Neutrophil Extracellular Traps and Platelet Activation for Identifying Severe Episodes and Clinical Trajectories in COVID-19.

The role of NETs and platelet activation in COVID-19 is scarcely known. We aimed to evaluate the role of NETs (citrullinated histone H3 [CitH3], cell-free DNA [cfDNA]) and platelet activation markers (soluble CD40 ligand [CD40L] and P-selectin) in estimating the hazard of different clinical trajectories in patients with COVID-19. We performed a prospective study of 204 patients, categorized as outpatient, hospitalized and ICU-admitted. A multistate model was designed to estimate probabilities of clinical transitions across varying states, such as emergency department (ED) visit, discharge (outpatient), ward admission, ICU admission and death. Levels of cfDNA, CitH3 and P-selectin were associated with the severity of presentation and analytical parameters. The model showed an increased risk of higher levels of CitH3 and P-selectin for ED-to-ICU transitions (Hazard Ratio [HR]: 1.35 and 1.31, respectively), as well as an elevated risk of higher levels of P-selectin for ward-to-death transitions (HR: 1.09). Elevated levels of CitH3 (HR: 0.90), cfDNA (HR: 0.84) and P-selectin (HR: 0.91) decreased the probability of ward-to-discharge transitions. A similar trend existed for elevated levels of P-selectin and ICU-to-ward transitions (HR 0.40); In conclusion, increased NET and P-selectin levels are associated with more severe episodes and can prove useful in estimating different clinical trajectories.

Endothelial Damage, Neutrophil Extracellular Traps and Platelet Activation in COVID-19 vs. Community-Acquired Pneumonia: A Case-Control Study.

COVID-19 has been a diagnostic and therapeutic challenge. It has marked a paradigm shift when considering other types of pneumonia etiology. We analyzed the biomarkers related to endothelial damage and immunothrombosis in COVID-19 in comparison to community-acquired pneumonia (CAP) through a case-control study of 358 patients with pneumonia (179 hospitalized with COVID-19 vs. 179 matched hospitalized with CAP). Endothelial damage markers (endothelin and proadrenomedullin), neutrophil extracellular traps (NETs) (citrullinated-3 histone, cell-free DNA), and platelet activation (soluble P-selectin) were measured. In-hospital and 1-year follow-up outcomes were evaluated. Endothelial damage, platelet activation, and NET biomarkers are significantly higher in CAP compared to COVID-19. In-hospital mortality in COVID-19 was higher compared to CAP whereas 1-year mortality and cardiovascular complications were higher in CAP. In the univariate analysis (OR 95% CIs), proADM and endothelin were associated with in-hospital mortality (proADM: CAP 3.210 [1.698-6.070], COVID-19 8.977 [3.413-23.609]; endothelin: CAP 1.014 [1.006-1.022], COVID-19 1.024 [1.014-1.034]), in-hospital CVE (proADM: CAP 1.623 [1.080-2.439], COVID-19 2.146 [1.186-3.882]; endothelin: CAP 1.005 [1.000-1.010], COVID-19 1.010 [1.003-1.018]), and 1-year mortality (proADM: CAP 2.590 [1.644-4.080], COVID-19 13.562 [4.872-37.751]; endothelin: CAP 1.008 [1.003-1.013], COVID-19 1.026 [1.016-1.037]). In conclusion, COVID-19 and CAP showed different expressions of endothelial damage and NETs. ProADM and endothelin are associated with short- and long-term mortality.

Acute and sustained increase in endothelial biomarkers in COVID-19.

Endothelial injury is related to poor outcomes in respiratory infections yet little is known in relation to COVID-19. Performing a longitudinal analysis (on emergency department admission and post-hospitalisation follow-up), we evaluated endothelial damage via surrogate systemic endothelial biomarkers, that is, proadrenomedullin (proADM) and proendothelin, in patients with COVID-19. Higher proADM and/or proendothelin levels at baseline were associated with the most severe episodes and intensive care unit admission when compared with ward-admitted individuals and outpatients. Elevated levels of proADM or proendothelin at day 1 were associated with in-hospital mortality. High levels maintained after discharge were associated with reduced diffusing capacity.

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 × 10(9) 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.

The administration of a loading dose of aspirin to patients presenting with acute myocardial infarction while receiving chronic aspirin treatment reduces thromboxane A2-dependent platelet reactivity.

Abstract The optimal dose of aspirin for patients presenting with acute myocardial infarction (AMI) while receiving chronic aspirin therapy has not been clearly established. We evaluated whether continued treatment with 100 mg of aspirin or a loading dose (200-500 mg) influences thromboxane A2 (TX) suppression or platelet reactivity. Sixty-four consecutive patients with AMI and 98 healthy subjects (82 aspirin-free and 16 receiving 100 mg daily for a week) were evaluated. Treatment was at the discretion of the attending physician. Collagen (1 µg/ml)-induced TX synthesis, (14)C-serotonin-release, platelet aggregation, and the PFA-100 assay were evaluated. The platelet TX synthesis of patients receiving a loading dose of aspirin was sixfold lower than that of patients receiving 100 mg of aspirin (p<0.005). This was associated with marked reductions in (14)C-serotonin-release and arachidonic-acid-induced aggregation and an increase in the PFA-100 closure time (p<0.01). Categorization of patients according to their TX synthesis (<95% or ≥ 95% inhibition vs. healthy aspirin-free subjects) revealed that 8% of the patients treated with loading doses had a poor response (<95% inhibition) vs. 53% of those treated with 100 mg (p<0.001). Patients with lower TX inhibition had higher serum NT-Pro-BNP (p<0.005), a marker of poor left ventricular systolic function. Administration of a loading dose of aspirin to patients with AMI during existing chronic aspirin treatment induced greater reductions in platelet TX synthesis and TX-dependent platelet reactivity than the continued treatment alone.

Assessment of platelet function in acute ischemic stroke patients previously treated with aspirin.

BACKGROUND: Platelet inhibition measured by platelet function tests could be critical to understand the reasons for early recurrence and to guide therapeutic recommendations. We assess the platelet function during the acute phase of ischemic stroke in patients pretreated with aspirin who continue their treatment with aspirin only, are started on clopidogrel only, or add clopidogrel to aspirin. METHODS: Sixty-four patients were taking aspirin before the stroke. Depending on the administered antiplatelet, 3 groups were defined: ASA: patients who continued on aspirin orally or intravenous acetylsalicylate of lysine, n = 30; CLO: patients who discontinued aspirin and were started on clopidogrel, n = 16; and ASA + CLO: patients who were prescribed both aspirin and clopidogrel, n = 10. Collagen-induced thromboxane A2 (TXA2) synthesis, ADP (adenosine diphosphate)-induced aggregation, and occlusion time (PF-100) were measured. RESULTS: CLO group only had a marked elevation of TXA2 (17.44 ± 15.62 ng/mL, P = .000) and a shortening of the platelet function analyzer (PFA)-100 closure time (157.13 ± 88 seconds, P = .047) compared with the other 2 groups (ASA: TXA2, .62 ± 1.59 ng/mL; ASA + CLO: TXA2 1.79 ± 4.59 ng/mL). They achieved a small (13%) but significant reduction of ADP-induced aggregation (87.00 ± 23.06 mm, P = .008) compared with the ASA group (102.82 ± 22.38 seconds). CONCLUSIONS: Stopping aspirin intake within the first 72 hours of the acute stroke drastically increases TXA2 synthesis. During the same time window, the freshly prescribed clopidogrel manages to reduce the ADP-induced aggregation only slightly (13%). This study offers analytic proof that the common practice of replacing aspirin with clopidogrel does not leave stroke patients fully protected during the first days after an ischemic stroke. Possible solutions could be to preserve aspirin during a few days or to use loading doses of clopidogrel at hospital admission.

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.

Neutrophil extracellular traps (NETs) in patients with STEMI. Association with percutaneous coronary intervention and antithrombotic treatments.

BACKGROUND: Neutrophil extracellular traps (NETs) are formed by DNA, histones and proteolytic enzymes, and are produced by activated neutrophils through different mechanisms. In turn, NETs can activate platelets and coagulation cascade favoring thrombotic processes. The aims of this study were to analyze levels and kinetics of NETs in ST-segment elevation myocardial infarction (STEMI) patients and correlate them with antithrombotic therapy and cardiovascular outcomes at follow-up. METHODS: 150 consecutive STEMI patients referred to primary percutaneous coronary intervention (pPCI) were included. Citrate anticoagulated blood was extracted immediately before pPCI, 30 min and 24 h after the procedure. As markers of NETS cell free DNA (cfDNA), nucleosomes and citrullinated Histone 3 (citH3) were determined. 46 healthy subjects were included as controls. Patients were follow-up for 1.4 ± 0.56 years. RESULTS: Before pPCI, NETs markers were elevated in STEMI patients compared to healthy controls (p < 0.05); these increased significantly 30 min post pPCI (p ≤ 0.001) and decreased at 24 h but remained elevated compared with the control group (p < 0.05). Patients treated with bivalirudin presented a lower increase of NETs 30 min post pPCI compared to patients treated with heparin (p < 0.05). Cardiovascular risk factors or type of stent implanted did not modify NETs levels. Cit3H (HR = 3.74; 95%CI 1.05-13.4; p = 0.042) and left ventricular ejection fraction ≤35% (HR = 6.84; 95%CI 2-23; p = 0.002) were independent predictors of composite endpoint of myocardial infarction, stroke, stent thrombosis and/or cardiovascular-cause death. CONCLUSIONS: NETs were elevated in STEMI patients, increased by pPCI and decreased thereafter. One of the most specific NETs markers was associated with cardiovascular outcomes.

Partial inhibition of platelet thromboxane A2 synthesis by aspirin is associated with myonecrosis in patients with ST-segment elevation myocardial infarction.

Heterogeneity in response to aspirin (ASA) treatment, or "aspirin resistance," could be of importance in patients with ST-segment elevation myocardial infarction (STEMI). Decreased effects of ASA in platelets could be due to partial inhibition of cyclo-oxygenase-1 (COX-1) or to COX-1-independent mechanisms. We evaluated the effect of ASA treatment in patients with STEMI for (1) platelet thromboxane A(2) (TXA(2)) synthesis, (2) platelet recruitment elicited by TXA(2)-dependent and -independent mechanisms, and (3) a possible association of these aspects of platelet reactivity with serum markers of myonecrosis. We studied 62 ASA-treated patients within 48 hours of onset of the acute event and 69 ASA-free and 10 ASA-treated controls. TXA(2) synthesis and platelet recruitment (fluid-phase proaggregate activity of cell-free releasate) were assessed after collagen stimulation (1 micro g/ml) of whole blood. Partial inhibition of TXA(2) by ASA was found in 21 patients (34%). This was associated with significant increases in troponin T, creatine kinase-MB mass, creatine kinase, and recruiting activity versus 41 patients with blocked TXA(2) production. This was independent of fibrinolysis, and platelet COX-2 expression was not augmented. TXA(2) blockade was achieved after subsequent daily treatments or platelet incubation with ASA in vitro, suggesting lower sensitivity of COX-1 to ASA. In addition, 28 patients (45%) had an abnormally increased recruiting activity despite TXA(2) blockade, which was also associated with increased myonecrosis. In conclusion, ASA resistance, elicited by TXA(2)-dependent and TXA(2)-independent mechanisms, was prevalent in patients with STEMI. This study describes, for the first time, the association of partial platelet TXA(2) inhibition with myonecrosis.

Vasoactive properties of antihypertensive lactoferrin-derived peptides in resistance vessels: Effects in small mesenteric arteries from SHR rats.

AIMS: Bovine lactoferrin (LF) hydrolysates and peptides identified thereof have shown antihypertensive effects in rat models, mainly but not exclusively by angiotensin-converting enzyme inhibition. In this study we aimed to assess the vasoactive effects and mechanisms of an ultrafiltered (<3kDa) pepsin LF hydrolysate (LFH) and a heptapeptide identified in a LF hydrolysate produced by yeast proteolysis (DPYKLRP) in peripheral resistance arteries from spontaneously hypertensive rats (SHRs). MAIN METHODS: We used a myograph system for isometric tension recording in isolated small mesenteric arteries from SHRs. Direct vasoactive effects of LFH (30-100µg/mL) and DPYKLRP (30-100µM) were assessed in arteries precontracted with phenylephrine (PE, 10µM) or KCl (120mM), and in PE-precontracted arteries preincubated (10min) with the NO synthase inhibitor L-NAME (0.1mM) or the cyclooxygenase inhibitor indomethacin (10µM). Indirect vasoactive effects of LFH (30-100µg/mL) or DPYKLRP (30-100µM) preincubation (10min) on the relaxant responses to the NO donor sodium nitroprusside (SNP, 0.01-10µM) or acetylcholine (Ach, 1-100µM) were also studied in PE-precontracted arteries. KEY FINDINGS: Both LHF and DPYKLRP elicited direct relaxation of mesenteric arteries, by a mechanism involving NO release, counteracting modulation by prostanoids and K+ efflux. Moreover, LF-derived peptides also showed indirect vasoactive effects by enhancing endothelium-dependent relaxation to Ach and endothelium-independent relaxation to SNP. SIGNIFICANCE: In conclusion, LF-derived peptides show ex vivodirect and indirect relaxing effects in small mesenteric arteries from SHRs. These vasoactive effects would reduce vascular peripheral resistance in vivo, and thus contribute to their antihypertensive effects.

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.

Regulation of Platelet Function by Acetylation/Deacetylation Mechanisms.

Reversible acetylation of histones is a well-known mechanism of epigenetic regulation of gene expression. More recently, studies have demonstrated that acetylation/deacetylation in several proteins regulate multiple aspects of cellular activity, especially those associated with energetic metabolism. Platelets are key participants in haemostasis and cardiovascular diseases. Although metabolic changes such as diabetes or lipidemia are well recognized risk factors for cardiovascular diseases, there is very little information about the relationship between metabolism and platelet reactivity. Recent studies have reported that different aspects of platelet function such as adhesion, aggregation, or granule release could also be regulated by acetylation of proteins. These cycles of acetylation/deacetylation are regulated by the contrasting action of acetyltransferases and deacetylases, which have been described by the presence of p300 and HDAC6, and sirtuins, respectively, in platelets. Remarkably, deacetylases, especially sirtuins, have been the subjects of intensive pharmaceutical research due to their implication in several physiological and pathological processes in organisms. The discovery of acetylation mechanisms in platelets opens new possibilities for the treatment and prevention of cardiovascular diseases through the regulation of acetylases/deacetylases in platelets. Therefore, the aim of this review is to present some recent reports concerning the role of acetylation of proteins in the control of platelet function, and the new possibilities of regulation of platelet function that this represent.

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

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 91.5 ng/ml ± 1.20, 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.

Pharmacological inhibition of platelet reactivity. Clinical and pharmacodynamic effects.

Platelets play an important role in both normal hemostasis and pathological thrombus formation. The key role of platelets in thrombosis is highlighted by the clinical benefit of treatment with antiplatelet drugs. Aspirin, either alone or in combination with clopidogrel in high-risk patients, is the most widely used antiplatelet agent. However, there is an individual response to these agents that may reduce the cardiovascular protection in patients who achieve a lower antiplatelet effect. Recently, P2Y12 receptor antagonists more potent than clopidogrel (e.g., prasugrel and ticagrelor) have been approved for patients with acute coronary syndromes and those undergoing percutaneous coronary interventions; these drugs provide greater platelet inhibition than clopidogrel. However, the increased effectiveness of these treatments has underscored the importance of carefully balancing the risks of ischemia and bleeding to achieve the best clinical outcomes. The increased knowledge of the molecular mechanisms of platelet activation has prompted a search for novel pharmacological targets for the inhibition of platelet reactivity. This article reviews the molecular mechanisms of action and limitations of use of current and emerging antiplatelet agents for treatment of cardiovascular disease.

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.

Serine/threonine phosphatases regulate platelet αIIbß3 integrin receptor outside-in signaling mechanisms and clot retraction.

AIMS: We studied the role of serine/threonine phosphatases (PSTPs) on αIIbß3 signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on αIIbß3 signaling for regulation of platelet aggregation and clot retraction. MAIN METHODS: We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies. KEY FINDINGS: We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out αIIbß3 activation, platelet aggregation, and cytoskeletal reorganization. Among markers of αIIbß3 outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr(759) of the ß3 chain of αIIbß3, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation. SIGNIFICANCE: The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.

TXA2 synthesis and COX1-independent platelet reactivity in aspirin-treated patients soon after acute cerebral stroke or transient ischaemic attack.

INTRODUCTION: The pharmacological target of aspirin is the inhibition of cyclooxygenase-1 (COX1) and thromboxane-A2 (TX) synthesis. Very few data are available on TX assessment in patients with stroke. We studied platelet TX synthesis, COX1-independent platelet reactivity, the influence of platelet-erythrocyte interactions and the potential association between platelet responses and the severity of stroke, evaluated with a clinical score (NIHSS). MATERIAL AND METHODS: We examined 157 aspirin-treated patients with acute stroke or TIA, 128 aspirin-free and 15 aspirin-treated healthy subjects (HS). Collagen-induced TX, platelet recruitment in whole blood and platelets ± erythrocytes (haematocrit 40%) were assessed in patients on daily-aspirin within three days from onset. Arachidonic-acid-, ADP-, thrombin-receptor activating peptide TRAP-, and collagen-induced aggregation were also evaluated. RESULTS: Partial TX inhibition (<95% inhibition vs aspirin-free controls) was observed in 13% of patients. This was associated with marked increases in COX1-dependent responses (arachidonic-acid- and collagen-induced aggregation and platelet recruitment; P<0.0001) but not with differences in ADP- or TRAP-induced aggregation. Partial TX inhibition was independently associated with severe stroke (NIHSS ≥ 12) at both admission (P<0.05) and discharge (P<0.05). Among patients with fully blocked TX, those with elevated COX1-independent platelet reactivity (mean+2SD of aspirin-treated HS) were most likely to suffer severe stroke (P<0.05). Platelet-erythrocyte interactions enhanced platelet reactivity in these patients by COX1-dependent and -independent mechanisms (P<0.0001). CONCLUSIONS: TX inhibition by aspirin varied across patients. Partial TX inhibition and COX1-independent platelet hyperfunction were associated with more-severe stroke.