犬心肌缺血早期血小板聚集功能和冠脉侧支循环的改变

本实验在18只麻醉开胸犬观察了急性心肌缺血早期血小板聚集功能和冠脉侧支循环功能的变化。实验结果如下:阻断冠脉后心肌缺血区血液中血小板聚集率(PAgR)增大,血小板计数(PC)减少。缺血50min时,PAgR增大58.7±5.6％,PC减少39.5±23.6％,与对照值有明显差异(均为P<0.01)。与此同时,在控制血压条件下,心肌缺血早期单位压力差下冠脉侧支血流量的变化与对照值无明显差异,而根据Wyatt等公式计算的流经缺血区末梢血管的有效侧支血流量明显降低,缺血50min时较对照值降低23.5±9.7％(P<0.05)。PAgR变化与有效侧支血流量改变呈明显负相关(r=-0.887,P<0.01);冠脉侧支指数与梗塞范围呈明显负相关(r=-0.847,P<0.01)。阻断冠脉前静脉注射血小板聚集功能抑制剂阿斯匹林,可明显减轻上述各项参数的异常变化。这些结果提示,心肌缺血早期血小板聚集功能的异常变化虽然对冠脉侧支血管的血流阻力影响较小,但却使流经缺血区末梢血管的有效侧支血流量明显减小,进而扩大梗塞范围。     

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats.

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.     

Influence of vitamin E on platelet aggregation and thrombocythemia in the rat.

Collagen-induced platelet aggregation was increased in 9-10 wk old vitamin E deficient rats although there was no difference in platelet count between deficient and control animals. With a more prolonged deficiency (at 15 wk) both platelet aggregation and platelet counts were elevated in the vitamin E deficient animals.     

Enhanced platelet aggregability in dogs following myocardial infarction

Coronary artery ligation in beagle dogs led to an abnormal platelet response that may be similar to that observed in patients with ischemic heart disease. Platelet rich plasma (PRP) obtained from these animals was markedly more responsive to collagen induced platelet aggregation than was PRP obtained from control animals which had undergone similar surgery but without having had a coronary artery ligated. Platelet sensitivity was followed after ligation for periods up to four months. The peak response to collagen occurred three weeks following surgery, remained high for three additional weeks and then gradually returned toward normal over the ensuing ten weeks. Crossover experiments employing platelets and platelet poor plasma (PPP) from ligated and control animals demonstrated that this observation was due to a plasma factor abnormality. Further studies established that BL-3459 and certain other inhibitors of platelet aggregation were capable of normalizing this exaggerated response to collagen.     

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3^?/?) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3^?/? mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.     

Studies on the biological effects of ozone: 10. Release of factors from ozonated human platelets

In a previous work we have shown that heparin, in the presence of ozone (O3), promotes a dose-dependent platelet aggregation, while after Ca2+ chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF), transforming growth factor beta1 (TGF-beta1) and interleukin-8 (IL-8) are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limb ischemia treated with O3 autohaemoteraphy (O3-AHT).     

Antioxidants alleviate nicotine-induced platelet aggregation in cerebral arterioles of mice in vivo

Experimental data on the effect of nicotine on cerebral microvessel thrombosis is lacking. Therefore, this study was carried out to elucidate the effects of nicotine on platelet aggregation in cerebral (pial) microcirculation of the mouse, and the possible protective effect of vitamins C and E. Male TO mice were divided into six groups, and injected i.p. with saline as a control, nicotine (1 mg/kg), vitamin C alone (100 mg/kg), vitamin E alone (100 mg/kg), nicotine plus vitamin C or nicotine plus vitamin E, all for one week before the experiment. After one week, platelet aggregation in cerebral microvessels of these groups of mice were studied in vivo. The appearance of the first platelet aggregation and total blood flow stop in arterioles and venules were timed in seconds. In the animals treated with nicotine, venules did not show any alteration in the platelet aggregation time in comparison to the control animals. However, in arterioles platelet aggregation time was significantly accelerated (p<0.001) in nicotine-treated animals as compared to controls. Both vitamins C and E prevented the shortening of arteriolar platelet aggregation time significantly (p<0.001) when applied with nicotine but not alone. It can be concluded that nicotine enhances the susceptibility to thrombosis in the cerebral arterioles in vivo and that vitamins C and E have alleviating effect on nicotine-induced thrombotic events in mice pial microvessels.     

Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

We treated four anesthetized dogs (Canis familiaris) with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous 111In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone (3 mg/kg) administered 5 min prior to ischemia and continuously (1 mg/kg/hr) throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude (% of baseline) when compared to controls (27-36% vs 9-14%, p less than 0.05). We estimated platelet accumulation as 111In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered 111In-labeled platelet accumulation when compared to controls (6158 +/- 2386 vs 9979 +/- 3852, mean +/- SEM). Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved.     

Effects of ovariectomy on aggregation,secretion, and metalloproteinases in porcine platelets

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.     

Increased plasma level of asymmetric dimethylarginine in hypertensive rats facilitates platelet aggregation: role of plasma tissue factor

This study was designed to explore the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in platelet aggregation in hypertension and its possible mechanisms. Spontaneously hypertensive rats (SHR) and L-NAME-induced hypertensive rats were orally administered with L-arginine (1 g/(kg·day) for 14 days. Systolic blood pressure, platelet aggregation, and plasma tissue factor (TF) level and activity were measured. The plasma concentration of ADMA in SHR was determined. In vitro, platelet-rich plasma isolated from Wistar rats was prepared in order to observe the effect of exogenous ADMA on platelet aggregation and TF level and (or) activity in platelet-rich plasma. In both types of hypertensive rats, systolic blood pressure, platelet aggregation, and the level and activity of plasma TF were elevated compared with corresponding control animals. Plasma ADMA level was also increased in SHR. Treatment with L-arginine, a competitor of ADMA, lowered blood pressure and inhibited platelet aggregation concomitantly with a decrease in plasma TF level and activity in both types of hypertensive rats. We also found that exogenous ADMA promoted platelet aggregation and increased TF level and (or) activity in platelet-rich plasma, an effect that was inhibited by pretreatment with L-arginine. Importantly, the enhanced platelet aggregation induced by exogenous ADMA was reduced by pretreatment with anti-TF antibody. The results suggest that endogenous ADMA may be involved in platelet hyperaggregation status in hypertension, and the facilitation of platelet aggregation by ADMA is related to upregulation of the level and activity of plasma TF.     

Improvement of platelet aggregation abnormalities in thrombocytosis after thrombocytopheresis

Platelet function tests were performed in three patients with thrombocytosis in myeloproliferative disorders before and after a swift reduction of platelet count by thrombopheresis. The decrease of platelet count obtained after the procedure was reversed in six days. In two patients with platelet aggregation defects, the normalization of aggregation abnormalities was observed after pheresis, followed by a progressive decrease of platelet response until the pre-pheresis values on 6th day. In the third patient with normal platelet aggregation, a progressive increase of platelet aggregation response was noted on the days following thrombopheresis with ischaemic symptoms of a foot toe. In all three patients, the changes of platelet aggregation were accompanied by a related increase of megathrombocytes. In the two patients with platelet aggregation abnormalities, plasma and platelet beta-thromboglobulin levels were related to changes in platelet count and aggregation.     

Antioxidant and antiplatelet effects of rosuvastatin in a hamster model of prediabetes

The objectives of this study were to determine the relationships among Type II diabetes (T2DM)-dependent elevations in platelet-derived reactive oxygen species (ROS), platelet-surface protein disulfide isomerase (psPDI) NO-releasing activity, and platelet aggregation and to evaluate the efficacy of rosuvastatin in normalizing these parameters in primary cells derived from a hamster model of prediabetic insulin resistance induced by fructose feeding. Platelets from rosuvastatin-treated non-fructose-fed (NFF) and fructose-fed (FF) hamsters were analyzed for aggregability and psPDI-denitrosation activity. Platelets from NFF animals treated with xanthine/xanthine oxidase (X/XO) were assessed for the same parameters and primary aortic endothelial cells (AEC) cultivated with a range of [rosuvastatin] +/- mevalonate were analyzed for ROS production. Platelets from FF hamsters displayed statistically significant enhanced ROS production, diminished psPDI-mediated NO-releasing activity, and hyperaggregability. Suggestively, platelets from NFF animals treated with X/XO displayed characteristics similar to platelets from FF animals. Rosuvastatin elicited a normalizing effect on all parameters measured in platelets from FF animals. Further, ROS production in primary AEC from FF animals could be blunted to that of NFF animals by concentrations of rosuvastatin in the range of those achieved in the bloodstream. Diminished psPDI-dependent NO-releasing activity and increased initial aggregation rates of FF platelets may result from elevated vascular ROS production under conditions of insulin resistance. Normalization of ROS production and platelet aggregation by rosuvastatin indicates its potential use as a vasculoprotective agent.     

Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by approximately 50% (P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.     

Relationships between Platelets and Blood Cells in Postprandial Glycemia in Healthy Individuals

The relationships between platelet aggregation and blood cell amount were studied in healthy subjects after fasting and after a standard carbohydrate breakfast. Positive relationships were revealed between certain parameters of platelet aggregation and the amounts of monocytes and eosinophils. Negative relationships were revealed between most parameters of platelet aggregation and the amount of platelets. Along with the well-known food leukocytosis and increased fibrinogen, postprandial conditions were characterized by an increase in certain parameters of platelet aggregation, destruction of the relationship between the fibrinogen concentration and platelet aggregation, impairment of the inverse relationship between the amount and aggregation of platelets, and moderate direct or inverse relationships between certain parameters of the first and the second stages of platelet aggregation and the erythrocyte parameters. As a disturbing factor, postprandial conditions activated platelet hemostasis by modulating the initial stages of aggregation and substantially changed the interrelations between blood cells.     

急性低氧对大鼠血液中儿茶酚胺及血小板聚集性的影响

健康ＳＤ雄性大鼠,体重２５０－３００ｇ,麻醉、气管插管,用人工呼吸机经气袋供气,自发吸入氧浓度为９％的氧氮混合气,用高效液相色谱－电化学联合检测法及电阻法检测循环血液中儿茶酚胺及全血血小权聚集性的动态变化。结果：急性低氧１５ｍｉｎ时血液中肾肾上腺素（Ａ）浓度及全血血小板聚集性显著增加（Ｐ〈０．０１）,而去甲肾上腺素（ＮＡ）浓度虽有所增加,但无统计学意义（Ｐ〉０．０５）;复氧１５ｍｉｎ时血液中儿茶酚     

Early recovery of microvascular perfusion induced by t-PA in combination with abciximab or eptifibatide during postischemic reperfusion

Background  

GPIIb/IIIa inhibitors abciximab and eptifibatide have been shown to inhibit platelet aggregation in ischemic heart disease. Our aim was to test the efficacy of abiciximab (Reo Pro) or eptifibatide (Integrilin) alone or in combination with plasminogen activator (t-PA) in an experimental model of ischemia reperfusion (I/R) in hamster cheek pouch microcirculation visualized by fluorescence microscopy. Hamsters were treated with saline, or abiciximab or eptifibatide or these drugs combined with t-PA infused intravenously 10 minutes before ischemia and through reperfusion. We measured the microvessel diameter changes, the arteriolar red blood cell (RBC) velocity, the increase in permeability, the perfused capillary length (PCL), and the platelet and leukocyte adhesion on microvessels.     

Platelet count and function at high altitude and in high-altitude pulmonary edema.

Platelet aggregation is the key process in primary hemostasis. Certain conditions such as hypoxia may induce platelet aggregation and lead to platelet sequestration primarily in the pulmonary microcirculation. We investigated the influence of high-altitude exposure on platelet function as part of a larger study on 30 subjects with a history of high-altitude pulmonary edema (HAPE) and 10 healthy controls. All participants were studied in the evening and the next morning at low altitude (450 m) and after an ascent to high altitude (4,559 m). Platelet count, platelet aggregation (platelet function analyzer PFA100; using epinephrine and ADP as activators), plasma soluble P (sP)-selectin, and the coagulation parameters prothrombin fragments 1+2 and thrombin-antithrombin complex were measured. High-altitude exposure decreased the platelet count, shortened the platelet function analyzer closure time by approximately 20%, indicating increased platelet aggregation, increased sP-selectin levels to approximately 250%, but left plasma coagulation unaffected. The HAPE-susceptible subjects were prophylactically treated with either tadalafil (a phosphodiesterase 5 inhibitor), dexamethasone, or placebo in a double-blind way. Subgroup analyses between these different treatments and comparisons of the seven placebo-treated individuals developing HAPE and controls revealed no differences in platelet count, platelet aggregation, or sP-selectin values. We conclude that exposure to high altitude activates platelets, which leads to platelet aggregation, platelet consumption, and decreased platelet count. These effects are, however, not more pronounced in individuals with a history of HAPE or actually suffering from HAPE than in controls and therefore may not be a pathophysiological mechanism of HAPE.     

Cholesterol-induced stimulation of platelet aggregation is prevented by a hempseed-enriched diet

Hypercholesterolemia indirectly increases the risk for myocardial infarction by enhancing the ability of platelets to aggregate. Diets enriched with polyunsaturated fatty acids (PUFAs) have been shown to reduce the detrimental effects of cholesterol on platelet aggregation. This study investigated whether dietary hempseed, a rich source of PUFAs, inhibits platelet aggregation under normal and hypercholesterolemic conditions. Male New Zealand white rabbits were fed one of 6 dietary interventions: regular control diet (RG); control diet + 10% hempseed (HP); control diet + 10% partially delipidated hempseed (DHP); control diet + 0.5% cholesterol (OL); control diet + 0.5% cholesterol + 10% hempseed (OLHP); control diet + 5% coconut oil (CO). After 8 weeks, blood was collected to measure ADP- and collagen-induced platelet aggregation and plasma levels of fatty acids, cholesterol, and triglycerides. The hempseed-fed animals (HP and OLHP) displayed elevated plasma levels of PUFAs and a prominent enhancement in 18:3n-6 (gamma-linolenic acid, GLA) levels, a unique PUFA found in hempseed. The cholesterol-supplemented groups (OL and OLHP) had significantly elevated plasma levels of cholesterol and triglycerides, but platelet aggregation was significantly augmented only in the OL group. The addition of hempseed to this diet (OLHP) normalized aggregation. The direct addition of GLA to the OL platelet samples blocked the cholesterol-induced stimulation of platelet aggregation. The results of this study demonstrate that when hempseed is added to a cholesterol-enriched diet, cholesterol-induced platelet aggregation returns to control levels. This normalization is not due to a reduction in plasma cholesterol levels, but may be partly due to increased levels of plasma GLA.     

PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.