In the present study, we aimed to investigate platelet activation induced by adenovirus type 3 (HAdV3) in vitro. Platelet-rich plasma (PRP) or whole blood was incubated with or without HAdV at various concentrations. Platelet aggregation, platelet counting, fibrinogen and expression of platelet membrane antigens (CD41a and CD62P) were determined following incubation with HAdV for different periods of time. The results demonstrated that HAdV at the concentrations of 109-1011 vp/ml enhanced adenosine diphosphate (ADP) or ristocetin-induced platelet aggregation, however did not alter the platelet count. Infection with HAdVs also reduced fibrinogen level. P-selectin and CD41a appeared rapidly on the surface after platelets were incubated with HAdVs in vitro for 30 min. In conclusion, HAdVs may induce activation of platelets and lead to a pre-thrombotic state of peripheral blood. This finding may aid in the development of measures to prevent severe HAdV infection.

Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. The purpose of this study was to characterize circulating platelets in diabetes and clarify their effects on endothelial function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Each experiment was performed by incubating carotid arterial rings with platelets (1.65×10(7) cells/mL; 30 min) isolated from STZ or control rats. Thereafter, the vascular function was characterized in isolated carotid arterial rings in organ bath chambers, and each expression and activation of enzymes involved in nitric oxide and oxidative stress levels were analyzed. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in carotid arteries treated with platelets isolated from STZ rats. Similarly, treatment with platelets isolated from STZ rats significantly reduced ACh-induced Akt/endothelial NO synthase signaling/NO production and enhanced TXB2 (metabolite of TXA2), while CD61 (platelet marker) and CD62P (activated platelet marker) were increased in carotid arteries treated with platelets isolated from STZ rats. Furthermore, the platelets isolated from STZ rats decreased total eNOS protein and eNOS dimerization, and increased oxidative stress. These data provide direct evidence that circulating platelets isolated from diabetic rats cause dysfunction of the endothelium by decreasing NO production (via Akt/endothelial NO synthase signaling pathway) and increasing TXA2. Moreover, activated platelets disrupt the carotid artery by increasing oxidative stress.

OBJECTIVE

The aim of the study was to evaluate the potential influence of ranibizumab and bevacizumab on platelet activation and aggregation, which are critical processes in the pathogenesis of arterial thromboembolic events (ATEs).

METHODS

For the assessment of platelet function, flow cytometry and aggregometry were employed. Platelets were isolated from healthy volunteers and exposed to ranibizumab (1 mg/ml and 150 ng/ml) and bevacizumab (2.5 mg/ml and 3 μg/ml) or their solvents for 10 and 30 min prior to the addition of TRAP (25 μM), PAR-4-AP (25 μM) or thrombin (0.02 U/ml). The surface expression of activated GP IIb/IIIa, P-selectin (CD62P) and platelet-bound stromal cell-derived factor-1 (SDF-1) was measured on resting (nonactivated) and activated platelets by flow cytometry. The platelet aggregation capacity was examined using light transmission aggregometry.

RESULTS

The expression of surface activation markers did not differ significantly between nonstimulated and TRAP-, PAR-4-AP- or thrombin-activated platelets after incubating with ranibizumab. However, GP IIb/IIIa, CD62P and SDF-1 were significantly downregulated in PAR-4-AP- and thrombin-activated platelets after exposure to bevacizumab 2.5 mg/ml. In addition, ranibizumab- and bevacizumab-FITC were significantly increased in all activated platelets. No significant differences were observed in the aggregation of activated platelets after incubation with ranibizumab or bevacizumab.

CONCLUSIONS

All ranibizumab concentrations as well as the bevacizumab concentration of 3 μg/ml had no influence on platelet activation and aggregation. Therefore, this in vitro study did not show any relationship between the exposition of activated platelets to ranibizumab or bevacizumab and the development of ATEs. However, the highest level of bevacizumab interfered with platelet activation, leading to downregulation of platelet activation markers. This observation might explain why the systemic treatment with high-dose bevacizumab could be associated with an increased risk of bleeding. Regarding the use of lower intravitreal dosages, further research should focus on the complex interactions between platelets and other cells, such as endothelial cells, which might stronger relate to a potentially increased risk of ATEs and depend on systemic vascular endothelial growth factor levels. Facing the different activation profiles, the diverse effects of the drugs on the cellular level have to be critically scrutinized for their clinical relevance.

Platelets were isolated from human blood by Percoll density gradient centrifugation in a low Ca2+/high Mg2+ buffer. The buffer reversibly inactivates the cells during separation. The purity of the isolated cells >> 99%) was determined by flow cytometry, their viability was confirmed by fluorescein diacetate hydrolysis, and their morphology was studied with TEM. Plasma proteins were adsorbed onto hydrophobic glass surface, and pure platelets were added and incubated for up to 30 min at 37 degree C. Platelet activation was determined by cell spreading, formation of microparticles and surface exposure of CD62P indicating the release of alpha-granules. Surface-immobilized IgG was shown to cause the release of microparticles and cell lysis, in accordance with data published by others. Surface-immobilized vWF was shown to induce CD62P exposure on the platelet cell surface. The specificity of this response was demonstrated by adsorbing plasma proteins from normal and factor VIII-deficient plasma.

Background: Therapy with irrevesible Bruton's tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) is associated with bleeding.

Objectives: To propose the predictive markers of such bleeding, as well as mechanisms responsible for decreased bleeding at later therapy stages. Patients/Methods We investigate platelet functional activity in 50 CLL and 16 MCL patients on ibrutinib using flow cytometry and light transmission aggregometry.

Results: Prior to treatment, both patient groups had decreased platelet counts, impaired aggregation with adenosine diphosphate (ADP), and decreased binding of CD62P, PAC1, and annexin V upon stimulation. Bleeding in patients treated with ibrutinib was observed in 28 (56%) CLL patients, who had decreased aggregation with ADP and platelet count before therapy. Their platelet count on therapy did not change, platelet aggregation with ADP steadily improved, and aggregation with collagen first decreased and then increased in anticorrellation with bleeding. Bleeding in MCL was observed in 10 (62%) patients, who had decreased dense granule release before therapy. ADP and ristocetin induced platelet aggregation in ibrutinib-treated MCL patients increased on therapy, while collagen-induced aggregation evolved similarly to CLL patients.

Conclusions: Our results suggest that ibrutinib-dependent bleeding in CLL patients involves of three mechanisms: decreased platelet count (the most important discriminator between bleeding and nonbleeding patients), impaired platelet response to ADP caused by CLL, and inhibition by ibrutinib. Initially, ibrutinib shifts the balance to bleeding, but then it is restored because of the improved response to ADP.

Keywords: Bleeding risk; Chronic lymphocytic leukemia; Flow cytometry; Ibrutinib; Platelet function.

Platelet activation and functional changes were investigated in acute lymphoblastic leukemia (ALL) to provide the basis for early diagnosis and evaluation of curative effect. Platelet parameters, immature platelet fraction (IPF%), immature platelet count (IPC), P-selectin (CD62p) (PAC-1) expression were detected in ALL, ALL-complete remission-induced (CR1), and normal groups with an automatic blood cell analyzer and flow cytometer. CD62p and PAC-1 were higher in the ALL group before adenosine-5-diphosphate (ADP) activation than in the normal group (P < 0.05); PAC-1 expression was higher and lower in the ALL-CR1 group than in normal and ALL groups (P < 0.05), respectively. CD62p and PAC-1 expression was lower in the ALL group than in the normal group after ADP activation (P < 0.05); PAC-1 expression was lower and higher in the ALL-CR1 group than in normal and ALL groups, respectively (P < 0.05). Platelet count (PLT), mean platelet volume (MPV), platelet hematocrit (PCT), and platelet distribution width (PDW) were lower in the ALL group than in the ALL-CR1 group (P < 0.05). PLT, MPV, and PCT did not differ between the ALL-CR1 group and the normal group (P>> 0.05). PDW did not differ statistically among all groups (P>> 0.05). IPF% and IPC values were higher and lower in the ALL group than in normal and ALL-CR1 groups (P < 0.05), respectively. These did not differ significantly between the normal group and the ALL-CR1 group (P>> 0.05). Therefore, ALL patients demonstrate platelet activation and platelet dysfunction; platelet parameters and membrane glycoprotein expression can be used to evaluate the effect of ALL.

The pathomechanisms of morbidity due to blood transfusions are not yet entirely understood. Elevated levels of red blood cell-derived microparticles (RMPs) are found in coagulation-related pathologies and also in stored blood. Previous research has shown that RMPs mediate transfusion-related complications by the intrinsic pathway. We hypothesized that RMPs might play a role in post-transfusion thrombotic complications by enhancing procoagulant activity also through the extrinsic pathway of coagulation.

In this laboratory study, blood from 18 healthy volunteers was stimulated with microparticles from expired stored red blood cells. Various clotting parameters were recorded. Flow cytometry, enzyme-linked immunosorbent assays, and real-time polymerase chain reaction were used to investigate possible mediating mechanisms.

The addition of RMPs shortened the clotting time from 194 to 161 seconds (p < 0.001). After incubation with RMPs, there was increased expression of tissue factor (TF) on monocytes and in plasma. TF messenger RNA expression increased in a time-dependent and concentration-dependent manner. There was a significant induction of interleukin-1β and interleukin-6. After stimulation with RMPs, there was a significant increase in the number of activated platelets, an increased percentage of PAC-1/CD62P (procaspase activating compound-1/platelet surface P-selectin) double-positive platelets, and an increased number of platelet-neutrophil duplets and platelet-monocyte duplets, indicating enhanced interaction of platelets with neutrophils and monocytes. Levels of CXCL-8 (C-X-C motif chemokine ligand 1) and interleukin-6 were significantly higher after treatment with RMPs.

Our results suggest that RMPs trigger coagulation through TF signaling, induce the secretion of proinflammatory cytokines, and induce cell-cell interaction between platelets and neutrophils. Thus, under certain conditions, RMPs could play a role in post-transfusion complications through these mechanisms.

Increased numbers of circulating microparticles (MPs) are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. Platelets and megakaryocytes are a major source of MP and are identified by presence of CD42b on the MP surface. MP shed from activated platelets can be identified by presence of P-selectin (CD62P). Tissue factor (TF) is the principal initiator of blood coagulation and its activity has been identified in MPs derived from patient plasma, which may contribute to thrombosis. Here, we have investigated by flow cytometry the expression of TF and CD62P on MP after exposure of diluted whole blood to TNF-activated endothelial cells (EC) both under static conditions and in our newly established model of flow. MPs were significantly increased in blood subjected to flow and this was further enhanced after exposure of blood to TNF-activated EC. MP surface expression of CD62P or TF was upregulated following exposure to TNF-activated EC under flow compared with flow with nonactivated EC or after static coculture with and without prior EC activation. These data strongly suggest that interactions of blood with inflamed EC can modulate production of CD62P and TF bearing MP under flow conditions, and thus may contribute to a prothrombotic environment.

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.

OBJECTIVE

The high glucose concentration observed in diabetic patients is a recognized factor of mitochondrial damage in various cell types. Its impact on mitochondrial bioenergetics in blood platelets remains largely vague. The aim of the study was to determine how the metabolism of carbohydrates, which has been impaired by streptozotocin-induced diabetes may affect the functioning of platelet mitochondria.

METHODS

Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Platelet mitochondrial respiratory capacity was monitored as oxygen consumption (high-resolution respirometry). Mitochondrial membrane potential was assessed using a fluorescent probe, JC-1. Activation of circulating platelets was monitored by flow cytometry measuring of the expressions of CD61 and CD62P on a blood platelet surface. To determine mitochondrial protein density in platelets, Western Blot technique was used.

RESULTS

The results indicate significantly elevated mitochondria mass, increased mitochondrial membrane potential (ΔΨm) and enhanced respiration in STZ-diabetic animals, although the respiration control ratios appear to remain unchanged. Higher ΔΨm and elevated mitochondrial respiration were closely related to the excessive activation of circulating platelets in diabetic animals.

CONCLUSIONS

Long-term diabetes can result in increased mitochondrial mass and may lead to hyperpolarization of blood platelet mitochondrial membrane. These alterations may be a potential underlying cause of abnormal platelet functioning in diabetes mellitus and hence, a potential target for antiplatelet therapies in diabetes.

Platelets play a vital role in hemostasis and thrombosis, and their demand and usage has multiplied many folds over the years. However, due to the short life span and storage constraints on platelets, it is allowed to store them for up to 7 days at room temperature (RT); thus, there is a need for an alternative storage strategy for extension of shelf life. Current investigation involves the addition of 50 mM N acetylcysteine (NAC) in refrigerated concentrates. Investigation results revealed that addition of NAC to refrigerated concentrates prevented platelet activation and reduced the sialidase activity upon rewarming as well as on prolonged storage. Refrigerated concentrates with 50 mM NAC expressed a 23.91 ± 6.23% of CD62P (P-Selectin) and 22.33 ± 3.42% of phosphotidylserine (PS), whereas RT-stored platelets showed a 46.87 ± 5.23% of CD62P and 25.9 ± 6.48% of phosphotidylserine (PS) after 5 days of storage. Further, key metabolic parameters such as glucose and lactate accumulation indicated reduced metabolic activity. Taken together, investigation and observations indicate that addition of NAC potentially protects refrigerated concentrates by preventing platelet activation, stabilizing sialidase activity, and further reducing the metabolic activity. Hence, we believe that NAC can be a good candidate for an additive solution to retain platelet characteristics during cold storage and may pave the way for extension of storage shelf life.

OBJECTIVE

To investigate the clinical effect of Erigeron injection (El) on positive expression rate of CD62p in platelet and content of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in serum of patients with acute cerebral infarction (ACI).

METHODS

Sixty-eight patients with ACI were randomly divided into the treated group (n = 35) and the control group (n = 33). Conventional treatment were given to both groups, and EI 40 ml/d were given additionally to the treated group, the treatment course for both groups was 15 days. The positive expression of platelet CD62p and the serum TNF-alpha and IL-6 in patients before and after treatment were determined with flow cytometric (FCM) and electrochemical-luminescence (ECL) techniques respectively.

RESULTS

The total curative effect in the treated group were significantly higher than that in the control group (P < 0.05). Levels of platelet CD62p and serum TNF-alpha and IL-6 in ACI patients before and after treatment were significant higher than those in the healthy group (P < 0.05), all the three parameters were significantly decreased after treatment, and the lowering in the treated group was more significant than that in the control group (P < 0.05).

CONCLUSIONS

The effect of El on ACI patients may relate to its action in down-regulating the expression of platelet CD62p, alleviating the immune response and inflammatory injury of central nervous system induced by cytokines.

High platelet reactivity and gastric mucosal injury after aspirin (ASA) treatment are associated with poor compliance and an increased risk of cardiovascular events. Panax notoginseng saponins (PNS) have been widely used for the treatment of coronary heart disease (CHD) in addition to antiplatelet drugs in China; however, the joint effect and possible mechanism of PNS in addition to ASA on platelet activation and gastric injury remain unclear. This study was designed to investigate the combinational effects of PNS with ASA, and to explore the underlying mechanism via arachidonic acid (AA) metabolism pathway using lipidomic analysis. In a randomized, assessor-blinded trial, 42 patients with stable coronary heart disease (SCHD) and chronic gastritis were randomly assigned to receive ASA (n = 21) or PNS + ASA (n = 21) for 2 months. Compared with ASA alone, PNS + ASA further inhibited CD62p expression, GPIIb-IIIa activation and platelet aggregation and led to increased platelet inhibition rate. PNS + ASA suppressed the activity of platelet cyclooxygenase (COX)-1, and decreased the production of TXB₂, PGD₂, PGE₂, 11-HETE, the downstream oxylipids of AA/COX-1 pathway in platelets, compared with ASA alone. The severity of dyspepsia assessment (SODA) results showed that patients in PNS + ASA group exhibited relieved dyspeptic symptoms as compared with those in ASA group, which might be associated with enhanced secretion of gastrin and motilin. In vivo study of myocardial infarction rats demonstrated that PNS attenuated ASA-induced gastric mucosal injury, which was related to markedly boosted gastric level of 6,15-diketo-13,14-dihydro-prostaglandin (PG)F1α, 13,14-dihydro-15-keto-PGE₂ and PGE₂ from AA/PG pathway in response to PNS + ASA compared with ASA alone. In summary, our study demonstrated that the combination of PNS and ASA potentiated the antiplatelet effect of ASA via AA/COX-1/TXB₂ pathway in platelets, and mitigated ASA-related gastric injury via AA/PG pathway in gastric mucosa.

Keywords: Arachidonic acid metabolism pathway; Aspirin; Gastric mucosal protection; Lipidomics; Panax notoginseng saponin; Platelet inhibition.

BACKGROUND

Thymic stromal lymphopoietin (TSLP) has been shown to be expressed in various inflammatory tissues, such as human atherosclerotic plaques. Many types of myeloid cells involved in atherosclerosis, including mast cells, lymphocytes, dendritic cells and monocytes/macrophages, present TSLP receptors (TSLPR). However, it is unknown whether platelets, which also play important roles in atherothrombosis, express TSLPR.

RESULTS

We applied flow cytometry and western blotting to show that TSLPR was expressed on the surface of human platelets. Following the addition of TSLP to platelets, the expression of CD62P, CD63, PAC-1 and p-Akt as well as aggregation and ATP release were increased significantly. A TSLPR antibody and a PI3K (phosphatidylinositol 3-kinase) enzyme inhibitor (LY294002) significantly inhibited the platelet activation induced by TSLP. The expression of TSLPR, CD62P and CD63 and the increment of the expression of CD62P and CD63 induced by TSLP in the acute coronary syndrome (ACS) group were markedly higher than those in the control group and the stable angina pectoris (SAP) group. The expression and the increment of the expression of CD62P and CD63 induced by TSLP were positively correlated with the expression of TSLPR.

CONCLUSIONS

Human platelets express functional TSLPR, which can be activated by TSLP to promote platelet activation. TSLP/TSLPR functions via activating the PI3K/AKT pathway, and this signalling pathway may be one of the mechanisms involved in thrombosis in ACS. In coronary disease patients, the determination of TSLPR in platelets may help to identify the risk of ACS.

OBJECTIVE

The purposes of this study were to (1) evaluate correlations among platelet, leukocyte, growth factor, and cytokine concentrations in canine platelet-rich plasmas (PRPs) produced from five different canine PRP-concentrating systems and (2) compare the effects of different activation protocols on platelet activation and growth factor release from one of these PRPs.

METHODS

PRP was made using blood from 15 dogs and each of 5 different PRP systems in a cross-over design. Complete blood counts were performed to quantify platelet and leukocyte concentrations. PRPs were activated, or not, according to manufacturer instructions, and transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor, and tumor necrosis factor-alpha (TNF-α) were quantified. Differences among platelet, leukocyte, and growth factor concentration were compared among the different systems. Correlations between platelet and anabolic growth factor concentrations were assessed. Subsequently, PRP was made from 12 additional dogs using one of the devices. Each PRP was divided into three aliquots that were activated with calcium chloride (CaCl2), human γ-thrombin (HGT), or not activated. Expression of CD62P and platelet-bound fibrinogen (CAP1) was quantified for each activation group. Concentrations of TGF-β1, PDGF-BB, and TNF-α were also quantified for each activation group and a fourth group that was frozen/thawed. Differences among activation groups were assessed by a Friedman test.

RESULTS

There were statistically significant differences among the PRPs made with difference devices with regard to platelet, leukocyte, TGF-β1, and PDGF-BB concentrations (p < 0.0001). There were weak to moderate correlations (R2 = 0.07-0.58) between platelet and anabolic growth factor concentrations but it appeared that activation had a greater effect on growth factor concentration than did cellular composition. Intentional platelet activation significantly increased CD62P and CAP1 expression as well as TGF-β1 and PDGF-BB concentrations in the one PRP in which all activation methods were assessed. Activation with HGT resulted in the greatest platelet activation, and CaCl2 and freeze/thaw elicited moderate increases in either growth factor release or CD62P and CAP1 expression.

CONCLUSIONS

There are positive correlations between platelet and anabolic growth factor concentrations in canine PRPs. However, intentional platelet activation has a greater effect on growth factor delivery than platelet concentration. Thrombin provides more robust activation than CaCl2.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4 degrees C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P(+) platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4 degrees C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4 degrees C, and analysed within 6 h.

Platelets were recently found to harbor infectious HIV virions in infected individuals who are on antiretroviral treatment with poor CD4+ T-cell recovery. In this study, we screened platelets from recently infected individuals, before and after antiretroviral therapy, for the presence of virus and examined platelet activation, as well as CD4+ T-cell recovery. This was followed by in vitro studies assessing platelet-CD4+ T-cell complex formation as a contributing factor to viral transmission. HIV+ platelets were detected in 10 of 10 acutely infected individuals with no prior history of antiretroviral therapy. The percentage of HIV+ platelets dropped significantly after 3 months of antiretroviral therapy in all of the study participants. These individuals also demonstrated significant recovery of CD4+ T cells. Interestingly, the percentage of HIV+ platelets correlated positively with viral load but not with CD4+ T-cell count. Furthermore, we found that platelet activation with soluble CD40L or thrombin receptor activator peptide 6 (TRAP6) increased platelet-virus interactions in vitro. TRAP6-mediated interactions were reduced by platelet antagonists, aspirin, and R406. We demonstrated that platelets transmit the virus to CD4+ T cells, and this transinfection was abolished by inhibiting platelet-T-cell complex formation via exposure to an anti-CD62P antibody. Additionally, treatment with TRAP6 significantly increased the transinfection, which was also inhibited by aspirin and R206. These results reveal that platelets have the potential to promote HIV viral spread during the acute stage of infection, by harboring infectious virus transmitting infection to susceptible CD4+ T cells through complex formation.

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Sepsis and related syndromes account for a high morbidity and mortality caused by the development of multiorgan failure. Pathogenesis of sepsis is complex, involving humoral as well as cellular factors. Since the role of platelets is still undefined in this concern, we investigated CD63, CD62P, CD36, and CD31 expression on platelets of patients in septic shock (n = 18) using a flow cytometric assay in whole blood. Samples were drawn within 24 hours of onset. We found thrombocytopenia accompanied by a significantly higher expression of CD63, CD62P, and CD31 and a significant downregulation of CD36 in comparison to healthy volunteers (n = 18). Changes in CD63 and CD62P expression indicates platelet activation. Because CD62P, CD36, and CD31 mediate interaction of platelets with leukocytes, subendothelial matrix and probably endothelial cells as well as platelet adhesion/aggregation, our findings suggest an involvement of platelets in leukocyte/endothelial cell interaction in septic shock. We suspect that thrombocytopenia is not due to bone marrow depression, but rather is due to consumption of highly activated platelets in the microcirculation. We feel that our observations may offer a rationale for potentially beneficial effects of antiplatelet therapy in sepsis; however, further studies have to evaluate its beneficial impact as well as its potential risk for bleeding complications.