Platelet Microvesicles Research Articles

Neuro-apoptogenic and blood platelet targeting toxins in benthic marine cyanobacteria from the Portuguese coast

Six strains of marine cyanobacteria, of which five benthic, were isolated from an area of the Portuguese coast with no known apparent toxic microbial bloom. Five strains were lethal for mice. Four of them produced lethargy and four lead to bleeding. One of the toxic strains was from a genus ( Aphanothece) not previously associated with toxin production. Extracts from four isolates induced SH-SY5Y-neuroblastoma cell apoptosis without affecting the viability of hepatocytes, NRK kidney cells, or fibroblasts. Aqueous extract from four isolates inhibited thrombin-induced blood platelet activation, with decreased P-selectin expression, platelet aggregation and shedding of platelet-derived micro-vesicles. Curiously, platelets treated with organic extracts from two of the cyanobacterial strains formed platelet micro-vesicles, expressed P-selectin on the surface and showed a distinct phosphotyrosine protein pattern, but failed to aggregate. We conclude that low-abundance marine cyanobacteria growing at low rates may be an important source for novel toxins that may be useful to dissect mammalian signalling pathways of apoptosis and platelet function.

Procoagulant potential of platelet α granules

In this brief review of the literature it is pointed out that during platelet activation and degranulation platelet α granules leave the platelet interior through blebs in platelet plasma membrane and through the tips of the pseudopods, and then accumulate in the external milieu. There they undergo disintegration and secondary adhesion to the platelet plasma membranes. During their disintegration they expose their tightly packed GPIIb–IIIa complexes, annexin V stainable aminophospholipids, factor V, and the membrane markers CD62 and CD63. There is also demasking of lysosomal acid phosphatase activity and microvesicle formation. Lysosomal nature of platelet α granules is mentioned. It is suggested that platelet α granules are the sole source of platelet procoagulant activity and platelet microparticles (MP) or microvesicles (MV). The implications of this concept for antiplatelet therapy are discussed. A relationship of this process to tissue factor exposure and apoptosis is suggested.

Platelet membrane fragments enhance the procoagulant effect of recombinant factor VIIa in studies with circulating human blood under conditions of experimental thrombocytopenia

The mechanism of action of recombinant factor VIIa (rFVIIa), which is being considered as an alternative treatment for the control of bleeding episodes in patients with thrombocytopenia, has not been fully characterized. This study was undertaken to explore the effects of rFVIIa and platelet microvesicles on hemostasis in an experimental model of thrombocytopenia. Damaged arterial segments were exposed to thrombocytopenic blood (shear rate 600 s −1) either with or without the addition of rFVIIa and/or platelet microvesicles. The presence of fibrin and platelets on the subendothelium were morphometrically quantified and immunolocalization techniques and electron microscopy were used for a more detailed analysis. Both rFVIIa and platelet microvesicles consistently improved fibrin formation on the damaged vascular subendothelium, and microvesicles were shown to be localized at different levels of the fibrin lattice. Further, under conditions of moderate thrombocytopenia, addition of platelet microvesicles potentiated the procoagulant action of rFVIIa. This effect may be due to the phospholipid surface provided by the platelet microvesicles. These studies support the concept that, under conditions of thrombocytopenia, both rFVIIa and platelet microvesicles enhance fibrin formation at sites of vascular damage.

The functional role of blood platelet components in angiogenesis.

The process of neovascularization greatly depends on the induction of the angiogenic phenotype of endothelial cells that is strictly controlled by humoral factors as well as by cellular communications in the vascular system. Although blood platelets contain several secretable pro- and antiangiogenic components, their overall role in angiogenesis remains poorly understood. In a mouse model of hypoxia-induced retinal angiogenesis, the situation of thrombocytopenia as well as inhibition of platelet aggregation by a highly specific alphaIIbbeta3-integrin antagonist or acetyl salicylic acid (Aspirin) administration, respectively, resulted in about 35-50% reduction of retinal neovascularization, compatible with a significant contribution of blood platelets in angiogenesis. Platelet remnants and microvesicles were found at sites of angiogenic sprouts. In vitro isolated platelets incorporated in a fibrin gel induced capillary sprouting of microvascular endothelial cells. Similarly, platelet releasate elevated the permeability of confluent endothelial cell monolayers to the same extent as hypoxia did. Platelet-derived VEGF as well as butanol-extractable lipid mediators were identified as predominant activators of angiogenesis, particularly of microvascular endothelial cell proliferation and migration. In addition, a synergistic effect between platelet-derived VEGF and bFGF in capillary sprouting and endothelial cell proliferation was found. Based on this proangiogenic role of platelets in neovascularization, anti-platelet substances can be considered as potent inhibitors of angiogenesis.

The formation of platelet microvesicles in septic pigs treated with different kinds of volume replacement

The formation of platelet microvesicles in septic pigs treated with different kinds of volume replacement

Thrombin receptor activating peptide (SFLLRN) potentiates shear-induced platelet microvesiculation

Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles . In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec -1 (shear stress ~375 dyne/cm 2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A 2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation. (J Lab Clin Med 2000;135:66-72)

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and α-Granules

AbstractPlatelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and α-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or α-thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and α-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains αIIb-β3 and β1, GPIbα, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Protein C Inhibitor Secreted from Activated Platelets Efficiently Inhibits Activated Protein C on Phosphatidylethanolamine of Platelet Membrane and Microvesicles

Protein C inhibitor (PCI) was detected in human platelets (2.9 ng/10(9) cells) and megakaryocytic cells (1.5 ng/10(6) cells). PCI mRNA was also detected in both platelets and megakaryocytic cells using nested polymerase chain reaction. PCI was found to be located in the alpha-granules of resting platelets. Approximately 30% of the total amount of PCI in platelets was released after stimulation with ADP, collagen, adrenalin, thrombin, or thrombin receptor-activating peptide. Secreted PCI was detected on the surface of activated platelets and phospholipid microvesicles. PCI secreted from thrombin receptor-activating peptide-stimulated platelets inhibited activated protein C (APC) efficiently. PCI significantly inhibited APC in the presence of phospholipid vesicles prepared using rabbit brain cephalin (RBC) or a mixture of 40% phosphatidylethanolamine (PE), 20% phosphatidylserine (PS), and 40% phosphatidylcholine (PC) with a second order rate constant of 1.0 x 10(6) M-1.min-1. Of these phospholipids, PE was critical for this inhibition. The dissociation constants of the binding of APC or PCI to solid phase phospholipids showed that APC binds more preferably to PE than to RBC or PS, and PCI to PE or RBC than to PS or PC. PCI binding to solid phase phospholipids depended on the presence of PE. RBC- or PE-bound PCI inhibited APC significantly but only weakly the gamma-carboxyglutamic acid domainless APC. The gamma-carboxyglutamic acid fragment of protein C suppressed the PCI-mediated inhibition of APC on solid phase RBC or PE. Most of the APC.PCI complex formed on solid phase RBC or PE was released into the soluble phase. These findings suggest that PCI secreted from activated platelets binds preferably to PE of platelet membrane and microvesicles and that it inhibits phospholipid-bound APC efficiently.

Impaired platelet function in analyzed by flow cytometry endotoxemic pigs

Endotoxin or bacterial lipopolysaccharide (LPS) is a structural component of Gram-negative bacteria. It is believed to be the major pathogenic factor of Gram-negative sepsis, and may result in intravascular coagulation and in a shock syndrome that is characterized by thrombocytopenia, leucopenia, hypotension, fever, reduced delivery of oxygen, metabolic acidosis and ultimately death. We have previously shown that both endotoxemic pigs and patients with Gram-negative sepsis have elevated levels of platelet microvesicles in their blood, which indicates platelet activation. In this study, we have used flow cytometry and fluorescein-labeled chicken anti-human fibrinogen to evaluate the in vivo effect of endotoxin on platelet function in a porcine model. Endotoxin infusion in pigs caused impaired platelet function when platelets were stimulated with adenosine-diphosphate in vitro ( P < 0.001). We also found a similarly decreased platelet function in patients with Gram-negative sepsis. Since flow cytometry is a rapid method for determination of platelet function, this method may turn out to be a useful tool in clinical situations. Our results may contribute to our understanding of the bleeding problems that may occur in septic shock and in disseminated intravascular coagulation. disseminated

Endotoxin induced platelet microvesicle formation measured by flow cytometry

Endotoxin (lipopolysaccharide, LPS) is a major component of the outermost membrane of gram-negative bacteria. Endotoxin is an important mediator of septic shock and it is involved in the development of disseminated intravascular coagulation (DIC), which is a dreaded complication of gram-negative bacterial infections. Platelet microvesicles are platelet derived vesicles that are formed during platelet activation. These microvesicles are too small to be detected by cell counters used in clinical laboratories, but they are active in haemostasis and may thus contribute to the development of DIC. We have used flow cytometry to study the in vivo effect of endotoxin on platelet microvesicle formation in clinical material and in a porcine model. We found increased levels of platelet microvesicles in patients with gram-negative bacterial sepsis. Endotoxin infusion in pigs caused microvesicle formation of up to four times the initial value. Thus, the formation of such microvesicles, which are associated with increased coagulation activity, may be initiated by endotoxin.

Platelet membrane glycoproteins and microvesicles in blood from postoperative salvage: a study in cardiac bypass patients.

Transfusion of blood collected by intraoperative and postoperative salvage systems has been linked to the development of thrombocytopenia and disseminated intravascular coagulation. Although functional defects have been reported in platelets from unwashed salvaged blood, platelet membrane glycoprotein (GP) composition, a potentially important determinant of function and survival, has not been studied. Platelets from 22 patients whose blood was salvaged at the completion of surgery were analyzed and compared to platelets obtained from the venous blood from the same patient. Platelet membranes were stained with fluorescein isothiocyanate-conjugated CD41a monoclonal antibody (anti-GPIIb/IIIa) to identify platelets, a phycoerythrin-conjugated monoclonal antibody, CD62 (anti-P-selectin) to identify activated platelets, and CD42b (anti-GPIb) or anti-GPIb/IX to assess GPIb. Samples were analyzed with a flow cytometer using software. Platelets obtained from salvaged blood demonstrated lower GPIb expression (CD42b and GPIb/IX monoclonal antibody binding), higher P-selectin expression, and greater numbers of platelet-derived microvesicles. The clinical significance of transfusing blood containing activated platelets and microvesicles merits investigation.

The effect of anticardiolipin antibodies lupus anticoagulant on the procoagulant activity of platelets and platelet microvesicles is dependent on β2-glycoprotein I: [formula omitted], T. Barbui, E.M. Bevers. Dept. of Haematology, Ospedali Riuniti, Bergamo, Italy and Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands

The effect of anticardiolipin antibodies lupus anticoagulant on the procoagulant activity of platelets and platelet microvesicles is dependent on β2-glycoprotein I: [formula omitted], T. Barbui, E.M. Bevers. Dept. of Haematology, Ospedali Riuniti, Bergamo, Italy and Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands

Platelet microvesicles adhere to subendothelium and promote adhesion of platelets

A role in hemostasis has been suggested for platelet membrane microvesicles (mv). The objectives of the studies reported here include functional analysis of platelet mv in models developed for study of platelet adhesion, as well as investigation of possible interactions between mv and intact platelets in these same adhesion models. Microvesicles were prepared from washed platelet concentrates by repeated freezing and thawing. Adhesion to subendothelium was measured quantitatively by radiolabelling mv with 111-In, and morphologically by scanning electron microscopy. Platelet mv adhered to subendothelium quantitatively over time. Using a modified Baumgartner chamber, we found adhesion of mv to subendothelium significantly increased with increasing shear rates. With this same model we found that prior exposure of subendothelium to mv greatly increased subsequent adhesion of platelets to the same everted vessel, compared to platelet adhesion in the absence of mv. All of these experiments were conducted with mv suspended in ACD/saline, indicating that plasma components are not essential for adhesion of mv. Our studies show that platelet mv adhere to subendothelium in much the same way as do platelets, and support the concept of a hemostatic role for mv in that they appear to increase platelet adhesion.

Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration.

Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration.