David E. Connor

The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib

It has been widely accepted that microparticles expose phosphatidylserine which in turn binds annexin V. It was the objective of this study to compare the antigenic characteristics and phospholipid-dependent procoagulant activity of annexin V positive and -negative subpopulations of platelet-derived microparticles. Annexin V positive and -negative microparticles were identified and characterised using flow cytometry and procoagulant activity was measured by a phospholipid-dependent assay (XACT). In unstimulated platelet-poor plasma, 80% of platelet-derived microparticles failed to bind annexin V. Varying the assay constituents (buffer, calcium and annexin V concentration) did not alter annexin V binding. The proportion of microparticles that bound annexin V was dependent upon the agonist, with physiological agonists such as collagen resulting in fewer annexin V binding microparticles than non-physiological agonists such as ionophore. CD42b (glycoprotein Ib) expression was significantly decreased and CD62p and CD63 expression were significantly increased in annexin V positive compared to annexin V negative subpopulations. There was no significant difference in CD41, CD61, CD42a and CD40L expression between annexin V positive and -negative subpopulations. A significant correlation between annexin V binding and XACT was found (p=0.033). Annexin V inhibited greater than 95% of phospholipid activity, suggesting that annexin V binding was a true reflection of procoagulant activity. The majority of platelet-derived microparticles in unstimulated plasma failed to bind annexin V and showed significantly increased levels of CD42b compared to annexin V positive events. Phospholipid-dependent procoagulant activity is limited to the annexin V positive subpopulation and is agonist-dependent. The significance of annexin V negative microparticles is unclear, however, it is possible that they possess other activities aside from procoagulant phospholipid activity.

The Lytic Effects of Detergent Sclerosants on Erythrocytes, Platelets, Endothelial Cells and Microparticles are Attenuated by Albumin and other Plasma Components in Vitro

OBJECTIVE To investigate the lytic effects of sodium tetradecyl sulphate (STS) and polidocanol (POL) on erythrocytes, platelets, endothelial cells and platelet-derived microparticle (PDMP) formation in vitro and the potential protective effects of serum albumin and agents such as procaine. MATERIALS AND METHODS The effects of sclerosants were studied in blood samples obtained from normal individuals. Absorbance densitometry was used to assess the lytic effects of sclerosants on blood cells and cultured human microvascular endothelial cells (HMEC) in plasma and in saline. PDMP were quantified by flow cytometry. RESULTS Haemolysis occurred in whole blood at sclerosant concentrations greater than 0.25% for STS and above 0.45% for POL. Similar concentrations of both agents caused platelet and endothelial cell lysis. Both sclerosants released PDMP at low concentrations but destroyed PDMP at higher concentrations. Albumin significantly reduced the lytic effect of both sclerosants on all cells but had a greater inhibitory effect on POL. Protamine at 0.01% had a neutralising effect on STS, whereas procaine and lignocaine showed no such activity. CONCLUSIONS Sclerosants at therapeutic concentrations lyse blood cells and endothelial cells in vitro. This effect is strongly reduced by serum albumin possibly contributing towards the low incidence of thromboembolic complications of sclerotherapy.

Detection of the procoagulant activity of microparticle-associated phosphatidylserine using XACT

One of the mechanisms by which platelet-derived microparticles elicit procoagulant activity is by an increased exposure of phosphatidylserine on their surface. We have previously demonstrated the utility of an activated factor X-based assay for the detection of procoagulant phospholipid activity [Xa clotting time (XACT)]. The objective of this study was to further characterize the specificity of the XACT to detect microparticle-associated procoagulant phospholipid activity. XACT testing for procoagulant phospholipid was measured using an ST4 machine and microparticle counting was performed using flow cytometry for Annexin V binding. Plasma microparticle counts were significantly correlated to XACT times (P = 0.0001). The XACT assay was insensitive to tissue factor, whereas the addition of microparticles to a whole blood sample shortened XACT times. Procoagulant phospholipid activity could be detected in both citrate and EDTA anticoagulated samples; however, XACT times and microparticle counts were more stable in EDTA anticoagulated samples over a 60 min period. The procoagulant phospholipid activity of microparticles generated by collagen stimulation was significantly impaired in EDTA anticoagulated samples when compared with citrate. Microparticles were capable of higher degrees of thrombin generation than equivalent concentrations of phosphatidylserine (as assessed by XACT times), suggesting that other factors bound to the microparticle surface enhance the procoagulant response. In conclusion, the XACT assay is a specific method for the detection of procoagulant phospholipid activity arising from phosphatidylserine on the microparticle surface; however, other factors presumably bound to the surface of the microparticle may also contribute to enhanced thrombin generation detectable by prothrombinase assays.

Platelet activation in acute pulmonary embolism

Summary.  Background: Platelet activation is implicated in thrombotic disorders, but has not been described in acute clinical pulmonary embolism (PE). Objectives: To investigate the natural history of platelet activation in PE and associated markers of inflammation, thrombosis and cardiac dysfunction. Methods: Thirty‐five consecutive patients (age 62 ±17 years) with acute PE were prospectively enrolled and followed for 6 months. Platelet activation was assessed by flow cytometry [measuring expression of platelet P‐selectin, conformational activation of glycoprotein IIb/IIIa complex (PAC‐1) and formation of platelet–leukocyte complexes] and by plasma soluble P‐selectin. Platelet activation, right ventricular (RV) function (assessed as RV ejection area by transthoracic echocardiography), D‐dimer and high‐sensitivity C‐reactive protein (hs‐CRP) were measured at presentation and repeated over 6 months follow‐up. Results: Soluble P‐selectin (56 ±19 ng mL−1, anovaP < 0.0001) and PAC‐1 (1.5 ± 1.8%, anovaP = 0.005) were mildly but significantly increased in patients with acute PE relative to healthy young men (soluble P‐selectin 33 ± 13 ng mL−1, P < 0.001; PAC‐1 binding 0.5 ± 0.6%, P < 0.01) and age‐matched controls (soluble P‐selectin 31 ± 9 ng mL−1, P < 0.001; PAC‐1 binding 0.4 ±0.4%, P < 0.05). Platelet P‐selectin expression and platelet–leukocyte complexes were not increased during acute PE. Echocardiographic RV ejection area correlated inversely with soluble P‐selectin (r = −0.47, P = 0.007) and positively with platelet P‐selectin (r = 0.49, P = 0.0007), suggesting P‐selectin is shed from activated platelets in proportion to the severity of RV dysfunction. Elevated soluble P‐selectin, D‐dimer and hs‐CRP demonstrated a time‐dependent return to normal during 6 months follow‐up. Conclusion: Platelet activation is evident after acute PE. Platelet activation correlates with the severity of RV dysfunction, and can persist for several months after acute PE.

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene.

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1FN) allele, followed by Clic1 knock‐out (Clic1−/−) mice by crossing Clic1FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y12 receptor signaling. genesis 48:127–136, 2010.

Nucleus pulposus cellular longevity by telomerase gene therapy.

Study Design. Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties. Objectives. To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner. Summary of Background Data. Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process. Methods. Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed. Results. Transfection was achieved successfully with 340% ± 7% (mean ± SD) relative telomerase activity in hTERT-transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions. Conclusions. Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency, or G1-cell cycle checkpoint control was evident.

Fructose metabolism by mature boar spermatozoa

In 1945, Mann showed fructose to be the principal sugar in semen. For over half a century the means by which fructose is metabolized by sperm has been assumed to be by an initial phosphorylation catalysed by hexokinase, but this has never been substantiated. In the present study, by comparing the metabolism of glucose and fructose by both whole boar sperm and hypotonically treated cells, it is confirmed that fructose is phosphorylated by hexokinase to produce fructose 6-phosphate.

Longitudinal changes in hemostatic parameters and reduced pulsatility contribute to non-surgical bleeding in patients with centrifugal continuous-flow left ventricular assist devices.

BACKGROUND Bleeding and thromboembolic events are identified complications in patients supported with newer centrifugal continuous-flow left ventricular assist devices (cfLVADs). Bleeding events have been associated with acquired von Willebrand syndrome (vWS) in these patients, though longitudinal changes and the effect of pulsatility remain unquantified. We evaluated longitudinal effects of third-generation cfLVADs on hemostatic biomarkers, non-surgical bleeding, and thromboembolic events. We investigated the association between pulsatility (as defined by aortic valve opening) on von Willebrand Factor (VWF) profile and bleeding. METHODS We prospectively studied 28 patients implanted with the HeartWare (HeartWare International, Framingham, MA) cfLVAD for up to 360 days. We performed bleeding and coagulation assays 8 times from pre-implant to Day 360 (D360) post-implant, including platelet aggregometry, VWF collagen binding activity-to-antigen (CBA/Ag) ratio, thromboelastography, soluble P-selectin, platelet-specific marker soluble glycoprotein VI (sGPVI), and platelet microparticles. Aortic valve opening was assessed by echocardiography at each assessment. Bleeding and thromboembolic events were documented. RESULTS Bleeding events occurred in 14 patients (50%). Maximal platelet inhibition occurred by D30. VWF profile impairment (VWF CBA/Ag < 0.8) was demonstrated in 89% of patients at D30, with subsequent recovery but further deterioration after D180. Bleeding was associated with elevated pre-implant sGPVI (p = 0.008). Pulsatility was associated with higher VWF CBA/Ag (p = 0.02) and a trend to less bleeding. CONCLUSIONS Third-generation cfLVADs were associated with longitudinal changes in hemostatic markers, and bleeding was associated with elevated pre-implant plasma sGPVI. Further, pulsatility may contribute to recovery of the VWF profile and potentially lower bleeding risk.

Low Concentration Detergent Sclerosants Induce Platelet Activation but Inhibit Aggregation due to Suppression of GPIIb/IIIa Activation in vitro

INTRODUCTION Sclerotherapy is associated with thromboembolic and ischemic neurological adverse events but the effects of sclerosants on platelet function are unknown. The aim of this study was to investigate the in vitro effects of detergent sclerosants Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on platelet activation and aggregation. MATERIALS AND METHODS Whole blood and platelet rich plasma samples were incubated with sclerosants. Platelet and platelet microparticle (PMP) counts were measured by flow cytometry. Platelet activation was examined by ELISA for soluble factors (sP-selectin, von Willebrand factor, sCD40L and serotonin) and by flow cytometry for membrane-bound markers (CD62p, CD63) and cytoplasmic calcium. Platelet aggregation was assessed by PFA-100®, light transmission and impedance (Multiplate®) aggregometry, and by flow cytometry for glycoprotein (GP) Ib and GPIIb/IIIa subunits, heterodimer expression and activation (PAC-1 binding). RESULTS Both agents lysed platelets at high concentrations (≥ 0.1%) but induced platelet activation at lower concentrations as evident by a rise in membrane-bound and soluble markers, cytoplasmic calcium and release of phosphatidylserine+PMP. Agonist-stimulated platelet aggregation was inhibited by both sclerosants. Membrane expression of GPIb and GPIIb/IIIa individual subunits or heterodimer was not affected by sclerosants but the activation of GPIIb/IIIa was suppressed. CONCLUSION Low concentration sclerosants activated platelets and released microparticles but inhibited platelet aggregation due to suppression of GPIIb/IIIa activation.

Flow cytometry demonstrates differences in platelet reactivity and microparticle formation in subjects with thrombocytopenia or thrombocytosis due to primary haematological disorders

BACKGROUND Traditional methods for the assessment of platelet function require a minimum number of platelets. As flow cytometry is independent of platelet number, we measured platelet activation and microparticle formation in thrombocytopenia and thrombocytosis. MATERIALS AND METHODS Blood was obtained from normal subjects or subjects with immune thrombocytopenic purpura (ITP), myelodysplasia (MDS) or essential thrombocythaemia (ET). Platelet activation and microparticle formation were assessed in resting and agonist stimulated samples. RESULTS Platelet activation was significantly decreased in MDS in agonist-stimulated platelets when compared to normals and ITP, however increased microparticle-to-platelet ratios were found. Absolute platelet-derived microparticle counts were significantly higher in ET when compared to normals, but there was no significant difference in microparticle-to-platelet ratios between ET and normals. CONCLUSIONS Decreased platelet activation was demonstrated in MDS when compared to normal subjects and ITP. Platelet-derived microparticle counts are increased in ET, reflecting increased platelet counts rather than an increase in platelet reactivity. Flow cytometric analysis of platelets may aid the diagnosis and management of these conditions.