No abstract

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

No abstract

A simple and rapid global haemostatic assay for determination of the overall hemostasis potential (OHP) in plasma represents a new approach in detecting alterations in the delicate balance between coagulation and fibrinolysis. The assay is based on repeated spectrophotometric registration of the fibrin-aggregation curve in platelet-poor plasma containing small amounts of exogenous thrombin, tissue-type plasminogen activator, and calcium. The overall coagulation potential and overall fibrinolytic potential are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The OHP assay was evaluated in connection with hypercoagulation in normal pregnancy, preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke, and vascular surgery as well as with hypocoagulability, especially in patients with hemophilia A or B. Preliminary results also indicate the possible usefulness of the assay in monitoring anticoagulant treatments. Large prospective clinical trials are needed before the method can be recommended for routine clinical application.

Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russell's viper venom time - dRVVT or activated partial thromboplastin time - aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs - dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2-1.4 LA could not be ruled out. Plasma concentrations varied between 8-172 µg/l for DAB, 8-437 µg/l for RIV and 36-178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.

Objectives: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. Material and method: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 mm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. Results: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. Conclusion: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups. Lupus (2012) 21, 802-805.

The present clinical and laboratory classification criteria for antiphospholipid syndrome (APS) were established in Sydney, Australia, in 2006. In this review, we focus on the obstetric subset of APS (OAPS), defined by persistent positivity for antiphospholipid antibodies together with either early recurrent pregnancy loss, early fetal death, stillbirth or premature birth <34 gestational weeks due to pre-eclampsia, eclampsia and placental insufficiency. It is important to diagnose these cases since most women suffering from OAPS can, when given appropriate treatment, have successful pregnancies. Furthermore, patients with OAPS may, depending on the antibody profile, be at enhanced risk of thrombotic events later in life. We present an update on the present knowledge of possible underlying pathogenesis, risk factors and risk estimations for adverse pregnancy outcomes before and during pregnancy, current treatment concepts, and long-term outcomes for women with OAPS and their children.

Our earlier investigations employing a flow measurement have yielded intriguing findings as to what governs the fibrin network porosity. To make the method suitable for use by more groups with various laboratory conditions, sample materials or study purposes, we simplified the essential equipment and thereby minimized the sample volume to 250 microl in comparison with the need for 3000 microl in the previous method. To assess whether the fibrin gel permeability depends on changes in thrombin generation potential and/or fibrinogen clotting property, different concentrations of thrombin with or without frozen-thawed platelets, serving as phospholipids, were used. The platelets and 0.05 IU/ml thrombin were added to plasma samples from patients with previous myocardial infarction. The fibrin gel permeability, expressed as Darcy constant (Ks), was decreased compared with that in controls, supporting findings about high risk of thromboembolism in this disease due to increases of thrombin activity and fibrinogen function. When 0.4 IU/ml thrombin was used in samples provided by 10 healthy individuals treated with acetysalicylic acid, Ks levels were increased during versus before therapy. Since almost no thrombin generation was found in the samples with the higher dose of exogenous thrombin, we considered that modifications in fibrinogen clotting property by acetysalicylic acid rendered the fibrin network more permeable. In summary, as the reproducibility remains satisfactory (coefficient of variation < 10%) despite aforementioned modifications in the equipment and reagents, any interested laboratory ought to be able to repeat the method. Assays of fibrin permeability in such a simple way may help to determine the fibrin clot stability in pathological/pharmacological studies, and probably serve as a tool to estimate thromboembolism risk in clinical materials, such as patients with cardiovascular diseases.