Antonio Moscardó

Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, αIIbβ3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets

BackgroundFibrinogen binding to the active conformation of the &agr;IIb&bgr;3 integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganization are important events in platelet function. Tyrosine phosphorylation of platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspects of platelet reactivity and their importance in cyclooxygenase (COX)-1–independent mechanisms in thrombin-stimulated human platelets. Methods and ResultsUsing washed platelets from normal donors and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conformational change of &agr;IIb&bgr;3, (2) actin polymerization and cytoskeletal reorganization, and (3) the quantity of tyrosine-phospho-rylated proteins associated with the reorganized cytoskeleton. The latter are important components of multimolecular signaling complexes. Concomitantly, platelet aggregation and secretion were significantly reduced. Aspirin did not affect receptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization. Importantly, aspirin significantly amplified the inhibitory effect of tyrphostin-A47 on all aspects of platelet reactivity that we evaluated. ConclusionsTyrosine protein phosphorylation is a regulatory control system of the inside-out mechanism of &agr;IIb&bgr;3 activation and cytoskeletal assembly in thrombin-stimulated human platelets. Inhibition of these aspects of platelet function with tyrphostin-A47 is amplified when platelets are treated with aspirin. Therefore, tyrosine phosphorylation is a major component of early signaling events and of COX-1–independent mechanisms of thrombin-induced platelet reactivity. The study results may indicate a novel target for therapeutic intervention.

Effect of Atorvastatin on Platelet Thromboxane A2 Synthesis in Aspirin-Treated Patients With Acute Myocardial Infarction

Inhibition of platelet thromboxane A(2) (TXA(2)) by aspirin is critical in patients with acute myocardial infarction (AMI), but some patients have persistent platelet TXA(2) production within 48 hours of the onset of AMI. Statins are known to reduce TXA(2) in aspirin-free patients with hypercholesterolemia. We hypothesized that treatment with aspirin plus atorvastatin could reduce persistent TXA(2) synthesis and aspirin resistance in patients with AMI. We evaluated platelet function in 184 aspirin-treated patients within 48 hours of the onset of AMI. Patients were divided into group A (treated with aspirin alone, n = 139) and group B (treated with aspirin plus atorvastatin, n = 45). We studied collagen-induced platelet TXA(2) synthesis, serotonin ((14)C-5HT) release and recruitment, and adenosine diphosphate-, arachidonic acid-, and collagen-induced platelet aggregation. Persistent TXA(2) synthesis was detected in 25% and 9% of groups A and B, respectively (p = 0.03). TXA(2), arachidonic acid-aggregation, and collagen-induced responses were significantly reduced in patients receiving dual treatment compared to those receiving aspirin monotherapy. Atorvastatin did not modify platelet reactivity in patients with efficiently blocked TXA(2) synthesis. These results strongly suggest a direct effect of the statin on platelet eicosanoid synthesis. This was confirmed in vitro by incubating washed aspirin-free and aspirin (1 muM)-treated platelets from normal subjects with 1 to 20 microM atorvastatin. Atorvastatin in vitro significantly reduced platelet TXA(2) synthesis and collagen-induced aggregation. In conclusion, atorvastatin combined with aspirin early in the onset of the acute event significantly reduced persistent TXA(2) and TXA(2)-dependent aspirin resistance. This could contribute to the clinical benefit of atorvastatin in patients with AMI.

Reduction of platelet cytosolic phospholipase A2 activity by atorvastatin and simvastatin: Biochemical regulatory mechanisms

UNLABELLED Statins have demonstrated effects beyond reducing cholesterol level that may contribute to their clinical benefit, including effects on platelet biochemistry and function. OBJECTIVES To explore and compare the antiplatelet effect of two lipophilic statins (atorvastatin and simvastatin) and one hydrophilic statin (pravastatin) concerning: a) collagen-induced platelet aggregation and thromboxane A2 (TXA2) synthesis; b) the additive effect of statins on TXA2 synthesis in platelets treated with a submaximally effective concentration of aspirin and c) the biochemical mechanisms involved. METHODS AND RESULTS Washed human platelets were incubated with statins (1-20μM), and stimulated with collagen (1μg/ml) or arachidonic acid (AA) (200μM) and TXB2 was quantified by ELISA. Incubation with simvastatin or atorvastatin reduced (36.2% and 31.0%, respectively) collagen-induced TXB2 synthesis (p<0.05) and platelet aggregation (p<0.001), whereas pravastatin had no effects. Simultaneous incubation with a submaximally effective concentration of aspirin (1μM) and atorvastatin or simvastatin significantly increased the inhibition of TXB2 synthesis by aspirin by 4.4- and 4.1-fold, respectively. Statins did not affect AA-induced TXB2 synthesis, excluding an effect on COX-1/TXA2 synthase activities. Atorvastatin and simvastatin concentration-dependently inhibited the collagen-induced increase in cytosolic calcium and the kinetics of cPLA2 phosphorylation. Lipophilic statins reduced phosphorylation of both ERK1/2 and p38 MAPK, which regulate cPLA2 phosphorylation and calcium movement. CONCLUSION We report for the first time a direct downregulation by atorvastatin and simvastatin of platelet cPLA2 activity through effects on calcium and MAPK, which reduce collagen-induced TXA2 synthesis. These mechanisms might contribute to their beneficial effects, even in aspirin-treated patients.

Regulation of cytosolic PlA2 activity by PP1/PP2A serine/threonine phosphatases in human platelets

Platelet thromboxane A2 (TXA2) synthesis is an important pathway of platelet reactivity. We report that in thrombin-stimulated platelets, PP1/PP2A serine/threonine phosphatases regulate phospholipase A2 (cPLA2) activity, which is required for TXA2 synthesis. Two mechanisms are involved: (a) constitutively active PP1/PP2A regulate cPLA2 phosphorylation, and (b) PP1/PP2A activity mediates agonist-induced increase in cytosolic Ca2+ ([Ca2+]i). Inhibition of PP1/PP2A with okadaic acid (OA) induces cPLA2 phosphorylation but reduces Ca2+ responses: release from intracellular stores and influx through the plasma membrane, particularly that mediated by store-mediated Ca2+ entry (SMCE). A significant correlation (r = 0.64) exists between OA-regulated [Ca2+]i and TXA2 synthesis. Okadaic acid-induced decrease in SMCE and the associated TXA2 synthesis are mediated by a reduction in protein-tyrosine phosphorylation. This reduction is not due to inhibition of tyrosine kinases but rather to an OA-mediated increase in tyrosine phosphatases. This is the first study to report that PP1/PP2A phosphatases are involved in the regulation of the two key elements in eicosanoid synthesis, [Ca2+]i and cPLA2 phosphorylation. Moreover, PP1/PP2A regulation of [Ca2+]i and tyrosine phosphorylation may be important for other calcium-dependent processes and/or signal transduction mechanisms in platelets.

Improvement of the circulatory function partially accounts for the neuroprotective action of the phytoestrogen genistein in experimental ischemic stroke

We tested the hypothesis that the phytoestrogen genistein protects the brain against ischemic stroke by improving the circulatory function in terms of reduced production of thromboxane A2 and leukocyte-platelet aggregates, and of preserved vascular reactivity. Ischemia-reperfusion (90 min-3 days, intraluminal filament) was induced in male Wistar rats, and functional score and cerebral infarct volume were the end points examined. Genistein (10mg/kg/day) or vehicle (β-cyclodextrin) was administered at 30 min after ischemia or sham-operation. Production of thromboxane A2 and leukocyte-platelet aggregates, as well as reactivity of carotid artery to U-46619 (thromboxane A2 analogue) and to platelet releasate was measured. At 3 days post-ischemia, both improvement in the functional examination and reduction in the total infarct volume were shown in the ischemic genistein-treated group. Genistein significantly reverted both the increased thromboxane A2 concentration and the increased leukocyte-platelet aggregates production found in samples from the ischemic vehicle-treated group. Both U-46619 and platelet releasate elicited contractions of the carotid artery, which were significantly lower in the ischemic vehicle-treated group. Genistein significantly restored both the decreased U-46619- and the decreased platelet releasate-elicited contractile responses. In conclusion, genistein protects the brain against an ischemia-reperfusion challenge, at least in part, by its beneficial effects on the circulatory function.

Residual cyclooxygenase-1 activity and epinephrine reduce the antiplatelet effect of aspirin in patients with acute myocardial infarction

Aspirin treatment is essential in patients with acute myocardial infarction (AMI) to block platelet thromboxane (TXA)₂ synthesis. Epinephrine is known to enhance platelet reactivity induced by other agonists and to be elevated in patients with AMI due to stress. Our objective was to study the influence of epinephrine on platelet TXA₂ synthesis in patients treated with aspirin for AMI at early onset (within 48 hours) and the potential biochemical mechanisms involved in the functional response. Washed platelets from 45 patients with AMI and 10 aspirin-free controls were stimulated with arachidonic acid (AA) or AA + epinephrine, and aggregation and TXA₂ synthesis were evaluated. Full platelet aggregation was recorded in 8/45 patients (18%) with a partial TXA₂ inhibition (86%) vs. the aspirin-free controls. Platelets from the remaining 37 patients did not aggregate to AA and had TXA₂ inhibition >95%. However, when platelets were simultaneously stimulated with AA + epinephrine, in 25/37 patients a large intensity of aggregation (73%) was observed and a 5.5-fold increase in TXA₂ synthesis, although this remained residual (<5% of aspirin-free controls). This residual-TXA₂ was critical in the functional response, as demonstrated by the complete inhibition by TXA₂ receptor blockade or additional aspirin in vitro. The phosphatidylinositol-3-kinase activity and the cytosolic calcium levels participated in this platelet response elicited by a receptor cooperation mechanism, while the Rho/p160(ROCK) pathway or the blockade of the ADP receptors (P2Y1, P2Y12) were without effect. Residual-cyclooxygenase -1 activity and epinephrine enhance TXA₂-dependent platelet function, which may reduce the clinical benefit of aspirin in patients with AMI.

Neutrophil extracellular traps are increased in patients with acute ischemic stroke: prognostic significance

Neutrophil extracellular traps (NETs) are networks of DNA, histones, and proteolytic enzymes produced by activated neutrophils through different mechanisms. NET formation is promoted by activated platelets and can in turn activate platelets, thus favoring thrombotic processes. NETs have been detected in venous and arterial thrombosis, but data in stroke are scarce. The aim of this study was to evaluate NETs in the plasma of patients with acute ischemic stroke and their potential association with baseline clinical characteristics, stroke severity, and one-year clinical outcomes. The study included 243 patients with acute ischemic stroke. Clinical and demographic data and scores of stroke severity (NIHSS and mRs) at onset and discharge were recorded. Markers of NETs (cell-free DNA, nucleosomes, and citrullinated histone 3 (citH3)), were determined in plasma. Patients were followed-up for 12 months after the ischemic event. NETs were significantly elevated in the plasma of patients with acute ischemic stroke when compared to healthy subjects. NETs were increased in patients who were over 65 years of age and in those with a history of atrial fibrillation (AF), cardioembolic stroke, high glucose levels, and severe stroke scores at admission and discharge. In multivariate analysis, elevated levels of citH3, the most specific marker of NETs, at onset were independently associated with AF and all-cause mortality at one-year follow-up. NETs play a role in the pathophysiology of stroke and are associated with severity and mortality. In conclusion, citH3 may constitute a useful prognostic marker and therapeutic target in patients with acute stroke.

Influence of COX-inhibiting Analgesics on the Platelet Function of Patients with Subarachnoid Hemorrhage

BACKGROUND Platelet function of patients with subarachnoid hemorrhage (SAH) may play an important part in both rebleeding and delayed cerebral ischemia, but little is known about aggregation pathways during the acute phase of stroke. Analgesics are used regularly in the first days after bleeding, and some can potentially inhibit the cyclooxygenase (COX) enzyme. We examined the platelet function of patients with SAH in order to describe their basal situation and determine whether the administration of intravenous nonsteroidal antiinflammatory drugs (NSAIDs) affected platelet aggregation. METHODS Arachidonic acid (AA)-induced aggregation and the platelet function analyzer (PFA)-100 test with collagen/epinephrine cartridges were used to study a group of SAH patients that was treated with dexketoprofen and dipyrone and to compare them to patients that had received no analgesia. RESULTS Ninety-six consecutive SAH patients prospectively enrolled in platelet studies. Twenty-seven patients were taking NSAIDs (10 on dexketoprofen and 17 on dipyrone), and there were 15 cases in the control group. AA-induced aggregation was 10% ± 3.2% for NSAIDs (mean ± standard error), specifically 17.2% ± 7% for dexketoprofen and 5.7% ± 1% for dipyrone. Aggregation in the control group was 72.4% ± 6% (P = .001). Both analgesics slowed the platelet plug formation during the PFA-100 test, with closure times of 237.2 ± 25 seconds for dexketoprofen and 198.4 ± 22 seconds for dipyrone and 138.1 ± 21 seconds in controls (P = .02). CONCLUSIONS The administration of COX-inhibiting analgesics leads to an hypoaggregability state in the first days of SAH. Further insight into their impact on complications such as rebleeding and delayed cerebral ischemia is needed in order to optimize the headache treatment of SAH.

Characterisation of DWI-MRI confirmed cerebral infarcts in patients with subarachnoid haemorrhage and their association with MMP-9 levels

Abstract Objectives: It has been suggested that metalloproteinase-9 (MMP-9) could predict the onset of cerebral vasospasm after subarachnoidal haemorrhage (SAH). The aim of this study was to analyse, in patients with SAH, the difference between patients with MRI ischaemic infarcts and patients without, and to investigate the role of metalloproteases as a prognostic factor for ischaemic infarcts. Methods: Sixty eight consecutive patients with SAH and diffusion-weighted magnetic resonance imaging (DWI-MRI) done 3 weeks after SAH. We define two groups, with and without DWI-MRI infarcts. Blood samples were taken at entry, 3 days and 1 week MMP-9 was determined through ELISA method. Results: Forty per cent were male, with a mean age of 54 ± 14 years. Twenty five patients, 36.8%, had DWI-MRI infarcts; in patients with MRI infarcts, SAH was more severe (Fisher = 4 52 vs 25.6%, P = 0.037), with more morbi-mortality (Rankin>3 48 vs 18.6%, P = 0.014), and more symptomatic vasospasm (28 vs 7%, P = 0.031). Levels of MMP-9 were higher than controls, but there were no significant differences between patients with and without infarcts (first determination no infarcts 39.40 ng/ml ± 35.40 vs infarcts 49.75 ng/ml ± 34.54, P>0.005, 3 days no infarcts 72.10 ng/ml ± 70.95 vs infarcts 62.28 ± 33.84, P>0.005, 1 week no infarcts 148.48 ng/ml ± 142.73 vs infarcts 91.51 ng/ml ± 41.20, P>0.005). Conclusion: Thirty eight percent in a well-studied series of patients with SAH have DWI-MRI infarcts; the infarcts were associated to SAH severity, SAH outcome and symptomatic vasospasm. Metalloproteinase-9 was higher in SAH patients than in controls, but it could not discriminate the infarct patients.

Platelet function in malignant hematological disorders.

Purpose of review In this article, we summarize the current knowledge on new roles played by platelets and their interactions with blood components, and their possible implications in malignant hematological disorders. Recent findings Recent reports in the literature are revealing that platelets are important partners in different aspects of physiology and pathophysiology beyond hemostasis and thrombosis, including but not restricted to inflammation, cancer or host defense. Moreover, several studies suggest that platelet interactions with other blood cells could regulate functional and biochemical responses of each other. Finally, platelet alterations in number as well as in function have been observed in different hematological disorders related with the action of treatments. Summary Common complications of leukemia are bleeding and thrombosis, in which the number and activity of platelets undoubtedly play an important role. Probably related with their apparent structural simplicity compared with other hematological cells, the interest in platelets in malignant hematological disorders has been mainly restricted to the determination of the number of circulating platelets. However, different studies have demonstrated that numbers of platelets between 6 and 80 × 109 platelets/l are a poor indicator of the risk of bleeding, as this number does not give any information on the functional activity of these platelets.