Abnormal pH-sensing of platelet Na+/H+ exchanger in patients with cardiac syndrome X

An enhanced activity of Na⁺/Li⁺ countertransport, studied as a surrogate of Na⁺/H⁺ exchanger, has been described in red blood cells of patients with cardiac syndrome X. In this study we investigated whether abnormalities in the activity of platelet Na⁺/H⁺ exchanger (NHE) also existed in syndrome X patients and whether such abnormality was associated with platelet activation. Platelet NHE activity was evaluated in 21 syndrome X patients and 18 controls by measuring the pH recovery in platelets after acid loading and/or thrombin stimulation. The linear correlation existing between the initial intracytoplasmic pH (pH_i) values and the maximal velocity of pH recovery allowed to calculate the values of slope and intercept at pH_i=6.6 (I_pH6.6) for each individual. Urinary excretion of the major TXB₂ metabolite, 11-dehydro-TXB₂ was measured in 15 syndrome X patients and 15 controls. The acidification-induced NHE activity resulted significantly higher in syndrome X patients compared to controls. Indeed, slope values were 0.75±0.29 and 0.5±0.23 min⁻¹ in patients and controls, respectively (P=0.01), while I_pH6.6 values were 0.24±0.1 and 0.17±0.1 ΔpH/min (P=0.04). The thrombin-stimulated NHE activity, however, was not different in the two groups and no significant difference in the urinary excretion of 11-dehydro-TXB₂ between patients and controls (median 920 vs. 765 pg/mg creatinine, respectively) (P=0.32) was also found. Thus our data demonstrate an alkaline shift in pH-dependence of platelet NHE of syndrome X patients. This abnormality does not seem to be associated with increased platelet activation.     

Spinal fusion using an autologous growth factor gel and a porous resorbable ceramic

Augmenting healing through a single application of an exogenous growth factor or bone morphogenetic protein is not a new concept. The use of autologous growth factors through platelet isolation and concentration provides multiple endogenous growth factors to the healing site. A posterolateral fusion model in aged sheep (5- to 6-year-old ewes) was used to examine the effects of the addition of growth factors through autologous platelet isolation on the biomechanic and histologic properties of the fusion using a resorbable coral bone graft substitute. At 6 months the combination of autologous growth factors to the Pro Osteon 500R plus aspirated bone marrow resulted in the greatest bending stiffness but not ultimate load. Autologous growth factors can be isolated from platelets and concentrated to provide multiple growth factors to the fusion site to aid in spinal fusion.     

Reduced basal nitric oxide bioavailability and platelet activation in young spontaneously hypertensive rats

OBJECTIVE: To investigate the role of basal nitric oxide (NO) bioavailability for platelet activation in young spontaneously hypertensive rats before onset of hypertension. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) in platelets was used as a sensitive monitor of in vivo NO bioavailability. METHODS AND RESULTS: Whole blood samples were taken from 10-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In vivo surface-expression of P-selectin and platelet-binding of fibrinogen were assessed by flow cytometry. Platelet VASP-phosphorylation at its serine 239 (Ser239) and serine 157 (Ser157) residues was assessed using specific antibodies to determine NO bioavailability in vivo, and compared with endothelial vasomotor function. The increment in vascular tone following inhibition of NO-synthase in slightly preconstricted aortic rings was reduced indicating less NO formation under physiological stimulation (WKY 71.1+/-4.1%; SHR 57.8+/-2.4%, P<0.05). In vivo platelet VASP-phosphorylation was significantly reduced at both phosphorylation sites in SHR (mean fluorescence for Ser239: WKY: 15.2+/-0.6; SHR: 11.7+/-0.5, P<0.01; Ser157: WKY: 53.0+/-3.0; SHR: 35.0+/-3.5, P<0.05). Surface-expression of P-selectin and membrane-bound fibrinogen were significantly enhanced in SHR compared with WKY (P-selectin: WKY: 23.2+/-3.4; SHR 58.3+/-7.9, P<0.001; platelet-bound fibrinogen: WKY: 8.6+/-0.5; SHR: 13.5+/-1.1, P<0.001). In vitro preincubation of platelets with the NO donor sodium nitroprusside normalized platelet surface-expression of P-selectin in SHR. CONCLUSION: Using VASP-phosphorylation as a sensitive monitor of in vivo NO bioavailability, these data provide evidence that reduced vascular NO formation in vivo contributes to increased platelet activation in young SHR.     

In vitro and in vivo effects of acetyldinaline on murine megakaryocytopoiesis

Purpose Acetyldinaline (CI-994) has shown preclinical efficacy in vitro and in vivo against solid tumor and leukemia cell lines. Since myelosuppression was the dose-limiting toxicity for acetyldinaline in preclinical and clinical studies, experiments were conducted to examine the in vitro and in vivo effects of acetyldinaline on murine megakaryocytic (CFU-meg) progenitor cells.Methods Bone marrow and spleen cells from untreated mice were continuously exposed in vitro to acetyldinaline or dinaline in clonal assays. For the in vivo study, BDF₁ mice were dosed orally with 50 mg/kg acetyldinaline every day for 14 days.Results Both acetyldinaline and dinaline induced an in vitro dose-dependent decrease in CFU-meg colonies derived from either the spleen or bone marrow. Splenic CFU-meg were more sensitive in vitro to acetyldinaline and dinaline than their marrow counterparts. In the in vivo experiments, platelet counts decreased throughout the 14-day dosing period and had returned to normal by day 18. Marrow and spleen CFU-meg declined after the first dose but had recovered by days 4 and 7, respectively. Elevated splenic CFU-meg counts were observed through day 20, 6 days after dosing ended. Recovery of platelet counts in treated mice was associated with increases in both marrow and splenic CFU-meg.Conclusions There was differential in vitro toxicity of acetyldinaline to murine CFU-meg derived from the bone marrow versus spleen. The in vitro assay predicted the more severe effect of acetyldinaline on splenic progenitors than on their marrow counterparts that was observed in the in vivo phase. In addition, megakaryocytopoiesis in the marrow showed evidence of recovery from drug toxicity in the face of continuing daily acetyldinaline treatments.     

The impact of peripheral arterial disease on circulating platelets

INTRODUCTION: To test the hypothesis that circulating platelets display evidence of reversible interactions with atherosclerotic lesions, platelet alpha-granule content and propensity for microaggregate formation were measured in samples from normal donors (n=65) and from patients with either peripheral arterial disease (n=47) or renovascular hypertension (n=22). To measure the effect of a defined arterial injury on platelet function, platelet samples were compared before and 30 min after elective angioplasty. MATERIALS AND METHODS: P-selectin was measured after strong stimulation of ultra-dilute platelets with thrombin (10 nM). Microaggregation was measured as a platelet count deficit in citrate-anticoagulated platelet-rich plasma (PRP) relative to that predicted from the count in EDTA-anticoagulated blood. RESULTS: Platelet alpha-granule P-selectin was significantly lower from platelets of patients compared to normal donors. In addition, platelets from patients have a significantly greater propensity to form microaggregates in citrate anticoagulant. In contrast to atherosclerotic renovascular hypertension, platelets from patients with fibromuscular dysplasia, a distinct non-inflammatory cause of arterial stenosis, do not differ significantly from normal donors. Other than the PRP platelet count, which rose transiently following angioplasty, other platelet measures were unchanged by the injury. CONCLUSIONS: Atherosclerotic arterial disease is associated with an increased share of platelets unable to express P-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. These platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process.     

Prothrombotic responses to exercise are little influenced by clopidogrel treatment

AIMS: Adenosine diphosphate (ADP) is involved in shear-induced platelet activation, which may be important for platelet responses to stress. We therefore tested the hypothesis that ADP receptor antagonism by clopidogrel treatment would attenuate exercise-induced platelet activation. METHODS AND RESULTS: Fifteen healthy volunteers performed exhaustive exercise without and with clopidogrel pretreatment (75 mg/day; 7 days) in a randomised crossover study. Filtragometry readings (reflecting platelet aggregability in vivo) and 11-dehydro-thromboxane B(2) (TxM) in plasma were determined before and after exercise. Platelet and leukocyte activity, platelet-platelet (PPA), and platelet-leukocyte aggregates (PLAs) in vivo and their responsiveness to agonist stimulation in vitro were assessed by flow cytometry. Clopidogrel treatment inhibited ADP-induced platelet P-selectin expression by 72% (54-85%). Exercise increased platelet aggregation (filtragometry and PPAs), elevated plasma TxM, increased single platelet P-selectin expression, elevated circulating PLAs, and enhanced ADP and thrombin-stimulated P-selectin expression. Clopidogrel prolonged filtragometry readings and attenuated agonist stimulated P-selectin expression at rest, but did not influence TxM in plasma or urine or attenuate platelet or leukocyte responses to exercise. Clopidogrel treatment did not influence plasma CD40L (ligand) at rest or after exercise. CONCLUSION: Clopidogrel treatment attenuates platelet activity in vivo at rest, but exercise counteracts the platelet stabilizing effects of clopidogrel. The hypothesis that ADP is involved in stress-induced platelet activation was not supported.     

Enhanced platelet serotonin 5-HT2A receptor binding in anorexia nervosa and bulimia nervosa.

Some evidence exists to suggest that serotonin 5-HT_2A receptor function is altered in anorexia nervosa and bulimia nervosa. In order to further investigate the 5-HT_2A receptor in eating disorders, platelet [³H]lysergic acid diethylamide ([³H]LSD) binding was studied in ten patients with anorexia nervosa, 23 patients with bulimia nervosa and 33 healthy controls. At admission, B_max for platelet [³H]LSD binding was significantly higher both in the anorexia nervosa group (30.6±4.2 fmol/mg protein; mean±S.D.) and in the bulimia nervosa group (30.8±7.6 fmol/mg protein) than in the control group (23.5 ±6.3 fmol/mg protein; p=0.01 and p=0.003, respectively). K_d was borderline significantly higher among anorexics (median 1.45 nM) and significantly higher among bulimics (median 1.66 nM) than among controls (median 0.95 nM; p=0.05 and 0.003, respectively). The Global Assessment of Functioning score and the body mass index were both significantly negatively correlated to K_d (r=−0.40; p=0.03 and r=−0.41 p=0.03, respectively), but not to B_max. The present study indicates that patients with anorexia nervosa as well as patients with bulimia nervosa have an enhanced 5-HT_2A receptor binding and provides further evidence for a serotonergic dysfunction in eating disorders.     

Lipopolysaccharide treatment reduces rat platelet aggregation independent of intracellular reactive-oxygen species generation

High production of reactive-oxygen species (ROS) by blood cells is involved in damage of the vascular endothelium and multiple organ dysfunction in sepsis. However, little is known about the intraplatelet ROS production in sepsis and its consequences on platelet reactivity. In this study, we evaluated whether the treatment of rats with lipopolysaccharide (LPS) affects platelet aggregation through intraplatelet ROS generation. Rats were injected with LPS (1?mg/kg, i.p.), and at 2 to 72?h thereafter, adenosine diphosphate (ADP) (3–10?µM) induced platelet aggregation was evaluated. Production of ROS in platelets was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Treatment of rats with LPS time-dependently inhibited ADP-induced platelet aggregation within 72?h. The inhibitory effect of LPS on platelet aggregation was further increased when the platelets were incubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 30?U/mL), polyethylene glycol-catalase (PEG-CAT; 1000?U/mL) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10?µM). The ROS production in non-stimulated platelets did not differ between control and LPS-treated rats. However, in ADP-activated platelets, generation of ROS was increased by 3.0- and 7.0-fold, as evaluated at 8 and 48?h after LPS injection, respectively. This increased ROS production was significantly reduced when platelets were incubated in vitro with DPI, PEG-SOD or PEG-CAT. In contrast, treatment of rats with N-acetylcysteine (150?mg/kg, i.p.) significantly reduced the inhibitory effect of LPS on platelet aggregation, and prevented the increased ROS production by in vivo LPS. Our results indicate that the increased intraplatelet ROS production does not contribute to the inhibitory effect of LPS on platelet aggregation; however, the maintenance of redox balance in LPS-treated rats is fundamental to restore the normal platelet response in these animals.     

Shorter PFA-100® closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA-100^® measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults, which may interfere with the evaluation of platelet and vWF function by means of the PFA-100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti-vWF antibody, and mixing experiments with neonatal platelet-rich and platelet-poor plasma were performed. The results showed that short cord blood PFA-100 CTs were caused by a constituent of neonatal platelet-poor plasma, probably the neonatal high multimeric vWF. Conclusion: This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA-100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.     

Computational Simulation of Platelet Deposition and Activation: I. Model Development and Properties

To better understand the mechanisms leading to the formation and growth of mural thrombi on biomaterials, we have developed a two-dimensional computational model of platelet deposition and activation in flowing blood. The basic formulation is derived from prior work by others, with additional levels of complexity added where appropriate. It is comprised of a series of convection-diffusion-reaction equations which simulate platelet-surface and platelet-platelet adhesion, platelet activation by a weighted linear combination of agonist concentrations, agonist release and synthesis by activated platelets, platelet-phospholipid-dependent thrombin generation, and thrombin inhibition by heparin. The model requires estimation of four parameters to fit it to experimental data: shear-dependent platelet diffusivity and resting and activated platelet-surface and platelet-platelet reaction rate constants. The model is formulated to simulate a wide range of biomaterials and complex flows. In this article we present the basic model and its properties; in Part II (Sorensen et al., Ann. Biomed. Eng. 27:449–458, 1999) we apply the model to experimental results for platelet deposition onto collagen. © 1999 Biomedical Engineering Society. PAC99: 8719Uv, 8380Lz, 8717Aa, 8710+e, 8768+z