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Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by the severe deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (< 10%). Rapid ADAMTS13 testing is crucial for an early diagnosis and optimal management of acute TTP. We evaluated the performance of the HemosIL AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, Massachusetts, United States), a fully automated chemiluminescent immunoassay with an analytical time of 33 minutes. A method comparison study was performed on 176 samples from 49 healthy donors and 127 TMA patients (109 TTP, 7 atypical hemolytic uremic syndrome, 11 other TMAs), comparing this new assay with an in-house FRETS-VWF73 assay and a commercial enzyme-linked immunosorbent assay (ELISA) (TECHNOZYM ADAMTS-13 Activity, Technoclone GmbH, Vienna, Austria). Agreement between methods was assessed with focus on ADAMTS13 activity less than 10%, the medical decision level relevant for TTP diagnosis. The HemosIL AcuStar ADAMTS13 Activity showed good correlation with both the FRETS-VWF73 (r = 0.96) and ELISA (r = 0.96) methods. Slope of the Passing–Bablok regression was 1.05 for FRETS-VWF73 and 1.02 for ELISA, and absolute bias at the medical decision level was +0.1 and +0.3%, respectively. The study also revealed high agreement with FRETS-VWF73 (kappa 0.97) and ELISA (kappa 0.98) methods in classifying TTP patients with a severe deficiency of ADAMTS13 activity. Because of its short turnaround time and full automation, the HemosIL AcuStar ADAMTS13 activity assay might become the assay of choice to rapidly test ADAMTS13 activity in plasma and thus establish the diagnosis of acute TTP in emergency settings.

The beneficial effects of restoration of coronary flow in patients with acute myocardial infarction may be hampered by inadequate tissue perfusion. Among other factors, it is likely that platelets contribute substantially to this phenomenon. Platelets may compromise blood flow at the microvascular level by forming a part of microemboli, by adhering to reperfused, capillary or venular endothelium or to attached leukocytes, by releasing substances producing vasoconstriction, or through toxic effects. Patients with acute coronary syndromes have an increased number of circulating activated platelets, and this systemic platelet activation has been related to the presence and extent of myocardial necrosis. The mechanisms of platelet deposition to reperfused microvessels are not fully understood, but likely involve the interaction between adhesion molecules such as selectins or glycoproteins expressed on these cells upon activation and their ligands on the surface of endothelial cells or polymorphonuclear leukocytes. While these interactions are potentially important therapeutic targets in acute myocardial infarction, reducing platelet deposition and increasing myocardial salvage by direct effects on the microvasculature is still challenging with the existing armamentarium of antiplatelet agents. This review summarizes the current knowledge on the mechanisms of platelet-mediated myocardial damage after reperfusion and the effects of pharmacological interventions aimed to reduce microvascular platelet deposition and platelet-mediated myocardial injury.

Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia–reperfusion injury. The addition of oleate–bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and 1H-nuclear magnetic resonance spectroscopy. Simulated ischaemia–reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7 ± 2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P= 0.02). The magnitude of LD accumulation was inversely correlated (r2= 0.68, P= 0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca2+-sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca2+ overload than control cells. These results suggest that LD exert a protective effect during ischaemia–reperfusion by sequestering free fatty acids and Ca2+.

Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca 2+ stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mM glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.

Gap junction-mediated communication can modulate cell death in different tissues. In myocardium, gap junction communication is altered during ischemia, which contributes to the development of arrhythmias, but still allows synchronization of the onset of rigor contracture in the progression of injury. During reperfusion, gap junction communication allows cell-to-cell spread of hypercontracture and cell death. Since the intracellular signal transduction systems involved in modulation of gap junction-mediated communication are activated during ischemic preconditioning, the hypothesis can be raised that gap junctions are end-effectors of preconditioning contributing to its protective effect on cell death. This paper reviews the available information supporting this hypothesis. It has been shown that ischemic preconditioning may influence gap junction-mediated intercellular communication by activation of different kinases, including PKC and MAPK cascades, and by preservation of cGMP among other mechanisms. Connexin phosphorylation by PKC, p38/MAPK, and PKG, tends to reduce intercellular communication. This effect of ischemic preconditioning seems to have no relevant consequences during prolonged ischemia, and does not significantly modify the time course of either electrical uncoupling or the frequency or temporal distribution of ventricular arrhythmias during this period. However, any modification of gap junction communication during initial reperfusion could contribute to the reduced extent of hypercontracture and cell death observed in preconditioned hearts. The potential role of gap junctions as effectors of ischemic preconditioning against lethal injury secondary to ischemia-reperfusion deserves to be investigated in depth.

Objectives We sought to assess the effect of glycoprotein (GP) IIb/IIIa blockade on myocardial platelet and polymorphonuclear leukocyte accumulation and on infarct size after coronary injury and transient coronary occlusion (CO) in pigs. Background It has been suggested that platelet GP IIb/IIIa blockade might reduce the severity of microvascular damage after reperfusion. Methods Sixteen thiopental-anesthetized, open-chest pigs, in whom platelets had been labeled with technetium-99m (99mTc) on the previous day, were submitted to catheter-induced left anterior descending coronary artery (LAD) injury followed by 55 min of CO and 5 h of reperfusion. Five minutes before reflow, the animals were blindly allocated to receive lamifiban (intravenous bolus of 250 μg/kg body weight and continuous infusion of 3 μg/kg per min) or saline. Results Lamifiban had a rapid and potent platelet anti-aggregatory effect, as demonstrated by significant prolongation of the bleeding time and profound (∼90%) inhibition of ex vivo platelet aggregation, and completely prevented the development of cyclic flow reductions of the LAD (0 vs. 5 ± 1, one of them followed by re-occlusion, in control animals, p = 0.005). However, compared with animals receiving placebo, those treated with lamifiban had a similar (p = NS) content of 99mTc platelets in the reperfused myocardium (288 ± 40% vs. 205 ± 27% of the value in the control region, respectively) and similar myeloperoxidase activity (0.50 ± 0.17 U/g vs. 0.47 ± 0.17 U/g, respectively) and infarct size (46.8 ± 12.0% vs. 49.8 ± 10.5% of the area at risk, respectively). Arteriolar platelet thromboemboli were very rarely seen on histologic analysis. Lamifiban did not modify platelet P-selectin expression in additional studies. Conclusions Platelet GP IIb/IIIa blockade has a potent antithrombotic effect at the culprit lesion, but does not significantly reduce the magnitude of microvascular platelet accumulation or myocardial damage after transient CO.

During myocardial ischemia, severe ATP depletion induces rigor contracture followed by intracellular Ca2+ concentration ([Ca2+]i) rise and progressive impairment of gap junction (GJ)-mediated electrical coupling. Our objective was to investigate whether chemical coupling through GJ allows propagation of rigor in cardiomyocytes and whether it persists after rigor development. In end-to-end connected adult rat cardiomyocytes submitted to simulated ischemia the interval between rigor onset was 3.7 ± 0.7 s, and subsequent [Ca2+]i rise was virtually identical in both cells, whereas in nonconnected cell pairs the interval was 71 ± 12 s and the rate of [Ca2+]i rise was highly variable. The GJ blocker 18α-glycyrrhetinic acid increased the interval between rigor onset and the differences in [Ca2+]i between connected cells. Transfer of Lucifer yellow demonstrated GJ permeability 10 min after rigor onset in connected cell pairs, and 30 min after rigor onset in isolated rat hearts submitted to nonflow ischemia but was abolished after 2 h of ischemia. GJ-mediated communication allows propagation of rigor in ischemic myocytes and persists after rigor development despite acidosis and increased [Ca2+]i.

We have studied the interaction between P1 and P2 purinoceptors in purified type-1 astrocyte cultures from postnatal days 7-8 rat cerebella using single cell microfluorimetry with fura-2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 +/- 0.3 microM. Costimulation of type-1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 microM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5'-N-ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 +/- 0.01 microM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 +/- 0.2 microM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type-1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of beta gamma subunits released after Gs stimulation.

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analogue used as a selective antagonist of adenosine receptors, caused apoptosis in a variety of leukaemia-derived cell lines as well as in cells from patients with myeloid leukaemia. Apoptosis was assessed by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, R3 and H4, in the nucleoplasm of cells. Cell cycle analysis indicated that apoptosis occurred irrespective of the cell cycle phase. DPCPX did not trigger apoptosis in resting human peripheral blood lymphocytes; neither did it potentiate the apoptotic effect of phytohemagglutinin (PHA), when these cells were activated by PHA. These results indicate that DPCPX may be useful in the therapy of proliferative disorders of the hematopoietic system.

The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH(4)), and renal (LLC-PK(1)) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH(4) cells and prevented the growth of LLC-PK(1) cells. The ion did not lead to the death of LLC-PK(1) cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.

1 A functional study of cell surface A2b adenosine receptors was performed on the T cell leukaemia line, Jurkat. 2 A2b receptors were coupled both to the adenylate cyclase system and to intracellular calcium channels. In fact, the agonist of A2b receptors, 5′-N-ethylcarboxamidoadenosine (NECA), led to a transient accumulation of intracellular calcium by an inositol phosphate-independent mechanism. 3 The NECA-induced accumulation of cGMP was not responsible for the calcium mobilization via A2b receptors. 4 The calcium response elicited by activation of A2b receptors was independent of that evoked by activation of the T cell receptor. 5 These findings not only delineate a novel transduction mechanism for adenosine but also support a specific role for adenosine in modulating signals elicited via the T cell receptor. British Journal of Pharmacology (1997) 122, 1075–1082; doi:10.1038/sj.bjp.0701495

SummaryPlatelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. This study aimed to investigate whether platelets from patients with AMI increase myocardial injury after ischaemia and reperfusion in isolated rat hearts and the modification of this effect by the P2Y12 receptor antagonist cangrelor and the glycoprotein IIb/IIIa receptor blocker abciximab. Isolated rat hearts were subjected to 40 minutes of global ischaemia and 60 minutes of reperfusion. Hearts (four simultaneous experiments per patient) were infused with platelets from nine AMI patients (seven thrombolysed, all on aspirin), untreated or incubated with 10 μM cangrelor or 5 μg/ml abciximab. Control experiments were performed using platelets from healthy volunteers and platelet-poor plasma. P-selectin expression on isolated platelets was higher in AMI patients than in controls and was not modified by the treatments. Control platelets or platelet-poor plasma did mild or no harm. In contrast, platelets from AMI patients significantly augmented myocardial injury, as demonstrated by worse left ventricular (LV) developed pressure, higher maximal LV end-diastolic pressure and coronary resistance, and greater lactate dehydrogenase release and infarct size. Both cangrelor and abciximab greatly attenuated these effects. In conclusion, activated platelets from AMI patients increase myocardial injury after ischaemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment might reduce infarct size by direct effects on reperfused myocardium in these patients.

Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia-reperfusion injury. The addition of oleate-bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and (1)H-nuclear magnetic resonance spectroscopy. Simulated ischaemia-reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7+/-2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P=0.02). The magnitude of LD accumulation was inversely correlated (r(2)=0.68, P=0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca(2+)-sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca(2+) overload than control cells. These results suggest that LD exert a protective effect during ischaemia-reperfusion by sequestering free fatty acids and Ca(2+).

Objective: Platelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. We aimed to investigate whether platelets from patients with AMI increase myocardial damage after transient ischemia in isolated rat hearts and the modification of this effect by the P2Y 12 receptor antagonist cangrelor and the GPIIb/IIIa receptor blocker abciximab. Methods: Platelets were obtained from 9 AMI patients (7 thrombolyzed, all on aspirin) within 24 h after symptom onset. Incubation with 100 μM cangrelor or 50 μg/ml abciximab resulted, respectively, in 78 ± 4 and 90 ± 2% inhibition of aggregation (optical aggregometry). Isolated rat hearts (four simultaneous experiments per patient) were subjected to 40 min of global ischemia and 60 min of reperfusion. Hearts received no additional intervention (Control) or were infused during the 5 min prior to ischemia with platelets (22.5x10 6 /min), either untreated or treated with cangrelor or abciximab. Results: P-selectin expression (flow cytometry) in isolated platelets before infusion was 31 ± 3% (P = NS between groups). Platelets augmented myocardial injury, as demonstrated by worse left ventricular developed pressure (LVDevP), higher left ventricular enddiastolic pressure (LVEDP) and coronary resistance, and greater LDH release and infarct size (TTC staining), and both cangrelor and abciximab greatly attenuated these effects (Table ). Conclusions: Activated platelets from patients with AMI increase myocardial injury after ischemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment may increase myocardial salvage by direct effects on the microcirculation in these patients.

Background Activated platelets might contribute to endothelial dysfunction in non-ischaemic territories during acute myocardial infarction. We assessed platelet deposition, coronary flow reserve and contractile function in remote cardiac regions after transient coronary occlusion and their association with systemic platelet activation. Materials and methods In 10 pigs (series A) subjected to 48-min occlusion of the left anterior descending coronary artery (LAD), 99mTc-platelet content in the right coronary artery (RCA) and its dependent myocardium was counted after reflow. In 10 pigs (series B) receiving the same occlusion of the RCA, the hyperaemic response at the LAD and systolic shortening in LAD-dependent myocardium were monitored after reperfusion. P-selectin expression on circulating platelets was assessed in both series by flow cytometry. Results In series A, platelet counts in the RCA and non-ischaemic myocardium were correlated with platelet content, polymorphonuclear leukocyte infiltration and infarct size in the reperfused zone, as well as with the percentage of P-selectin-positive platelets after reflow. In series B, a transient reduction in peak hyperaemic response in the LAD and sustained contractile dysfunction in non-ischemic myocardium were observed after releasing the RCA occlusion, these changes being also correlated with platelet activation status. Conclusions Ischaemic injury triggers macro- and microvascular platelet deposition and causes an impairment in coronary flow reserve and contractile function in distant regions of the heart, which are related to activation of circulating platelets.

An unexpected high incidence of thromboembolic complications has been described in patients with systemic autoimmune diseases treated with CD40L immunotherapy. Since activated platelets express CD40L, we aimed to investigate the effects of CD40L mAb in platelet aggregation induced by physiological stimuli. Optical aggregometry was performed on platelet-rich plasma and washed platelets obtained from systemic venous blood (0.38% citrate) of anesthetized pigs. CD40L mAb clone 5c8, used in clinical trials for autoimmune diseases, was used. In platelet-rich plasma, CD40L mAb neither induced platelet aggregation per se, nor significantly affected maximal aggregation or slope of ADP-induced aggregation curves. However, it dose-dependently inhibited spontaneous deaggregation observed in ADP-stimulated samples. This effect was not observed with an irrelevant isotype-matched immunoglobulin. The stabilizing effect on platelet aggregates was neither glycoprotein IIb/IIIa-mediated nor Ca2+-dependent but was abolished by acetylsalicylic acid pretreatment. F(ab')2 fragments did not stabilize ADP-induced platelet aggregates but inhibited the stabilizing effect of CD40L mAb. Similar results were obtained with washed platelets, although higher amplification of ADP-induced aggregation was observed. In conclusion, CD40L expression produced by physiological or pathophysiological platelet activation can sustain a pro-aggregatory effect of CD40L mAb by a mechanism involving mAb Fc domain. These results could help to explain the mechanism of CD40L mAb-induced thromboembolic complications.

Thrombin exerts multiple actions on cardiomyocytes leading to increased intracellular Na + and Ca 2+ concentrations, and to activation of a Ca 2+ -independent PLA2, and has been proposed to favor the genesis of arrhythmias and ischemic injury in acute coronary syndromes. However, the influence of thrombin on cardiomyocyte cell death during ischemia–reperfusion has not been studied. A beneficial influence of low thrombin concentrations has been described in other cell types. HL-1 cardiomyocytes were subjected to simulated ischemia (SI) and reperfusion (SR) and cell death was assessed by means of LDH release to the incubation media. Thrombin dose-dependently increased cell death in normoxic cells, in cells subjected to SI, and in cells subjected to SR (by 20 ± 8%, 95 ± 32% and 35 ± 9%, respectively, at 100 U/ml). The effects of thrombin were associated to increased cytosolic Ca 2+ overload, mimicked by 100 µM PAR-1 agonist peptide SFLLRNPNDKYEPF, and reversed by the direct thrombin inhibitor lepirudin (IC50 = 1.3 ± 0.2 µg/ml). The presence of thrombin during simulated ischemia– reperfusion increases cardiomyocyte cell death by a mechanism that involves activation of PAR-1 receptors and can be prevented by the direct thrombin inhibitor lepirudin. © 2005 Elsevier Ltd. All rights reserved.

Objective: Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca2+-dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GCNO) to the cell membrane has been recently proposed to play a role. The aim of this study was to determine the possible functional relevance of GCNO bound to the cardiomyocyte membrane. Methods: Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GCNO activity was assayed in parallel experiments. Results: In untreated cardiomyocytes, approximately 30% of β1-and α1-subunits of GCNO and a similar proportion of GCNO activity were found in the particulate fraction. The dependence of GCNO activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 caused GCNO to translocate to the sarcolemma, increased GCNO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca2+-dependent changes on the phosphorylation status of GCNO, since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca2+/calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GCNO activity but not with their effects on cytosol-associated GCNO. Conclusion: These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GCNO differs from that of cytosolic GCNO, and 3) membrane association may have a crucial role in determining the response of cells to NO.

Objectives The goal of this study was to assess whether selectin blockade reduces myocardial platelet deposition and platelet-mediated injury after transient ischemia. Background Selectins participate in platelet adhesion to reperfused endothelium. Methods Thiopental-anesthetized, open-chest pigs were subjected to mechanical injury of the left anterior descending coronary artery followed by a 48-min occlusion and 2 (n = 20) or 4 (n = 16) h of reperfusion. Fifteen minutes before occlusion, animals were blindly allocated to receive a continuous intravenous infusion of the selectin blocker fucoidan (30 μg/kg/min, plus a 1-mg/kg bolus in the latter group) or saline. In isolated rat hearts infused with thrombin-activated platelets, the effects of fucoidan (30 μg/ml) administered during reperfusion after 40 min of global ischemia were also analyzed. Results Fucoidan did not prevent the development of cyclic reductions in coronary flow, but reduced the content of 99mTc-labeled platelets in reperfused myocardium after 2 h of reperfusion (23.4 ± 3.3 vs. 42.1 ± 8.3 × 106platelets/g in treated and untreated animals, p = 0.03) and attenuated the impairment in the coronary flow reserve and reduced infarct size after 4 h (53 ± 2% vs. 73 ± 5% of the ischemic region, respectively, p = 0.003). Treated animals showed a trend toward less neutrophil infiltration early after reperfusion, but not after 4 h. In isolated hearts, fucoidan improved functional recovery and reduced coronary resistance and lactate dehydrogenase release, lacking any beneficial effects if given in the absence of platelets. Conclusions The results suggest that selectin-dependent adhesion is a prominent mechanism of platelet deposition in reperfused cardiac microvessels and highlight its potential as a therapeutic target in patients with acute myocardial infarction.

Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca(2+) stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mm glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.

Platelets (Plt) accumulate in reperfused myocardium but their effect on myocardial necrosis has not been established. We tested the hypothesis that the effect of Plt depends on their activation status. Pig Plt were obtained before 48 min of coronary occlusion (pre-CO-Plt), 10 min after reperfusion (R-Plt), or after a 60-min sham operation (sham-Plt). Plt were infused into isolated rat hearts ( n = 124) and subsequently submitted to 60 min of ischemia and 60 min of reperfusion. P-selectin expression was higher ( P = 0.02) in R-Plt than in pre-CO-Plt or sham-Plt. Lactate dehydrogenase (LDH) release during reperfusion was similar in hearts receiving pre-CO-Plt, sham-Plt, or no Plt, but R-Plt increased LDH release by 60% ( P = 0.004). Activation of pre-CO-Plt with thrombin increased P-selectin expression and LDH release ( P < 0.001), and these results were unaffected by tirofiban. There was a close correlation between P-selectin expression and LDH release ( r = 0.84; P < 0.001), and myocardial Plt accumulation ( r = 0.85; P < 0.001). We conclude that the deleterious effect of Plt on reperfused myocardium depends on their activation status as represented by P-selectin expression, which is enhanced by ischemia-reperfusion.

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC (S-nitroso-N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid approximately 90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (approximately 50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.

Few human pathologies are as complex as atherosclerosis. Due to its importance it has been and continues to be an object of intense research. It is widely accepted that the initial events in atherogenesis are endothelial dysfunction and subsequent infiltration of the vascular wall by inflammatory cells. However, the problem becomes more complicated the more we try to understand the evolution of the disease. Thus, multiple molecular mediators involved at different levels in the interaction between environmental factors and cell proliferation or cell death within the lesions have been identified, and these determine, in the final analysis, their progression or destabilization. 1 In this regard, medical literature dating back more than a century has already described the presence of small blood vessels in diseased arterial walls. The suggestion that neovascularization could play a role in the progression of atherosclerotic lesions also dates back a long time. 2 However, this hypothesis received little attention until recently, when the identification of some of the principal factors involved in angiogenesis, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor, 3 helped to deepen our understanding of the mechanisms and consequences of neovascularization in arterial lesions. Similarly, there have been rapid advances in research on the application of these findings aimed at stimulating the development of new blood vessels in ischemic tissue through the direct administration of growth factors or gene transfer. 3 The promising results of such

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analog used as selective antagonist of adenosine receptors, caused apoptosis in a human leukemia T cell line. Jurkat cells treated with DPCPX underwent apoptosis as demonstrated by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. Cell cycle and cell sorting analyses indicated an arrest of cells in G(2)/M followed by the appearance of apoptotic cells in G(1) and G(2)/M phases. The mechanism of programmed cell death does not seem to be mediated by signal transduction events at the plasma membrane since it did not involve activation of cell membrane receptors and modification of the intracellular levels of Ca(2+) or cAMP. Apoptosis by incorporation into DNA of a derivative of DPCPX is suggested in basis of the presence of radioactivity label in the DNA obtained from cells preincubated with [(3)H]DPCPX.