Formation Of Platelet-monocyte Complexes Research Articles

Distinct phenotypes of platelet, monocyte, and neutrophil activation occur during the acute and convalescent phase of COVID-19

SARS-CoV-2 has spread rapidly worldwide, causing the COVID-19 pandemic. Platelet activation and platelet-leukocyte complex formation are proposed to contribute to disease progression. Here, we report platelet and leukocyte activation during acute and convalescent COVID-19 in patients recruited between May-July 2020. Blood samples were analyzed by flow cytometry and ELISA using paired comparison between inclusion (day 0) and 28 days later. The majority of patients were mildly or moderately ill with significantly higher cytokine levels (IL-6 and IL-10) on day 0 as compared with day 28. Platelet activation and granule release were significantly higher on day 0 compared with day 28, as determined by ADP- or thrombin-induced surface CD62P expression, baseline released CD62P, and thrombin-induced platelet-monocyte complex formation. Monocyte activation and procoagulant status at baseline and post activation were heterogeneous but generally lower on day 0 compared with day 28. Baseline and thrombin- or fMLF-induced neutrophil activation and procoagulant status were significantly lower on day 0 compared with day 28. We demonstrate that during the acute phase of COVID-19 compared with the convalescent phase, platelets are more responsive while neutrophils are less responsive. COVID-19 is associated with thromboembolic events where platelet activation and interaction with leukocytes may play an important role.

Characterization of platelet signature on monocytes isolated from HIV infected individuals

Abstract Background Higher levels of platelet-monocyte complexes (PMCs) are detected in human immunodeficiency virus (HIV)-infected individuals. Such complexes are implicated in expansion of monocytes exhibiting inflammatory phenotype, and their translocation into tissues, thus promoting development of cerebrovascular and cardiovascular disorders. Here we have recruited small sub-set of HIV-infected individuals to investigate how platelets cause epigenetic changes in monocytes of HIV-infected individuals. We posit that the formation of PMCs facilitates monocyte differentiation via exchange of biomaterial between activated platelets and monocytes during HIV-infection. Methods We performed ATAC-seq to measure changes in chromatin accessibility from two types of monocytes, PMC-derived or non-PMC-derived, isolated from HIV-infected persons. Results Using ATAC-Seq data, we were able to detect distinct changes in chromatin accessibility patterns within the genomic regions of proinflammatory mediators in PMC-derived monocytes. Chromatin accessibility of inflammatory genes was dramatically increased in PMC-derived monocytes. This suggested to us a possibility of active exchange of molecules that causes such alterations in monocytes. Conclusions Increases in chromatin accessibility of pro-inflammatory genes occur in the PMC-derived monocytes in persons with HIV. This change is likely due to the transfer of platelet antigens to monocytes via cell-cell interaction, suggesting a novel mechanism through which platelets trigger inflammatory response in monocytes. Thus, our initial findings pave a way that has a strong diagnostic as well as therapeutic value.

Combined Aerobic and Resistance Exercise Training Reduces Acute Exercise‐Induced Platelet‐Monocyte Complex Formation in Overweight, Postmenopausal Women

Inactivity‐related diseases such as cardiovascular disease (CVD) involve a complex interplay among endothelial cells, leukocytes and activated platelets in response to vascular injury. Activated platelets are involved in atherogenesis and can promote an inflammatory phenotype of both the vasculature and leukocytes, particularly monocytes. Platelet‐monocyte complexes (PMCs) are markers of in vivo platelet activation and may be early indicators of subclinical inflammation.PURPOSETo examine the influence of combined aerobic and resistance exercise training on platelet activation, as measured by PMCs, and to characterize the acute exercise‐induced response of PMCs in a population at risk for CVD.METHODSThirty‐eight overweight‐obese (BMI: 32.8 ± 4.6 kg×m−2, age: 64 ± 5.1 years) post‐menopausal women were randomly assigned to either an exercise (EX, n=20) or education control (ED, n=18) group. EX performed 2 sets of 8 resistance exercises and 25 minutes of treadmill walking three times a week for 12 weeks while ED attended education classes (CPR certification, invited authors, health‐related talks) twice weekly for the same duration. Before (BT) and after (AT) the intervention, EX performed a single exercise bout during which blood was obtained pre‐exercise, immediately post‐exercise, 1‐hour and 2 hours post‐exercise. Blood was obtained at the same time points in resting ED. PMCs were identified via flow cytometry and analyzed in each monocyte phenotype. Monocyte phenotypes were defined as: Mon1 (CD14++CD16−), Mon2 (CD14++CD16+), and Mon3 (CD14loCD16++). All events positive for both CD14 and CD42a (marker for platelets) were considered PMCs.RESULTSExercise training resulted in a significant increase in VO2max (BT: 21.1 ± 0.85, AT: 23.7 ± 0.90 ml×kg−1×min−1, p < 0.001) and leg press strength (BT: 70 ± 4.1, AT: 99 ± 5.9 kg, p < 0.001), while no changes in fitness were observed in ED. Exercise training (EX: BT = 27.6 ± 2.7, AT = 23.7 ± 2.9 %) prevented the observed increase in percent PMC in ED (ED: BT = 20.9 ± 2.9, AT = 26.4 ± 3.2 %, p = 0.029). We observed a group x training interaction where exercise training reduced mean PMC percentage across all time points (EX: BT = 27.6 ± 2.4, AT = 22.1 ± 2.8 %, p = 0.034) while ED did not change (ED: BT = 20.2 ± 2.5, AT = 23.6 ± 2.9 %). PMCs formed with Mon1 monocytes followed a similar response (p = 0.031), but no significant changes were observed with PMCs formed in Mon2 or Mon3. A single exercise bout did not influence PMCs in any phenotype.CONCLUSIONOur results indicate that 12 weeks of combined exercise training reduces PMC formation in obese, postmenopausal women; however, a single bout of exercise did not significantly change percent PMC. The increase in PMCs in ED after the intervention is difficult to explain, but may be related to the time of year of the BT and AT experimental trials. These data may provide evidence that exercise training reduces CVD risk via reductions in platelet activation.Support or Funding InformationTCU Invests in Scholarship Grant; TCU Research and Creative Activities FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Intravenous and Oral N-Acetylcysteine Treatment in a Patient with Sickle Cell Disease at Disease Baseline

Background: We previously showed that patients with sickle cell disease (SCD) have elevated levels of von Willebrand factor (VWF) with high adhesive activity and the level of total active VWF correlated with hemolysis at disease baseline. These findings suggest that VWF plays a role in the pathophysiology of SCD [Chen et al, Blood. 2011; 117:3680‐3]. We also showed that N-acetylcysteine (NAC), a mucolytic and antioxidant drug, can reduce the level of large VWF multimers and platelet adhesion in human plasma in vitro and in mice in vivo [Chen et al, JCI, 2011, 121:522]. Based on these findings, we hypothesized that administration of NAC in SCD patients might reduce VWF multimers in vivo and reduce oxidative stress by scavenging reactive oxygen species (ROS). We started a pilot study to evaluate the effects of intravenous (IV) and oral NAC administration in SCD patients at disease baseline (NCT01800526). In this report, we administered NAC to one patient by IV and orally and compared the effect on biomarkers by the two routes of NAC administration.Patient and protocol: The subject is a 51-year-old male with SS genotype, a history of leg ulcers and acute chest syndrome, and who was not taking hydroxyurea at the time of the study. NAC was infused at a low dose (150 mg/kg over 8 hours) and infusion was repeated at high dose (300 mg/kg over 8 hrs) after 4 weeks. Blood samples were collected before (0), during (4 hr) and after the NAC infusions (8, 24 and 72 hr). Five months after the high dose IV infusion, during which parameters returned to baseline levels, oral NAC was administered for 4 weeks at 2.4g/day. Weekly blood samples were collected four weeks before the start of oral NAC, during oral NAC, and one week after oral NAC administration. We measured RBC parameters and RBC fragments, dense cells and percentage of platelet-monocyte complexes (PMC) by flow cytometry. We also analyzed VWF antigen, VWF multimers, ADAMTS13 antigen and activity levels in plasma. We also measured total, oxidized and reduced forms of NAC, cysteine and glutathione levels in plasma and whole blood by mass spectrometry.Results: 1) The subject did not experience adverse side effect during and after the treatment period. 2) RBC, hemoglobin and hematocrit levels did not significantly change with either IV or oral NAC treatment. Oral and IV NAC decreased dense cell and RBC fragments. NAC reduced the number of dense cells 13% during low dose IV, 79% during high dose IV and 73% with the oral administration. Compared to baseline, RBC fragments decreased 40% during low dose IV, 28% during high dose IV, and 27% with the oral NAC treatment. 3) NAC reduced formation of PMC from 71% to 24% (low dose IV), from 72% to 35% (high dose IV), and from 62% to 27% (oral). 4) VWF multimer size significantly decreased during low and high dose NAC infusions but did not change with oral NAC administration. VWF antigen and ADAMTS13 activity levels did not change with either routes of NAC administration. 5) The concentration of NAC in whole blood increased during treatments: elevated to 0.5 mM and 1 mM at the end of low- and high-dose IV infusions, respectively. At the conclusion of oral administration, the NAC concentration was 4 µM. Total glutathione concentrations in whole blood did not change significantly during and after NAC treatment at either dose or by either route. Free Cys concentrations in plasma increased compared to respective baseline levels 10-fold for low dose infusion (from 10 µM to 101 µM), 20-fold for high dose infusion (from 9 µM to 200 µM), but the level did not change significantly for oral administration (from 5 µM to 7 µM). Protein-bound Cys levels in plasma as markers of oxidative stress were significantly decreased with low- and high-dose IV infusion. However, the level did not change significantly with oral NAC administration.Summary: Both IV and oral administration of NAC were safe and well tolerated in the patient with SCD. Both IV and oral administration reduced the number of dense cells, RBC fragmentation, and formation of PMCs. IV, but not oral NAC, reduced VWF multimer size. These finding suggest that IV administration resulted in higher concentration and had more biologic effects; however both approaches may have a role in treatment of patients with SCD. DisclosuresKonkle:Sangamo: Research Funding; Gilead: Consultancy; Shire: Research Funding; Genentech: Consultancy; CSL Behring: Consultancy; Bioverativ: Research Funding; BioMarin: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding.

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications

A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease

▪We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD.VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522).In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry.Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood.Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. DisclosuresNo relevant conflicts of interest to declare.

Intracoronary platelet and monocyte activation status within platelet-monocyte complexes are determinants of inflammation in ST elevation myocardial infarction1.

Platelet Monocyte Complexes (PMCs) are commonly expressed in coronary artery disease but their pathologic significance in ST elevation myocardial infarction (STEMI) is unclear. This study evaluates the relationship between locally activated PMCs and intracoronary inflammation in stable and unstable coronary disease. Micro catheter aspirated blood samples of 15 STEMI and 7 stable angina patients are collected from the coronary artery (CA), aorta (AO) and right atrium (RA). Samples are labelled with monoclonal antibodies and prepared for flow cytometry. CD 14 and CD 61 double positive cells are identified as PMC. P-selectin expression is identified by additional CD62P positivity and TF expression by additional CD142 positivity. Plasma TNF-alpha and IL-6 are measured using ELISA and CRP is measured in plasma using a high sensitivity automated microparticle enhanced latex turbidimetric immunoassay. No site-specific difference is seen in overall PMC expression in STEMI or stable angina. Surface P-selectin expression in STEMI [median (IQR)] is significantly higher in CA [35.01 (23.15-56.99)] compared with AO [15.99 (10.3-18.85)] or RA [14.02 (10.42-26.08)] (p = 0.003). Intracoronary PMC correlates significantly with intracoronary TNF-alpha (r = 0.87, p = 0.001) and intracoronary IL-6 (r = 0.76, p = 0.03). Bound monocytes within P-selectin positive and tissue factor positive complexes correlate positively with intracoronary TNF-alpha (r = 0.81, p = 0.008 & r = 0.80, p = 0.009 respectively) and IL-6 (r = 0.54, p = 0.16 & r = 0.71, p = 0.05 respectively). No such correlation is observed in the peripheral circulation of STEMI and stable angina patients. Inflammation is not attributable to PMC formation per se. However, increased intracoronary P-selectin expression by activated platelets and tissue factor expression by activated monocytes within the complexes are determinants of local intracoronary inflammatory burden in STEMI.

Contribution of Platelet CX 3 CR1 to Platelet–Monocyte Complex Formation and Vascular Recruitment During Hyperlipidemia

The chemokine receptor CX(3)CR1 is an inflammatory mediator in vascular diseases. On platelets, its ligation with fractalkine (CX(3)CL1) induces platelet activation followed by leukocyte recruitment to activated endothelium. Here, we evaluated the expression and role of platelet-CX(3)CR1 during hyperlipidemia and vascular injury. The existence of CX(3)CR1 on platelets at mRNA and protein level was analyzed by RT-PCR, quantitative (q)PCR, FACS analysis, and Western blot. Elevated CX(3)CR1 expression was detected on human platelets after activation and, along with increased binding of CX(3)CL1, platelet CX(3)CR1 was also involved in the formation of platelet-monocyte complexes. Interestingly, the expression of CX(3)CR1 was elevated on platelets from hyperlipidemic mice. Accordingly, CX(3)CL1-binding and the number of circulating platelet-monocyte complexes were increased. In addition, CX(3)CR1 supported monocyte arrest on inflamed smooth muscle cells in vitro, whereas CX(3)CR1-deficient platelets showed decreased adhesion to the denuded vessel wall in vivo. Platelets in hyperlipidemic mice display increased CX(3)CR1-expression and assemble with circulating monocytes. The formation of platelet-monocyte complexes and the detection of platelet-bound CX(3)CL1 on inflamed smooth muscle cells suggest a significant involvement of the CX(3)CL1-CX(3)CR1 axis in platelet accumulation and monocyte recruitment at sites of arterial injury in atherosclerosis.

Detection of circulating platelet–monocyte complexes in persons infected with human immunodeficiency virus type-1

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30-60 min. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16⁺ inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet-monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16⁺ or CD16⁻ monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16⁺ PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.

Low-dose sirolimus-eluting hydroxyapatite coating on stents does not increase platelet activation and adhesion ex vivo

We previously found paclitaxel-eluting polymer-coated stents causing more human platelet-monocyte complex formation than bare metal stents in vitro. Presently, we examined patterns of platelet activation and adhesion after exposure to 6 nanofilm HAp-coated (HAp-nano) stents, 6 HAp-microporous-coated (HAp-micro) stents, 5 HAp sirolimus-eluting microporous-coated (HAp-SES) stents and 5 cobalt-chromium stents (BMS) deployed in an in vitro flow system. Blood obtained from healthy volunteers was circulated and sampled at 0, 10, 30 and 60 min. By flow cytometry, there were no significant differences in P-Selectin expression between the 4 stent types (HAp-nano = 32.5%; HAp-micro = 42.5%, HAp-SES = 10.23%, BMS = 7% change from baseline at 60 min, p = NS); PAC-1 antibody binding (HAp-nano = 11.8%; HAp-micro = 2.9%, HAp-SES = 18%, BMS = 6.4% change from baseline at 60 min, p = NS) or PMC formation (HAp-nano = 21.6%; HAp-micro = 4%, HAp-SES = 6.6%, BMS = 17.4% change from baseline at 60 min, p = NS). The 4 stent types did not differ in the average number of platelet clusters >10 μm in diameter by SEM (HAp-nano = 2.39 ± 5.75; HAp-micro = 2.26 ± 3.43; HAp-SES = 1.93 ± 3.24; BMS = 1.94 ± 2.41, p = NS). The majority of the struts in each stent group were only mildly covered by platelets, (HAp-nano = 80%, HAp-micro = 61%, HAp-SES = 78% and BMS = 52.1%, p = NS). The HAp-microporous-coated stents (ECD) attracted slightly more proteinaceous material than bare metal stents (HAp-micro = 35% struts with complete protein coverage, P < 0.0001 vs. other 3 stent types). In conclusion, biomimetic stent coating with nanofilm or microporous hydroxyapatite, even when eluting low-dose sirolimus, does not increase the platelet activation in circulating human blood, or platelet adhesion to stent surface when compared to bare metal stents in vitro.

Might platelet-leucocyte complexes be playing a role in major vascular involvement of Behçet's disease? A comparative study

We determined platelet-leucocyte complexes, which play roles in the thrombosis-inflammation relationship, in Behçet's disease patients with and without major vascular involvement (MVI) and in healthy controls. We included 36 Behçet's disease patients (22 male, 14 female, mean age: 34.4 +/- 8.3 years) and 20 healthy individuals (14 male, six female, mean age: 31.8 +/- 4.4 years). Whole blood count, CRP and ESR were determined in both groups. Clinical data about the patients were obtained from medical charts. Individuals with hypertension, diabetes, coronary artery disease, and smokers were excluded. Behçet's disease patients with MVI were taken as a separate group (8 male, 5 female, mean age: 37 +/- 8 years). MVI was defined as the presence of pulmonary arterial aneurysm, deep venous thrombosis, vena cava inferior or superior thrombosis, or venous sinus thrombosis. Flow cytometry was used to determine platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), basal and adenosine diphosphate (ADP)-stimulated platelet CD62P expression. Behçet's disease patients with MVI had significantly higher PNC than Behçet's disease patients without MVI and healthy controls (P values = 0.01). PMC levels in Behçet's disease patients with MVI were significantly higher than in healthy controls (P = 0.01). The groups were similar in basal and ADP-stimulated platelet CD62P expression (P values >0.05). Basal and ADP-stimulated CD62P expression, PMC and PNC were not significantly different between active Behçet's disease versus inactive Behçet's disease patients. The evaluated parameters were similar in Behçet's disease patients with and without uveitis, and pathergy-positive and pathergy-negative groups. Our results might suggest that the formation of PMC and PNC might play a role in thrombosis and MVI of Behçet's disease.

Patterns of activation and deposition of platelets exposed to the polymeric surface of the paclitaxel eluting stent

The interaction of platelets with the polymeric surface of drug eluting stents has not been fully described in the literature. Our aim was to analyze the patterns of activation and deposition of platelets exposed to two different stent platforms; (a) the polymeric surface of the paclitaxel eluting stent (Taxus((R)) stent, PES,) and (b) the metallic surface of a stent with identical structural design (Express((R)) stent, BMS). Platelet activation was tested by deploying stents in an in vitro flow chamber model. Anticoagulated blood of 25 healthy volunteers was circulated (flow rate 10 ml/min for 60 min) into the flow chamber system. P-selectin expression, glycoprotein IIb/IIIa activation (PAC-1 binding) and platelet-monocyte complexes (PMC) formation were evaluated at 0, 10, 30 and 60 min. Surface platelet deposition was assessed by surface electron microscopy in stents implanted in the in vitro system for 60 min and in stents implanted in normal porcine coronary arteries for 24 h. Platelet activation evaluation showed a higher P-Selectin expression (92.9% of baseline in PES versus 68.3 % in BMS, P = 0.01) and higher PMC formation (125.7 % of baseline in PES versus 75.6% in BMS, P < 0.01) in the PES compared to the BMS control group. PAC-1 binding levels did not differ among groups. In the in vitro study, SEM analysis of the stent surface showed no statistical differences on platelet deposition between the groups. In addition, presence of proteinaceous material was more frequently seen on the BMS group (moderate to complete coverage = 80% in BMS versus 26% in PES, P < 0.01). In the in vivo study, complete platelet coverage was similar between groups (PES = 7% versus BMS = 8%, P = NS). However, there was an overall trend towards less platelet deposition on the BMS surface (mild and moderate coverage = 83%, 9% in BMS versus 49%, 44% in PES, P < 0.001 for both) but thrombus formation was not observed in either group. The polymeric surface of the PES appears to induce a higher degree of platelet activation and deposition compared to the BMS surface. The biological implications of these findings on the patterns of vascular healing need to be further studied in vivo. Condensed Abstract The interaction of human platelets with the surface of drug eluting stents has not been fully characterized. Patterns of platelet activation and adhesion were evaluated in vitro and in vivo after exposing platelets to the surface of the paclitaxel-eluting stent and identical bare metal stent. The degree of PMC formation and P-selectin expression was increased in PES compared to BMS. In the in vivo study, complete platelet coverage was similar between groups. There was an overall trend towards less platelet deposition on the BMS surface, however, thrombus formation was not observed on either surface. The polymeric surface of the PES appears to induce a higher degree of platelet activation and deposition compared to the BMS surface.

Elevated platelet and leukocyte response to oral bacteria in periodontitis.

Periodontitis is associated with an increased risk for cardiovascular diseases (CVD), but the underlying mechanisms are poorly understood. Recently, we showed that platelets from periodontitis patients are more activated than those from controls. Given the regularly occurring bacteremic episodes in periodontitis patients, we hypothesized that platelets and/or leukocytes from periodontitis patients are more sensitive to stimulation by oral bacteria, in particular the known periodontal pathogens, than platelets from control subjects. Three-color flow cytometry analysis was performed to quantify activation of platelets (P-selectin, PAC-1, CD63) and leukocytes (CD11b) in whole blood from patients with periodontitis (n = 19) and controls (n = 18), with and without stimulation by oral bacteria. Phagocytosis was assessed by using green-fluorescent protein (GFP)-expressing Aggregatibacter actinomycetemcomitans (Aa). Neutrophils and monocytes were activated by all species of oral bacteria tested, but no differences were observed between patients and controls. In response to several species of oral bacteria, platelets from periodontitis patients showed, compared with controls, increased exposure of P-selectin (P = 0.027) and increased formation of platelet-monocyte complexes (P = 0.040). Platelet-leukocyte complexes bound and/or phagocytosed more GFP-Aa than platelet-free leukocytes (for neutrophils and monocytes, in both patients and controls, P < 0.001). In periodontitis, increased platelet response to oral bacteria is paralleled by increased formation of platelet-leukocyte complexes with elevated capacity for bacterial clearance. We speculate that activated platelets and leukocytes might contribute to increased atherothrombotic activity.

Increased circulating platelet–neutrophil, platelet–monocyte complexes, and platelet activation in patients with ulcerative colitis: A comparative study

It is reported that the incidence of thromboembolism is increased in ulcerative colitis (UC), and hypercoagulability persists even when patients are in remission. We evaluated the association of inflammatory response parameters with UC activity, and activation parameters of the platelets, endothelium, and the coagulation system in UC. Eighteen UC patients and 19 healthy subjects were included in the study. The patients' clinical features were recorded down; whole blood counts and acute phase parameters were evaluated. UC patients were divided into two as active (9 patients) and inactive (9 patients) according to combined clinical activity index (CAI) and endoscopic activity index (EAI) scores. In all subjects, platelet CD62P expression, platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), and platelet microparticles (PMP) were determined by flow cytometry. E-selectin, thrombin-antithrombin complex (TAT) levels in plasma, and sCD40L levels in serum were determined by ELISA. In both active and inactive UC patients, platelet CD62P expression, the percentages of PMC, and PNC were significantly higher than those in the control group (P< 0.01). PMP level was higher in the control group than in inactive UC patients (P = 0.001). sCD40L level was significantly higher in active UC group than in the control group (P < 0.01). EAI score correlated significantly with PMP (r = 0.5, P = 0.04) and sCD40L (r = 0.48, P = 0.044); CAI score had a negative correlation (r = -0.68, P = 0.002) with sE-selectin level. In addition to increased CD62P expression and sCD40L, increased formation of PMC and PNC suggests a role for platelet-leukocyte complex formation together with platelet activation in thromboembolic events observed in UC.

Platelet binding to monocytes increases the adhesive properties of monocytes by up-regulating the expression and functionality of β1 and β2 integrins.

Human monocytes adhere to activated platelets, resulting in the formation of platelet-monocyte complexes (PMC). Complex formation depends on the interaction between platelet-displayed P-selectin and the specific ligand for P-selectin on leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1). We have recently shown that monocytes within PMC have increased adhesive capacity to the activated endothelium. To better understand the effect of platelet binding on the capacity of monocytes to adhere to activated endothelium, the P-selectin-PSGL-1 interaction-induced changes in integrin functionality were studied. The binding of platelets to monocytes via P-selectin-PSGL-1 interactions was shown to increase expression and activity of α4 β1 and αM β2 integrin, with a concomitant decrease in L-selectin expression. Furthermore, the binding of platelets to monocytes resulted in increased monocyte adhesion to intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and fibronectin. Platelet binding was also responsible for an increase in monocyte transendothelial migration. Similar effects were observed after engagement of PSGL-1 with specific antibodies or with P-selectin immunoglobulin protein. Our data suggest that platelets, by binding via P-selectin to PSGL-1 on monocytes, induce up-regulation and activation of β1 and β2 integrins and increased adhesion of monocytes to activated endothelium. Hence, monocytes within PMC are in a higher state of activation and may have, therefore, an increased atherogenic capacity.

Platelet Binding to Monocytes Induces Changes in Integrin Functionality Promoting Monocyte Adhesion and Transendothelial Migration. Do Platelets Migrate Along?.

Monocyte adhesion to and transmigration across the endothelium are essential steps in atherogenesis. We have shown that the adhesive interactions between monocytes and the activated endothelium are increased when platelets bind to the monocyte surface and form platelet-monocyte complexes (PMC). PMC formation is dependent on interactions between platelet-displayed P-selectin and PSGL-1 on the monocyte surface. To better understand the effect of platelet binding on the capacity of monocytes to adhere to activated endothelium the P-selectin-PSGL-1 interaction - induced changes in integrin functionality were studied. The binding of platelets to monocytes via P-selectin-PSGL-1 interactions was shown to increase expression and activity of α4 beta;1 - and αM β2 - integrins which, resulted in increased monocyte adhesion to ICAM-1, VCAM-1, fibronectin and subsequently to stimulated endothelial cells. Platelet binding also induced monocyte migration (up to a 3-fold increase), compared to monocytes without platelets on their surface. To investigate the role of platelets in this process we determined the fate of platelets (within the PMC) during monocyte transendothelial migration. After forming PMC by mixing freshly isolated and fluorescently labeled platelets and monocytes, PMC were seeded on endothelial cells cultured on top of a fibrin gel. The cells were allowed to migrate across the endothelial layer into the gel where afterwards the position of platelets and monocytes was analyzed by confocal laser scanning microscopy. We found that the platelets were retained at the endothelium, suggesting that they detach from the PMC and are left behind on the endothelial layer upon monocyte transendothelial migration. In line with this we observed that the monocytes that were in the fibrin gel, underneath the endothelial layer, did not carry any platelets. This was confirmed by PMC migration over endothelial layered transwell filters since almost only monocytes were found in the lower compartment after migration. After testing different migration barriers, platelets seem to be shed from the monocyte surface upon monocyte migration by mechanical stress rather than only endothelial interaction. Our data suggest that monocytes bound to platelets are in a higher state of activation and have an increased atherogenic capacity. Furthermore, platelets seem to mainly play a role in the monocyte recruitment to the endothelium because once the monocytes cross the endothelial layer, platelets detach from their surface. Altogether, our findings, by showing that PMC have a strong atherogenic capacity, might be helpful in finding new therapeutic ways to prevent atherosclerosis and inflammation.

Platelet and Inflammatory Alterations Associated with Late Restenosis in Patients Undergoing Angioplasty and Brachytherapy.

Restenosis limits long-term success of coronary angioplasty (PTCA). Intracoronary brachytherapy, while thought to provide a solution, has also been associated with late thrombotic occlusion. We hypothesized that platelets, with their associated hemostatic, immune modulatory, and inflammatory properties, are associated with these adverse reactions. Whole blood samples were collected from consented patients undergoing de novo PTCA with stent placement (n=57), or PTCA for stent restenosis followed by intracoronary β-radiation (n=18) or γ-radiation (n=22) at baseline (BL) and at 12-24 hrs, 4–6 wks, 6 months and 1 yr post-procedure. All patients were treated with heparin, a GPIIb/IIIa inhibitor, clopidogrel and aspirin prior to PTCA. Inflammatory activation was assessed in terms of plasma IL-6 and C-Reactive Protein (CRP) levels. Platelet activation was assessed in terms of platelet P-selectin expression and the formation of platelet-monocyte complexes using flow cytometry. A significant increase in the levels of platelet-monocyte complexes (β>de novo PTCA>γ) and IL-6 (β=γ>de novo stent) 12–24 hours post-PTCA has previously been shown while levels of P-selectin (+) platelets and CRP were not significantly different between the 3 groups at any time point [Circ. 106(19):II-621, 2002]. This patient population is now further analyzed in relation to the occurrence of restenosis during the 1-year post-procedure follow-up period. Clinical follow-up was available on 76 of 97 patients (78%).Thirty-five of 76 patients exhibited some degree of late restenosis as detected by angiogram. The groups of patients with or without restenosis during the 1-year follow-up were evenly matched in terms of the incidence of diabetes (50% vs. 49%), hypertension (94% vs. 95%) and hyperlipidemia (85% vs. 90%). Patients experiencing restenosis during the follow-up period exhibited similar IL-6 levels as those patients who did not have restenosis. Although CRP levels were higher at BL and 12–24 hrs post-procedure in patients experiencing late restenosis (9.7±2.2 and 12.7±2.9 mg/ml, respectively) compared to patients without late restenosis (6.1±1.0 and 9.1±1.4 mg/ml, respectively), this difference did not reach statistical significance. Levels of platelet-monocyte complexes were increased relative to BL at 12–24 hours post-procedure (18.5±3.3% vs. 39.5±4.2%, p<0.05 vs. BL) in patients with restenosis at follow-up as well as in those without restenosis (15.5±2.4% vs. 39.4±3.5%, p<0.05 vs. BL).Platelet activation, measured as the percentage of P-selectin (+) platelets, was higher in patients experiencing restenosis at follow-up (p<0.05 vs. no restenosis). At baseline and at 12-24 hours post-procedure, the percentage of activated platelets was approximately 2-fold higher in patients who would subsequently experience restenosis (BL: 1.7±0.4 vs. 0.7±0.1%; 12–24 hrs: 2.5±0.8 vs. 1.3±0.3%). While observed with all patients, this finding was more pronounced in de novo stent patients (BL: 2.1±0.1 vs. 0.8±0.2%; 12–24 hrs: 3.4±1.5 vs. 0.9±0.3%) compared to those receiving brachytherapy. Despite potent, multi-targeted anti-platelet therapy, significant post-procedural platelet activation was observed in patients undergoing PTCA with or without subsequent brachytherapy. The data suggest that enhanced platelet activation may contribute to the restenotic process. Whether the increased platelet activation observed in the restenotic patients is due to antiplatelet drug resistance remains to be determined.

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients.

Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg(-1) over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 +/- 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 +/- 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.

Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation.

Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. PMCs in the circulation might be proatherogenic, and prevention of their formation is a possible therapeutic goal.

Platelet–monocyte complex formation: effect of blocking PSGL-1 alone, and in combination with αIIbβ3 and αMβ2, in coronary stenting

Background: Binding of platelet P-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) is an initial event in the interactions between platelets and monocytes. Platelet–monocyte complexes (PMCs) have been implicated in several vascular disease processes, including acute coronary syndromes (ACS) and complications after percutaneous coronary intervention (PCI). We investigated the effect of ex vivo blockade of PSGL-1, alone and in combination with blockade of the αMβ 2 (Mac-1) and α IIbβ 3 (GP IIb/IIIa) integrins, on PMC formation. Methods and results: Dual-label flow cytometry was used to detect PMCs in the blood of 10 volunteers and 10 patients undergoing PCI who received intravenous GP IIb/IIIa antagonists. PSGL-1 blockade, both prior to and after platelet stimulation, markedly reduced the formation of PMCs. Concomitant ex vivo blockade of the αMβ 2 and α IIbβ 3 integrins did not result in further decreases of PMCs compared to PSGL-1 blockade alone. Antagonism of PSGL-1 also led to near elimination of leukocyte–platelet interactions under flowing conditions. Conclusion: Blockade of PSGL-1 alone is sufficient to inhibit and reverse the formation of PMCs following platelet stimulation. Concurrent antagonism of PSGL-1 and the α IIbβ 3 and αMβ 2 integrins was not more effective than inhibition of PSGL-1 alone. These results suggest that platelet–monocyte complex formation is mostly dependent on PSGL-1.