Aspirin and the in vitro linear relationship between thromboxane A2‐mediated platelet aggregation and platelet production of thromboxane A2

Summary. Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A₂ formation is non‐linear and TXA₂ generation must be inhibited by at least 95% to inhibit TXA₂‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA₂ generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA₂ production in response to arachidonic acid (0.03–1 mmol L^?1), collagen (0.1–30 μg mL^?1), epinephrine (0.001–100 μmol L^?1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L^?1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA₂ production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC₅₀ values that did not differ. A linear relationship existed between aggregation and TXA₂ production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R² 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA₂ generation and TXA₂‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.     

Persistent production of platelet thromboxane A2 in patients chronically treated with aspirin

BACKGROUND: Patients treated with aspirin may have a reduced sensitivity to its antiplatelet effect. The mechanism accounting for such a reduced sensitivity might involve an impaired interaction of aspirin with cyclooxygenase-1 (COX)-1. OBJECTIVE: We sought to investigate whether platelets from patients under chronic treatment with aspirin still produce TxA2 and whether there is any relationship between the eventual persistent TxA2 formation and platelet aggregation. Finally, whether platelet-derived TxA2 can be inhibited by in vitro addition of aspirin. METHODS: Collagen-induced platelet aggregation and thromboxane-A2 (TxA2) were measured in 196 patients treated with aspirin (100-330 mg day(-1)) because of previous vascular events or presence of risk factors of atherosclerosis. RESULTS: Collagen-induced TxA2 production of the entire cohort was 128.7 +/- 21.6 pg 10(-8) cells, and was significantly correlated with platelet aggregation (Spearman's correlation coefficient = 0.44; P < 0.0001). Patients in the highest quartile of TxA2 showed higher platelet response to collagen (P < 0.0001) when compared with those in the lowest quartile. In a subgroup of 96 patients, platelets were treated in vitro with a TxA2 receptor antagonist (13-azaprostanoic acid) or aspirin before stimulation with collagen. 13-APA acid significantly inhibited platelet aggregation. Aspirin reduced (-72.9%) TxA2 production in patients with TxA2 values above the median but it was ineffective in those with TxA2 values below the median. CONCLUSION: In some patients chronically treated with aspirin platelet production of TxA2 may persist and account for enhanced platelet aggregation. Incomplete inhibition of COX-1 seems to be implicated in persistent TxA2 production.     

The P2Y1 receptor plays an essential role in the platelet shape change induced by collagen when TxA2 formation is prevented.

ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.     

The endocannabinoid 2‐arachidonoylglycerol activates human platelets through non‐CB1/CB2 receptors

Summary. Background: The endocannabinoid 2‐arachidonoylglycerol (2‐AG) is an endogenous lipid that acts through the activation of G‐protein‐coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2‐AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2‐AG also binds to platelet surface and leads to cell activation. Objective: To investigate the biological consequence of 2‐AG interactions with human platelets and to clarify the role of cannabinoid receptors. Methods: Gel‐filtered platelets were stimulated with 2‐AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA‐2 loaded platelets. Thromboxane A₂ (TxA₂) generation was evaluated as Thromboxane B₂ accumulation with a commercial EIA assay. Results: 2‐AG induced platelet shape change, aggregation and secretion with a dose‐dependent mechanism that required engagement of platelet TxA₂ receptors. 2‐AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA₂. Indeed 2‐AG was able to induce a robust generation of TxA₂ through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2‐AG. Moreover, neither CB₁ and CB₂ proteins nor CB₁/CB₂ mRNAs were detected in platelets. Conclusions: 2‐AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non‐CB₁/CB₂ receptor‐mediated mechanism.     

TRA-418, a novel compound having both thromboxane A2 receptor antagonistic and prostaglandin I2 receptor agonistic activities: its antiplatelet effects in human and animal platelets

Summary.  TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A₂ (TP) receptor antagonistic and prostaglandin I₂ (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC₅₀ values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 µg kg min⁻¹ or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.     

Increased biosynthesis of thromboxane A2 by diabetic platelets

Abstract. Because ADP has been reported to produce a secondary wave of platelet aggregation in diabetic subjects, and since ADP is known to enhance normal platelet biosynthesis of pro-aggregating thromboxane A₂, we tested whether or not the reported increased sensitivity of diabetic platelets to ADP may also result in increased platelet biosynthesis of thromboxane A₂. To test this hypothesis, ¹⁴C-arachidonic acid (¹⁴C-AA) was incubated in vitro with washed human platelets' in the presence or absence of ADP. These studies included platelets isolated from thirty normal volunteers, twenty-six diabetic subjects with and without known vascular complications, eighteen non-diabetic pregnant females and fourteen pregnancy-induced diabetic females. Data from these studies demonstrated: (i) a significant increase in the capability of diabetic platelets in response to ADP to biosynthesize thromboxane A₂ from arachidonic acid when compared to platelets from normal controls (P < 0.001); (ii) a significant increase in thromboxane A₂ biosynthesis by platelets from pregnancy-induced diabetic subjects over nondiabetic pregnant females (P < 0.001); (iii) a two-fold increase in thromboxane A₂ biosynthesis by platelets from diabetic subjects with vascular complications when compared to those without vascular complications. Although our data also showed approximately a twofold increase in thromboxane A₂ biosynthesis by platelets from diabetic subjects with greater than 10 years of the disease when compared to diabetic subjects with less than 10 years, these latter results were, however, not statistically significant. Results from these studies suggest that a relationship may exist between the markedly increased ADP-induced platelet aggregation in diabetes mellitus and the vascular complications associated with this disease. Whether or not increased capacity of the diabetic platelet to biosynthesize pro-aggregating thromboxane A₂ in response to ADP or other pro-aggregating agents is per se a triggering factor in occlusive vascular diseases reported in diabetic subjects must await further studies.     

Renal thromboxane A2 synthesis in the Lyon hypertensive rat

The aim of the present study was to assess the mechanisms by which norepinephrine (NE) increased the synthesis of prostanoids and revealed a hyperactivity of the Thromboxane (Tx) A2 synthase in the Lyon genetically hypertensive (LH) rat kidney. To this purpose, the effects of NE (1.2 x 10(-8) to 9.6 x 10(-7) M) on renal function and prostanoid synthesis were assessed in isolated perfused kidneys following beta-adrenoceptor blockade by sotalol (10(-5)M) and compared to those of equipressor concentration of an alpha 2-adrenoceptor agonist, BHT 933 (3.5 x 10(-4) M) and angiotensin II (AII) (7.7 x 10(-9) M). Kidneys were isolated from eight week-old male LH rats and from their normotensive (LN) and low blood pressure (LL) controls and perfused in a single pass system. In baseline conditions, sotalol did not modify renal function or urinary prostanoids in any of the three strains. Following NE stimulation, it potentiated the increase in renal vascular resistance of LL and LN controls but not that of LH rats. The pressure-natriuresis and the urinary prostanoids remained unchanged. BHT 933 elicited a weak stimulation of prostanoid release while AII markedly increased it and revealed, as did NE, the hyperactivity of the TxA2 synthase. It is concluded that the NE-induced stimulation of prostanoid synthesis does not involve beta-adrenoceptors and is unrelated to the associated hemodynamic changes. These results also demonstrate that the increased renal synthesis of TxA2 observed in LH rat kidney is not a specific response to alpha-adrenoceptor stimulation and is likely to involve activation of the phosphoinositide pathway.     

血浆血栓素A2和前列环素在不稳定型心绞痛发病中的意义

目的：探讨血管活性物质血栓素Ａ２（ＴＸＡ２）和前列环素（ＰＧＩ２）在不稳定型心绞痛发病中的作用。方法：测定５３例冠状动脉粥样硬化性心脏病（冠心病）患者血浆血栓素Ｂ２（ＴＸＢ２）和６酮前列腺素Ｆ１α（６ｋｅｔｏＰＧＦ１α）含量的变化。结果：不稳定型心绞痛３２例与稳定型心绞痛２１例比较，不稳定型心绞痛组血浆ＴＸＢ２的含量显著升高，为稳定型心绞痛组的３．９１倍（Ｐ＜０．００１）；ＴＸＢ２／６ｋｅｔｏＰＧＦ１α比值也明显高于稳定型心绞痛组（Ｐ＜０．００１）。结论：冠心病心绞痛发作时存在的血浆ＴＸＢ２含量异常增高，ＴＸＢ２与６ｋｅｔｏＰＧＦ１α平衡失调，导致冠状动脉舒缩障碍、痉挛等改变而致心绞痛发生。     

Bleeding tendency and impaired platelet function in a patient carrying a heterozygous mutation in the thromboxane A2 receptor

Summary. Background: Thromboxane A₂ receptor (TXA₂R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA₂R abnormality and investigate the mechanism for the impaired response to TXA₂. Patient: The proband (OSP‐2, PT) was a 7‐year‐old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm )‐induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA₂R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA₂R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA₂R. We then examined α_IIbβ₃ activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α_IIbβ₃ and Rap1B activation in the proband. In addition, platelet secretion as monitored by P‐selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y₁₂ has been shown to play a critical role in the sustained α_IIbβ₃ activation (Kamae et al. J Thromb Haemost 2006; 4 : 1379). As expected, small amounts of exogenous ADP (0.5 μm ) partially restored the sustained α_IIbβ₃ activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA₂R mutation is, at least in part, as a result of the decrease in ADP secretion.     

血栓素A2、前列环素与阻塞性睡眠呼吸暂停低通气综合征及其合并高血压的相关性研究

目的探讨阻塞性睡眠呼吸暂停低通气综合征(OSAHS)及其合并高血压患者血浆血栓素B2(TXB2)、6-酮前列腺素F1α(6-Keto-PGF1α)水平的变化.方法检测OSAHS及其合并高血压患者血浆TXB2、6-Keto-PGF1α水平并与正常人进行对照,将检测结果与睡眠呼吸监测指标进行相关性分析.结果 OSAHS患者血浆TXB2及TXB2/ 6-Keto-PGF1α(T/P)比值明显高于正常对照组[(77.37±20.13) ng/L vs (47.97±12.05) ng/L,(5.47±3.83) vs (1.73±0.99)](P<0.01),6-Keto-PGF1α浓度明显低于正常对照组[(20.07±12.11) ng/L vs (31.27±9.41) ng/L](P<0.01),且OSAHS合并高血压组与单纯OSAHS组比较血浆TXB2及T/P比值比较差异有统计学意义[(141.78±21.66) ng/L vs (77.37±20.13) ng/L,(11.06±6.97) vs (5.47±3.83)](P<0.01);血浆TXB2、6-Keto-PGF1α水平与OSAHS的病情严重程度有相关性.结论 OSAHS及其合并高血压患者存在TXA2与PGI2的失衡,这种变化在OSAHS合并高血压患者中更为明显.     

Increased production of platelet thromboxane B2 in non-insulin-dependent diabetes. Relationship to vascular complications

Abstract. Platelet thromboxane B₂ production was studied in forty-seven non-insulin-dependent diabetics by incubating platelets with increasing concentrations of arachidonic acid. In comparison with thirty-two healthy subjects, diabetics showed increased thromboxane B₂ production at 0·7 mmol/l (mean: 236 pmol/10⁸ platelets, SEM 201–277; v. 135, 105–174; P < 0·05) and at 1·0 mmol/l (673, 613–739; v. 405, 377–486, P < 0·01) but not at 0·5 mmol/l. Patients were subdivided according to the presence or absence of vascular complications. Patients without microangiopathy showed significantly greater thromboxane B₂ production than healthy subjects at all the arachidonic-acid concentrations ( P < 0·02 or less). Patients with microangiopathy had platelet thromboxane production similar to that observed in healthy subjects at all the arachidonic-acid concentrations ( P > 0·30) but significantly lower than that of non-microangiopathic patients at 0·5 ( P < 0·01) and at 0·7 mmol/l arachidonic acid ( P < 0·05). These results indicate that non-insulin-dependent diabetics have increased production of platelet thromboxane B₂ only when they do not have microvascular complications.     

Vascular reactivity to angiotensin II alone or combined with a thromboxane A2 mimetic in the isolated perfused kidney of Lyon hypertensive rats

The aim of this study was to evaluate whether thromboxane A₂‐prostaglandin H₂ (TP) receptor activation potentiates the renal vasoconstrictor effect of Angiotensin II (Ang II) in genetically hypertensive rats of the Lyon strain (LH). Concentration‐response curves (CRCs) to Ang II (5 pM to 10 nM ), to the specific TP receptor agonist U46619 (7.5–960 nM ) and to a mixture of Ang II + U46619 (fixed molar ratio of 1 : 9) were obtained in single‐pass perfused kidneys isolated from 8 week‐old LH and low blood pressure (LL) control rats. Baseline vascular resistance was significantly increased in LH compared to LL kidneys. Comparison of the CRCs obtained for Ang II and U46619 showed that, in both strains, Ang II was about 100 times more potent than U46619. For both drugs, the pD₂ or slope values did not differ among the two strains. Co‐activation of TP receptors, analyzed with the method of Pöch and Holzmann, tended to potentiate the effects of Ang II in LH but not in LL kidneys. In conclusion, isolated perfused kidneys of LH rat did not exhibit an increased vascular sensitivity to acute infusion of Ang II or U46619 compared to control LL ones. In addition, the results suggest that the interactions between Ang II and TP receptor agonist may differ among the two strains.     

Antithrombotic effects of S 18886, a novel orally active thromboxane A2 receptor antagonist

Summary. Platelet activation and thrombus formation play a critical role in the onset of acute coronary syndromes. Thromboxane A₂ (TxA₂) is among the different chemical modulators released by activated platelets. TxA₂ is considered one of the most powerful agonists for platelet activation. In addition, TxA₂ exerts a vasoconstrictor effect by serving as an agonist of the thromboxane receptor (TP) on the vascular smooth muscle cell membranes. The putative effect of TxA₂ on thrombosis is demonstrated by the clinical effectiveness of acetylsalicylic acid (ASA) in the prevention of acute coronary syndromes. Among the clinically used antiplatelet agents, clopidogrel has shown to be slightly more effective than ASA in the prevention of atherothrombotic events in patients with peripheral arterial disease, and is one of the most widely used after aspirin. The aims of the study were to study the antithrombotic effects of escalating doses of the TP‐receptor antagonist, S 18886 and to compare its effects with those achieved by the administration of ASA (5 mg kg^?1 day^?1), and clopidogrel (3 mg kg^?1 day^?1). The study was undertaken at high and low shear rate conditions using the Badimon perfusion chamber in a porcine model. Antithrombotic effects were assessed as changes on platelet and fibrin(ogen) deposition. The doses of 30 and 100 µg kg^?1 day^?1 were selected based on a previous platelet aggregation study. S 18886 shows a dose‐dependent antithrombotic response. The dose of S‐100 develops similar antithrombotic effects to those of clopidogrel and superior to those of aspirin. The antithrombotic effects were statistically significant at both studied shear rate conditions. Therefore, the orally active TP‐receptor antagonist, S 18886, appears to be a new and effective agent to prevent atherothrombotic complications.     

Cisplatin-induced renal effects and thromboxane A2 receptor blockade

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A₂ receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 ± 25 mL min⁻¹ ( P  = 0.049 vs. baseline) and effective renal plasma flow by 94 ± 150 mL min⁻¹ ( P  = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 ± 18 mL min⁻¹ ( P  = 0.050 vs. baseline) and in effective renal plasma flow by 26 ± 63 mL min⁻¹ ( P  = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.     

Residual platelet thromboxane A2 and prothrombotic effects of erythrocytes are important determinants of aspirin resistance in patients with vascular disease

Background: Permanent inactivation of cyclooxygenase‐1 and inhibition of platelet thromboxane A₂ (TxA₂) constitute the main mechanisms underlying the prevention of vascular disease by aspirin. Methods and Results: We studied platelet TxA₂ synthesis and its impact on platelet reactivity and platelet–erythrocyte [platelet‐rich plasma (PRP)–RBC] interactions in 533 aspirin‐treated patients with vascular disease. Seventy aspirin‐free and 16 aspirin‐treated normal subjects were evaluated as controls. Collagen (1 μg mL^?1)‐induced platelet activation (¹⁴C‐5HT release) and recruitment (proaggregatory activity of cell‐free releasates from activated platelets) were assessed in PRP, PRP + RBC, and whole blood (WB). TxA₂ was quantified in releasates from WB. Aspirin inhibited TxA₂ synthesis and platelet function in all patients, but to different degrees. Forty‐two patients (8%) displayed partial (<95%) inhibition of TxA₂ relative to that of aspirin‐free controls. They produced >3.5 ng mL^?1 TxA₂ and had higher platelet reactivity than 491 patients who had undetectable TxA₂ or produced residual TxA₂ (R‐TxA₂; ≤3.5 ng mL^?1). Patients with R‐TxA₂ were distributed into TxA₂ quartiles. Patients in the third and fourth quartiles had significantly elevated ¹⁴C‐5HT release in PRP, which was markedly amplified in PRP + RBC and WB. TxA₂ in the fourth quartile translated into increased platelet aggregation and recruitment. Significant correlations were found between R‐TxA₂ and platelet hyperfunction. Conclusion: Biochemical markers (TxA₂ synthesis, ¹⁴C‐5HT release) and biological assays (platelet aggregation and recruitment) used to monitor the aspirin effect in a large population of patients presenting with vascular disease have evidenced the importance of R‐TxA₂ and the prothrombotic effects of RBC in aspirin resistance.     

前列环素和血栓素A2在急性坏死性胰腺炎并发肾损害发病机制中的作用

目的：探讨前列环素（ＰＧＩ２）、血栓素Ａ２（ＴＸＡ２）在急性坏死性胰腺炎并发肾损害发病机制中的作用。方法：采用胰胆管结扎、胰腺被膜下注射５％牛磺胆酸钠复制急性坏死性胰腺炎肾损害大鼠模型,以放射免疫方法动态测定术后２４、４８小时肾静脉血浆和肾组织中血栓素Ｂ２（ＴＸＢ２）、ＰＧＩ２水平,并观察尿素氮（ＢＵＮ）、血肌酐（ＳＣｒ）的改变。结果：随着急性坏死性胰腺炎病程进展,肾静脉血浆和肾组织中ＴＸＢ２、６酮前列腺素１α（６ｋｅｔｏＰＧＦ１α）水平逐渐升高,ＴＸＢ２水平升高较６ｋｅｔｏＰＧＦ１α为甚,ＴＸＢ２／６ｋｅｔｏＰＧＦ１α比值增大,ＢＵＮ、Ｃｒ亦显著提高。结论：ＴＸＡ２和ＰＧＩ２平衡紊乱是急性坏死性胰腺炎并发肾损害的主要因素之一。     

Platelet and vessel associated prostacyclin and thromboxane A2/prostaglandin endoperoxide receptors

Synthetic stable analogues of thromboxane A2 (TXA2), cyclic endoperoxides (PGH2) and prostacyclin (PGI2) opened up new opportunities for investigating the mechanisms of action of these compounds. They proved to be useful pharmacological probes for characterizing PGI2 and TXA2/PGH2 receptors. Over the past few years, new synthetic antagonists with high specificity allowed the modulation of biological responses to endogenous eicosanoids. These compounds will, therefore, considerably promote our understanding of the biological function and significance of arachidonate metabolites. The present review summarizes current concepts that have arisen concerning platelet and vascular PGI2 and TXA2/PGH2 receptors, their transmembrane signal transduction, as well as their possible implications in the pathophysiology of cardiovascular disease.     

Increased platelet sensitivity and thromboxane B2 formation in type-II hyperlipoproteinaemic patients

Platelet aggregation induced by collagen, ADP and epinephrine, was monitored in 150 type-II patients (115 type IIA and 35 type IIB) and compared with a reference group of normolipidaemic controls; in addition, malondialydehyde formation and thromboxane B2 were examined in a subsample of the type-IIA patients. Threshold aggregatory concentrations were significantly lower in the whole group of type-II patients for all three aggregating agents; no difference in terms of aggregatory response was detected between platelets from type-IIA and -IIB patients. Only 56% of type-II patients, however, exceeded the 95th percentile of the threshold aggregatory concentrations in controls. The formation of malondialdehyde in platelet-rich plasma stimulated with thrombin and collagen, was significantly higher in platelets from type-IIA patients. The production of thromboxane B2 by platelets, from endogenous arachidonic acid in type-IIA patients, was significantly higher and exceeded the highest level found in controls.     

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background:  Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives:  We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results:  Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcγRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10-kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL⁻¹ soluble GPVI by ELISA (normal plasma, ∼15 ng mL⁻¹) and IgG purified from patient plasma caused FcγRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcγRIIa on normal platelets. C onclusions:  In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.