Inorganic polyphosphate (polyP) physiology 35 Caveats in studies of the physiological role of polyphosphates in coagulation

Abstract Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation

Формирование рельефа поверхности при алмазном выглаживании упругим инструментом

Материалы XIII Междунар. науч.-техн. конф. (науч. чтения, посвящ. 125-летию со дня рождения П. О. Сухого), Гомель, 22 окт. 2020 г

Contact activation: important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents

Background A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect. Objectives To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin). Results Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation. Conclusions Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs

Quantification of Platelet Contractile Movements during Thrombus Formation

Imaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads

The aim of the present study was to set up and evaluate a novel method for studies of platelet adhesion and activation in blood and platelet suspensions such as platelet concentrate (PC) samples using protein-coated polystyrene beads and flow cytometry. To demonstrate its usefulness, we studied PCs during storage. PCs were prepared by aphaeresis technique (n = 7). Metabolic variables and platelet function was measured on day 1, 5, 7 and 12 of storage. Spontaneous and TRAP-6-induced adhesion to fibrinogen- and collagen-coated beads was analyzed by flow cytometry. P-selectin and phosphatidyl serine (PS) expression was assessed on platelets bound to beads as well as on non-adherent platelets. Platelet adhesion to fibrinogen beads had increased by day 12 and adhesion to collagen beads at day 7 of storage (p < 0.05). TRAP-6 stimulation significantly increased the platelet adhesion to fibrinogen beads (p < 0.05) as well as the P-selectin and PS exposure on platelets bound to beads (p < 0.01) during the first 7 days of storage, but by day 12, significant changes were no longer induced by TRAP-6 stimulation. We demonstrate that our adhesion assay using protein-coated polystyrene beads can be used to assess the adhesion properties of platelets during storage without the addition of red blood cells. Therefore it may offer a useful tool for future studies of platelet adhesive capacity in transfusion medicine and other settings

Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders

<p>Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (<i>p</i> = 0.00008, hazard ratio 8.7; 95% CI 2.5–40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.</p

In vitro and in vivo evaluation of chemically modified degradable starch microspheres for topical haemostasis.

Degradable starch microspheres (DSMs) are starch chains cross-linked with epichlorhydrin, forming glycerol-ether links. DSMs have been used for many years for temporary vascular occlusion and drug delivery in treatment of malignancies. They are also approved and used for topical haemostasis by absorbing excess fluid from the blood and concentrating endogenous coagulation factors, thereby facilitating haemostasis. This mechanism of action is not sufficient for larger bleedings in current chemical formulations of DSMs, and modification of DSMs to trigger activation of platelets or coagulation would be required for use in such applications. Chemical modifications of DSMs with N-octenyl succinic anhydride, chloroacetic acid, acetic anhydride, diethylaminoethyl chloride and ellagic acid were performed and evaluated in vitro with thrombin generation and platelet adhesion tests, and in vivo using an experimental renal bleeding model in rat. DSMs modified to activate platelets in vitro were superior in haemostatic capacity in vivo. Further studies with non-toxic substances are warranted to confirm these results and develop the DSM as a more effective topical haemostatic agent

Gradients in surface nanotopography used to study platelet adhesion and activation

Gradients in surface nanotopography were prepared by adsorbing gold nanoparticles on smooth gold substrates using diffusion technique. Following a sintering procedure the particle binding chemistry was removed, and integration of the particles into the underlying gold substrate was achieved, leaving a nanostructured surface with uniform surface chemistry. After pre-adsorption of human fibrinogen, the effect of surface nanotopography on platelets was studied. The use of a gradient in nanotopography allowed for platelet adhesion and activation to be studied as a function of nanoparticle coverage on one single substrate. A peak in platelet adhesion was found at 23% nanoparticle surface coverage. The highest number of activated platelets was found on the smooth control part of the surface, and did not coincide with the number of adhered platelets. Activation correlated inversely with particle coverage, hence the lowest fraction of activated platelets was found at high particle coverage. Hydrophobization of the gradient surface lowered the total number of adhering cells, but not the ratio of activated cells. Little or no effect was seen on gradients with 36 nm particles, suggesting the existence of a lower limit for sensing of surface nano-roughness in platelets. These results demonstrate that parameters such as ratio between size and inter-particle distance can be more relevant for cell response than wettability on nanostructured surfaces. The minor effect of hydrophobicity, the generally reduced activation on nanostructured surfaces and the presence of a cut-off in activation of human platelets as a function of nanoparticle size could have implications for the design of future blood-contacting biomaterials