Soluble CD62P Research Articles

The role of α1AMP-activated protein kinase (α1AMPK) in vascular dysfunctions induced by sepsis

Sepsis is a pathological circumstance whose outcome is associated with organ dysfunction and death. Vascular hyperpermeability and haemostasis impairment, both play a key role in the onset of organ failure. Our previous work highlighted the protective role of α1AMPK against sepsis-induced hyperpermeability. However, the impact of α1AMPK on haemostasis has never been investigated in this condition. This study aims to characterize the interaction of α1AMPK with hemostasis in sepsis. C57BL6 J mice and α1AMPK wild-type (WT) and knock-out (KO) mice were challenged with intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli O55B5 (10 mg.kg-1) during 6 hours. Plasmatic markers of coagulation, platelet and NETosis activation were measured by ELISA and membrane markers of platelet activation were analysed by flow cytometry. The dsDNA analysis of NETosis was performed by fluorometry. Ex vivo platelets activation was assessed by flow cytometry on platelets α1AMPK WT and KO, incubated in a medium with 5 μg/mL of LPS. In C57BL6 J mice, haemostasis was activated 6 hours after LPS administration, as reflected by the increase in fibrinogen, plasminogen activator inhibitor and tissue plasminogen activator. In addition, platelets isolated from LPS-treated mice were activated as demonstrated by the increased exposure of CD62P and αIIbβIII on platelet surface and soluble CD62P detection in plasma. Finally, we measured a significant increase in myeloperoxidase, neutrophil elastase and plasmatic dsDNA, all markers of NETosis. Same parameters were measured in α1AMPK WT and KO mice. We brought out an increase of fibrinolysis impairment and heightened a trend of NETosis in α1AMPK undergoing sepsis. Furthermore, platelets of α1AMPK KO mice exhibited a more important activation ex vivo, in response to LPS. These results suggest that α1AMPK is involved in the regulation of hemostasis disturbances following sepsis.

Soluble CD40L and CD62P levels differ in single-donor apheresis platelet concentrates and buffy coat-derived pooled platelet concentrates.

Platelet storage lesions are structural and biochemical changes in platelet concentrates (PCs), and depend on variables in collection and processing, as well as secondary procedures and storage conditions; such lesions can be mitigated by the use of platelet additive solutions (PASs). This study investigated release of the inflammatory markers sCD40L and sCD62P by single-donor apheresis platelet concentrates (SDA-PCs) and buffy coat-derived pooled platelet concentrates (PPCs) before and after storage. SDA-PC and PPC samples (n = 9089) processed by various methods and stored for different durations were obtained following production in one regional setting, the French National Blood Service. Soluble factors were quantified in PC supernatants immediately after processing and at the time of delivery, using biological testing technology (Luminex). SDA-PCs appeared more activated than PPCs at the end of the production step (i.e., prior to storage); however, proinflammatory soluble factors exhibited greater increases in PPCs than in SDA-PCs during storage. In SDA-PCs, PAS-D (65%) led to reduced secretion of sCD62P, but favored secretion of sCD40L, compared with the alternative PAS-E. These data stress the importance of the production (processing) steps of PC manufacture and of storage. The extent to which they affect patient outcomes awaits further investigation in clinical studies.

Evolution of platelet functions in cirrhotic patients undergoing liver transplantation: A prospective exploration over a month.

This prospective observational study was designed to analyze platelet functions across time in 50 patients scheduled for liver transplantation (LT) secondary to decompensated cirrhosis or hepatocellular carcinoma. Platelet functions were assessed before LT (pre-LT), one week (D7) and 1 month (D28) after LT. Platelet count significantly increased from pre-LT time to day 28 as well as circulating CD34+hematopoietic stem cells. To avoid any influence of platelet count on assays, platelet function was evaluated on platelet-rich-plasma adjusted to pre-LT platelet count. Although platelet secretion potential did not differ between time-points, as evaluated by the expression of CD62P upon strong activation, platelet aggregation in response to various agonists significantly increased along time, however with no concomitant increase of circulating markers of platelet activation: platelet microvesicles, platelet-leukocyte complexes, soluble CD40L and soluble CD62P. In the multivariate analysis, hepatic function was associated with platelet count and function. A lower platelet aggregation recovery was correlated with Child C score. History of thrombosis or bleeding was associated with respective higher or lower values of platelet aggregation. This longitudinal analysis of platelet functions in LT patients showed an improvement of platelet functions along time together with platelet count increase, with no evidence of platelet hyperactivation at any time-point.

Assessment of soluble platelet CD40L and CD62P during the preparation process and the storage of apheresis platelet concentrates: Absence of factors related to donors and donations

Platelet transfusions may be associated with certain adverse effects in recipients, potentially caused by the presence of biological response modifiers contained in the platelet concentrates. The aim of this study is to identify the parameters that reflect platelet activation during both the preparation process and the storage of platelet concentrates. A total of 3,949apheresis platelet concentrate samples were studied with regard to parameters related to the donor as well as to the preparation process and their storage. Key glycoproteins characteristic of platelet activation, i.e. soluble CD40L and CD62P, were quantified in platelet concentrate supernatants on completion of their processing and during storage, using Luminex technology. We observed an increase in soluble factors over time. However, the different parameters studied in connection either with the donors or with the donations, such as (i) donor gender, (ii) donor blood group, (iii) time of collection and (iv) type of apheresis separator, do not seem to have any effect on platelet activation or the release of soluble CD40L and CD62P.

In vitro quality of apheresis platelets divided into paediatric-sized units and stored in PVC bags plasticised with TOTM, BTHC or DINCH.

Two of the predictive factors of the quality of small volumes of platelets suitable for paediatric use are bag size and material. This study evaluated the storage properties of paediatric platelet aliquots in TOTM-, BTHC- or DINCH-PVC bags. (i) Three apheresis platelet concentrates (PC) were pooled and split into three units. One was retained as an adult unit (control; polyolefin bag). The second and third units were split into four MacoPharma TOTM-PVC and BTHC-PVC paediatric bags, respectively. (ii) Two apheresis PC were pooled and split into two units. One PC was retained as an adult unit, and the other was split into four Fresenius DINCH-PVC paediatric bags. Testing was performed on storage for pH, blood gases, hypotonic shock response, soluble CD62P, LDH, glucose and lactate, ATP, CD62P, CD63, platelet-derived microparticles and annexin V. The volumes, platelet yields and pH of all paediatric units met local specifications. The TOTM-PVC bag showed no worse quality than the adult bag up to day 7 for all parameters studied, and it maintained pH higher than BTHC-PVC and DINCH-PVC over storage. The BTHC-PVC bag was shown to be the most gas permeable; however, it had the highest glucose consumption rates and the highest platelet activation. All bags showed an acceptable in vitro quality. Overall, the TOTM-PVC paediatric bag showed better platelet quality compared to the other storage bags, whereas storage in the BTHC-PVC bag resulted in poorer platelet quality.

Maximising platelet availability by delaying cold storage.

Cold-stored platelets may be an alternative to conventional room temperature (RT) storage. However, cold-stored platelets are cleared more rapidly from circulation, reducing their suitability for prophylactic transfusion. To minimise wastage, it may be beneficial to store platelets conventionally until near expiry (4 days) for prophylactic use, transferring them to refrigerated storage to facilitate an extended shelf life, reserving the platelets for the treatment of acute bleeding. Two ABO-matched buffy-coat-derived platelets (30% plasma/70% SSP+) were pooled and split to produce matched pairs (n = 8 pairs). One unit was stored at 2-6°C without agitation (day 1 postcollection; cold); the second unit was stored at 20-24°C with constant agitation until day 4 then stored at 2-6°C thereafter (delayed-cold). All units were tested for in vitro quality periodically over 21 days. During storage, cold and delayed-cold platelets maintained a similar platelet count. While pH and HSR were significantly higher in delayed-cold platelets, other metabolic markers, including lactate production and glucose consumption, did not differ significantly. Furthermore, surface expression of phosphatidylserine and CD62P, release of soluble CD62P and microparticles were not significantly different, suggesting similar activation profiles. Aggregation responses of delayed-cold platelets followed the same trend as cold platelets once transferred to cold storage, gradually declining over the storage period. The metabolic and activation profile of delayed-cold platelets was similar to cold-stored platelets. These data suggest that transferring platelets to refrigerated storage when near expiry may be a viable option for maximising platelet inventories.

Modeling the effect of platelet concentrate supernatants on endothelial cells: focus on endocan/ESM-1.

Platelets (PLTs) are prone to activation and the release of biologic response modifiers (BRMs) under storage conditions. The transfusion inflammatory reaction in the vascular compartment involves endothelial cell activation due to cell-cell interactions and BRMs infused with the blood products. Endocan/ESM-1 is a proteoglycan secreted by endothelial cells under the control of proinflammatory cytokines. We aimed to measure endocan activity in supernatants of PLT components (PCs), implicated in serious adverse reactions (SARs) or not (no.AR), sampled at different stages during storage. PLT function, by quantification of soluble CD62P, and their ability to produce endocan were assessed. Functional testing of PC supernatants was performed on EA.hy926 endothelial cells in vitro by exposing them to PC supernatants from each group (no.AR or SARs); EA.hy926 activation was evaluated by their production of interleukin (IL)-6 and endocan. PLT endocan secretion was not induced in response to PLT surface molecule agonists, and no significant correlation was observed between sCD62P and endocan concentration after PLT activation. However, we observed a significant increase in the secretion of IL-6 and endocan after EA.hy926 activation by all PC supernatants. IL-6 and endocan secretion were significantly higher for cells stimulated with SAR than those stimulated with no.AR PC supernatants, as well as cell apoptosis. The correlation between the secretion of endocan and that of IL-6 by endothelial cells suggests that endocan can be used as a predictive marker of inflammation for the quality assessment of transfusion grade PLTs.

Storage of apheresis platelet concentrates after manual replacement of >95% of plasma with PAS 5.

Recently, a glucose- and bicarbonate-containing additive solution termed PAS 5 demonstrated acceptable 7-day platelet storage after >95% plasma replacement with PAS on the day of collection (Day 0). In this study, we examined platelet storage in >95% PAS 5 after manual washing of Day 1 apheresis platelets in plasma collected using either the Amicus or Trima plateletpheresis devices. Triple platelet donations in plasma were obtained from Amicus (n=10) and Trima (n=10) plateletpheresis devices and stored overnight before being centrifuged and manually processed into three units with the following storage media: 100% plasma, >95% PAS 5 or 65% PAS 5/35% plasma. Platelet units were sampled on Days 1, 5 and 7 of storage using a range of tests recommended by the UK guidelines. The majority of in vitro assay results for platelets in PAS 5 were similar to results in paired 100% plasma platelets (controls). The pH of PAS 5 stored platelet units was above the UK recommended guidelines of 7·4 by Day 5. PAS 5 platelets were no more activated than controls as evidenced by comparable soluble P-selectin levels and CD62p and CD42b expression. PAS 5 platelets also exhibited adhesion and aggregation profiles higher than (Day 1) or comparable to (Days 5 and 7) controls as measured by Impact R. The 7-day in vitro storage parameters investigated were comparable between >95% PAS 5 and 100% plasma platelets derived from both Amicus and Trima plateletpheresis devices, with the exception that lactose dehydrogenase release rate and pH were significantly higher in PAS 5 units.

Endothelial cell injury with inflammatory cytokine and coagulation in patients with sepsis.

Current studies on CD62P have focused mainly on cardiovascular diseases, while only few studies have evaluated the effects of CD62P on the development of sepsis and the association between endothelial cell injury with inflammation and coagulation. This study attended to explore the association between endothelial cell injury with inflammation and coagulation by evaluating the expression of soluble CD62P (s-CD62P) in plasma and its mechanism in patients with sepsis, thus to provide the evidence of effective treatment of sepsis with anti-adhesion therapy targeted CD62P. A total of 70 critically ill patients with systemic inflammatory response syndrome (SIRS) admitted to intensive care unit (ICU) between September 2009 and February 2010 were enrolled for a prospective and control study. According to the diagnostic criteria of sepsis/SIRS, the patients were divided into two groups: a sepsis group (n=38) and a SIRS group (n=32). Another 20 healthy volunteers served as a control group. Patients in the sepsis group and SIRS group were matched by clinical signs of high blood pressure, diabetes and its complications. The demographics of the patients including age, sex, body mass index (BMI), smoking and alcohol addict were compared among the groups. Six mL peripheral blood samples were collected within 24-hour admission in ICU for enzymelinked immunosorbent assay (ELISA) to detect the plasma levels of s-CD62P, TNF-α, and hs-CRP. And variables of coagulation function such as platelet (PLT), prothrombin (PT), activated partial thromboplastin time (APTT), D-dimer and antithrombin-III (AT-III) were analyzed during 24 hours after admission to ICU. Meanwhile sequential organ failure assessment (SOFA) score of critically ill patients was evaluated. Data were expressed as mean±standard deviation and were statistically analyzed by using SPSS 17.0 statistical software. The differences in plasma levels of s-CD62P of patients in each group were analyzed by ANOVA and the Kruskal-Wallis test. The relations between s-CD62P and inflammatory cytokines as well as with coagulation were determined by Pearson's product moment correlation coefficient analysis. Changes were considered as statistically significant if P value was less than 0.05. Compared with the control group and SIRS group, the sepsis group demonstrated significantly higher levels of s-CD62P, TNF-α and highly sensitive C-reactive protein (hs-CRP) (P<0.05). The plasma levels of D-dimer, PT, and APTT in the sepsis and SIRS groups were significantly higher than those in the control group, while the platelet count and the activity of AT-III were obviously lower (P<0.05). In the sepsis group, the plasma levels of hs-CRP and TNF-α were positively correlated with PT, APTT, and D-dimer, and negatively correlated with AT-III and PLT (P<0.05). The plasma levels of s-CD62P were significantly correlated with the plasma levels of TNF-α, hs-CRP, D-dimer, PT, and APTT, whereas they were correlated negatively well with PLT and AT-III (P<0.05). The concentration of plasma s-CD62P is elevated as a early biomarker in patients with sepsis, and it serves as one of the pathogenic factors responsible for endothelial cell damage. Coagulation and mediators of inflammation promote each other, aggravating the severity of sepsis. Plasma s-CD62P may be an important factor for the development of coagulation and inflammatory reaction.

Circulating levels of soluble EMMPRIN (CD147) correlate with levels of soluble glycoprotein VI in human plasma

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147), which binds to the platelet-specific collagen receptor glycoprotein (GP) VI, is expressed in a range of cell types including platelets and leukocytes, and has been implicated in neoplastic disease and atherosclerotic coronary disease. Both CD147 and GPVI can be shed from cell membranes and detected in plasma. However, while the relationship between soluble CD147 (sCD147), soluble GPVI (sGPVI) and standard markers of platelet activation has received little attention, such analysis may help reveal pathways mediating release of sCD147. We investigated the relationship between sCD147 and platelet markers including sGPVI, soluble and platelet-bound CD62P (P-selectin), active αIIbβ3 (assessed by PAC-1 binding) and platelet CD147 in 25 patients with stable angina pectoris (SAP), 13 patients with no coronary artery disease (CAD) and 10 healthy donors. Plasma levels of sCD147 significantly correlated with sGPVI (r = 0.46, p = .004), but did not correlate with any other platelet markers examined. Linear regression analysis identified that sCD147 levels could be predicted by sGPVI levels (β = .445, p = 0.003) and age (β = 0.304, p = 0.038), but were independent of potential clinical confounders such as CAD, diabetes and medication usage. As sCD147 strongly correlates with platelet-specific sGPVI, a common platelet source and/or mechanism of release may contribute to sCD147 levels in vivo.

Endothelial cell injury correlates with inflammatory cytokine and coagulation in the patients with sepsis

Objective To observe the clinical findings about the endothelial cell injury related to the genesis of inflammatory cytokines and coagulation.Methods A total of 70 critically ill patients with SIRS (systemic inflammatory response syndrome) admitted to intensive care unit (ICU) between September 2009 and February 2010 were enrolled for a prospective and control study.According to diagnostic criteria of Sepsis/SIRS,the patients were divided into two groups:sepsis group (n =38) and SIRS group (n =32),and another 20 healthy volunteers served as control group.Patients in the sepsis group and SIRS group were matched by clinical signs of high blood pressure,diabetes and its complications.The demographics of patients including age,sex,body mass index (BMI),smoking and alcohol addict were comparable among the different groups.The 6 ml peripheral blood samples were collected within 24 h after admission to ICU for enzyme-linked immunosorbent assay (ELISA) to detect the plasma levels of s-CD62P,TNF-α,and hsCRP.And variables of coagulation function such as platelet (PLT),prothrombin time (PT),activated partial thromboplastin time (APTT),D-dimer and antithrombin-Ⅲ (AT-Ⅲ) were analyzed during 24 h after admission to ICU.Meanwhile sequential organ failure assessment (SOFA) score of critically ill patients was evaluated.Data were expressed in mean ± standard deviation and were statistically analyzed by using SPSS 17.0 statistical software.The differences in plasma levels of s-CD62P of patients in each group were analyzed by ANOVA and Kruskal Wallis test.The relationship between s-CD62P and inflammatory cytokines as well as with coagulation were determined by Pearson correlation analysis.Changes were considered as statistically significant if P value was less than 0.05.Results ① Compared with control group and SIRS group,the levels of s-CD62P,TNF-α and high sensitive C-reactive protein (hs-CRP) were significantly higher in sepsis group (P ＜ 0.05).② The plasma levels of D-dimer,PT,APTT in sepsis group and SIRS group were significantly higher than those in control group,while the platelet count (PLT) and the activity of AT-Ⅲ were obviously lower (P ＜ 0.05).③ In sepsis group,the plasma levels of hs-CRP and TNF-α positively correlated with PT,APTT,D-dimer,and negatively correlated with AT-Ⅲ,PLT (P ＜ 0.05).④ Plasma levels of s-CD62P were significantly correlated with plasma levels of TNF-α,hs-CRP,D-dimer,PT,APTT,whereas correlated negatively well with PLT,AT-Ⅲ (P ＜ 0.05).Conclusions The plasma s-CD62P concentration is elevated as a early biomarker in patients with sepsis,and it acted as one of pathogenic factors responsible for endothelial cell damage.Coagulation and mediators of inflammation promotes each other,aggravating the severity of the sepsis.The plasma s-CD62P may be the important factor associated with initiation of coagulation development and inflammatory reaction. Key words: Sepsis; Endothelial cell injury; Plasma soluble CD62P; Inflammatory factor; Coagulation

Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients

Objectives Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro-inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers. Methods Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB 2, and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated. Results No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P = 0.009; P = 0.04; P = 0.004, respectively). In both studied groups serum level of TxB 2 and platelet aggregation under AA stimulation were lower than before treatment (P < 0.001). Conclusion Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.

Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke

Although the platelet activation profile after stroke is a well-known issue, the platelet reactivity assessed prospectively after ischaemic stroke still remains equivocal. The aim of this study was to evaluate the reactivity of platelets in response to stimulation with thrombin receptor-activating peptide (TRAP) at 1, 10 and 90 days after ischaemic stroke and to compare it with results obtained in control groups. We determined the increment in surface expression of CD62P, CD40L and monocyte- and granulocyte-platelet aggregate formation using five-colour flow cytometry in 86 subjects after an ischaemic event, in 62 disease controls, and in 38 healthy volunteers. We assessed the plasma levels of CD62P and CD40L soluble forms. In patients after stroke a significantly lower increment in CD62P surface expression (p < 0.01) and higher increments in both CD40L platelet surface expression (p < 0.01) and monocyte-platelet aggregate percentage (p < 0.01) were found at every studied time point, as compared with the control groups. Plasma levels of soluble CD62P (sCD62P) and soluble CD40L (sCD40L) were increased in stroke subjects in both the acute and the subacute phase of the stroke and they dropped to levels observed in controls at day 90 after the ischaemic incident. In all studied groups a positive correlation was noted between plasma levels of sCD62P and sCD40L. In conclusion, while at 3-month follow-up the levels of soluble forms normalize in stroke patients, the profile of platelet reactivity in response to activation with TRAP differs from that observed in the controls despite the secondary stroke prevention.

Platelet proinflammatory activity in clinically stable patients with CF starts in early childhood

Background Early onset chronic inflammation is present in CF. Platelets may contribute to inflammation by cytokine release and interaction with leukocytes. Methods Parameters of platelet proinflammatory function (soluble CD62P, soluble CD40L, the percentage of platelet–leukocyte aggregates, platelet CD62P) and platelet procoagulatory function (PAC-1-binding to activated integrin α IIbβ 3 and expression of integrin α IIbβ 3 = CD41a) were measured in patients and controls. Results Levels of sCD62P, sCD40L were increased in CF irrespective of age and activity of inflammation. The number of platelet–leukocyte aggregates was elevated in older CF patients. PAC-1-binding to platelets decreased with growing activity of inflammation. Exocytosis of CD41a upon platelet activation was reduced. Conclusion In CF, platelet proinflammatory activity is increased at very young age already and might promote inflammation and tissue damage. On the other hand, platelets seem to downregulate the activation of their most important integrin (α IIbβ 3) for clot formation.

Expression of EMMPRIN (CD147) on circulating platelets in vivo.

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). 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. 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. 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.

P10-09. Altered release of RANTES from human, normal platelets: contribution of distinct HIV-1MN gp41 peptides

Methods We used platelets sampled from healthy, HIV-1,2 negative, normal blood donors. Platelet-Rich Plasma (PRP) was incubated after addition thrombin receptor agonist peptide or HIV env glycoprotein. PRP were centrifuged and platelet free supernatant was stored. Two recombinant gp120MN (produced in insect cells using the baculovirus expression system) and synthetic peptides of gp41MN, and mAbs to gp41: D50 and 2F5, were all obtained through the AIDS Research and Reference Reagent Program. Soluble CD62p and RANTES production in cultures were measured by specific ELISA.

Donor platelets stored for at least 3 days can elicit activation marker expression by the recipient's blood mononuclear cells: an in vitro study

Recent studies have demonstrated that infused platelets (PLTs) can promote inflammation. The objective of this study was to evaluate the impact of storage of transfusion-grade PLTs on the peripheral blood mononuclear cells (PBMNCs) of the recipient. An in vitro cell model system was established to measure the degree of activation of donor PLTs during 5 days of their storage and then to measure immune cell activation by detecting marker expression in coculture experiments. The level of soluble CD62p increased significantly by Day 3, and membrane expression of CD62p increased significantly from Day 2, indicating some degree of PLT activation over time during storage (p < 0.05). Donor PLTs and PBMNC subsets (monocytes, B cells, and T cells) from recipients were cocultured for 48 hours. The number of PLT-PBMNC subset doublets detected by flow cytometry was correlated with the PLT storage time after Day 3 (p < 0.05), indicating consistent binding of PLTs to PBMNCs. The results of these experiments showed that there was a consistent and significant increase in expression of conventional activation markers of T cells, B cells, and monocytes compared with appropriate controls (p < 0.05 to <0.01). The results of this study indicate that, from Day 3 onward, activation markers are consistently expressed on PLTs. From these results, we conclude that activated PLTs may affect PBMNC interactions in recipients.

Mechanisms of Platelet Activation by Antithymocyte Globulin (ATG).

We have previously shown that polyclonal rabbit ATG from Fresenius (ATG-F) can induce non-overt DIC in patients undergoing hematopoietic stem cell transplantation (HSCT) [Weber et al. 2003]. The compensated state of the coagulopathy suggested that the severe drop in platelet counts (>60%) observed in patients with normal pretreatment levels was not solely due to thrombin generation and platelet consumption. We thus further investigated the mechanisms of ATG-F-induced thrombocytopenia, hypothesizing that ATG-F had direct platelet-stimulating activity. Using an indirect flow cytometric binding assay we found that, compared to control IgG, ATG-F dose-dependently (1–100 μg/ml) bound to the surface of resting and TRAP-activated platelets, showing an up to 50fold increase in MFI at 50–100 μg/ml. In a washed platelet system, in which baseline positivity for CD62P was generally <15%, ATG-F alone had no effect on platelet aggregation. However, when platelets were primed with low concentrations of ADP (1–2 μM) or epinephrine (0.5 μM), ATG-F induced strong and stable aggregation of up to 80–100% in a dose-dependent manner (25–100 μg/ml), whereas control IgG did not. The priming effect of ADP was dependent on both P2Y1 and P2Y12 as evidenced by respective ADP receptor antagonists. Preincubation of platelets with inhibitory CD32 mAb completely abolished ATG-F-induced platelet aggregation, suggesting that clustering of, and signalling through, FcγRIIA was crucial for this platelet response. Similarly, ATG-F-derived F(ab′)2 had no effect on ADP-triggered platelet aggregation. Furthermore, preliminary studies indicated that responsiveness of platelet donors to ATG-F was associated with the FcγRIIA-R/R131 polymorphism, which has previously been shown to confer increased in vitro platelet responsiveness to heparin/PF4 antibodies from patients with HIT. In a 14C-serotonin release assay, in which prolonged incubation at 370C under shaking conditions increases baseline platelet activation and ensures constant platelet-platelet contacts, ATG-F (100 μg/ml), but not control IgG, induced strong FcγRIIA-dependent dense-granule release of up to 80%. In contrast, using diluted platelet-rich plasma under static conditions, ATG-F had only minor effects on platelet activation, increasing CD62P+ platelets from 2±1 to 15±5% (n=5). In a cohort of 16 consecutive HSCT patients receiving ATG-F, two patients were identified with normal pretreatment platelet counts and soluble CD62P (sCD62P) plasma levels of >51 ng/ml (mean+2SD of 23 controls). In these patients, an ATG-F-induced drop in platelet count to 38 and 19% of baseline was associated with a 1.7 and 2.2fold increase in sCD62P, respectively, indicating further platelet activation. In summary, strong binding of ATG-F to resting platelets may accelerate their clearance by the reticulo-endothelial system, thus contributing to the pathophysiology of thrombocytopenia. While ATG-F alone had negligible effects on resting platelets, it significantly enhanced activation of prestimulated platelets. Patients with normal-to-high platelet counts and evidence of in vivo platelet activation may especially be prone to this potentially hazardous side effect.

72. Intravascular Interaction between Adenovirus and Platelets Promotes Adenovirus Sequestration, Degradation and Toxicity

Intravascular (iv) delivery of adenovirus (Ad) vectors is hindered by acute toxicity that is triggered by trapping of virus in the reticuloendothelial system (RES) within minutes of administration, and subsequent production of pro-inflammatory cytokines/chemokines. While Ad localizes to tissue macrophages and endothelial cells of the liver, spleen and lung within minutes of iv delivery, the mechanism of this reticuloendothelial targeting is unknown. We have identified a novel pathway by which platelets mediate trapping of Ad in the RES. Rapidly after iv administration fiber independent interaction between Ad and circulating platelets can be seen by Southern blot, by transmission electron microscopy (TEM) of isolated platelets and blood cell fractionation of mice receiving radiolabeled Ad. Blood platelet levels drop after Ad administration, while serum levels of the platelet activation marker soluble CD62p rise. Within 5 minutes of administration, Ad and platelets co-localize in the liver sinusoids along with endothelial and Kupffer cells. TEM showed Ad particles inside degranulated platelets and platelet aggregates within the liver sinusoids. Ad particles could also be seen inside Kupffer cells. Immunohistochemistry (IHC) showed widespread co-labelling of platelets (CD41), Kupffer cells (F4/80) and Ad (hexon) throughout the liver. In the spleen, Ad genomes were found within 5 minutes and Ad particles were detected in CD41 and F4/80 rich regions of the marginal zone by IHC. In the lung, Ad genomes were found within 5 minutes. Platelet accumulation was seen by IHC after delivery of Ad vectors, with platelet-endothelial interactions visible in the pulmonary vasculature by TEM. To determine the contribution of Ad-platelet interactions towards toxicity, mice were pre-injected with a platelet depleting antibody prior to virus delivery. At 6 hours post virus delivery, vector genomes in liver, lung and spleen were detected by Southern blot, and circulating serum cytokine levels were determined. Serum levels of the pro-inflammatory cytokines/chemokines IFN-|[gamma]|, IL-6 and MCP- 1 were lower in platelet depleted mice. The effect of Ad neutralizing antibodies on Ad-platelet interactions was also determined in mice that had been pre-immunized with the same vector prior to iv Ad vector administration. Early after vector administration significantly lower levels of Ad genomes were associated with circulating platelets in pre-immunized mice. While the levels of Ad deposition in the liver were unchanged in pre-immunized mice, there was reduced Ad deposition within the lungs of pre-immunized mice. Taken together these data suggest that rapid interaction between Ad and platelets occurs after iv administration. Platelet-Ad interactions apparently play a role in RES sequestration and degradation of Ad, and subsequent toxicity. We are currently attempting to identify the structural components involved in Ad-platelet interactions in vivo and investigating the contribution of this interaction to sequestration, degradation and toxicity, after Ad administration.

Cigarette smoke metal‐catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules

Smoking is a significant risk factor for development of atherosclerosis. However, the pathophysiology of smoking-mediated vessel wall damage is not understood. With tools ranging from analytical chemistry to cell biology, we show that cigarette smoke contains metals that catalyze the direct oxidation of cellular proteins by smoke oxidants. Oxidation of cellular proteins causes a loss of microtubule function, culminating in microtubule depolymerization and proteasome-dependent degradation of alpha-tubulin. As a consequence of the microtubule collapse, cytoskeletal structures as well as intermediate filaments break down, leading finally to a contraction of vascular endothelial cells. We observed a smoke extract-induced, calpain-dependent degradation of the intracellular form of platelet-endothelial cell adhesion molecule 1/CD31, as well as a release of P-selectin/CD62P, IL-6, and IL-8 from endothelial cells into the supernatant. Increased levels of soluble CD62P and IL-6 are well known to be associated with smoking in humans. Increased permeability of the vascular endothelium is a crucial event in atherogenesis. This work highlights the compounds and mechanisms by which cigarette smoke induces leakiness of the vascular endothelium.