Plasma levels of P-selectin are determined by platelet turn-over and the P-selectin Thr715Pro polymorphism

BACKGROUND: Plasma levels of soluble P-selectin (sP-selectin) are often used to demonstrate platelet activation. METHODS: We determined sP-selectin in a variety of disorders characterized by high or low platelet counts and compared their levels with those in healthy subjects. Furthermore, we determined the Thr715Pro polymorphism in all subjects. RESULTS: Total concentrations of sP-selectin were clearly associated with levels of platelet counts. Thus, calculation of sP-selectin per platelet showed that these levels in patients with thrombocytopenia due to marrow failure and in patients with increased platelet counts were similar to those in controls. Only patients with an increased platelet turn-over had elevated sP-selectin per platelet. While carriers of the Pro715 polymorphism had lower sP-selectin levels than non-carriers, this genetic disposition was over-ruled in patients with increased platelet turn-over. CONCLUSION: For the demonstration of platelet activation it is preferable to define sP-selectin based on platelet counts under the consideration of the Pro715Thr polymorphism.     

Platelet P-selectin and platelet mass, volume and component in sickle cell disease: relationship to genotype

BACKGROUND AND PURPOSE: Excess platelet activation (e.g. increased soluble P selectin [sPsel] and beta thromboglobulin [beta-TG]) is well established in sickle cell disease (SCD) and may contribute to the prothrombotic/hypercoagulable state and vascular occlusion characteristic of the disease. We hypothesised altered whole platelet P-selectin (pPsel), and morphological platelet indices mass, volume and component in SCD and two of its major genotypes. METHODS: We recruited 35 SCD patients [mean age 31 years, 54% men]. Of these, 16 had homozygous sickle cell (HbSS) disease and 19 had sickle-haemoglobin-C (HbSC) disease. Patients were compared with 29 subjects with normal haemoglobin (HbAA) matched for age and ethnicity. Platelet mass, volume and component were measured by flow cytometry, pPsel in platelet lysate, sP-sel and beta-TG by ELISA. RESULTS: SCD patients had lower pP-sel and mean platelet volume (MPV) but elevated platelet component (MPC), and, as expected, elevated platelet count, and sP-sel (all p<0.05) compared to HbAA subjects. In both groups, pPsel correlated with MPV, and MPV correlated positively with mean platelet mass (MPM) and negatively with MPC. sPsel correlated with platelet count only in SCD, not in the controls. Platelet count alone was different (higher) in HbSS compared to HbSC, and sPsel correlated with platelet count only in HbSC disease, not in HbSS disease. CONCLUSION: Patients with SCD have various abnormalities in their platelets regardless of genotype: there are more numerous platelets, which are smaller, contain less P selectin per cell, but have a higher concentration of granules than those of HbAA subjects. These differences may mark and/or promote the prothrombotic state in SCD.     

Inhibition by ticlopidine of Paf-acether-induced in vitro aggregation of rabbit and human platelets

Ticlopidine was incubated $\text{in vitro}$ with rabbit or human washed platelets and aggregations were triggered by submaximal concentrations of adenosine-5′-diphosphate (ADP), arachidonic acid (AA) and Paf-acether (platelet-activating factor), the mediators of the three known pathways of platelet activation. Inhibition of Paf-acether-induced rabbit platelet aggregation was proportionnal to the concentrations of Ticlopidine used. The same range of inhibition by Ticlopidine was observed when aggregations were triggered by the two other agonists. Human platelet aggregation induced by Paf-acether was also inhibited by Ticlopidine. Inhibition was increased when platelets were rendered insensitive to ADP and AA. Our results show that Ticlopidine inhibits human and rabbit platelet aggregation triggered by Paf-acether through a mechanism not related to the inhibition of the ADP and prostaglandin pathways.     

Inhibition of platelet aggregation in whole blood by adrenoceptor antagonists

It has recently become possible to study platelet aggregation in whole blood which may more closely resemble the in-vivo situation as the platelets are left in their natural milieu with red and white cells present which themselves can influence aggregation. The effects of 4 adrenoceptor antagonists on platelet aggregation in whole blood were studied in-vitro using the Clay-Adams Ultra Flo 100 whole blood platelet counter. Labetalol, pindolol and propranolol inhibited aggregation to 0.5 microgram/ml collagen in a dose dependent manner, and were synergistic with prostacyclin in inhibiting collagen induced aggregation. These 3 drugs also promoted reversal of aggregation induced by 10 microM ADP, but only inhibited 0.5 mM arachidonic acid induced aggregation at high drug concentrations. Atenolol had no effect on either collagen, ADP or arachidonic acid induced aggregation. The anti-platelet effect of these drugs may be of value in the treatment of vascular disease.     

Inhibition of rabbit platelet phosphodiesterase activity and aggregation by calcium channel blockers

Rabbit platelet cyclic AMP phosphodiesterase is inhibited by the three calcium channel blockers nifedipine, diltiazem, and verapamil with IC50's of 100 microM, 100 microM and 420 microM, respectively. Also, platelet aggregation induced by 4 microM ADP is inhibited by those compounds. Verapamil is the most potent aggregation inhibitor with an IC50 of 260 microM while diltiazem and nifedipine have IC50's of 630 microM and 840 microM, respectively. All three compounds display a maximum inhibition of 80-85%. Diltiazem and PGD2 potentiate the antiaggregatory activity of each other in that the inhibitions occurring in the presence of the combination of the two (at varying concentrations) exceed the calculated sums of the inhibitions produced by each alone. On the other hand, the antiaggregatory activities of verapamil or nifedipine, are additive with that of PGD2 in that no significant differences exist between the observed inhibitions produced by the combinations and the calculated summed values of the individual inhibitions. Our data suggest, therefore, that in addition to lowering intracellular calcium ions, which are required for platelet aggregation, the three calcium channel blockers inhibit the breakdown of cyclic AMP thereby promoting antiaggregation.     

P-选择素基因S290N和P-选择素糖蛋白配体-1基因M62I多态性与缺血性脑梗死血小板白细胞聚集体的相关性探讨

目的探讨血小板白细胞聚集体在缺血性脑梗死中的变化以及与P-选择素基因S290N和P-选择素糖蛋白配体-1基因M62I多态性的关系。方法选取58例缺血性脑梗死患者作为病例组,20例正常人群作为对照组。病例组分为大动脉粥样硬化性脑梗死(LAA)19例,小动脉闭塞性脑梗死(SAO)39例。运用流式细胞技术检测所有受试者的血小板白细胞聚集体(PLA)、血小板单核细胞聚集体(PMA)、血小板淋巴细胞聚集体(PLy A)和血小板中性粒细胞聚集体(PNA)水平。并运用基因测序方法检测P-选择素基因S290N和P-选择素糖蛋白配体-1基因M62I多态性。结果 PMA%在病例组与对照组、LAA与对照组、SAO与对照组间差异有统计学意义(P=0.000,P=0.018,P=0.000)。PNA%在病例组与对照组、LAA与对照组间差异有统计学意义(P=0.045,P=0.002)。PNA%在LAA组SELP基因S290N位点SS和SN基因型间差异有统计学意义(P=0.008)。PLA%在LAA组PSGL-1基因M62I位点MM、MI和Ⅱ基因型间差异有统计学意义(P=0.046)。结论 PMA和PNA与缺血性脑梗死发病有关,SELP基因S290N位点SN基因型以及PSGL-1基因M62I位点MM基因型会增加LAA发生风险。     

Abciximab treatment in vitro after aspirin treatment in vivo has additive effects on platelet aggregation, ATP release, and P-selectin expression

To prevent arterial thrombosis, abciximab is administered together with aspirin. However, whether or not there are benefits to combine abciximab with aspirin is not yet well defined. Healthy volunteers were studied for the effect of aspirin+abciximab using sodium arachidonate and adenosine diphosphate (ADP) alone or in combination to induce platelet activation/aggregation. Abciximab produced complete inhibition of platelet aggregation induced with ADP but only 40% inhibition of aggregation induced by 0.75-mmol/l sodium arachidonate. Abciximab added in vitro to platelet-rich plasma (PRP) from platelets from aspirin-treated donors produced an almost complete inhibition of platelet aggregation. Aspirin, and abciximab alone, did not inhibit adenosine triphosphate (ATP) release as thoroughly as aspirin+abciximab did. Abciximab (3–5 μg/ml) produced inhibition of P-selectin expression induced with 5 (from 46.2±6.0% to 27.4±7.0%, P=.002) and 20-μmol/l ADP (from 53.1±8.1% to 35.1±11.0%, P=.019), but no effect was observed when 0.75-mmol/l sodium arachidonate was used (P=.721). Aspirin diminished P-selectin expression in sodium arachidonate-stimulated platelets (from 77.7±11.8% to 40.2±3.6%, P<.0001) in non-aspirinated and platelets from aspirin-treated donors, respectively. Abciximab (3, 4, and 5 μg/ml) added to platelets from aspirin-treated donors decreased P-selectin expression in platelets stimulated with sodium arachidonate from 40.2±8.6% to 25.6±11.5% (P=.027), to 20.5±3.5% (P<.0001), and to 22.5±1.8% (P<.0001). We concluded that the antiplatelet effect of abciximab is greatly increased by aspirin.     

Storage of platelets for tests of platelet function: effects of pH on platelet aggregation and liberation of beta-thromboglobulin

Platelet aggregation responses are influenced by conditions of storage of platelet-rich plasma (PRP). The aim of the present study was to further define the necessity for pH control during storage of PRP for tests of platelet function. Aliquots of citrated PRP were maintained at different pH levels by alteration of the CO2 content of the atmosphere in an incubation chamber. At intervals over 2-2 1/2 hours, plasma beta-thromboglobulin and 14C-serotonin were measured as well as platelet aggregation induced by ADP and collagen. At each time a dose response curve was studied for aliquots stored at each pH level. When two aliquots were maintained at different pH levels in the range 6.85-7.90, there was a significant increase in aggregation at the higher pH, even when the pH difference was as small as 0.2 units. In this range, pH did not influence the rate of deterioration of the aggregation response, but when pH was above 8.0, there was marked deterioration of the response. Increased pH was associated with an increase in plasma levels of beta-thromboglobulin and 14C-serotonin, which was more marked when pH was above 8.0. It appears that increases in pH are harmful to platelets and even small pH changes should be avoided during storage of platelet-rich plasma for tests of platelet function.     

替格瑞洛与西洛他唑对氯吡格雷抵抗的急性缺血性脑卒中患者的疗效及安全性比较

目的 探讨替格瑞洛与西洛他唑对氯吡格雷抵抗的急性缺血性脑卒中(AIS)患者的疗效及安全性的影响。方法 将80例对氯吡格雷抵抗(血小板聚集率>50%)AIS患者按照数字表法随机分为替格瑞洛组(入组40例,完成37例)和西洛他唑组(入组40例,完成39例); 在AIS常规治疗的基础上替格瑞洛组将氯吡格雷换用替格瑞洛治疗(90 mg/次,2次/d); 西洛他唑组将氯吡格雷换用西洛他唑治疗(100 mg/次,2次/d)。于改变治疗方案前及改变治疗方案后1、3、6、12个月分别检测血小板聚集率(PIR),观察2组治疗12个月内的缺血事件、出血事件及药物的不良反应。结果 改变治疗方案后12个月替格瑞洛组总有效率显著高于西洛他唑组(z=-2.086,P=0.037)。替格瑞洛组缺血事件发生率低于西洛他唑组(χ²=4.057,P=0.034); 替格瑞洛组的出血事件发生率高于西洛他唑组(χ²=4.501,P=0.034); 替格瑞洛组的呼吸困难发生率高于西洛他唑组(χ²=4.505,P=0.034); 替格瑞洛组的其他不良反应发生率高于西洛他唑组(χ²=4.021,P=0.045)。改变治疗方案后1、3、6、12个月替格瑞洛组患者的PAR低于西洛他唑组(F=15.320,P=0.000)。结论 对氯吡格雷抵抗的AIS患者,替格瑞洛比西洛他唑的血小板抑制作用更强,缺血事件发生率更低,但出血事件、呼吸困难及其他不良反应的发生率更高,因此对于血栓风险较高、出血风险较低的患者,建议换用替格瑞洛; 对于血栓风险较低、出血风险较高的患者,建议换用西洛他唑。     

Inhibition of platelet thromboxane synthesis by 7-(1-imidazolyl) heptanoic acid: dissociation from inhibition of aggregation

The effects of 7-(1-imidazolyl)heptanoic acid (7-IHA), a thromboxane synthetase inhibitor, and indomethacin on platelet aggregation and immunoreactive thromboxane B₂ (iTXB₂) synthesis were studied in human platelet rich plasma. The ID₅₀ for inhibition of arachidonic acid induced iTXB₂ synthesis by 7-IHA was 1.9 μM but the ID₅₀ for inhibition of platelet aggregation was estimated to be 1.35 mM. 7-IHA (0.5 mM) inhibited arachidonic acid induced iTXB₂ synthesis to values less than that obtained in the presence of indomethacin (10 μM); but did not inhibit aggregation, while indomethacin completely inhibited platelet aggregation. 7-IHA had no effect on platelet fatty acid cyclooxygenase; did not promote synergism with ADP or arachidonic acid; arachidonic acid plus ADP or stable PGH₂ analogs. We conclude: 1) that 7-IHA is a specific inhibitor of platelet thromboxane synthesis which at the same concentrations does not inhibit arachidonic acid induced platelet aggregation and 2) that inhibition of platelet TXA₂ synthesis by thromboxane synthetase inhibitors may not necessarily inhibit arachidonic acid induced platelet aggregation.     

The effects of glucosamine on platelet aggregation

The effects of glucosamine on platelet aggregation induced by a variety of agents were investigated. When compared with the effects of N-acetyl glucosamine (NAG), an acetylated derivative of glucosamine (1), it became apparent that the N-acetyl group was important in stimulating $\text{Staphylococcus aureus}$ induced platelet aggregation. The absence of the N-acetyl group from NAG changes it to glucosamine which is an inhibitor rather than a stimulator of $\text{S. aureus}$ induced platelet aggregation and release. Glucosamine also inhibits collagen, epinephrine, and ADP induced platelet aggregation and release.     

Factors influencing shear-induced platelet alterations: platelet lysis is independent of platelet aggregation and release

To gain further insight into the mechanisms involved in fluid shear-induced platelet alterations, we examined conditions and factors that might affect shear-induced platelet reactions in human citrated platelet-rich plasma (C-PRP) under well-defined laminar flow conditions at shear stresses between 0 and 160 dyn/cm² using a Couette rotational viscometer. Prevention of excessive alkalinization of C-PRP during shear due to CO₂ loss did not appreciably affect shear-induced platelet aggregation (PAG), adenine nucleotide (AN) release or platelet lysis. Shear-induced PAG and AN release were significantly greater in C-PRP stored and sheared at 24°C as compared to C-PRP stored at 24°C or 37°C and sheared at 37°C whereas platelet lysis was not affected by temperature. When C-PRP was sheared in the presence of EDTA or EGTA, shear-induced PAG up to shear stresses of 80 dyn/cm² was almost completely suppressed whereas AN release and lysis were unaffected. Exposure of C-PRP to PGE₁ and theophylline before shear virtually abolished shear-induced PAG and AN release at shear stresses up to 80 dyn/cm² but had no demonstrable effect on shear-induced platelet lysis. These findings seem to indicate that ADP released from platelets by shear and extracellular Ca⁺⁺ or in the presence of PGE₁ and theophylline. These findings seem to indicate that the structural and biochemical changes associated with shear-induced PAG and release in our system do not predispose platelets to shear-induced lysis.     

Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry

Introduction

Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed.

Material and methods

We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow and RISTOhigh). Measurements of haematological values were performed employing Sysmex K-4500.

Results

We found no association between platelet aggregation and age (p > 0.57 for all agonists, except RISTOlow: p = 0.05). Platelet aggregation was significantly higher in women compared to men for all agonists (p < 0.0003), except RISTOlow (p = 0.05). A reference interval is presented as 95% confidence interval suitable for any age and both sex. Day-to-day variation was < 11% for all agonists except for RISTOlow. No association was found between platelet aggregation and haematocrit or red blood cell count after adjusting for age and gender except for RISTOhigh. A positive significant association was found between platelet count and platelet aggregation (p < 0.04). Finally, a significant positive association was found between platelet aggregation and white blood cell count for all agonists (p < 0.05) except RISTOlow and RISTOhigh (p > 0.05).

Conclusion

Reference intervals for platelet aggregation in healthy individuals (age: 17 to 66 years) were established in hirudin whole blood measured by Multiplate® employing the most commonly used agonists.     

Effects of N-acetyl glucosamine on platelet aggregation

N-acetyl glucosamine (NAG) alone did not aggregate platelets but in low concentrations shortened the delay phase of the $\text{Staphylococcus aureus}$ induced platelet aggregation in a concentration dependent manner, which suggests that NAG may be one of the components of the $\text{S. aureus}$ cell wall responsible for platelet aggregation. NAG, on the other hand, inhibited collagen induced platelet aggregation and the secondary platelet aggregation induced by epinephrine and ADP. This inhibition appears to be due to impurities that occur at higher levels in some NAG lots than in others. NAG has also been shown to decrease the length of time before the release of ATP from platelets when $\text{S. aureus}$ is the aggregating agent.     

Further studies on the role of red blood cells in spontaneous platelet aggregation

The influence of red blood cells on platelet aggregation has recently been a subject of considerable interest. We have studied the effect of red cells on spontaneously formed platelet aggregates in rotating vials at 37 degrees C. Platelet aggregation was quantified by measuring the fall in number of single platelets with a whole blood platelet counter. Autologous packed red cells, platelet rich plasma and platelet free plasma were used to reconstitute aliquots of blood with constant platelet count but 0-60% haematocrit (Hct). The fall in platelet count was minimal at zero Hct, increased markedly with the Hct in the anaemic range and less markedly in the normal to polycythaemic ranges of Hct. Scanning electron microscopic observation of whole blood showed the presence of small platelet aggregates after about 3 mins rotation and very large aggregates after about 12 mins. ADP from red cells has been implicated in triggering platelet aggregation in whole blood. Whether aggregates are formed as a result of ADP leaking from the red cells or by their jostling physical action on the platelets is discussed. The marked effect of the red cells on spontaneous platelet aggregation however, justifies the manipulation of the Hct as a useful therapeutic option in the control of thrombotic and bleeding tendencies.     

Inhibition of human platelet aggregation by vitamin K

The effect of several vitamin K (Vit K) analogues on the aggregation of human platelets was examined. The analogues were potent inhibitors of aggregation induced by ADP, thrombin, collagen and arachidonate but were less active against aggregation induced by the calcium ionophore A23187. Vit K3 also prevented platelet membrane phosphatide breakdown induced by collagen. These effects were not due to a direct inhibition of enzymes involved in the liberation of arachidonate or its subsequent transformation. The analogues exerted no effects on enzymes regulating intraplatelet cAMP. However, these effects could be overcome by increasing extracellular Ca++ levels, indicating a possible interaction with Ca++ regulation in platelets.     

Prevention of the platelet alpha-granule release reaction by membrane-active drugs

A range of membrane-active drugs were tested for their ability to prevent beta-thromboglobulin and platelet factor 4 release from freshly collected blood platelets. While all the drugs tested could inhibit collagen-induced platelet aggregation, only a few, notably procaine and the anti-malarial drugs chloroquine, hydroxychloroquine, camoquine and quinacrine (mepacrine), effectively prevented the alpha-granule release reaction.