Generation Of Procoagulant Microparticles Research Articles

The Human Platelet as an Innate Immune Cell: Interactions Between Activated Platelets and the Complement System.

Platelets play an essential role in maintaining homeostasis in the circulatory system after an injury by forming a platelet thrombus, but they also occupy a central node in the intravascular innate immune system. This concept is supported by their extensive interactions with immune cells and the cascade systems of the blood. In this review we discuss the close relationship between platelets and the complement system and the role of these interactions during thromboinflammation. Platelets are protected from complement-mediated damage by soluble and membrane-expressed complement regulators, but they bind several complement components on their surfaces and trigger complement activation in the fluid phase. Furthermore, localized complement activation may enhance the procoagulant responses of platelets through the generation of procoagulant microparticles by insertion of sublytic amounts of C5b9 into the platelet membrane. We also highlight the role of post-translational protein modifications in regulating the complement system and the critical role of platelets in driving these reactions. In particular, modification of disulfide bonds by thiol isomerases and protein phosphorylation by extracellular kinases have emerged as important mechanisms to fine-tune complement activity in the platelet microenvironment. Lastly, we describe disorders with perturbed complement activation where part of the clinical presentation includes uncontrolled platelet activation that results in thrombocytopenia, and illustrate how complement-targeting drugs are alleviating the prothrombotic phenotype in these patients. Based on these clinical observations, we discuss the role of limited complement activation in enhancing platelet activation and consider how these drugs may provide opportunities for further dissecting the complex interactions between complement and platelets.

Venous gas emboli are involved in post-dive macro, but not microvascular dysfunction.

Previous studies have shown vascular dysfunction of main conductance arteries and microvessels after diving. We aim to evaluate the impact of bubble formation on vascular function and haemostasis. To achieve this, we used a vibration preconditioning to influence bubble levels without changing any other parameters linked to the dive. Twentty-six divers were randomly assigned to one of three groups: (1) the "vibrations-dive" group (VD; n = 9) was exposed to a whole-body vibration session 30min prior the dive; (2) the "diving" group (D; n = 9) served as a control for the effect of the diving protocol; (3) The "vibration" protocol (V; n = 8) allowed us to assess the effect of vibrations without diving. Macro- and microvascular function was assessed for each subject before and after the dive, subsequently. Bubble grades were monitored with Doppler according to the Spencer grading system. Blood was taken before and after the protocol to assess any change of platelets or endothelial function. Bubble formation was lower in the VD than the diving group. The other measured parameters remained unchanged after the "vibration" protocol alone. Diving alone induced macrovascular dysfunction, and increased PMP and thrombin generation. Those parameters were no longer changed in the VD group. Conversely, a microvascular dysfunction persists despite a significant decrease of circulating bubbles. Finally, the results of this study suggest that macro- but not microvascular impairment results at least partly from bubbles, possibly related to platelet activation and generation of pro-coagulant microparticles.

Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA.

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

Effects of peroxisome proliferator-activated receptor-γ agonists on the generation of microparticles by monocytes/macrophages

Microparticles are membrane vesicles shed by cells upon activation and/or apoptosis. Microparticles are involved in several processes, including blood coagulation and thrombosis. In addition to their role in the regulation of lipid metabolism, peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists exert other effects, both dependent on and independent of PPAR-γ activation. Some PPAR-γ agonists have been linked to an increased risk of thrombotic diseases. We aimed to investigate the potential role of PPAR-γ agonists on the generation of procoagulant microparticles by human monocytes/macrophages. Monocytes/macrophages were isolated from the buffy coats of normal donors. Cells were incubated with three structurally unrelated PPAR-γ agonists, namely, rosiglitazone, pioglitazone, and 15-deoxy-Δ(12,14)-prostaglandin J(2). Microparticle generation was assessed as phosphatidylserine concentration by a prothrombinase assay, after capturing the microparticles onto annexin V-coated wells. Intracellular calcium concentration was assessed by a fluorescent probe. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by western blot. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused a dose-dependent, significant increase in intracellular calcium mobilization and tissue factor-bearing microparticle generation. EGTA inhibited microparticle generation. The specific PPAR-γ inhibitor, GW9662, also inhibited microparticle generation. Finally, rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2) caused phosphorylation of ERK; inhibition of ERK by PD98059 inhibited microparticle generation. The PPAR-γ agonists rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused an increase in procoagulant, tissue factor-bearing microparticle generation by human monocytes/macrophages. The effect was dependent on ERK phosphorylation and partly mediated through intracellular calcium mobilization; however, direct activation of the PPAR-γ ligand was also involved.

Enhanced efficacy of recombinant FVIII in noncovalent complex with PEGylated liposome in hemophilia A mice

AbstractRecombinant FVIII formulated in PEG-ylated liposomes (rFVIII-PEG-Lip) was reported to increase the bleed-free days from 7 to 13 days (at 35 IU/kg rFVIII) in severe hemophilia A patients. To understand the underlying mechanism, we sought to recapitulate its efficacy in hemophilia A mice. Animals treated with rFVIII-PEG-Lip achieved approximately 30% higher survival relative to rFVIII after tail vein transection inflicted 24 hours after dosing. The efficacy of rFVIII-PEG-Lip represents an approximately 2.5-fold higher “apparent” FVIII activity, which is not accounted for by its modestly increased (13%) half-life. The enhanced efficacy requires complex formation between rFVIII and PEG-Lip before the administration. Furthermore, PEG-Lip associates with the majority of platelets and monocytes in vivo, and results in increased P-selectin surface expression on platelets in response to collagen. Rotational thromboelastometry (ROTEM) analysis of whole blood from rFVIII-PEG-Lip–treated animals at 5 minutes up to 72 hours after dosing recapitulated the 2- to 3-fold higher apparent FVIII activity. The enhanced procoagulant activity is fully retained in plasma unless microparticles are removed by ultracentrifugation. Taken together, the efficacy of rFVIII-PEG-Lip is mediated mainly by its sensitization of platelets and the generation of procoagulant microparticles that may express sustained high-affinity receptors for FVIII.

Generation of Procoagulant Microparticles in Cerebrospinal Fluid and Peripheral Blood After Traumatic Brain Injury

Traumatic brain injury (TBI) can induce cell damage. Procoagulant microparticles (MPs) are reliable markers of cell stimulation. The aim of this study was to investigate the generation of procoagulant MPs in the cerebrospinal fluid (CSF) and plasma of patients with severe TBI. CSF and plasma MPs of 16 patients with severe TBI were quantified by functional prothrombinase assay (i) on the day of the trauma, (ii) during a 10-day follow-up and compared with control samples. The cellular origin of MP was determined after capture with specific antibodies. The CSF and plasma of patients with severe TBI revealed a significantly increased generation of MP compared with control samples on the day of the trauma (CSF: 4.5 +/- 1.8 vs. 0.83 +/- 0.28 nanomolar PhtdSer equivalent; p = 0.01 and plasma 4.1 +/- 3.7 vs. 2.3 +/- 0.19 nanomolar PhtdSer equivalent; p = 0.02). Procoagulant MPs were mainly of platelet and endothelial origin in CSF. MPs decreased significantly in the CSF 10 days after TBI. In CSF, a sustained generation of procoagulant MP was evidenced in two patients presenting a poor clinical outcome. In the blood flow, elevated amounts of procoagulant MPs were detected in three patients presenting disseminated intravascular coagulopathy during the follow-up. Procoagulant MP testifying to platelet and endothelial activation are produced in the CSF and in the plasma after severe TBI. A sustained generation of procoagulant MP in the CSF could contribute to a poor clinical outcome.

Generation of procoagulant microparticles in cerebrospinal fluid and in blood after traumatic brain injury

Generation of procoagulant microparticles in cerebrospinal fluid and in blood after traumatic brain injury

Potential role of P-selectin glycoprotein ligand-1 in haematological diseases

PSGL-1 is a major counterreceptor of all three types of selectins that is expressed in several leukocyte subsets. Data presented, here prove that this mucin may be implied in haematological disorders. We established on normal peripheral blood and in samples derived from 20 AML patients that PSGL-1 is differently expressed in various leukocyte subsets. Myeloblasts appearing in acute myeloid leukaemia patients express significantly less PSGL-1 (12 000 ? 5300) than mature neutrophils (p< 0.001). In monocytic leukaemias, however, the amount of PSGL-1 on monocytic precursors is displayed in a fairly broad range which was not significantly different from that of mature monocytes (p=0.084). Monoblasts/promononocytes possess more PSGL-1 than myeloblasts and the expression pattern is completely non-overlapping. This would imply a differential expression of PSGL-1 during myeloid haemopoietic development and suggests, that the quantitation of surface PSGL-1 may help in differentiating myeloblasts from monoblasts by immunophenotyping in different AML subsets. PSGL-1 has also a certain role in the generation of procoagulant microparticles (MP) as in the PSGL-1 knockout mouse the MP number failed to increase with age and the MP contained significantly less tissue factor than wild type mice. Since PSGL-1 P-selectin interaction is crucial in generating a procoagulant effect we tested the hypothesis that the administration of a P-selectin IgG chimera (Psel-Ig) corrects bleeding tendency in a murine haemophilia model and in human haemophilic blood. The addition of Psel-Ig resulted in significant improvement of the bleeding tendency in mice and in the generation of MP in human hemophilic blood. Thus, the Psel-Ig can become an alternative route to control bleeding tendency in coagulopathies.