Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo

BACKGROUND: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)). METHODS AND RESULTS: Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro. CONCLUSIONS: beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.     

Vitronectin binds to activated human platelets and plays a role in platelet aggregation.

Vitronectin (Vn) is a multifunctional 75-kD glycoprotein that is present in plasma and the extracellular matrix. Vn functions as a complement regulatory protein in plasma, and promotes the growth and attachment of cells in tissue culture. Recent cDNA cloning reveals that like other adhesive proteins, Vn contains the sequence Arg-Gly-Asp and binds to some members of the integrin class of adhesive membrane receptors. In liposomes, the platelet membrane glycoprotein complex IIb/IIIa binds Vn, as well as fibrinogen, von Willebrand factor, and fibronectin. We examined the binding of purified Vn to resting and stimulated human platelets. Vn bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a Kd of 320 nM and 8,000 sites per platelet. Epinephrine or ADP stimulation led to specific binding with KdS of 93 and 116 nM, respectively. Binding was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and by monoclonal and polyclonal antibodies to GPIIb/IIIa. Endogenous platelet Vn stores were identified in immunoblots of gel-filtered platelets and the surface expression of endogenous platelet Vn was thrombin inducible. Monoclonal as well as polyclonal antibodies to Vn inhibited platelet aggregation, suggesting that Vn plays a role in the formation of stable platelet aggregates.     

Iron-induced platelet aggregation measurement: a novel method to measure platelet function in stenting for ST segment elevation myocardial infarction

Background Iron and (stainless) steel are potent platelet aggregation activators, and may be involved in stent thrombosis, a serious complication after intracoronary stenting. Current platelet function tests are suboptimal, because of inappropriate agonists and/or lack of reproducibility. We tested the feasibility and reproducibility of a novel platelet function test using stainless steel as an agonist and compared it with other platelet function tests. Materials and methods In 111 patients with acute ST segment elevation myocardial infarction (STEMI), duplo measurements of iron (Fe)‐induced platelet aggregation (FIPA) were performed after clopidogrel, acetylsalicylic acid and/or tirofiban treatment. Within 1 h, citrated blood samples drawn from the femoral sheath before primary percutaneous coronary intervention were added to 100 mg of low carbon steel and after 5 s mixing with vortex, the samples were incubated for 15 min. The ratio between the non‐aggregated platelets in the agonist sample and platelets in a reference sample was calculated as the platelet aggregation inhibition. Results FIPA measurement was highly reproducible (correlation coefficient (R) = 0·942, P < 0·001 between duplo samples). FIPA correlated well with adenosine diphosphate‐induced platelet aggregation (R = 0·83, P < 0·001) but weakly with platelet function analyser‐100 bleeding time (R = 0·56, P < 0·001). FIPA could be measured in patients in which platelet aggregation could not be measured by platelet function analyser‐100 or after adenosine diphosphate. Conclusion This study showed good reproducibility of a novel platelet function test using stainless steel as an agonist and showed correlation with validated platelet function tests. We found that the novel platelet function test is a suitable test for measurement of platelet aggregation inhibition in patients undergoing stenting for STEMI, even when they are taking multiple antiplatelet regimens.     

Migraine: possible role of shear-induced platelet aggregation with serotonin release

Background.- Migraine patients are at an increased risk for stroke, as well as other thromboembolic events. This warrants further study of the role of platelets in a proportion of migraine patients. Objective.- To extend the "platelet hypothesis" using literature data and observations made in a rat model of shear stress-induced platelet aggregation. Such aggregation causes release of serotonin, leading to vasoconstriction during sufficiently strong aggregation and to long-lasting vasodilation when aggregation diminishes. This vasodilation also depends on nitric oxide and prostaglandin formation. Results.- A role for platelet aggregation in a number of migraineurs is indicated by reports of an increased platelet activity during attacks and favorable effects of antiplatelet medication. We hypothesize that in those patients, a migraine attack with or without aura may both be caused by a rise in platelet-released plasma serotonin, albeit at different concentration. At high concentrations, serotonin may cause vasoconstriction and, consequently, the neuronal signs of aura, whereas at low concentrations, it may already stimulate perivascular pain fibers and cause vasodilation via local formation of nitric oxide, prostaglandins, and neuropeptides. Platelet aggregation may be unilaterally evoked by elevated shear stress in a stenotic cervico-cranial artery, by reversible vasoconstriction or by other cardiovascular abnormality, eg, a symptomatic patent foramen ovale. This most likely occurs when a migraine trigger has further enhanced platelet aggregability; literature shows that many triggers either stimulate platelets directly or reduce endogenous platelet antagonists like prostacyclin. Conclusion.- New strategies for migraine medication and risk reduction of stroke are suggested.     

Decreased collagen-induced platelet aggregation and increased platelet arginine levels in migraine: a possible link with the NO pathway

We studied whole blood platelet aggregation induced by collagen, platelet activating factor (PAF) and measured basal platelet L-arginine (L-arg) levels, as an indirect index of the nitric oxide (NO) pathway in migraine. Migraine, both with and without aura groups, showed a reduced aggregation to collagen, but not to PAF, compared with control subjects. Platelet L-arg levels were significantly increased in migraine with aura sufferers, whereas the plasma levels were in the same range in migraineurs and controls. Platelet hyperesponsiveness to collagen stimulation in migraine may be linked to an increased availability of the amino acid precursor and an abnormal NO synthesis.     

急性脑梗死患者血小板参数及聚集率变化的临床意义

目的：探讨血小板最大聚集率（MAR）、血小板计数（PLT）、血小板比容（PCT）、血小板分布宽度（PDW）、血小板平均体积（MPV）与急性脑梗死病程的关系,为其临床早期诊断和治疗提供依据。方法选取该院107例急性脑梗死（ACI）患者,根据头部 CT 或 MRI 病灶大小,梗死体积按 Pullicino 公式（长×宽×层数/2）计算进行分组：大梗死组（病灶体积大于10 cm3）、中梗死组（病灶体积4～10 cm3）和小梗死组（病灶体积小于4 cm3）,40例健康对照组,分别用 PL-11血小板分析仪检测经 PLR-06血小板诱聚剂诱导前后的 MAR、PLT、PDW、MPV、PCT,对测定结果进行统计分析。结果（1）与对照组比较,经 PLR-06血小板诱聚剂诱导前,大脑梗死组 PLT、PCT、PDW、MPV 明显升高（P ＜0．01）;中、小脑梗死组 PLT、PCT、PDW、MPV 升高（P ＜0．05）。（2）与对照组比较,经 PLR-06血小板诱聚剂诱导后,各脑梗死组 MAR 升高（P ＜0．05）;PLT 差异无统计学意义（P ＞0．05）;大、中脑梗死组 PCT、PDW、MPV 升高（P ＜0．05）;小脑梗死组 PCT、PDW、MPV 差异无统计学意义（P ＞0．05）。结论血小板聚集率、数量及体积的改变与急性脑梗死的发生和发展密切相关,监测它们的变化对预防和治疗急性脑梗死具有重要的临床意义。     

The impact of different antimigraine compounds on platelet and erythrocyte aggregation

Clinical and experimental data suggest that ergotamine compounds and triptans may contribute to vascular events such as myocardial infarction and stroke. The role of blood cell aggregation in this context is, however, not clarified. We aimed to evaluate the impact of different acute antimigraine compounds on platelet and erythrocyte aggregation in a human ex vivo experimental design. In 20 healthy subjects without migraine and in 20 healthy subjects with migraine without aura, platelet and erythrocyte aggregation were measured before and after intake of placebo, acetylsalicylic acid, ergotamine tartrate, zolmitriptan and sumatriptan. Platelet aggregation was measured by the so-called platelet reactivity index. Erythrocyte aggregation was measured by photometric assessment in an aggregometer. Ergotamine tartrate induced a significant increase of platelet aggregation, whereas acetylsalicylic acid induced a significant decrease in both subject groups. After placebo, after sumatriptan and after zolmitriptan, no significant changes of platelet aggregation were noted. Erythrocyte aggregation was affected by neither compound. We can conclude that platelet aggregation, but not erythrocyte aggregation, is increased after intake of ergotamine tartrate. This may in part contribute to vascular side-effects of this compound. Acetylsalicylic acid and the triptans appeared to be safe with respect to platelet and erythrocyte aggregation.     

A humanized monoclonal antibody that inhibits platelet‐surface ERp72 reveals a role for ERp72 in thrombosis

Essentials

• ERp72 is a thiol isomerase enzyme.
• ERp72 levels increase at the platelet surface during platelet activation.
• We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti‐ERp72).
• Anti‐ERp72 inhibits platelet functional responses and thrombosis.

Summary

Background

Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulfide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface, including PDI, ERp5 and ERp57, and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis.

Aim

We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation; however, its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis.

Methods

Using HuCAL technology, fully humanized Fc‐null anti‐ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti‐ERp72) selected for further testing in platelet functional assays.

Results and conclusions

Anti‐ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation, revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti‐ERp72 into mice protected against thrombosis.     

Acid citrate dextrose (acd) formula a as a new anticoagulant in the measurement of in vitro platelet aggregation

To evaluate whether the use of ACD Formula A may affect in vitro platelet function, blood samples were obtained from 21 healthy blood donors and anticoagulated in ACD (acid-citrate dextrose, NIH Formula A), Na citrate 3.8%, and K₃EDTA. Platelet count, mean platelet volume, and in vitro platelet aggregation were evaluated on each sample. No significant difference was observed in platelet count and mean platelet volume among the different samples. Conversely, the ACD treated platelets showed a higher reactivity to the agonists as demonstrated by a significant increase of the maximum percentages of aggregation induced by ADP, epinephrine, and collagen, as well as a significant decrease of secondary aggregation thresholds to ADP and epinephrine. In conclusion, it may be speculated that ACD Formula A is capable of better maintaining the intraplatelet signal transduction mechanisms during PRP preparation, thus improving the overall responsiveness of platelets.©1995 wiley-Liss, inc.     

丁胺卡那霉素对EDTA依赖性凝集血小板的解离及其机制

目的研究丁胺卡那霉素对EDTA抗凝剂依赖的聚集血小板的解离作用和机制,为血常规标本中血小板凝聚提供可靠的解决方法。方法在EDTA依赖的假性血小板减少症(PTCP)患者的EDTA-K2抗凝血样本中,于不同时间段加入不同浓度丁胺卡那霉素进行凝集血小板的解离试验,通过血小板计数和涂片观察解离效果;用流式细胞仪检测血小板膜表面CD41、CD61、CD62p、PAC-1和IgG的表达百分率。结果抽血后1h内加入丁胺卡那霉素对血小板凝集的解离作用明显,血小板计数可恢复到即时检测的水平,血小板CD62p、PAC-1和IgG的表达量被显著抑制,而CD41和CD61未受明显影响。结论PTCP患者血常规样品抽血后1h内加入丁胺卡那霉素能有效解离凝集的血小板,作用机制可能与抑制患者血小板膜表面CD62p、PAC-1和IgG的表达有关;此法有助于解决EDTA所致的血小板计数的假性减少。     

脑安胶囊不同制剂对大鼠血小板聚集的影响

目的观察两种不同工艺制剂的脑安胶囊(新型及传统型)对血小板聚集的影响,为研制活血化瘀类中药的新工艺提供了依据。方法大鼠60只,单纯随机分为6组,每组10只,分别为空白对照组(生理盐水),新型脑安胶囊低剂量组(40mg/kg)和高剂量组(80mg/kg)、传统型脑安胶囊低剂量组(40mg/kg)和高剂量组(80mg/kg),阿司匹林组(300mg/kg);实验药物经口灌胃,1次/d,连续给药3d后取血测定血小板的聚集率,并计算出聚集抑制率。结果新型脑安胶囊高剂量、传统型脑安胶囊高剂量和阿司匹林对血小板聚集抑制率分别为66.4%,57.4%和49.5%,经统计学分析,两种脑安胶囊高剂量对血小板聚集抑制率均超过阿司匹林(t=2.7-5.1,P<0.01,P<0.05),其中新型脑安胶囊高剂量与传统型脑安胶囊高剂量对血小板聚集抑制率相比较差异亦有显著性意义(P<0.01)。结论新型脑安胶囊对血小板的抑制作用明显高于传统型脑安胶囊。     

Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease

Summary. Background: Previous studies have demonstrated considerable variation in the antiplatelet effect of aspirin. Objectives: To investigate the impact of platelet turnover on the antiplatelet effect of aspirin in patients with stable coronary artery disease (CAD) and to identify determinants of platelet turnover. Methods: Platelet turnover was evaluated by measurements of immature platelets and thrombopoietin in 177 stable CAD patients on aspirin monotherapy, including 85 type 2 diabetics and 92 non‐diabetics. Whole blood platelet aggregation was determined using the VerifyNow^® Aspirin test and multiple electrode aggregometry (MEA, Multiplate^®) induced by arachidonic acid (AA) (1.0 mm ), adenosine diphosphate (ADP) (10 μm ) and collagen (1.0 μg mL^?1). Results: Immature platelet levels significantly correlated with MEA (r = 0.31–0.36, P‐values < 0.0001) and the platelet activation marker sP‐selectin (r = 0.19, P = 0.014). Contrary to the VerifyNow^® test, MEA significantly correlated with variations in platelet count (r = 0.45–0.68, P‐values < 0.0001). Among patients with residual platelet reactivity according to AA, there were significantly more diabetics (61% vs. 41%, P = 0.027) and higher levels of sP‐selectin (77.7 ± 29 vs. 70.2 ± 25 ng mL^?1, P = 0.070) and serum thromboxane B₂ (0.81 [0.46; 1.70] vs. 0.56 [0.31; 1.12] ng mL^?1, P = 0.034). In a multivariate regression analysis, immature platelet levels were determined by thrombopoietin levels (P < 0.001), smoking (P = 0.020) and type 2 diabetes (P = 0.042). Conclusions: The antiplatelet effect of aspirin was reduced in CAD patients with an increased platelet turnover. Once‐daily dosing of aspirin might not suffice to adequately inhibit platelet aggregation in patients with an increased platelet turnover.     

心痛宁对大鼠血小板聚集及膜流动性的影响

目的 观察心痛宁对大鼠血小板聚集性及膜流动性的影响。方法 将Wistar大鼠随机分为心痛宁高剂量组（15g/kg日生药）及低剂量组（7.5g/kg日生药）、阿司匹林组（5mg/kg/日）、未用药组及正常对照组,共灌胃七日。然后以皮下注射肾上腺素（0.9mg/kg)加冰水刺激法造模,18小时后采血,以比浊法测血小板聚集率、荧光偏振法测血小板膜流动性。结果 心痛宁可抑制血小板聚集率的升高、防止血小板膜流动性下降,其作用与阿司匹林无显著性差异。     

慢性肺原性心脏病急性加重期血浆内皮素-1与血小板功能关系的探讨

目的：探讨血浆内皮素１（ＥＴ１）、血小板聚集及血小板内游离Ｃａ２＋在慢性肺原性心脏病（肺心病）急性加重期的意义及其相互关系。方法：用放射免疫分析法测定两组（４２例慢性肺心病患者急性加重期和２５例健康者）血浆ＥＴ１含量，用比浊法测定两组血小板聚集率和用Ｆｕｒａ２／ＡＭ荧光技术测定血小板内游离Ｃａ２＋含量。结果：慢性肺心病急性加重期ＥＴ１水平明显升高（Ｐ＜０．０１），血小板聚集率增高（Ｐ＜０．０１），血小板内游离Ｃａ２＋含量显著增加（Ｐ＜０．０１）。另外还观察到ＡＤＰ诱导的血小板聚集率与血小板内游离Ｃａ２＋含量之间呈正相关（ｒ＝０．７３８，Ｐ＜０．０１），而血浆ＥＴ１水平与血小板聚集率和血小板内游离Ｃａ２＋含量之间无明显相关性。结论：血浆ＥＴ１在慢性肺心病急性加重期的发病中可能起一定作用。此期血小板的功能处于活跃状态，易于聚集，形成血栓，加重病情；而血浆ＥＴ１对血小板功能可能无影响。     

Evaluation of the clinical utility of platelet aggregation studies in the long-term follow-up of patients with atherosclerotic vascular disease.

The present study was designed to evaluate the usefulness of laboratory monitoring of antiplatelet therapy by means of a multiparametric evaluation of in vitro platelet aggregation tests in the attempt to individually optimize a given therapeutic regimen. The presence of a condition of hyperaggregability was shown in approximately 80% of patients with different forms of atherosclerotic vascular disease not undergoing any therapeutic regimen with antiplatelet agents. Conversely, a significant decrease in platelet activity was observed in patients undergoing different therapies based on acetylsalicylic acid (ASA), ticlopidine, or indobufen. The similar antiaggregatory effect of low-dose vs. high-dose ASA therapies was also shown. Dipyridamole alone showed no antiaggregatory effect, which, in turn, was reached only by the addition of ASA. Nevertheless, the association of ASA plus dipyridamole did not show any stronger antiplatelet effect than ASA alone. The evaluation of in vitro platelet activity in a group of patients treated with picotamide failed to show any significant change in comparison with the untreated group, probably due to the short half-life of picotamide in man and/or to its capability of reversibly antagonizing the action of thromboxane at receptor level. The evaluation of a long-term follow-up of 90 patients treated with different antiplatelet agents supports the idea that a multiparametric analysis of in vitro platelet aggregation may provide valuable help in monitoring and optimizing a given therapeutic regimen.