen and platelet reactivity were no different at any time point. Fibrinolytic proteins changed over time, manifested by decreased PAI-1 antigen and activity levels at 24 h.Based upon the parameters measured, bedrest causes no increase in hemostatic function. In fact, bedrest causes the potential for enhanced fibrinolysis, that differs from that previously reported for normal activity over 24 h. This may represent a protective mechanism to counter the effects of stasis from bedrest.

SummaryPlatelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B2 in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca 2+ ]o) that is present in citrated PRP. In fact, low [Ca 2+ ]o artifactually potentiates the platelet TxB2 production in vitro. Forty patients with diabetes mellitus and 37 matched Controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of 14C-serotonin and TxB2 production were induced in platelet rieh plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p

SummaryThe effect of type IIB von Willebrand’s factor (vWF) on platelet cytosolic Ca2+ ion concentration, measured by means of the probe fura 2, was investigated. Seven patients with type IIB von Willebrand disease (vWD) were studied. Addition of type IIB vWD plasma to platelet suspensions induced a cytosolic calcium increase accompanied by platelet aggregation. Both processes were completely abolished by addition of the calciumchelating agent EGTA, indomethacin, peptide RGDS, and monoclonal antibodies blocking the vWF binding site on GPIb-IX (LJIB1) or the cytoadhesive receptor on GPIIb-IIIa (LJCP8). The ADP-scavenger apyrase and the protein kinase C-inhibitor staurosporine partially inhibited the rate of the cytosolic calcium increase. No direct correlation between the extent of Ca2+ rise and the phenotypic expression of IIB vWD, such as the degree of spontaneous platelet aggregation or thrombocytopenia was apparent. It is suggested that aggregation and cytosolic Ca2+ increase in platelets exposed to plasma from type IIB vWD patients is mediated by a self-potentiating mechanism involving both GPIb and GPIIb-IIIa receptors as well as the thromboxane biosynthetic pathway.

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

SummaryIn peripheral thrombolysis adjuvant anti-platelet therapy may help to lyse otherwise resistant thrombus, thereby increasing the number of patients successfully treated and reducing the “time to lysis”. If continued after lysis it may help to prevent early rethrombosis. In this pilot study 21 patients undergoing peripheral thrombolysis with streptokinase were randomised to receive the thromboxane receptor antagonist sulotroban or placebo. The dose of sulotroban given was 2 mg/min (four patients), 4 mg/min (five patients) or 8 mg/min (four patients), eight patients received placebo. The clinical and laboratory effects of the treatment were monitored.Thrombolysis was achieved more quickly in patients receiving sulotroban, however, there was no difference between groups in the number of patients in whom recanalisation was achieved (six of eight receiving placebo and eight of 13 receiving sulotroban) or in the number of cases of early rethrombosis. During lysis there was an increase in plasma beta-thromboglobulin with similar levels being found in patients receiving sulotroban and streptokinase and those receiving streptokinase alone. No other major changes in platelet function during lysis were seen in patients receiving streptokinase alone. Sulotroban significantly reduced platelet aggregtion and 14C-5HT release in response to several platelet agonists. With the thromboxane mimetic U46619 the degree of inhibition of aggregation and 14C-5HT release depended on the dose of sulotroban used. High levels of inhibition were associated with an excess of haemorrhagic complications especially in combination with a low plasma fibrinogen level.We conclude that the use of low dose sulotroban in combination with streptokinase merits further study and may have a role in accelerating lysis. However, the combination of high dose sulotroban with streptokinase causes an increased incidence of bleeding. Monitoring platelet function during such therapy may identify patients likely to bleed.

SummaryIn a group of 6 patients with lupus anticoagulant (LA) and antiphospholipid (aPL) antibodies detected by ELISA ovemight urine and blood were simultaneously collected. A significantly increased urinary excretion of the platelet-derived thromboxane (TX) metabolite 11-dehydro-TXB2 was found in this group, as compared to 12 healthy individuals. In contrast, a small but significant reduction of the vascular prostacyclin (PGI2) metabolite 2,3-dinor-6-keto-prostaglandin F1α was observed.To further elucidate the effect of these antibodies on platelet activation we isolated the F(ab’)2 fragments from IgG of the 6 patients and 5 Controls, and we evaluated the effect of these fragments on the responses of isolated normal platelets to thrombin.Patients’ F(ab’)2 increased platelet aggregation and serotonin release of platelets stimulated by low dose thrombin (0.01 U/ml). At threshold thrombin concentration (0.05 U/ml) an enhanced TXB2 production was also observed.In summary, our results show, in addition to the altered TXA2/PGI2 balance observed in vivo, a direct stimulatory effect of aPL antibodies on platelet activation in vitro. This effect is related to recognition of phospholipid epitopes on platelets as shown by its neutralization upon preincubation with phospholipids. This phenomenon may be relevant for the thrombotic tendency of these patients.

SummarySmoking is a risk factor for the development of atherosclerotic cardiovascular disease, in men as well as in women. An increased urinary excretion of the thromboxane metabolite 2,3-dinor-thromboxane B2 (Tx-M) has been observed in smokers of both genders, suggesting that cigarette smoking may facilitate cardiovascular disease via an action on the platelets. The present study addressed the hypothesis that the increased Tx-M excretion in female smokers reflects a true facilitation of platelet reactivity in vivo, rather than an increased destruction of the platelets. In healthy female volunteers (aged 20–46 years, 18 smokers and 17 non-smokers) platelet life-span and indices of platelet activity were determined, together with plasma levels of plasminogen activator inhibitor-1 (PAI-1), fibrinogen, peripheral blood cell counts and hematocrit. The urinary excretion of Tx-M was higher in smokers than in non-smokers (361 vs. 204 pg/mg creatinine, respectively, p

SummaryPotential pharmacokinetic and pharmacodynamic interactions between two oral doses of GR32191 (40 and 80 mg), a new thromboxane antagonist, and heparin (5,000 IU bolus + 1,000 IU/h for 3 h) were studied in eighteen healthy male volunteers using two separate double-blind, randomised, placebo-controlled, cross-over studies.Mean (range) bleeding time values were 8.4 min (7.5–9.7) during heparin/placebo, 12.1 min (9.2–18.6) (GR32191/placebo) and 16.3 min (11.5–21.4) (GR32191/heparin) in the 40 mg study, while these values were 8.7 min (5.5–15.5), 16.0 min (9.3 – >36.0) and 23.8 min (10.7 – >36.0), respectively in the 80 mg study. Compared to screening values, the combination of 80 mg of GR 32191 and heparin had a greater effect on the bleeding time than the sum of the prolongations after the separate treatments (p = 0.05). In the 40 mg study this was not the case. Pharmacokinetics of heparin (as assessed by plasma anti-Xa and antithrombin activity) and GR32191 were unaltered during coadministration of the two drugs. GR32191 did not influence the effects of heparin on APTT. Heparin slightly diminished the inhibition of collagen induced platelet aggregation by 80 mg of GR32191 and the U-46619 (thromboxane A2-mimetic) induced platelet aggregation remained unchanged. Overall fibrinolytic activity (as evaluated by the fibrin plate test) was similar during all three treatments in the study with 80 mg.The combination of 80 mg of GR32191 and heparin caused a prolongation of the bleeding time which was more than expected on the basis of their individual effects.

SummaryMass contents of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) were measured in U46619-stimulated human platelets. 1 µM of U46619 induced maximum responses in aggregation, 5-hydroxytryptamine (5HT) secretion and increase in intracellular free Ca2+ concentration ([Ca2+]i). Aggregation was almost comparable to that induced by maximal dose (1 U/ml) of thrombin, while 5HT release was almost half. The initial [Ca2+]i peak in response to U46619 was about half of thrombin stimulation. Production of IP3 and DG was, however, less than one tenth of that seen in thrombin stimulation. The profile (time course and concentration-dependency) of IP3 formation did not correlate with that of [Ca2+]i, suggesting that U46619 stimulates IPs-dependent and -independent Ca2+ mobilization. DG production was small but sustained for more than 5 min. These findings support the recent hypothesis that aggregation is regulated by a delayed accumulation of DG. The low level of 5HT secretion could be explained by the low production of second messengers, IP3 and DG.

SummaryIndividualised antiplatelet therapy and platelet function testing have attracted considerable clinical interest, but several aspects of test performance have not been thoroughly evaluated. We investigated repeatability and concordance of light transmission aggregometry (LTA) induced with arachidonic acid (AA) 1.0 mM, PFA-100® induced with collagen/epinephrine, multiple electrode aggregometry (MEA) induced with AA 0.5 or 0.75 mM and VerifyNow® Aspirin. Patients with stable coronary artery disease (n=43) and healthy individuals (n=21) were included. All tests were performed in duplicate at baseline in healthy individuals and in duplicate for four days in all study participants during aspirin treatment. Serum and urinary thromboxane metabolites were measured several times to evaluate cyclooxygenase-1 inhibition by aspirin. MEA was most sensitive for aspirin as treatment induced a 12-fold difference in AA-induced platelet aggregation. Coefficients of variation for duplicate measurements at baseline (0.4–12%), during aspirin treatment (3–46%) and for day-to-day variability (3–37%) differed markedly between tests and were lowest for VerifyNow®. The prevalence of aspirin low-responsiveness also differed between tests (0–9%) and the agreement was low: kappa≤0.21 for all tests compared with AA-induced LTA (reference test), which correlated best with VerifyNow® (r=0.43, p

SummaryAspirin ‘resistance’ (AR) is a phenomenon of uncertain etiology describing decreased platelet inhibition by aspirin. We studied whether (i) platelets inAR demonstrate increased basal sensitivity to a lower degree of stimulation and (ii) platelet aggregation with submaximal stimulation could predict responses to aspirin. Serum thromboxane B2 (TxB2) levels and platelet aggregation with light transmission aggregometry (LTA ) were measured at baseline and 24 hours after 325 mg aspirin administration in 58 healthy subjects. AR was defined as the upper sixth of LTA (≥ 12%) to 1.5 mM AA. Baseline platelet aggregation with sub-maximal concentrations of agonists [ADP 2 µM, arachidonic acid (AA) 0.75 mM, collagen 0.375 and 0.5 µg/ml] was greater in AR subjects compared with non-AR subjects, but not with higher concentrations (ADP 5 µM and 20 µM, AA 1.5 mM and collagen 1 µg/ml). Post-aspirin platelet aggregation was elevated in AR subjects with both submaximal and maximal stimulation. Baseline and post-aspirin serumTxB2 were higher inAR subjects and decreased further with ex-vivo COX -1 inhibition, suggesting incompletely suppressed COX -1 activity. Pre-aspirin platelet aggregation to 0.75 AA demonstrated a dichotomous response with 29/58 subjects having aggregation ≤15% and 29/58 subjects having aggregation ≥75%. In the high aggregation group 28% had AR compared to 6% in the non-AR group (p=0.04). In conclusion, platelets in AR subjects demonstrate increased basal sensitivity to submaximal stimulation, which could predict responses to antiplatelet therapy.

SummaryThe combination of thromboxane synthase inhibition with thromboxane receptor antagonism has been shown to result in a strong inhibition of platelet aggregation and a prolongation of the bleeding time (Gresele et al., J. Clin Invest 1987;80: 1435–45).Ridogrel is a single molecule that efficiently achieves both inhibitions in human volunteers. The present study was performed in patients with obstructive peripheral arterial disease and elevated plasma β-thromboglobulin levels. Patients were treated with either 2 × 300 mg ridogrel or 2 × 300 mg placebo per day for 2½ days, according to a double blind randomised parallel design. Plasma β-thromboglobulin decreased significantly throughout active treatment starting within 2 h after administration; serum and urinary immunoreactive TxB2 levels and urinary 11-dehydro- TxB2 excretion were significantly lower and serum PGE2 and 6-keto-PcF1α levels significantly higher with ridogrel; no changes were observed in the placebo-treated group.In conclusion this study demonstrates a reduction of platelet activation in vivo by ridogrel.