A deleterious effect of aspirin on the antithrombotic properties of endothelial cells is not observed with platelets previously exposed to aspirin: studies in a flow system

We have explored both the independent and combined effects of aspirin on cultured endothelial cells and platelets, and its influence on platelet deposition onto an extracellular matrix. Blood was circulated through a flat perfusion chamber with two coverslips placed sequentially with respect to blood flow. The first coverslip (upstream) was covered with a cultured endothelial cell monolayer, and the second (downstream) coated with extracellular matrix obtained after endothelial cell removal. Platelet interaction was measured on the second coverslip. Treatment of endothelial cells on the first coverslip with 100 μM aspirin strongly reduced 6-keto-PGF(1a) levels recovered in the perfusates (118.3 ± 35.8 vs 1038.0 ± 308.5 pg/ml) and significantly increased platelet deposition on the downstream coverslip (% covered surface: 38.6 ± 6.4% vs 14.6 ± 1.8%; P < 0.001). Increased platelet deposition (% covered surface: 24.9 f 3.1%; P < 0.01) was observed in perfusions performed with blood containing aspirinized platelets, in the presence of intact endothelial cells. Treatment with aspirin of both platelets and endothelial cells had no additional effect on platelet adherence. Pretreatment of cultured endothelial cells with aspirin did not influence the adhesive properties of their underlying extracellular matrix. Our results indicate that, although endothelial cell cyclooxygenase is important in regulating platelet adhesion, its blockade seemed to have minimal effect on platelet deposition once platelet-cyclooxygenase was already inhibited.     

Effects of aspirin and indomethacin separately in red blood cells and platelets. Modulation of the adhesive and cohesive functions of platelets under flow conditions

We treated red cells and platelet sepatately in vitro with aspirin or indomethacin to inhibit platelet function. The excess of drug was removed by profuse washing prior to blood reconstitution. We examined the influence of such treatments on platelet interaction with vascular subendothelium employing a perfusion system. Treatment of red cells or platelets with aspirin showed a similar pattern of platelet deposition onto subendothelium. However, platelet adhesion was significantly increased in treated red cells (21.7 ± 2.7% vs 13.7 ± 1.9% in controls; P < 0.05) whereas thrombus was significantly decreased in treated platelets (6.2 ± 1.62% vs 13.8 ± 1.7% in controls; P < 0.05). Treatment of red cells or platelets with indomethacin strongly inhibited platelet interaction. Thrombus and covered surface were decreased in experiments with treated red cells (2.3 ± 0.73% and 14.9 ± 2.3%, respectively; P < 0.05). Adhesion, thrombus and covered surface were decreased in experiments with treated platelets (4.3 ± 0.7%, 2.3 ± 0.9%, 9.2 ± 1.5%, respectively; P < 0.05). Platelet aggregation experiments performed with aspirin-treated red cells showed a progressive inhibition of platelet function. Testing levels of drug in plasma samples from the perfusates showed that levels of drug were very similar to those obtained if treated red blood cells were not washed after treatment. All these results suggest that red cells retained some quantities of drug. Our data highlight the potential effect of red cells interfering with platelet function inhibitors.     

Effects of aspirin and indomethacin separately in red blood cells and platelets. Modulation of the adhesive and cohesive functions of platelets under flow conditions

We treated red cells and platelet sepatately in vitro with aspirin or indomethacin to inhibit platelet function. The excess of drug was removed by profuse washing prior to blood reconstitution. We examined the influence of such treatments on platelet interaction with vascular subendothelium employing a perfusion system. Treatment of red cells or platelets with aspirin showed a similar pattern of platelet deposition onto subendothelium. However, platelet adhesion was significantly increased in treated red cells (21.7 ± 2.7% vs 13.7 ± 1.9% in controls; P < 0.05) whereas thrombus was significantly decreased in treated platelets (6.2 ± 1.62% vs 13.8 ± 1.7% in controls; P < 0.05). Treatment of red cells or platelets with indomethacin strongly inhibited platelet interaction. Thrombus and covered surface were decreased in experiments with treated red cells (2.3 ± 0.73% and 14.9 ± 2.3%, respectively; P < 0.05). Adhesion, thrombus and covered surface were decreased in experiments with treated platelets (4.3 ± 0.7%, 2.3 ± 0.9%, 9.2 ± 1.5%, respectively; P < 0.05). Platelet aggregation experiments performed with aspirin-treated red cells showed a progressive inhibition of platelet function. Testing levels of drug in plasma samples from the perfusates showed that levels of drug were very similar to those obtained if treated red blood cells were not washed after treatment. All these results suggest that red cells retained some quantities of drug. Our data highlight the potential effect of red cells interfering with platelet function inhibitors.     

Low-dose aspirin does not lower in vivo platelet activation in healthy smokers

Smoking causes atherosclerosis, and smokers have increased thromboxane (TXA₂) formation. As aspirin inhibits TXA₂ production we speculated that smokers would preferentially profit from inhibition of the TXA₂ pathway by aspirin. Increased expression of P-selectin, a constituent of the alpha-granules of platelets, and increased levels of circulating (c)P-selectin in plasma are markers for platelet activation. The aim of this study was to compare P-selectin expression on platelets between smokers and nonsmokers, and to compare with placebo the effect of 2 weeks administration of 100 mg/d aspirin on platelet activation in smokers. Smokers exhibited higher P-selectin expression on platelets than non-smokers (2.7 ± 1.8% v 1.6 ± 0.6%, P = 0.018), thus confirming increased platelet activation. Aspirin did not reduce platelet activation as demonstrated by unchanged P-selectin expression on platelets and cP-selectin plasma levels.     

Comparison of aspirin and indobufen in healthy volunteers

The aim of this study is to quantify the extent and recovery of platelet inhibition after administration of indobufen and aspirin in healthy volunteers. Indobufen inhibits platelet aggregation by reversibly inhibiting the platelet cyclooxygenase enzyme, thereby suppressing thromboxane synthesis. Twenty healthy volunteers completed the study and received aspirin (200?mg/day for 2 weeks) followed by a 4-week washout period and then indobufen (200?mg twice a day for 2 weeks). The percent (%) inhibition of platelet aggregation (IPA) was assessed using arachidonic acid (0.5?mg/ml) and adenosine diphosphate (5?µM) at 4, 12, 24 and 48 hours after last dose of each drug. IPA assessed using arachidonic acid as the agonist was similar at 4 hours after the last dose of indobufen (81.07?±?9.36%) and aspirin (96.99?±?0.29%, p?=?0.10), but significantly lower at 12 hours (74.04?±?9.55% vs. 97.94?±?0.28%, p?=?0.02), 24 hours (33.39?±?11.13% vs. 97.48?±?0.32%, p?p?p?=?0.002). Indobufen (200?mg twice a day) caused equivalent initial inhibition of platelet aggregation to aspirin (200?mg daily), and the anti-aggregation effect diminished faster than after aspirin.     

Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro

Abstract  

Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B₂ (TxB₂) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p < 0.001). Larger platelets also synthesized more TxB₂ than small platelets both before (1348 ± 276 pg/mL vs. 1023 ± 214 pg/mL, respectively, p = 0.01) and after aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin.     

Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4?ng/ml DDAVP. Fluorophor-labeled platelets (10⁶/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320?s^?1). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.     

Effects of low‐dose aspirin on endothelial function in hypertensive patients

Background: It has been reported that administration of low‐dose aspirin significantly reduces the frequency of major cardiovascular events in patients with hypertension and coronary artery disease. It is generally considered that the preventative effects of long‐term aspirin administration on major cardiovascular events are due to the inhibition of platelet aggregation. Hypothesis: It is not known whether administration of low‐dose aspirin restores endothelium‐dependent vasodilatation, and this study was undertaken to prove or disprove this question in patients with hypertension. Methods: Flow‐mediated endothelium‐dependent dilatation and glyceryl trinitrate‐induced endothelium‐independent dilatation were investigated in 18 hypertensive patients and 10 normotensive control subjects. In the hypertensive patients, flow‐mediated dilatation was investigated and cyclic guano‐sine monophosphate plasma (cGMP) was measured before and at 8 weeks after the administration of 162 mg of aspirin. Results: Flow‐mediated dilatation before aspirin administration was more reduced in the hypertensive patients than in the control subjects (6.4 ± 2.0% vs. 11.3 ± 2.3%, p< 0.0001). Glyceryl trinitrate‐induced dilatation before aspirin administration was similar in hypertensive patients and control subjects. Flow‐mediated dilatation after aspirin administration was improved compared with that before aspirin administration (10.4 ± 3.5% vs. 6.4 ± 2.0%, p<0.0004). The cGMP product after aspirin administration was significantly higher than that before aspirin administration. Conclusions: Administration of low‐dose aspirin may restore the endothelium‐dependent vasodilatation in hypertensive patients. Furthermore, increased nitric oxide production may play a partial role in the improvement in endothelial function induced by administration of low‐dose aspirin.     

Platelet-derived microparticle levels are significantly elevated in patients treated by elective stenting compared to subjects with diagnostic catheterization alone

Significant platelet reactivity with endothelial damage may occur during percutaneous coronary intervention such as balloon angioplasty and elective stenting in patients with stenotic or occluded arteries in coronary artery disease. Activated platelets express several surface epitopes to aggregate and form heterotypic interactions with leukocytes and endothelial cells, secrete biomarkers to enhance their prothrombotic properties and also generate increased level of microparticles (PMPs). These events may contribute to subacute stent thrombosis induced by the procedure-mediated trauma. Our aim was to study the level of PMPs and other platelet activation markers at an early time point after stenting with bare metal stent in patients on aspirin antiplatelet pre-treatment, and these results were compared to data obtained from subjects with diagnostic catheterization alone. We found that at 15 minutes after the completion of stenting, the levels of PMPs (557 ± 83/µl versus 325 ± 42/µl), the platelet P-selectin expression (2.7 ± 0.3% versus 1.99 ± 0.1%) and the ratio of platelet-monocyte heterotypic aggregates (48 ± 4% versus 38 ± 3%) were significantly (p < 0.05) elevated in patients compared to unstented subjects, but no difference was found in soluble P-selectin values. We suggest that the observed cellular changes are early and sensitive markers to detect the platelet-activating effect of stent implantation.     

In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets

Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.     

Response to aspirin and clopidogrel in patients scheduled to undergo cardiovascular surgery

Background Recent data indicate that among patients undergoing percutaneous coronary intervention low platelet response to aspirin is associated with clopidogrel low response. It is unclear whether these findings extend to other patient populations. We, therefore, aimed to evaluate the relation between response to aspirin and clopidogrel among patients scheduled to undergo cardiac or vascular surgery. Methods Patients who were scheduled for cardiac or vascular surgery and had taken aspirin 81–325 mg daily for at least a week and clopidogrel 75 mg daily for at least 3 days underwent blood testing for platelet function. One hundred patients were included in the current analysis. Platelet function was evaluated by the modified TEG platelet mapping assay with addition of ADP or arachidonic acid (AA), and by the PFA-100 assay with collagen-epinephrine (CEPI) or collagen-ADP (CADP) cartridges. Low response to aspirin or clopidogrel was defined as inhibition ≤20% for TEG-AA or TEG-ADP, respectively. Results Thirteen patients (13%) were low responders to aspirin and 34 (34%) were low responders to clopidogrel. Eight patients were low responders to both drugs. There were no differences in clinical characteristics between drug low responders versus sensitive patients. Aspirin low responders had lower TEG-ADP inhibition (19.5 ± 6 vs. 35.8 ± 3%, P = 0.03) and tended to have lower PFA-CADP time (84.7 ± 7 vs. 105.6 ± 5 s, P = 0.1) than aspirin sensitive patients. Clopidogrel low responders had lower TEG-AA inhibition (58 ± 6 vs. 75.1 ± 4%, P = 0.01) and PFA-CEPI time (168 ± 13 vs. 200.4 ± 10 s, P = 0.07) than clopidogrel sensitive patients. Conclusions In patients scheduled to undergo cardiovascular surgery low response to aspirin is associated with low response to clopidogrel.     

Platelets decrease albumin permeability of pulmonary artery endothelial cell monolayers

Since platelets may modulate endothelial cell permeability, we examined the effects of platelets on 125I-albumin permeability of cultured bovine pulmonary artery endothelial cell monolayers. The experimental system consisted of endothelial cells grown to confluence on a gelatinized polycarbonate filter. We quantified the diffusive flux of 125I-albumin from luminal chamber to the abluminal chamber. Washed human platelets added to the monolayers decreased the albumin flux in a concentration-dependent manner, with a 65% decrease occurring at the highest concentration of platelets (5 x 10(7) platelets) added to the 700-microliters luminal chamber. In contrast, neither paraformaldehyde-fixed platelets nor fresh red blood cells changed 125I-albumin permeability. Platelets had no effect on 125I-albumin permeability across the gelatinized filters without endothelial cells present. Supernatants of platelet lysates also reduced albumin flux. The effect produced by intact platelets or platelet lysate was not influenced by the presence of ketanserin (a serotonin receptor antagonist), propranolol (a beta-adrenergic receptor antagonist), or aspirin (an inhibitor of cyclooxygenase). Platelets activated by thrombin did not produce an effect that was different from the effect produced by intact platelets. The activity of the supernatant of platelet lysate remained in the aqueous phase after ether extraction. The results indicate that the platelet-mediated decrease in endothelial cell permeability to 125I-albumin is the result of a hydrophilic platelet-derived factor(s) and not secondary to mechanical obstruction of endothelial "leaks" by the platelets.     

The effect of preoperative aspirin administration on postoperative level of von Willebrand factor in off-pump coronary artery bypass surgery

The effect of preoperative administration of aspirin on endothelial function in the patients undergoing off-pump coronary artery bypass (OPCAB) surgery is still unclear. Fifty consecutive patients undergoing OPCAB between May 2006 and May 2007 were equally divided into two groups — one without preoperative aspirin (group A; the first 25 patients) and the other with preoperative aspirin (group B; the next 25 patients). We investigated the degree of postoperative endothelial dysfunction by measuring the von Willebrand factor activity, which is a possible indicator of endothelial damage. The level of von Willebrand factor was not different between groups before surgery (group A 166% ± 53% vs group B 181% ± 62%; P = 0.39). Immediately after surgery it was significantly higher than before surgery in group A (231% ± 79%; rate of increase 1.24 ± 0.58), but not in group B (183% ± 77%; rate of increase 1.03 ± 0.55) (P < 0.02). The level was still significantly higher in group A than in group B on postoperative day 1 (group A 294 ± 66 vs 254 ± 51; P = 0.03), but there was no difference between groups on postoperative day 6. Although the frequency of blood transfusion was higher in group B, there was no difference in the amount of intraoperative bleeding between the groups. Preoperative use of aspirin before OPCAB could suppress the postoperative increase in von Willebrand factor, a possible indicator of endothelial damage, only in the early postoperative phase.     

Prevention of arterial thrombosis by a monoclonal antibody against the 100 to 109 amino acid sequence stretch of the beta-subunit of the human platelet fibrinogen receptor: A comparative study with low dose aspirin

Objectives. The aim of this study was to compare, in dogs, the antithrombotic activity of aspirin and the murine monoclonal antibody P37, which inhibits platelet aggregation and fibrinogen binding to activated platelets.Background. The antithrombotic activity of P37 has been somewhat predictable, given its in vitro platelet antiaggregating activity and localization at or very near the fibrinogen binding site in the platelet fibrinogen receptor, the glycoprotein IIb/IIIa or integrin alphaIIb-beta₃.Methods. The monoclonal antibody P37 of the immunogammaglobulin-1 isotype was prepared according to previously described immunization and fusion protocols and screening assays. To compare its antiaggregating capacity with that of aspirin, experimental thrombosis was induced in all dogs by means of direct current applied to the carotid artery. Autologous platelets had previously been labeled with indium-111 oxine. The dogs were assigned to three groups: group I (n = 18) was the control group; group II (n = 12) was treated orally with 5 mg of aspirin/kg body weight per day for 7 days before induction of thrombosis, and group III (n = 10) was treated intravenously with a single dose of P37 (0.8 mg/kg).Results. The indium-111 oxine activity deposited in the thrombi was 12.94 ± 12.83% (mean ± SD) in group I, 3.55 ± 2.99% in group II and 0.03 ± 0.03% in group III. The differences between groups were always statistically significant (p < 0.05).Conclusions. We conclude that a single dose (0.8 mg/kg) of P37 in a canine model of arterial thrombosis is ~100 times more efficient than the administration of aspirin (5 mg/kg per day) in preventing platelet deposition during thrombus formation.     

Antiplatelet Versus Warfarin Therapy: Platelet, Neutrophil, and Thrombus Deposition for Intracoronary Stents in a Porcine Model

Antiplatelet therapy may offer an advantage over warfarin in reducing adverse clinical events after intracoronary stem implantation. The mechanism of this effect has not been elucidated but platelet adhesion may play a predominant role in the process of subacute stent thrombosis. This study compared the effect on platelet, neutrophil, and thrombus deposition of three different anticoagulation regimens (aspirin/warfarin vs aspirin/ticlopidine vs aspirin alone) after intracoronary stenting in juvenile swine. Thirty stents were deployed in 15 juvenile farm swine randomized to one of 3 anticoagulation protocols: aspirin 325 mg/day; aspirin 325 mg/day and ticlopidine 500 mg/day; aspirin 325 mg/day and warfarin 0.1 mg/kg/day. Autologous platelets were labeled using ¹¹¹Indium-oxime and a slotted tube metal stent was deployed using a high pressure balloon. Platelet deposition in the stented segment was determined at 24 hours. Each segment was analyzed by light microscopy to document an injury score; mean and maximum thrombus deposition per strut were measured; and neutrophils were counted on each metal strut and in the vessel wall. Platelet deposition at 24 hours was significantly higher in the aspirin/warfarin group (3.69 ± 1.16 ± 10⁸ plts/cm²) than in the aspirin/ticlopidine (1.74 ± 0.45 ± 10⁸ plts/cm², P = 0.0009) or aspirin (2.42 ± 2.13 ± 10⁸ plts/cm², P = 0.03) groups. There was no significant difference between the aspirin and aspirin/ticlopidine groups (P = 0.9). Mean thrombus area per strut was significantly greater in the aspirin/warfarin group (0.027 ± 0.006 mm²) compared to the aspirin/ticlopidine group (0.017 ± 0.002 mm², P = 0.002). The majority of the thrombus (79%) but minority of neutrophils (24%) were found on the stent struts. In this coronary stent model, warfarin given with aspirin was associated with greater thrombogenicity and more platelet deposition than either aspirin/ticlopidine or aspirin alone.     

The Neonatal Platelet: Evaluation of Platelet Malonyl Dialdehyde Formation as an Indicator of Prostaglandin Synthesis

Summary . It is recognized that neonates exhibit a transient thrombocytopathy. Whether the cause of the platelet abnormality is due to a storage pool defect, or an‘aspirin-like’abnormality with inhibition of platelet prostaglandin synthesis is unclear. In an attempt to assess neonatal platelet prostaglandin synthesis, platelet rich plasma (PRP) from 10 normal control maternal–neonatal pairs was obtained contemporaneously with PRP from donors who had ingested 600 mg aspirin within 12-18 h prior to study. When platelet malonyl dialdehyde production in the presence of the sulphydryl inhibitor N-ethyl maleimide (NEM), or thrombin, was measured as an indicator of platelet prostaglandin synthesis, a significant decrease was observed in neonatal platelets (2.46 ± 0.61 and 0.90 ± 0.19 nmol/10⁹ platelets respectively) when compared to those of the paired maternal or adult controls (3. 23 ±0.31 and 1.30 ± 0.17 nmol/10⁹ platelets). As expected, a marked decrease in platelet malonyl dialdehyde production in response to NEM or thrombin was also seen in the platelets from the donors who had ingested aspirin (0.25±0.14 and 0.10 ± 0.09 nmol/10⁹ platelets). When compared to the normal maternal control PRP both the neonatal and aspirin donor PRPs demonstrated abnormalities in second phase platelet aggregation in response to ADP and epinephrine, and an abnormal response to collagen. However, mutual correction and secondary irreversible aggregation was obtained when both these abnormal PRPs were mixed in varying proportions. No mutually corrective effect was seen using neonatal PRP obtained from infants where maternal documentation of aspirin ingestion was present within 48 h prior to delivery, or when normal neonatal PRP was preincu-bated with aspirin in vitro prior to mixing with PRP from donors who had recently ingested aspirin. These findings provide evidence that the neonatal platelet dysfunction cannot be attributed to an abnormality in prostaglandin synthesis or an‘aspirin-like’defect. Although platelet malonyl dialdehyde formation in response to NEM and thrombin was decreased when compared to the adult the amount synthesized by the neonatal platelet in response to external stimuli appears to be     

The thrombolytic effect of aspirin in animal model

Background The aspirin induced platelet aggregation has been reported to be mediated through the inhibition of platelet prostaglandin synthesis. This compound has also been recently reported to stimulate nitric oxide synthesis in platelets. Since nitric oxide has been reported to produce fibrinogen/fibrinolytic effect, investigation was carried out to determine fibrinolytic effect of in vivo exposure of platelets to aspirin in normal volunteers on the fibrinolysis of the clotted platelet-rich plasma in vitro. The thrombolytic effect of aspirin in situ was also carried out by injecting aspirin solution in the mice with ADP induced formed thrombi in the coronary artery. Methods and Results It was found that the clotted platelet-rich plasma prepared from the volunteers (n = 10, F = 5, M = 5) who ingested 150 mg aspirin, began to undergo spontaneous and progressive fibrinolysis for 200 min at 37°C with the generation of fibrin degradation products in the lysate. No such fibrinolysis could be seen in control experiments. When platelet thrombi were produced in the coronary artery of mice by injecting ADP, and these animals subsequently received intravenous injection of aspirin (4 μM final), they not only survived (P < 0.0001, n = 10) the thrombogenic assault but the lysis of the platelet thrombi was also noted in the post mortem examination. The thrombolytic effect of aspirin was found to be comparable to that of streptokinase in these animals. Conclusions Aspirin, through the stimulation of NO synthesis, may produce thrombolysis in vivo.     

血小板二磷酸腺苷受体H2单体型与阿司匹林抗血小板功能学监测关联的研究

目的探讨血小板二磷酸腺苷受体(P2Y12)H2单体型对中国老年汉族患者阿司匹林抑制血小板聚集功能及血小板计数的影响。方法入选北京万寿路地区服用阿司匹林老年汉族患者431例,使用美国Sequenom系统单核苷酸多态性分型技术对P2Y12H1/H2单体型进行鉴定。采用花生四烯酸诱导的光比浊和血栓弹力图法、二磷酸腺苷诱导的光比浊和血栓弹力图法、血小板活化标记物血小板激活复合物1、CD62P等血小板功能检测,对P2Y12H2单体型与血小板计数及阿司匹林抗血小板聚集功能进一步行相关分析。结果 431例患者中,P2Y12H1/H1基因型285例,H1/H2基因型136例,H2/H2基因型10例。H1/H1、H1/H2、H2/H2基因型血小板计数分别为(216.09±58.76)×109/L,(195.06±55.16)×109/L,(164.90±46.12)×109/L,差异有统计学意义(P<0.01)。H1/H2+H2/H2与H1/H1比较,经年龄、性别校正后,多因素回归分析仍提示血小板计数与H2相关联(P=0.005)。结论 P2Y12H2单体型对阿司匹林的抗血小板作用无明显影响。携带P2Y12H2单体型患者的血小板计数明显低于H1型患者,提示P2Y12H2单体型可能是正常人群血小板计数低下的一个基因学标志。     

Exposure to platelets promotes functional properties of endothelial progenitor cells

Recent evidence indicates that endothelial progenitor cells (EPCs) have an important role in the process of repair following vascular injury, and that platelets mediate their recruitment to sites of injury. Platelets and EPCs can interact and bind directly. However, there is limited information on the effect of platelets on EPC function following this interaction. We, therefore, aimed to assess the in vitro effect of platelets on functional properties of EPCs. Human EPCs were isolated from donated Buffy coats and purified on a magnetic separation column specific for CD133. They were incubated either on fibronectin matrix, or co-incubated with washed platelets (isolated from healthy volunteers), for 7 days. Number of EPC colony forming units (CFU) was quantified, and endothelial cell lineage confirmed by immunostaining. Functional properties of the cultured cells were evaluated by MTT—proliferation assay and migration assay using the Boyden chamber. Co-incubation of EPCs with platelets compared to incubation of EPCs alone (on fibronectin matrix) resulted in higher number of CFUs after 7 days (6.5 ± 1.3 vs. 3.5 ± 0.5 CFUs/well, respectively, P = 0.005). In addition, co-incubation of EPCs with platelets versus EPCs alone was associated with higher proportion of living cells, by the MTT assay (0.2 ± 0.01 vs. 0.12 ± 0.04 MTT 570 nm respectively, P = 0.003), and higher number of migrated EPCs, assessed by the migration assay (1400 ± 212 vs. 580 ± 180 migrated cells/2000 cells, respectively, P < 0.0001). In vitro exposure to platelets promotes the capacity of EPCs to form colonies, proliferate and migrate. Therefore, the interaction with platelets appears to augment EPC functional properties.     

Procoagulant activity of endotoxin-treated human endothelial cells exposed to native human flowing blood

It has been reported that cultured endothelial cells become procoagulant when exposed to endotoxin. This prompted us to investigate whether human endothelial cells treated with endotoxin could promote the generation of fibrin when exposed to human flowing blood. For this purpose we used a parallel-plate perfusion chamber in which confluent cultured endothelial cells from human umbilical veins were exposed for five minutes to directly drawn human nonanticoagulated blood, at wall shear rates of 100, 650, and 2600 sec-1. Fibrin deposition was assessed by morphometry. No fibrin deposition occurred on normal endothelial cells. In contrast, cells incubated with endotoxin for 4 or 18 hours induced fibrin deposition, but only at a shear rate of 100 sec-1. Since some extracellular matrix was exposed between the cells, we investigated whether extracellular matrix played a role in fibrin formation. When the endothelial cells incubated or not with endotoxin were removed by EDTA, the exposed extracellular matrix perfused with blood at 100 sec-1 supported platelet and fibrin deposition in both cases. This suggests that the effect of endotoxin on endothelial cells was not due to extracellular matrix alteration but only to cellular activation or secretion of procoagulant substances. We conclude that human endothelial cells treated with endotoxin can trigger fibrin formation and deposition at their surface when exposed to flowing blood at low shear rate.