BACKGROUND

Pathogen contamination, causing transfusion-transmitted diseases, is an ongoing concern in transfusion of cellular blood products. In this explorative study, the pathogen-inactivating capacity of UVC irradiation in platelet (PLT) concentrates was investigated. The dose dependencies of inactivation of several viruses and bacteria were compared with the effect on PLT quality.

METHODS

The potential of UVC irradiation was studied with a range of lipid-enveloped (LE) and non-lipid-enveloped viruses (NLE) and bacteria. LE viruses were bovine viral diarrhea virus (BVDV), human immunodeficiency virus (HIV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and vesicular stomatitis virus (VSV). NLE viruses were canine parvovirus (CPV) and simian virus 40 (SV40). Bacteria were Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Bacillus cereus. After spiking and irradiation, samples were tested for residual infectivity and reduction factors (RFs) were calculated. Furthermore, the effect of UVC irradiation on PLT quality was determined by measuring in vitro quality variables.

RESULTS

A UVC dose of 500 J per m(2) resulted in acceptable PLT quality (as measured by pH, lactate production, CD62P expression, and exposure of phosphatidylserine) and high RFs (>4 log) for CPV, TGEV, VSV, S. epidermidis, S. aureus, and E. coli. Intermediate RFs (approx. 3 log) were observed for BVDV, PRV, and B. cereus. Low RFs (approx. 1 log) were found for HIV and SV40. No differences in virus reduction were observed between cell-free and cell-associated virus.

CONCLUSIONS

UVC irradiation is a promising pathogen-reducing technique in PLT concentrates, inactivating bacteria, and a broad range of viruses (with the exception of HIV) under conditions that have limited effects on PLT quality. Further optimization of the UVC procedure, however, is necessary to deal with blood-borne viruses like HIV.

The formation of platelet-neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet-neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet-neutrophil microaggregate formation in developing hemodialysis-associated complications.

Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin αIIβI was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet-neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the αIIβI-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet-neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulation of platelets, and inhibits the formation of platelet-neutrophil aggregates. These immuno-regulatory effects of C1q may have a crucial role in innate immunity and inflammation.

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.

OBJECTIVE

To investigate whether platelets are activated in asthmatics with increased release of preformed mediators and to investigate the influence of oral administration of theophylline on them.

METHODS

Comparison of the intracellular free calcium concentration ([Ca2+]i) in platelets as an indicator of platelet activation, CD62P expression on platelets, and the chemokine regulated upon activation in normal T cells expressed and presumably secreted (RANTES) level in platelet-rich buffer supernatants between asthmatics and normal subjects.

METHODS

The respiratory outpatient clinics, Hiroshima University, Japan.

METHODS

Twenty-five normal volunteers, 19 asthmatics taking no oral drugs associated with asthma treatment (group A), and 18 asthmatics taking oral theophylline (group B).

RESULTS

While the resting [Ca2+]is in platelets were similar among the three groups, the [Ca2+]is in group A were significantly higher than those in normal subjects (p<0.05) and group B (p<0.01) after thrombin or 9,11-epithia-11,12-methano-thromboxane A2 (STA2) stimulation in the absence of external Ca2+. The CD62P expression level and RANTES level in group A after STA2 stimulation were significantly higher than those in normal subjects and group B (p<0.05).

CONCLUSIONS

We conclude that agonist-mediated activation of platelets is augmented in asthmatics resulting in enhanced release of chemokine such as RANTES, which could be suppressed by oral administration of theophylline.

Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the alpha-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment.

BACKGROUND

Platelet and neutrophil activation plays a crucial role in reperfusion injury. To determine whether platelet and neutrophil activation occurs in the coronary circulation after cold cardioplegic arrest and reperfusion, we studied 22 patients undergoing coronary artery bypass or valve procedures, or both procedures.

METHODS

Blood was sampled from the coronary sinus and the radial artery (A) before bypass; (B) immediately after cross-clamp release; and (C) 5 minutes after cross-clamp release; and was analyzed for surface markers of platelet (CD62P) and neutrophil (CD11b) activation.

RESULTS

During bypass, platelet activation increased significantly (p < 0.01) over prebypass values, but no difference was seen between arterial and coronary sinus samples. Neutrophil activation also increased significantly (p < 0.001) during bypass, but there was no difference between arterial and coronary sinus samples.

CONCLUSIONS

Cellular activation occurs locally in the coronary circulation during bypass, but no more so after cold cardioplegic arrest and reperfusion.

Specific molecules including inflammatory cell adhesion molecules mediate attachment of blood leukocyte and platelets to the endothelium and mononuclear cell migration into the arterial intima. However, the clinical significance of soluble cell adhesion molecules very early in the course of acute coronary syndrome is not known. We assayed platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), intercellular adhesion molecule-1 (ICAM-1, CD54), and P-selectin (CD62P) in plasma obtained from 20 patients within 3 h after the onset of acute myocardial infarction (AMI); 16 patients with unstable angina pectoris; 20 patients with stable angina pectoris, and 28 controls. Blood samples were obtained on hospital admission and again 1 week after onset of AMI and unstable angina, and on admission in patients with stable angina and controls. Plasma PECAM-1 concentration (ng/ml) on admission was higher in patients with AMI (25.6+/-4.7) and unstable angina (24.7+/-4.4) than in stable angina (20.5+/-4.4) and control (18.8+/-3.8) groups. In both AMI and unstable angina, plasma PECAM-1 had decreased significantly by 1 week (AMI, 20.8+/-4.0; unstable angina, 21.0+/-4.1). Plasma ICAM-1 concentration (ng/ml) on admission was higher in patients with AMI (254+/-70), unstable angina (264+/-78), and stable angina (245+/-68) than in controls (201+/-56), but did not differ between the three coronary syndromes. Plasma P-selectin concentration did not differ between the four groups, including controls. Therefore, soluble PECAM-1 concentration may be a sensitive markers providing early diagnostic aid in acute coronary syndromes.

OBJECTIVE

Platelets play a key role in haemostasis and wound healing, contributing to formation of vascular plugs. They are also involved in formation of atherosclerosic plaques. Some traditional diets, like the Mediterranean diet, are associated with a lower risk of cardiovascular disease. Components in these diets may have anti-platelet functions contributing to their health benefits.

METHODS

We studied the effects of alperujo extract, an olive oil production waste product containing the majority of polyphenols found in olive fruits, through measurement of effects on platelet aggregation and activation in isolated human platelets, and through identification of changes in the platelet proteome.

RESULTS

Alperujo extract (40 mg/L) significantly decreased in vitro ADP- (p = 0.002) and TRAP- (p = 0.02) induced platelet activation as measured by the flow cytometry using the antibody for p-selectin (CD62p), but it did not affect the conformation of the fibrinogen receptor as measured by flow cytometry using the antibodies for anti-fibrinogen, CD42a and CD42b. Alperujo extract (100 mg/L) inhibited both collagen- and TRAP-induced platelet aggregation by 5% (p < 0.05), and a combination of hydroxytyrosol and 3,4-dihydroxyphenylglycol were, at least partly, responsible for this effect. Proteomic analysis identified nine proteins that were differentially regulated by the alperujo extract upon ADP-induced platelet aggregation. These proteins represent important mechanisms that may underlie the anti-platelet effects of this extract: regulation of platelet structure and aggregation, coagulation and apoptosis, and signalling by integrin αIIb/β3.

CONCLUSIONS

Alperujo extract may protect against platelet activation, platelet adhesion and possibly have anti-inflammatory properties.

OBJECTIVE

To investigate the inflammatory response within intact carotid plaques from carotid eversion endarterectomy (CEE) to determine the relationship between immunohistological plaque morphology and ischaemic cerebrovascular symptoms.

METHODS

Intact CEE plaques from 71 patients with high-grade (>70%) stenosis undergoing CEE (group I, symptomatic, n=42; group II, asymptomatic, n =29) and 12 normal postmortem arteries (control group) were analysed with specific antibodies to inflammatory cells (T-Lymphocytes (CD3, CD4), cytotoxic T-cells (CD8), B-lymphocytes (CD20), natural killer cells (CD57), macrophages (CD68)), endothelial adhesion molecules (ICAM-1 (CD54), P-selectin (CD62P), E-selectin (CD62E), VCAM-1 (CD106) and T-lymphocyte co-stimulatory molecule (CD40)) and procoagulatory modulators (thrombomodulin (CD141), tissue factor (CD142)). Both groups were matched for gender, age, risk factors, degree of carotid artery stenosis. Plaques were measured using a semiquantitative score system in a blinded fashion by two observers. Statistical analysis of the group differences were performed by using the Kruskal-Wallis test and the Multitest Procedure with Permutation-Testing. Significance was taken as a p<0.05.

RESULTS

There were significantly more inflammatory cells, an overexpression of P-selectin and the procoagulatory markers thrombomodulin and tissue factor in symptomatic compared to both asymptomatic plaques and the ones of the control group. In both groups there was no significance for ICAM-1, VCAM-1, macrophages and co-stimulatory molecule CD40. There was also no significance for any factor between the asymptomatic and the control group. However, the differences between the symptomatic and the asymptomatic group were highly significant for all factors.

CONCLUSIONS

These data suggest that structural changes and inflammatory damage within the individual plaque seems to be a critical step in promoting plaque rupture with embolic sequelae.

Our objective was to evaluate the hemocompatibility of biodegradable stent fibers, employing a closed-loop circulation system filled with human blood. We also investigated the effects of the anti-inflammatory and anti-proliferative drugs curcumin and paclitaxel, incorporated into stent fibers. Fresh whole blood was circulated in four parallel closed-loop systems: the empty tube circuit (control) and tubes containing either a PLLA fiber coil (PLLA), a curcumin-loaded PLLA coil (C-PLLA) or a paclitaxel-loaded PLLA coil (P-PLLA). The influence of PLLA fiber, alone or loaded with drug incorporated during melt-extrusion, on leukocyte and platelet adhesion and activation was determined by flow cytometry. The effects of blood flow and fiber properties on cell deposition were assessed by scanning electron microscopy (SEM). The flow cytometry results clearly demonstrated that PLLA triggers blood cell activation at the site of deployment, as shown by increases in CD11b, CD62P and leukocyte-platelet aggregates, compared to controls. Curcumin and paclitaxel treatments both significantly reduced leukocyte and platelet activation and adhesion to PLLA fibers, as shown by flow cytometry and SEM. Activated leukocytes and platelets revealed significantly lower CD11b and CD62P receptor binding for C-PLLA compared with PLLA alone, and slightly lower for P-PLLA. Reductions in platelet-leukocyte aggregates were observed as well. In addition, there was less leukocyte and platelet adhesion to C-PLLA, compared with PLLA fiber controls, as shown by SEM. A continuous linear thrombus, composed of platelets, leukocytes, red blood cells and fibrin was occasionally detected along the line of tangency between the coil and the tube wall. Flow separation and eddying, proximal and distal to the line of tangency of coil and tube, is thought to contribute to this deposit. Curcumin was more effective than paclitaxel in reducing leukocyte and platelet activation and adhesion to PLLA stent fibers in this setting. However there was evidence of paclitaxel degeneration during melt extrusion that may have inhibited its effectiveness. Incorporation of the anti-inflammatory and anti-proliferative drug curcumin into bioresorbable stent fibers is proposed to prevent thrombosis and in-stent restenosis.

Platelet activation and circulating platelet-leucocyte complexes increase in vascular ischemic events and autoimmune inflammatory diseases. Platelet activation markers and platelet-leucocyte complexes were evaluated in primary Raynaud's phenomenon (RP) and in RP secondary to systemic sclerosis (SSc). Whole-blood flow cytometry was utilized to quantify CD62P, platelet microparticles (PMP), platelet-monocyte complexes (PMC) and platelet-neutrophil complexes (PNC) in primary RP and in SSc patients with secondary RP. SSc patients with secondary RP had significantly higher platelet CD62P expression than primary RP patients and controls (P = 0.017 and 0.004, respectively). Primary and secondary RP patients had higher mean PMC and PNC levels than controls (all P < or = 0.001). PMP level in SSc patients with pulmonary hypertension was significantly higher than in others (P = 0.048). All parameters were similar in SSc patients with and without digital ulcers, aspirin-users and nonusers (P>> 0.05). CD62P level decreased significantly after iloprost administration in four patients with digital ulcers (16.1 +/- 17.4 vs 7.4 +/- 3.8%, P = 0.03). Our results suggest there is platelet-leucocyte complex formation in RP, and, despite antithrombotic therapy, platelet activation and platelet-leucocyte interaction are ongoing in SSc. This is important as it might have potential therapeutic implications with respect to using antiplatelet drugs in SSc.

OBJECTIVE

Endothelial dysfunction (ED) plays a role in diabetic cardiovascular complications. Hyperglycemia increases cytockines involved in vascular inflammation. Inhibition of phosphodiesterase type 5 (PDE5) exerts a relaxation on corpora cavernosa and has cardioprotective properties. The effect of chronic sildenafil treatment, on ED markers and metabolic parameters in a non-randomized study on men with type 2 diabetes (T2DM), was investigated.

METHODS

Twenty-eight T2DM patients (61.2 ± 7.8 years, hemoglobin A1c (HbA1c) 7.9 ± 1.3%, duration of diabetes 11.5 ± 7.8 years) were treated with sildenafil 100 mg/d for 3 months. Baseline and postprandial glycemia, insulin, HbA1c, HOMA index, lipids, glomerular filtration rate, homocysteine were assessed at each visit. P-selectin (CD62P), CD14/42b, CD14/41, ICAM (CD54), PECAM (CD31) and CD11b/CD18, were evaluated, after monocyte isolation with flow-cytometry, before and after treatment.

RESULTS

After 3 months, sildenafil decreased P-selectin (p < 0.05), post-prandial glycemia (p < 0.01), HbA1c (p < 0.01), low-density lipoprotein cholesterol (p < 0.01) and increased high-density lipoprotein (p < 0.05).

CONCLUSIONS

PDE5 inhibition, in T2DM patients, reduces the endothelial function marker P-selectin and exerts a beneficial effect on glycometabolic control.

In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

OBJECTIVE

We sought to determine whether platelet activity in patients with heart failure is related to an ischemic versus nonischemic etiologic condition, clinical disease severity, or adverse clinical outcomes.

BACKGROUND

Platelet activity may affect outcome in patients with heart failure. A prospective evaluation of the relation of baseline platelet function to etiologic condition, New York Heart Association (NYHA) class, and clinical outcomes has not been previously reported.

METHODS

Ninety-six consecutive outpatients with ambulatory heart failure with an ejection fraction <0.40 and NYHA Class II to IV symptoms who presented to the Duke Heart Failure Clinic and 14 healthy control subjects formed the study groups. Baseline characteristics and blood analyzed for thromboxane (Tx) B2, 6-keto PGF(1alpha), platelet contractile force, adenosine diphosphate/collagen shear-induced closure time, whole blood aggregation and CD41, CD31, CD62p, and CD51/CD61 by flow cytometry were determined. Survival status and hospitalizations were determined in the heart failure patient cohort.

RESULTS

The median age of patients was 65 years (22% female, 64% white). An ischemic etiologic condition was present in 61% of patients. The population had mild to moderate heart failure: NYHA class I (1%), II (41%), III (46%), and IV (12.5%) and severe ventricular dysfunction (median ejection fraction = 0.20). There were 39 clinical events (7 deaths, 3 cardiac transplants, 29 other first hospitalizations) in 305 median days of observation. Platelet activity, indicated by whole blood aggregation with 5 micromol adenosine diphosphate (P =.04) and Tx B2 (P =.01), was higher in patients with heart failure. Whole blood aggregation was greater than the 90th percentile in 22% of patients with heart failure versus 7% of control subjects. Platelet function did not differ for any of the markers between the ischemic and nonischemic groups and was not affected by antecedent aspirin. There was no relation of NYHA class or the occurrence of events to platelet activity.

CONCLUSIONS

Platelet activity is heightened in 22% of outpatients with stable heart failure symptoms and is not affected by antecedent aspirin therapy. The degree of platelet activation is similar in ischemic and nonischemic patients with heart failure and is not related to clinical disease severity. Current methods to assess platelet activation do not appear to predict outcome.

BACKGROUND

Platelets play an important role in atherosclerosis and thromboembolic events. We examined the relationship between platelet activity and outcomes after an ischemic stroke.

METHODS

Using flow cytometry, we serially measured the fractions of circulating platelet activity (CD62p expression) after an ischemic stroke in early (<48 h), recent (day 7), convalescent (day 21) and chronic (day 90) phases in 92 consecutive patients with an ischemic stroke. Patients were classified into high (CD62p expression >3.16%) and low (CD62p expression < or =3.16%) platelet activity groups according to the median value of CD62p expression in the early phase of a stroke.

RESULTS

The composite end point--death, recurrent stroke and severe neurological impairment (alive in care), defined as a score of >13 on the National Institutes of Health Stroke Scale--within the first 30 days and at an interval of 8.2 +/- 1.5 months of follow-up was determined for each group. In the first 30 days, the composite end point occurred in 37.0% of patients in the high platelet activity group as compared with 6.5% in the low platelet activity group (p = 0.0004). At a mean follow-up of 8.2 +/- 1.5 months, the composite end point occurred in 36.6% of patients in the high platelet activity group as compared with 10.9% in the low platelet activity group (p = 0.0044). Multiple stepwise logistic regression analysis displayed that high platelet activity (p = 0.011), age (p = 0.013) and the presence of coronary artery disease (p = 0.021) were independently associated with adverse outcomes at the intermediate-term follow-up.

CONCLUSIONS

Results of this study showed that high platelet activity is strongly associated with adverse clinical outcomes after an early ischemic stroke.

Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.

Tumor-associated thrombosis is the second leading risk factor for cancer patient death, and platelets activity is abnormal in cancer patients. Discovering the mechanism of platelet activation and providing effective targets for therapy are urgently needed. Cancer cell- derived IgG has been reported to regulate development of tumors. However, studies on the functions of cancer cell-derived IgG are quite limited. Here we investigated the potential role of cancer cell-derived IgG in platelet activation. We detected the expression of CD62P on platelets by flow cytometry and analyzed platelet function by platelets aggregation and ATP release. The content of IgG in cancer cell supernatants was detected by enzyme-linked immune sorbent assay. The distribution of cancer-derived IgG in cancer cells was analyzed by immunofluorescence assay. Western blot was performed to quantify the relative expression of FcγRIIa, syk, PLCγ2. The interaction between cancer cell-derived IgG and platelet FcγRIIa was analyzed by co-immunoprecipitation. The results showed that higher levels of CD62P were observed in cancer patients' platelets compared with that of healthy volunteers. Cancer cell culture supernatants increased platelet CD62P and PAC-1 expression, sensitive platelet aggregation and ATP release in response to agonists, while blocking FcγRIIa or knocking down IgG reduced the activation of platelets. Coimmunoprecipitation results showed that cancer cell-derived IgG interacted directly with platelet FcγRIIa. In addition, platelet FcγRIIa was highly expressed in liver cancer patients. In summary, cancer cell-derived IgG interacted directly with FcγRIIa and activated platelets; targeting this interaction may be an approach to prevent and treat tumor-associated thrombosis.

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive experiments, as the binding protein for VacA. Plasmon resonance confirmed their binding, and dot blot analysis revealed that the peptide sequence AA 321-340 of multimerin 1 is the binding site for VacA. In conclusion, we propose a new interaction between multimerin1 and VacA , which may give another insight into H. pylori-induced platelet activations under H. pylori infection.

OBJECTIVE

EMMPRIN (CD147) is a matrix metalloproteinase inducer present on leukocytes and recently identified on platelets in vitro. We examined platelet CD147 expression in vivo and in correlation with markers of platelet activation and coronary artery disease (CAD).

METHODS

This prospective observational study involved 70 subjects (55 patients with CAD and 15 controls). Platelet CD62P expression, PAC-1 expression, platelet-leukocyte aggregates and CD147 (both platelet and leukocyte) expression were assessed by flow cytometry, and soluble CD62P expression was assessed by enzyme-linked immunosorbent assay. A full blood count and high-sensitivity C-reactive protein test were performed.

RESULTS

CD147 was expressed on 20.45% +/- 1.63% (mean +/- standard error of the mean) of circulating platelets, whereas CD62P and PAC-1 were expressed on 0.87% +/- 0.12% and 0.90% +/- 0.27% of platelets, respectively. Platelet CD147 expression correlated with CD62P expression (r = 0.359, P = 0.002), PAC-1 expression (r = 0.428, P < 0.001), leukocyte CD147 expression (monocyte, r = 0.416, P = 0.001; granulocyte, r = 0.434, P < 0.001), C-reactive protein level and neutrophil/lymphocyte ratio (NLR). CAD patients had significantly higher CD147 mean fluorescence intensity than controls on circulating platelets (2.41 +/- 0.14 vs. 2.87 +/- 0.09, P = 0.014), monocytes (8.57 +/- 1.20 vs. 12.3 +/- 0.57, P = 0.006) and granulocytes (4.30 +/- 0.65 vs. 6.50 +/- 0.34, P = 0.005). Age adjustment eliminated the association between platelet CD147 expression and CAD, but the association between leukocyte CD147 expression and CAD persisted. According to multivariate analysis, the independent predictors of platelet CD147 expression were monocyte CD147 expression, NLR and age.

CONCLUSIONS

Platelet CD147 expression is evident in vivo and correlates moderately with traditional platelet activation markers and leukocyte CD147 expression. Platelet CD147 expression shows a stronger association with age, and leukocyte CD147 expression a stronger association with clinical CAD, suggesting differences in the regulation of platelet and leukocyte CD147 expression in vivo.

Infection with group B streptococcus (GBS) is the most common cause of early onset neonatal sepsis in many countries, leading to neonatal morbidity and mortality. There is much evidence for a direct involvement of platelets in the pathogenesis of inflammation and sepsis. Several bacteria are known to directly interact with platelets leading to activation and aggregation, a phenomenon also observed with GBS. Here, we demonstrate that GBS rapidly bound to platelets; however, only strains isolated from septic patients bound fibrinogen on their surface and induced platelet thromboxane synthesis, platelet aggregation, and P-selectin (CD62P) expression. In contrast, GBS strains isolated from healthy newborns or healthy pregnant women induced only shape change, but not platelet thromboxane synthesis, platelet aggregation, or CD62P expression. All GBS strains investigated were able to activate FcgammaRIIA receptor signaling pathways including phospholipase C gamma2 (PLCgamma2), as well as calcium/calmodulin-dependent myosin kinase II (CaMKII) and phosphorylation of myosin light chain (MLC). In contrast, protein kinase C (PKC) was exclusively activated by GBS strains isolated from septic patients, and p38 mitogen activated protein kinase (p38 MAP kinase) was preferentially activated by septic GBS strains. Furthermore, stress signaling kinase SEK1/MKK4 and focal adhesion kinase (FAK) were activated by all tested GBS strains in a FcgammaRIIA-independent way. This study demonstrates that septic, but not colonizing, GBS strains bind fibrinogen on their surface, and that septic GBS strains influence platelet function not only via the FcgammaRIIA receptor, but also via pathways distinct from IgG-mediated signalling. These mechanisms lead to platelet aggregation and secretion, thereby possibly modulating the pathophysiologic course of GBS infections.

The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na⁺/H⁺ exchanger (NHE) was also explored.

MAC and PLT were isolated from peripheral blood of healthy subjects and incubated with/without SGLT2i [empagliflozin (EMPA) and dapagliflozin (DAPA) 1-100 μM] to assess their effects on SA (100 μM)-induced readouts of inflammation, oxidant stress and apoptosis in MAC and on expression of PLT activation markers by flow-cytometry after ADP-stimulation. Potential NHE involvement was tested with amiloride (aspecific NHE inhibitor) or cariporide (NHE1 inhibitor). Differences among culture conditions were identified using one-way ANOVA or Friedman test.NHE isoforms (1,5-9), but not SGLT2 expression, were expressed in MAC and PLT. EMPA and DAPA (100 μM) significantly reduced SA-induced inflammation (IL1β, TNFα, MCP1), oxidant stress (SOD2, TXN, HO1), but not apoptosis in MAC. EMPA and DAPA (both 1 μM) reduced PLT activation (CD62p and PAC1 expression). SGLT2i effects were mimicked by amiloride, and only partially by cariporide, in MAC, and by both inhibitors in PLT.EMPA and DAPA ameliorated lipotoxic damage in stearate-treated MAC, and reduced ADP-stimulated PLT activation, potentially via NHE-inhibition, thereby pointing to plaque stabilization and/or thrombosis inhibition as potential mechanism(s) involved in SGLT2i-mediated cardiovascular protection.

BACKGROUND

In addition to their cholesterol lowering ability, statins have proven pleiotropic effects in the cardiovascular system. Chronic inflammation with interactions between platelets and endothelial cells leads to an upregulation of activity markers of atherosclerosis. The purpose of this study was to investigate the effects of simvastatin and atorvastatin on platelets and endothelial cells in an in vitro endothelial cell model.

RESULTS

After a 24 h incubation period with either simvastatin or atorvastatin (1 μmol/L), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide (LPS), and were then incubated in direct contact with activated platelets. Platelet surface expression of CD40L and CD62P and expression of ICAM-1, VCAM-1, uPAR and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MMP-1. The increased expression of VCAM-1 and uPAR on endothelial cells by stimulation with LPS and by direct contact with activated platelets was significantly reduced to a similar extent through pre-incubation with both atorvastatin and simvastatin (p < 0.05). Platelets without endothelial cell contact, but in direct contact with either statin, showed similar significant reductions in surface expression of CD40L (p < 0.005).

CONCLUSIONS

These effects may explain the ability of statins to reduce the progression of atherosclerosis in addition to their cholesterol-lowering properties.

We evaluated platelet and endothelial activation parameters in psoriatic arthritis (PsA), a disease reported to be associated with the development of endothelial dysfunction and increased atherosclerotic complications. Twenty patients with PsA, eight psoriasis and 20 healthy controls were included into the study. The patients' clinical features and acute phase parameters were assessed. In all patients and controls, platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), and basal and ADP-stimulated P-selectin expression were determined with flow cytometry; soluble E-selectin (sE-selectin) and soluble CD40L (sCD40L) were determined with ELISA. Patterns of joint involvement and degrees of skin involvement in PsA patients were assessed. PMC in PsA patients were significantly higher than in the control group (p = 0.02). PNC were not significantly different among the three groups (p values>> 0.05). sE-selectin levels in both PsA and psoriasis groups were significantly higher than in healthy controls (p values, respectively, <0.001 and 0.023). Basal and ADP-stimulated CD62P expression and sCD40L level were similar in all groups (p values>> 0.05). Polyarticular PsA patients had significantly higher sCD40L than oligoarticular plus spondylitic PsA groups (p = 0.04). sCD40L level was higher in active PsA group than in inactive PsA group (p = 0.03). Groups with limited and extensive skin involvement did not differ significantly in the evaluated parameters. C-reactive protein (CRP) level in PsA patients correlated with sCD40L (r = 0.69, p = 0.012), basal CD62P expression (r = 0.89, p < 0.001) and ADP-stimulated CD62P expression (r = 0.73, p = 0.001). Endothelial activation might be have a role in the pathogenesis of both psoriasis and PsA. Among parameters of platelet activation, only PMC might play a role in the pathogenesis of PsA.