Sustained Platelet Activation Research Articles

Preferential and sustained platelet activation in COVID-19 survivors with mental disorders

Pre-existing mental disorders are considered a risk factor for severe COVID-19 outcomes, possibly because of higher vascular burden. Moreover, an unconventional platelet activation characterizes COVID-19 and contributes to inflammatory and thrombotic manifestations. In the light of the inflammation theory of mental disorders, we hypothesized that patients with mental disorders could be sensitive to the SARS-CoV-2 elicited platelet activation. We investigated platelet activation in 141 COVID-19 survivors at one month after clearance of the virus, comparing subjects with or without an established pre-existing diagnosis of mental disorder according to the DSM-5. We found that platelets from patients with a positive history of psychiatric disorder underwent unconventional activation more frequently than conventional activation or no activation at all. Such preferential activation was not detected when platelets from patients without a previous psychiatric diagnosis were studied. When testing the effects of age, sex, and psychiatric history on the platelet activation, GLZM multivariate analysis confirmed the significant effect of diagnosis only. These findings suggest a preferential platelet activation during acute COVID-19 in patients with a pre-existing psychiatric disorder, mediated by mechanisms associated with thromboinflammation. This event could have contributed to the higher risk of severe outcome in the psychiatric population.

High platelet adrenergic activity and concomitant activation of the pituitary/medullar axis as alarming laboratory parameters in ACS survivors-the STRESS-AMI study.

Kinetics of stress-related biological parameters were determined in acute coronary syndrome (ACS) patients undergoing complex cardiovascular rehabilitation. We determined platelet functionality in the absence/presence of a selective alpha-2 adrenergic receptor inhibitor, atipemazole parallel with salivary cortisol levels at enrolment, and at 3- and 12-months follow-up in 75 ACS patients with percutaneous coronary intervention. Pharmacological/non-pharmacological secondary prevention methods have been efficiently applied. Baseline aggregometry indicated platelet hyperactivity, decreasing gradually and being significantly reduced late, at 12 months (p < 0.05). Cortisol levels followed similar kinetics (p < 0.05). Baseline epinephrine-induced aggregations (EIA) significantly correlated with most of the other platelet agonists, even at subsequent time-points. Patients with upper-quartile EIA at enrolment (EIA-UQ) had significantly higher ADP- and collagen-induced aggregations at enrolment, at 3- and 12-months follow-up as well, indicating that high adrenergic response in the acute phase is accompanied by general platelet hyperactivity and predicts sustained platelet activation. In the EIA-UQ group higher cardiac biomarker release, elevated C-reactive protein and cortisol levels, and lower baseline left ventricular ejection fraction were detected.Atipemazole significantly reduced platelet aggregation induced by several platelet agonists, being most potent and comparable to full in vitro P2Y12 inhibition on collagen-induced aggregations (p < 0.05), indicating that catecholamines might serve as promt/long-term modulators of platelet function. Despite effective CCR programme and dual antiplatelet therapy, prolonged activation of sympathetic neuroendocrine system and general platelet hyperactivity can be detected up to one year in ACS patients with high adrenergic platelet activity. Moreover, initial high adrenergic activity is accompanied by clinical parameters associated to increased cardiovascular risk, therefore early identification of these patients might support complex optimal long-term therapy.

Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear

BackgroundCollagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. ObjectivesTo determine the importance of GPVI clusters for thrombus formation in whole blood under shear. MethodsWe utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. ResultsAutomated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. ConclusionLabeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.

Autoimmunity as a sequela to obesity and systemic inflammation.

The rising prevalence of obesity presents a world-wide challenge as it is associated with numerous comorbidities including cardiovascular disease, insulin resistance and hypertension. Obesity-associated illnesses are estimated to cause nearly 4 million deaths globally per year, therefore there is a critical need to better understand associated pathogenesis, identify new therapeutic targets, and develop new interventions. Emerging data identify a key role for chronic inflammation in mediating obesity related disease states and reveal higher incidence of autoimmune disease development. Of the multiple potential mechanisms linking obesity and autoimmunity, the strongest link has been shown for leptin, a hormone secreted at high levels from obese white adipose tissue. Numerous studies have demonstrated that leptin enhances activation of both arms of the immune system, while its absence protects against development of autoimmunity. Other potential newly discovered mechanisms that contribute to autoimmune pathogenesis are not directly connected but also associated with obesity including sustained platelet activation, gut dysbiosis, and aging. Here we review how obesity instigates autoimmunity, particularly in the context of immune cell activations and adipokine secretion.

Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization.

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05–0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Interactions of HIV and Antiretroviral Therapy With Neutrophils and Platelets

Neutrophils are important components of the innate immune system that mediate pathogen defense by multiple processes including phagocytosis, release of proteolytic enzymes, production of reactive oxygen species, and neutrophil extracellular trap formation. Abnormalities of neutrophil count and function have been described in the setting of HIV infection, with the majority of antiretroviral agents (ARVs), excluding zidovudine, having been reported to correct neutropenia. Questions still remain, however, about their impact on neutrophil function, particularly the possibility of persistent neutrophil activation, which could predispose people living with HIV to chronic inflammatory disorders, even in the presence of virally-suppressive treatment. In this context, the effects of protease inhibitors and integrase strand transfer inhibitors, in particular, on neutrophil function remain poorly understood and deserve further study. Besides mediating hemostatic functions, platelets are increasingly recognized as critical role players in the immune response against infection. In the setting of HIV, these cells have been found to harbor the virus, even in the presence of antiretroviral therapy (ART) potentially promoting viral dissemination. While HIV-infected individuals often present with thrombocytopenia, they have also been reported to have increased platelet activation, as measured by an upregulation of expression of CD62P (P-selectin), CD40 ligand, glycoprotein IV, and RANTES. Despite ART-mediated viral suppression, HIV-infected individuals reportedly have sustained platelet activation and dysfunction. This, in turn, contributes to persistent immune activation and an inflammatory vascular environment, seemingly involving neutrophil-platelet-endothelium interactions that increase the risk for development of comorbidities such as cardiovascular disease (CVD) that has become the leading cause of morbidity and mortality in HIV-infected individuals on treatment, clearly underscoring the importance of unraveling the possible etiologic roles of ARVs. In this context, abacavir and ritonavir-boosted lopinavir and darunavir have all been linked to an increased risk of CVD. This narrative review is therefore focused primarily on the role of neutrophils and platelets in HIV transmission and disease, as well as on the effect of HIV and the most common ARVs on the numbers and functions of these cells, including neutrophil-platelet-endothelial interactions.

Platelet and Endothelial Activation as Potential Mechanisms Behind the Thrombotic Complications of COVID-19 Patients

The authors hypothesized that the cytokine storm described in COVID-19 patients may lead to consistent cell-based tissue factor (TF)-mediated activation of coagulation, procoagulant microvesicles (MVs) release, and massive platelet activation. COVID-19 patients have higher levels of TF+ platelets, TF+ granulocytes, and TF+ MVs than healthy subjects and coronary artery disease patients. Plasma MV-associated thrombin generation is present in prophylactic anticoagulated patients. A sustained platelet activation in terms of P-selectin expression and platelet-leukocyte aggregate formation, and altered nitric oxide/prostacyclin synthesis are also observed. COVID-19 plasma, added to the blood of healthy subjects, induces platelet activation similar to that observed invivo. This effect was blunted by pre-incubation with tocilizumab, aspirin, or a P2Y12 inhibitor.

GENETIC OVERLAP AND CAUSATION: RECONCEPTUALIZING LINKS BETWEEN MENTAL AND PHYSICAL HEALTH

Vascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline.A total of 143 individuals (70.4 ± 6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry.Significant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean + 2SD of the Grade 0 group, and the odds ratio of the Grade 2 + 3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score.Elevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.

GAS6/TAM Pathway Signaling in Hemostasis and Thrombosis.

The GAS6/TYRO3-AXL-MERTK (TAM) signaling pathway is essential for full and sustained platelet activation, as well as thrombus stabilization. Inhibition of this pathway decreases platelet aggregation, shape change, clot retraction, aggregate formation under flow conditions, and surface expression of activation markers. Transgenic mice deficient in GAS6, or any of the TAM family of receptors that engage this ligand, exhibit in vivo protection against arterial and venous thrombosis but do not demonstrate either spontaneous or prolonged bleeding compared to their wild-type counterparts. Comparable results are observed in wild-type mice treated with pharmacological inhibitors of the GAS6-TAM pathway. Thus, GAS6/TAM inhibition offers an attractive novel therapeutic option that may allow for a moderate reduction in platelet activation and decreased thrombosis while still permitting the primary hemostatic function of platelet plug formation.

CD62-mediated activation of platelets in cerebral white matter lesions in patients with cognitive decline

BackgroundVascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline. MethodsA total of 143 individuals (70.4±6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry. ResultsSignificant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean+2SD of the Grade 0 group, and the odds ratio of the Grade 2+3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score. ConclusionElevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.

Abstract 57: Platelet Extracellular-signal-regulated Kinase 5 is a Redox Switch which Regulates Myocardial Infarct Expansion via Matrix Metalloproteinases

Background: Dysregulated platelet activation in an ischemic microvascular environment may play a role in myocardial infarction (MI). Platelet receptor signaling is well-characterized, but mechanisms of receptor-independent activation, such as by reactive oxygen species (ROS) generated in ischemic conditions, are less well understood. We discovered that ERK5, a nuclear protein which is ROS-activated in others cells, is abundantly present in platelets. We investigated whether ERK5 could regulate platelet activation and thrombosis in healthy and diseased states. Methods: Human and mouse platelets were stimulated with agonists including ADP, U46619, TRAP, convulxin, or ROS (H 2 O 2 or 5% O 2 ). ERK5 activity was assessed by immunoblotting. Platelet activation was assessed via fluorescent-activated cell sorting (FACS) for P-selectin or activated GPIIb/IIIa. Intravascular thrombus (pulmonary embolus) or mesenteric thrombus (oxidative injury) formation was assessed by ex vivo fluorescent imaging and in vivo intravital microscopy, respectively. MI was performed in wild-type (WT) and in platelet specific ERK5 deficient (ERK5 -/- ) mice by LAD coronary artery ligation. Left ventricular (LV) function was determined by echocardiography. Matrix metalloproteinase (MMP) activity was determined by in-gel zymography. Results: Human and platelet ERK5 was activated by ROS and via the thrombin and thromboxane receptors, but not via the purinergic or collagen receptors. Murine in vivo thrombosis was regulated by platelet ERK5 only if the injury involved oxidative stress. MI in mice promoted sustained platelet activation over one week in an ERK5-dependent manner. Following MI, platelet ERK5 -/- mice had less reactive platelets, less platelet MMP activity and thromboxane production, attenuated MMP activity in the LV, less remodeling with smaller infarcts, and enhanced myocardial systolic performance. Conclusions: ERK5 is an ischemic sensor in platelets which regulates ongoing platelet activation after MI as well as remodeling via myocardial microvasculature. These observations may explain ischemic microvascular aberrations like the no-reflow phenomenon following percutaneous coronary intervention, suggesting a novel pharmacologic target.

Analysis of Protease Activated Receptor 4 (PAR4) By Histidine Hydrogen Deuterium Exchange

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered ligand that interacts with a binding pocket on the receptor. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. We now describe the first biophysical studies examining this unique mode of activation using PAR4. PAR4 is one of the thrombin receptors on platelets and is required for sustained platelet activation. Recent data from our laboratory and others suggest that PAR4 has the potential to be a therapeutic target. To examine the tethered ligand activation mechanism in general and further examine PAR4 as a target, we have expressed and purified PAR4 that does not contain additional stabilizing sequences from Sf9 cells for biophysical studies. The current study uses histidine-hydrogen deuterium exchange (His-HDX). His-HDX is a structural mass spectrometry technique that relies on the slow exchange of the C2 carbon on the imidazole ring of histidine. PAR4 has nine histidine residues that are well spaced throughout the protein, which gives us a global view of solvent accessible and non-accessible regions. Unique peptides were generated that contained each of the histidine residues (His136, His159, His180, His229, His240, His269, His280, His306, and His380). These peptides were used to determine the t1/2 for each His residue in full length or thrombin cleaved PAR4. The t1/2 values for His159 and His269 were greater than 40 hours in both states indicating that these two histidine residues are not exposed to solvent in either case. In contrast, the thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have a decrease in solvent accessibility upon thrombin treatment. These residues are spread throughout the protein, which indicates that PAR4 undergoes a global conformational change and the overall structure becomes more rigid. In agreement with these data, thrombin cleaved PAR4 is more resistant to protease digestion by chymotrypsin. The half-life was extended 4-fold over the uncleaved PAR4. We are also able to make conclusions regarding specific regions of PAR4. His229 is located in extracellular loop 2 near the proposed ligand-binding site and has a 2-fold increase in t1/2 following thrombin treatment. These data suggest that the tethered ligand may be binding to this region and protecting it from exchange. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysical approaches. These studies shed light on the overall conformational changes that follow activation of PARs by a protease. Finally, the His-HDX used for purified PAR4 lays important groundwork that will allow us to examine PAR4 and other platelet GPCRs in their native membrane environment using this and related mass spectrometry approaches. DisclosuresNo relevant conflicts of interest to declare.

The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

Mean Platelet Volume and Long-Term Mortality in Patients Undergoing Percutaneous Coronary Intervention

Increased platelet activity is associated with adverse cardiovascular events. The mean platelet volume (MPV) correlates with platelet activity; however, the relation between the MPV and long-term mortality in patients undergoing percutaneous coronary intervention (PCI) is not well established. Furthermore, the role of change in the MPV over time has not been previously evaluated. We evaluated the MPV at baseline, 30 days, 60 days, 90 days, 1 year, 2 years, and 3 years after the procedure in 1,512 patients who underwent PCI. The speed of change in the MPV was estimated using the slope of linear regression. Mortality was determined by query of the Social Security Death Index. During a median of 8.7 years, mortality was 49.3% after PCI. No significant difference was seen in mortality when stratified by MPV quartile (first quartile, 50.1%; second quartile, 47.7%; third quartile, 51.3%; fourth quartile, 48.3%; p= 0.74). For the 839 patients with available data to determine a change in the MPV over time after PCI, mortality was 49.1% and was significantly greater in patients with an increase (52.9%) than in those with a decrease (44.2%) or no change (49.1%) in the MPV over time (p <0.0001). In conclusion, no association was found between the baseline MPV and long-term mortality in patients undergoing PCI. However, increased mortality was found when the MPV increased over time after PCI. Monitoring the MPV after coronary revascularization might play a role in risk stratification.

Platelets release matrix metalloproteinase-2 in the coronary circulation of patients with acute coronary syndromes: possible role in sustained platelet activation

To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of β-thromboglobulin (β-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A₂ (sPLA₂), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both β-TG and sPLA₂ correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

CalDAG-GEFI is at the nexus of calcium-dependent platelet activation

AbstractThe importance of the second messengers calcium (Ca2+) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca2+ sensor(s). We here identify Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca2+-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A2 (TxA2) release. CalDAG-GEFI–dependent TxA2 generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca2+-induced integrin activation, TxA2 generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI– and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.

Sustained platelet activation following intracoronary beta irradiation

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Uniform platelet activation exists before coronary stent implantation despite aspirin therapy

Background Platelets play an important role in the natural history of coronary artery disease. Enhanced platelet aggregation and receptor expression unquestionably occur after coronary stent implantation; however, the functional characteristics of platelets before stenting have not been fully elucidated. Methods Platelets were assessed before intervention by platelet-rich plasma aggregation (PA) with 5 mmol adenosine diphosphate (ADP) and 1 mg/mL collagen; whole blood aggregation (WBA) by 1 mg/mL collagen; shear-induced closure time (CT); contractile force (CF); and expression of 9 surface receptors by flow cytometry in 126 patients undergoing elective coronary artery stent placement. All patients received aspirin for at least 7 days. The data were compared with those from 64 healthy volunteers. Results Each test revealed sustained platelet activation in patients undergoing coronary stenting compared with control values. These differences were significant for collagen-induced PA (P =.031); CF (P =.0001); expression of glycoprotein (GP) IIb/IIIa (P =.0001); P-selectin (P =.0008); platelet/endothelial cell adhesion molecule (PECAM)-1 (P =.0001); CD107a (P =.0001); CD107b (P =.0004); and CD63 (P =.009). Conclusion Platelets are indeed activated before coronary stenting despite antecedent therapy with aspirin. (Am Heart J 2001;142:611-6.)

PTCA: periprocedural platelet activation. Part II of the Duesseldorf PTCA platelet study (DPPS)

Percutaneous transluminal coronary angioplasty (PTCA) with its various manipulations could create a dangerous, sudden haemostatic response. This study was performed to investigate PTCA-induced periprocedural changes in platelet activation and its consequences. Twenty-five consecutive patients admitted for elective PTCA were preclassified as having or not having circulating activated platelets. Blood samples were taken for platelet activation marker analysis before, six times during and 2 h after PTCA. Intravascular platelet activation was analysed by flow cytometry to measure activation-dependent surface markers thrombospondin, P-selectin (CD62) and lysosomal GP53 (CD63). PTCA was associated with a significant reduction of peripheral platelet count. The initiation of the PTCA procedure led to a significant loss of more than 50% of the degranulated, activated platelets. After PTCA, the number of degranulated, activated platelets uniformly increased. We conclude that PTCA can induce consumption, particularly of preactivated platelets, and lead to sustained platelet activation after the procedure. This might explain why preactivated patients are at increased risk of suffering periprocedural ischaemic events and why increased thrombogenicity favours acute flow disruption and the progression of coronary stenosis at the lesion site.

Thromboxane metabolite excretion in patients with hand-arm vibration syndrome.

As chronic exposure to hand-held vibrating tools may cause endothelial injury, a subsequent sustained platelet activation with the increased release of vasoconstricting thromboxane A2 (TxA2) could be of pathophysiological importance in vibration-induced Raynaud's phenomenon. Therefore, the aim of this study was to elucidate whether or not hand-arm vibration syndrome is accompanied by increased endogenous TxA2 biosynthesis. The study involved 64 men, aged 23-61 years, stratified according to the exposure to vibrating tools, the presence of Raynaud's phenomenon, and smoking habit. Forty of them were car mechanics and 24 were age-matched healthy volunteers who served as controls. The assessment of platelet TxA2 formation in vivo was performed by quantification of the urinary excretion of its major metabolite, 2,3-dinorthromboxane B2 (2,3-dinor-TxB2), employing gas chromatography-mass spectrometry. The average urinary excretion rate of 2,3-dinor-TxB2 in patients with Raynaud's phenomenon was 296 +/- 42 pg/mg creatinine and did not differ significantly from the corresponding values in controls (328 +/- 62 pg/mg creatinine) or individuals exposed to vibrating tools, but without any signs of vasospastic disease (232 +/- 29 pg/mg creatinine). The only statistically significant difference was found between smokers and non-smokers (P < 0.001), a finding confirming the existence of chronic platelet dysfunction in cigarette smokers. The present data indicate that chronic exposure to vibrating tools, with or without Raynaud's phenomenon, is not associated with an enhanced platelet function as monitored by the urinary excretion of 2,3-dinor-TxB2. Hence, a possible vibration-induced vascular injury does not seem to provide a stimulus sufficient to induce a persistent platelet activation.