Joanne E. Joseph

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.

Increased circulating platelet–leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis

It is possible that platelet activation may play a pathogenic role in the increased risk of thrombosis associated with antiphospholipid antibodies (APA). In this study, levels of in vivo platelet activation were measured in 20 patients with primary antiphospholipid syndrome (PAPS) and 30 systemic lupus erythematosus (SLE) patients (14 of whom had secondary APS) using sensitive flow cytometry. Soluble P‐selectin levels were also assayed. Platelet CD63 expression was significantly higher in PAPS than normal controls (P = 0·007), as well as SLE patients with and without secondary APS (P = 0·03 and P = 0·002 respectively). PAC‐1 binding was significantly higher in PAPS than the control group (P = 0·007) and SLE patients without APS (P = 0·015). Platelet–leucocyte complexes were significantly higher in SLE patients than both PAPS and the control group, and platelet–monocyte complexes were significantly increased in PAPS compared with the control group. (Platelet–leucocyte complexes were also significantly higher than controls in 10 rheumatoid arthritis (RA) patients without APA). Soluble P‐selectin levels were significantly higher in PAPS and SLE patients than the control group. Platelet CD62p expression, annexin V binding and platelet microparticle numbers were not increased in PAPS or SLE patients. We conclude that there is evidence of increased platelet activation in PAPS and SLE, and this is important to note as it may have potential therapeutic implications with respect to use of antiplatelet agents in these patients.

Performance of the platelet function analyser PFA-100 in testing abnormalities of primary haemostasis.

The PFA-100 device is a new instrument for the in-vitro testing of platelet function. Primary haemostasis is stimulated by recording the closure time taken for platelets to seal a 150 microm aperture in the centre of a membrane coated with collagen and either epinephrine or ADP. Patients with type 3 von Willebrands disease (n = 4) all had infinitely prolonged closure times (> 200 s) with both types of cartridge. A patient with afibrinogenemia exhibited only slightly prolonged closure times of 111 and 166 s for the ADP and epinephrine membranes, respectively. Patients with Glanzmanns thrombasthenia (n = 6) and Bernard Soulier syndrome (n = 2) had grossly prolonged closure times (> 200 s) with both types of cartridges. These results confirmed that the PFA-100 system was highly dependent on normal von Willebrand factor, glycoprotein Ib and glycoprotein IIb/IIIa levels but not on plasma fibrinogen. Patients with storage pool disease (n = 6) and Hermansky Pudlak syndrome (n = 7) had prolonged closure times with the epinephrine cartridge. There was no evidence of enhanced platelet function in patients with antiphospholipid syndrome, in sickle-cell disease or thalassemia. However, ingestion of aspirin resulted in a near consistent and significant prolongation of the closure time for the epinephrine cartridge but not for the ADP cartridge in both normal subjects and patients. The test offers a reliable, reproducible, rapid and simple means of assessing high-shear platelet function in vitro.

New oral anticoagulants: a practical guide on prescription, laboratory testing and peri-procedural/bleeding management

New oral anticoagulants (NOAC) are becoming available as alternatives to warfarin to prevent systemic embolism in patients with non‐valvular atrial fibrillation and for the treatment and prevention of venous thromboembolism. An in‐depth understanding of their pharmacology is invaluable for appropriate prescription and optimal management of patients receiving these drugs should unexpected complications (such as bleeding) occur, or the patient requires urgent surgery. The Australasian Society of Thrombosis and Haemostasis has set out to inform physicians on the use of the different NOAC based on current available evidence focusing on: (i) selection of the most suitable patient groups to receive NOAC, (ii) laboratory measurements of NOAC in appropriate circumstances and (iii) management of patients taking NOAC in the perioperative period, and strategies to manage bleeding complications or ‘reverse’ the anticoagulant effects for urgent invasive procedures.

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.

Timely diagnosis and management of heparin-induced thrombocytopenia in a frequent request, low incidence single centre using clinical 4T's score and particle gel immunoassay

Clinicians must promptly decide which patients suspected of having heparin‐induced thrombocytopenia (HIT) warrant a change in anticoagulation. This single‐centre series of 246 HIT testing referrals assessed the combination of clinical score (thrombocytopenia, timing, thrombosis, other causes of thrombocytopenia not evident; 4T’s), Diamed ID‐Heparin‐PF4 immunoassay (PaGIA) and 14C Serotonin Release Assay (SRA) to develop a practical and safe diagnostic strategy for HIT. A total of 142/256 (58%) referrals were in patients with a low 4T’s score, with 12/246 (5%) in the high scoring group. PaGIA was positive in 24/246 (9·7%) patients, whilst SRA was positive in 9/246 (3·6%). The overall positive predictive value of a positive PaGIA test alone was 37·5%, however this reached 80% for the high scoring group. Both negative PaGIA and low clinical score independently had negative predictive values of 100%. We subsequently developed an algorithm that, when applied to this cohort, would have resulted in 18/246 patients (7%) definitely requiring alternative anticoagulation, whilst a further 7/246 (2·8%) patients would have been considered on an individual basis. Ultimately (based on SRA) this would have resulted in 16/246 (6·5%) patients unnecessarily having a change in their anticoagulation, with 9/246 (3·6%) patients being ‘correctly treated’. The combination of 4T’s scoring and PaGIA permitted a practical and safe approach to rapid HIT diagnosis and management.

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.

New oral anticoagulants: a practical guide on prescription, laboratory testing and peri-procedural/bleeding management. Australasian Society of Thrombosis and Haemostasis.

New oral anticoagulants (NOAC) are becoming available as alternatives to warfarin to prevent systemic embolism in patients with non‐valvular atrial fibrillation and for the treatment and prevention of venous thromboembolism. An in‐depth understanding of their pharmacology is invaluable for appropriate prescription and optimal management of patients receiving these drugs should unexpected complications (such as bleeding) occur, or the patient requires urgent surgery. The Australasian Society of Thrombosis and Haemostasis has set out to inform physicians on the use of the different NOAC based on current available evidence focusing on: (i) selection of the most suitable patient groups to receive NOAC, (ii) laboratory measurements of NOAC in appropriate circumstances and (iii) management of patients taking NOAC in the perioperative period, and strategies to manage bleeding complications or ‘reverse’ the anticoagulant effects for urgent invasive procedures.

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.