The acute effect of beta-guanidinopropionic acid versus creatine or placebo in healthy men (ABC-Trial): A randomized controlled first-in-human trial.

AIMS: Increasing evidence indicates that the ATP-generating enzyme creatine kinase (CK) is involved in hypertension. CK rapidly regenerates ATP from creatine phosphate and ADP. Recently, it has been shown that beta-guanidinopropionic acid (GPA), a kidney-synthesized creatine analogue and competitive CK inhibitor, reduced blood pressure in spontaneously hypertensive rats. To further develop the substance as a potential blood pressure-lowering agent, we assessed the tolerability of a sub-therapeutic GPA dose in healthy men. METHODS: In this active and placebo-controlled, triple-blind, single-centre trial, we recruited 24 healthy men (18-50 years old, BMI 18.5-29.9 kg m-2 ) in the Netherlands. Participants were randomized (1:1:1) to one week daily oral administration of GPA 100 mg, creatine 5 g, or matching placebo. The primary outcome was the tolerability of GPA, in an intent-to-treat analysis. RESULTS: Twenty-four randomized participants received the allocated intervention and 23 completed the study. One participant in the placebo arm dropped out for personal reasons. GPA was well tolerated, without serious or severe adverse events. No abnormalities were reported with GPA use in clinical safety parameters, including physical examination, laboratory studies, or 12-Lead ECG. At day 8, mean plasma GPA was 213.88 (SE 0.07) in the GPA arm vs. 32.75 (0.00) nmol l-1 in the placebo arm, a mean difference of 181.13 (95% CI 26.53-335.72). CONCLUSION: In this first-in-human trial, low-dose GPA was safe and well-tolerated when used during 1 week in healthy men. Subsequent studies should focus on human pharmacokinetic and pharmacodynamic assessments with different doses.

Toll-Like Receptor Signalling Is Not Involved in Platelet Response to Streptococcus pneumoniae In Vitro or In Vivo.

Streptococcus (S.) pneumoniae strains vary considerably in their ability to cause invasive disease in humans, which is at least in part determined by the capsular serotype. Platelets have been implicated as sentinel cells in the circulation for host defence. One of their utensils for this function is the expression of Toll-like receptors (TLRs). We here aimed to investigate platelet response to S. pneumoniae and a role for TLRs herein. Platelets were stimulated using four serotypes of S. pneumonia including an unencapsulated mutant strain. In vitro aggregation and flow cytometry assays were performed using blood of healthy volunteers, or blood of TLR knock out and WT mice. For in vivo pneumonia experiments, platelet specific Myd88 knockout (Plt-Myd88-/-) mice were used. We found that platelet aggregation was induced by unencapsulated S. pneumoniae only. Whole blood incubation with all S. pneumoniae serotypes tested resulted in platelet degranulation and platelet-leukocyte complex formation. Platelet activation was TLR independent, as responses were not inhibited by TLR blocking antibodies, not induced by TLR agonists and were equally induced in wild-type and Tlr2-/-, Tlr4-/-, Tlr2/4-/-, Tlr9-/- and Myd88-/- blood. Plt-Myd88-/- and control mice displayed no differences in bacterial clearance or immune response to pneumonia by unencapsulated S. pneumoniae. In conclusion, S. pneumoniae activates platelets through a TLR-independent mechanism that is impeded by the bacterial capsule. Additionally, platelet MyD88-dependent TLR signalling is not involved in host defence to unencapsulated S. pneumoniae in vivo.

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.

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.

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

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.

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 alpha(IIb)beta(3)), 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 microM) and prolactin (50 and 1,000 microg/l). The presence of the prolactin receptor was investigated by Western blot and flow cytometry. In response to either 50 or 1,000 microg/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.

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.

Predilution versus postdilution during continuous venovenous hemofiltration: a comparison of circuit thrombogenesis.

During continuous venovenous hemofiltration, predilution can prolong circuit survival time, but the underlying mechanism has not been elucidated. The aim of the present study was to compare predilution with postdilution, with respect to circuit thrombogenesis. Eight critically ill patients were treated with both predilutional and postdilutional continuous venovenous hemofiltration in a crossover fashion. A filtration flow of 60 ml/min was used in both modes. We chose blood flows of 140 and 200 ml/min during predilution and postdilution, respectively, to keep the total flow through the hemofilter constant. Extracorporeal circuit pressures were measured hourly, and samples of blood and ultrafiltrate were collected at five different time points. Thrombin-antithrombin complexes and prothrombin fragments F1 + 2 were measured by ELISA, and platelet activation was assessed by flow cytometry. No signs of thrombin generation or platelet activation were found during either mode. During postdilution, baseline platelet count and maximal prefilter pressure had a linear relation, whereas both parameters were inversely related with circuit survival time. In summary, predilution and postdilution did not differ with respect to extracorporeal circuit thrombogenesis. During postdilution, baseline platelet count and maximal prefilter pressure were inversely related with circuit survival time.

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.