Marianne C. L. Schaap

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease

It has long been known that patients with sickle cell disease have ongoing activation of their coagulation system, which is exacerbated during painful occlusive crises. In this paper, the authors explore the role of the increased numbers of erythrocyte derived microparticles in this phenomenon and suggest that a surprisingly large proportion of this is dependent on Factor XI. See related perspective article on page 1481. Background Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease. Design and Methods In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in 25 consecutive patients with sickle cell disease. Results The majority of microparticles originated from platelets (GPIIIa,CD61) and erythrocytes (glycophorin A,CD235), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=−0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.023). Conclusions We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles.

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

INTRODUCTION Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. MATERIALS AND METHODS Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. RESULTS Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. CONCLUSIONS This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes.

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.

Cell-derived vesicles exposing coagulant tissue factor in saliva

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

Activated complement components and complement activator molecules on the surface of cell-derived microparticles in patients with rheumatoid arthritis and healthy individuals

Objectives: In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell-derived microparticles of RA patients and healthy individuals. Methods: Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex- and age-matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). Results: Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. Conclusions: This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid.

Synthesis of platelet-activating factor by human blood platelets and leucocytes. Evidence against selective utilization of cellular ether-linked phospholipids

Synthesis of platelet activating factor (PAF) in blood platelet suspensions may be due to leucocyte contamination. We therefore investigated PAF synthesis in human blood platelet suspensions and granulocyte- (PMN)-enriched leucocyte suspensions upon stimulation by thrombin and Ca2+-ionophore A23187, both in the presence and absence of the presumed PAF catabolism inhibitor phenylmethylsulfonyl fluoride (PMSF). PAF synthesis was measured by aggregation of washed rabbit platelets and by [3H]acetate incorporation. In contrast to A23187, thrombin was unable to stimulate PAF synthesis by leucocytes. As thrombin did induce PAF synthesis by platelet suspensions, this was evidently not due to leucocyte contamination. A23187 also induced PAF synthesis by platelets, but this was dependent upon the platelet isolation method and possibly associated activation. The ratio of [3H]acetate incorporation into 1-alkyl- versus 1-acyl-2-acetylglycerophosphocholine upon stimulation of non-PMSF-treated leucocytes and platelets amounted to 12.8 and 1.2, respectively. These values are at least 10-fold higher than the ratio of 1-alkyl versus 1-acyl species in the cellular phosphatidylcholine precursor for PAF. By PMSF pretreatment, the distribution of incorporated [3H]acetate between 1-ether- and 1-ester-linked species became similar to that in the precursor phosphatidylcholines of the respective cell type, due to increased recovery of [3H]acetate in the acyl compounds. Both leucocyte and platelet homogenates rapidly degraded acylacetylglycerophosphocholine to (acetyl)glycerophosphocholine, and this deacylation was inhibited by PMSF pretreatment of the cells. We conclude that upon cell stimulation a phospholipase A2 converts both alkylacylglycerophosphocholine and diacylglycerophosphocholine to the 2-lysoanalogs in a ratio similar to the occurrence of the parent compounds. The acetyltransferase subsequently acetylates both compounds to acylacetylglycerophosphocholine and alkylacetylglycerophosphocholine (PAF), respectively. Deacylation of the 1-ester-linked species, either before or after acetylation, gives the impression of selective utilization of 1-ether-linked species for PAF production. It is only after inhibition of the deacylation by pretreatment of the cells with PMSF that a mainly nondiscriminative use of 1-ether- and 1-ester-linked species by both phospholipase A2 and acetyltransferase becomes evident.

Prolactin does not affect human platelet aggregation or secretion

Platelets play an important role in the development of plaque formation and in the events after rupture of the atherosclerotic plaque, leading to atherothrombosis. Multiple hormones, either in excess or when deficient, are involved in the development of atherothrombotic disease, but, to which extent such hormones affect platelet function, is still controversial. It was the objective of this study to assess the ability of the pituitary hormone prolactin to affect platelet functions. Venous blood was collected from six healthy males. Platelet activation was studied by (i) flow cytometry in whole blood (exposure of P-selectin as a measure of platelet secretion, and binding of PAC-1 as a measure of ligand- binding conformation of αIIbβ3), and by (ii) optical aggregation and whole blood aggregation. All studies were performed without and with exposure to several concentrations of ADP (0.1, 0.5 and 1.0 μM) and prolactin (50 and 1,000 μg/l). The presence of the prolactin receptor was investigated by Western blot and flow cytometry. In response to either 50 or 1,000 μg/l prolactin, no evidence of platelet activation or aggregation was found. In addition, ADP-induced platelet activation or aggregation was not enhanced by prolactin. Finally, prolactin receptors could not be detected on the surface of platelets. The present data indicate that prolactin does not directly modulate platelet function.

Synergistic effects of platelet-activating factor and other platelet agonists in human platelet aggregation and release: The role of adp and products of the cyclooxygenase pathway

The synergistic effects of platelet-activating factor (PAF) with ADP, collagen, thrombin, A23187, adrenaline, sodium arachidonate and ristocetin in human platelet aggregation and beta-thromboglobulin (beta-TG) release were investigated in citrated platelet-rich plasma (PRP). Synergism in both aggregation and release was present with all agonists except ristocetin. Upon oral intake of aspirin (ASA) the PAF-induced irreversible aggregation as well as the synergistic irreversible aggregation became reversible. Both prior to and after ASA ingestion ADP removal by creatine phosphate/creatine phosphokinase (CP/CPK) resulted in a reduced, reversible platelet aggregation induced by PAF alone or in combination with the other agonists. The ADP-removal and ASA-ingestion also strongly inhibited the beta-TG release. The synergistic aggregation and release were also inhibited by ASA and indomethacin in vitro as well as by the competitive ADP-inhibitor ATP. It is concluded that not only the activation of human platelets by low doses of PAF itself, but also the synergism of PAF and other platelet agonists is highly dependent upon ADP and products of the cyclooxygenase pathway.

Platelet-activating factor: mediator of the third pathway of platelet aggregation? A study in three patients with deficient platelet-activating factor synthesis.

Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

Objectives To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Methods Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Results Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. Conclusions In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.