Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

Abstract

circulating microparticles (Mps) are procoagulant due to the surface containing phosphatidylserine (pS), which facilitates coagulation. We investigated if Mps improve hemostasis in HA plasma models. Mps isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (SteD) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of Mps. Mps partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. in summary, our results demonstrate that pS positive Mps could improve hemostasis in HA plasma models. Hemophilia A (HA) is an inherited bleeding disorder, which is caused by a deficiency in coagulation factor VIII (FVIII) 1 and characterized by insufficient thrombin generation and fibrin formation 2. Patients with HA are characterized as having: severe (<1% of normal FVIII activity), moderate (1-5%) or mild (5-40%) HA. Prophylactic FVIII replacement therapy is recommended for severe and some moderate HA patients, in order to transform the bleeding phenotype from severe to non-severe 3. The major concerns of current therapy to HA are the development of FVIII inhibitors, and the need of frequent injections due to the short half-life time of drugs 4. Moreover, because of the high cost of therapy, HA patients in many developing countries do not have access to prophylaxis 5. Finding novel potential adjunctive therapies for HA is therefore of interest. Circulating microparticles (MPs) are small (0.1-1 µm) membrane vesicles originating from many different cells by membrane blebbing after activation, apoptosis, or high shear stress 6. MPs, as their parental cells, may provide cell surface component and participate in the coagulation process 7. In in vitro studies with plasma from healthy individuals, MPs enhance thrombin generation, fibrin clot structure and clot stability 8,9. Elevated levels of total MPs, especially tissue factor (TF) positive MPs, have been associated with cardiovascular disease and cancer 10. Few studies have investigated the role of MPs in HA. Levels of MPs in plasma have been found to be higher in untreated HA patients compared with healthy individuals 11. One previous clinical study of plasma from on-demand-treated severe HA patients showed that the level of MPs decreased after FVIII treatment, and was inversely correlated with thrombin generation and fibrin formation. These findings suggest that MPs may participate

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis

“…Moreover, circulating PEVs have the ability to incorporate into the fibrin clots ( 35 ). We have recently confirmed the close interaction between PEVs and fibrin fibers, particularly at branch points and junctions ( 24 ). This finding may suggest intensified fibrin formation around the surface of PEVs, following the activation of platelets and the propagation phase of thrombin generation.…”

“…points and junctions(24). This finding may suggest intensified fibrin formation around the surface of PEVs, following the activation of platelets and the propagation phase of thrombin generation.…”

“…Conjugate isotype-matched immunoglobulins (IgG1-FITC, IgG1-PE, and IgG1-APC) with no reactivity against human antigens were used as negative controls to define the positive and negative gates. The absolute number of EVs was calculated by means of the following formula: Concentration (μl) = (Events reading × 550)/(44 × 20), where 550 μl is the total volume of sample in the tube analyzed, 44 μl is the volume of the analyzed sample over 90 s, and 20 μl is the volume of the plasma sample added in the tube ( 24 ). The concentration of EVs is expressed as 10 6 EVs/l, and the intra and interassay coefficients of variation for EV measurements were less than 9%.…”

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks

“…Fibrin clots at identical concentrations were also prepared for STORM imaging, and fiber diameters were calculated from reconstructed STORM images and were used as a standard for evaluating the accuracy of the diameter values obtained using the turbidimetric approaches. STORM imaging and Simulated Emission Depletion Microscopy (STED), another super-resolution microscopy technique, have been previously used to determine fiber diameter from plasma [ 49 , 50 , 51 ] and from purified fibrinogen [ 52 ], providing fiber diameters over a range of 100–400 nm, matching those previously reported from SEM imaging [ 34 ]. However, super-resolution fluorescence imaging, unlike SEM imaging, does not require drying of the samples, which could cause changes in the morphology of the fibers.…”

Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding

“…A freeze-dried platelet product derived from group O platelets has been reported to have a PS-rich surface that amplifies hemostasis and is currently in clinical trials ( 63 ). Another recent study has shown that plasma-derived PS-positive microparticles improved clot quality in blood samples from patients with hemophilia; however, this was only demonstrated in vitro as such particles cannot be administered in vivo due to a systemic procoagulant risk ( 64 ).…”