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Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1bfl/flGPIbα-Cre+ mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl3-induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1bfl/flGPIbα-Cre+ mice, revealed that the reduced thrombus size post-FeCl3-injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1bfl/flGPIbα-Cre+ mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1bfl/flGPIbα-Cre+ mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear.

Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s−1). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.

BACKGROUND: The collagen receptor glycoprotein VI (GPVI) is an attractive antiplatelet target due to its critical role in thrombosis but minor involvement in hemostasis.OBJECTIVE: To investigate GPVI receptor involvement in platelet activation by collagen-I and atherosclerotic plaque using novel blocking and non-blocking anti-GPVI nanobodies (Nbs).METHODS: Nb effects on GPVI-mediated signaling and function were assessed by western blot and whole blood thrombus formation under flow. GPVI clustering was visualized in thrombi using fluorescently labeled Nb28.RESULTS: Under arterial shear, inhibitory Nb2 blocks thrombus formation and platelet activation on collagen and plaque, but only reduces adhesion on plaque. In contrast, adhesion on collagen, but not plaque, is decreased by blocking integrin α2β1. Adhesion on plaque is maintained despite inhibition of integrins αvβ3, α5β1, α6β1, and αIIbβ3. Only combined αIIbβ3 and α2β1 blockade inhibits adhesion and thrombus formation to the same extent as Nb2 alone. Nb2 prevents GPVI signaling, with loss of Syk, Lat, and PLCɣ2 phosphorylation, especially to plaque stimulation. Non-blocking fluorescently labeled Nb28 reveals distinct GPVI distribution patterns on collagen and plaque, with GPVI clustering clearly apparent on collagen fibers and less frequent on plaque. Clustering on collagen fibers is lost in the presence of Nb2.CONCLUSIONS: This work emphasizes the critical difference in GPVI-mediated platelet activation by plaque and collagen; it highlights the importance of GPVI clustering for downstream signaling and thrombus formation. Labeled Nb28 is a novel tool for providing mechanistic insight into this process and the data suggest Nb2 warrants further investigation as a potential anti-thrombotic agent.

Background Collagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen-mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis-related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure. Objectives Our aim was to characterize mechanisms by which fibrin-GPVI interactions trigger GPVI shedding. Methods Platelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin-mediated platelet responses. Results Fibrin induced αIIbβ3-independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre-treatment with inhibitors of Src family kinases but were divalent cation- and metalloproteinase-dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen-related peptide caused αIIbβ3-dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity. Conclusions Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

Anti-platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown.To assess how clinical and platelet parameters, anti-PF4 antibody levels, and patient serum reactivity changes during follow-up after VITT presentation.We describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199-280) after vaccination. We measured anti-PF4 antibodies and PF4 levels in patient serum during follow-up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time.Anti-PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9-2.6 OD) and remain relatively high (1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow-up and no patients had recurrent thrombosis. Patients' platelets during follow-up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow-up serum alone was weaker at stimulating donor and patient platelets. However, follow-up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50.Explanations for reduced platelet activation during follow-up, despite similar total anti-PF4 antibody levels, remains unclear. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.

BackgroundAnti-Platelet Factor 4 (PF4) IgG antibodies that activate platelets via FcγRIIa have been shown to be an important part of the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). There is now extensive literature on its presentation and initial management. There is no literature however on what happens to these patients following discharge.MethodsWe collected clinical data and samples from seven patients presenting with VITT and followed them up for 82-145 days. We also collected clinical samples from them at last follow-up. Testing for anti-PF4/heparin antibodies was done using an anti-PF4/heparin enzymatic immunoassay. Flow Cytometry was used to look at FcγRIIa levels on patient platelets. Light Transmission Aggregometry with patient serum and healthy donor / patient platelets was used to analyse platelet responsiveness, in the presence and absence of PF4.FindingsAll patients were discharged on direct oral anticoagulants. Two patients remain completely symptom free, three have ongoing headaches, two have residual neurological deficits. Two patients developed mild thrombocytopenia and worsening headache (but without cerebral venous sinus thrombosis) and were retreated, one of these with rituximab. All patients, except the one treated with rituximab, had similar anti-PF4 antibody titres at 80-120 days to their levels at diagnosis. Platelets from patients at follow-up had normal levels of FcγRIIa and had normal responses to thrombin and collagen-related-peptide. Patient serum from diagnosis strongly activated healthy donor platelets in the presence of PF4. Serum from follow-up was much weaker at stimulating platelets, even in the presence of PF4.InterpretationThis study shows that despite similar PF4 antibody titres at diagnosis and during follow-up, there are further differences in patient serum, that are not apparent from currently used testing, that result in lower levels of platelet activation during the follow-up period. Further understanding of these factors are important in order to assess duration of anticoagulation for these patients.FundingThis work was supported by an Accelerator Grant (AA/18/2/34218) from the British Heart Foundation (BHF) and by a National Institute for Health Research (NIHR) grant.Key pointsPF4 antibody titres do not reduce up to 4-months post ChAdOx1 nCoV-19 in patients with VITTDespite similar PF4 antibody titres, diagnostic serum is more potent at activating platelets in the presence of PF4 than follow-up serum.

In this work, we propose a label-free COSI system to quantify morphological changes and platelet activity along non-patterned collagen fibers within millisecond in microfluidics channels under flow at sub-platelet imaging resolution.

In this work, we propose a label-free COSI system to investigating morphological changes and platelet-platelet interactions within a thrombus during embolism events to interrogate prothrombotic events within a microfluidics channel under flow.

Digital Holographic Microscopy (DHM) has emerged as a powerful imaging tool due to its ability to provide quantitative sample morphological data in a label-free manner. Off-axis holography further opens possibilities of numerical approaches to image analysis and data processing. However, this involves computationally heavy workflows that limit the tool’s usability in biomedical research. In this paper, a software package is developed for off-axis hologram reconstruction and processing. The software can perform a variety of bioimaging operations including imaging, region of interest selection, automated reconstruction, and data extraction, with a user-friendly graphical user interface. Originally programmed in MATLAB, the software can be installed to use in common lab-based Microsoft Windows computers. The software can be easily adopted to work with customized off-axis DHM setups, aiming to increase the efficiency of DHM’s bioimaging workflow in the community. To demonstrate this software, blood platelet experiments were conducted to show quantitative volume change of thrombus formation.

Platelets are small anucleate cells that are essential for many biological processes including hemostasis, thrombosis, inflammation, innate immunity, tumor metastasis, and wound healing. Platelets circulate in the blood and in order to perform all of their biological roles, platelets must be able to arrest their movement at an appropriate site and time. Our knowledge of how platelets achieve this has expanded as our ability to visualize and quantify discreet platelet events has improved. Platelets are exquisitely sensitive to changes in blood flow parameters and so the visualization of rapid intricate platelet processes under conditions found in flowing blood provides a substantial challenge to the platelet imaging field. The platelet's size (~2 μm), rapid activation (milliseconds), and unsuitability for genetic manipulation, means that appropriate imaging tools are limited. However, with the application of modern imaging systems to study platelet function, our understanding of molecular events mediating platelet adhesion from a single-cell perspective, to platelet recruitment and activation, leading to thrombus (clot) formation has expanded dramatically. This review will discuss current platelet imaging techniques in vitro and in vivo, describing how the advancements in imaging have helped answer/expand on platelet biology with a particular focus on hemostasis. We will focus on platelet aggregation and thrombus formation, and how platelet imaging has enhanced our understanding of key events, highlighting the knowledge gained through the application of imaging modalities to experimental models in vitro and in vivo. Furthermore, we will review the limitations of current imaging techniques, and questions in thrombosis research that remain to be addressed. Finally, we will speculate how the same imaging advancements might be applied to the imaging of other vascular cell biological functions and visualization of dynamic cell-cell interactions.

The application of advanced optical imaging technology for platelet biology is in its infancy. In this talk, we shall introduce the potential impact of optical imaging in understanding the roles of platelets in thrombus formation and in hemostasis. We will summarize how techniques in optical imaging has enabled exploration of morphological, molecular and fluidic parameters that determine platelet adhesion, activation and consequent roles in thrombus formation. Finally, we discuss the convergence of multiple imaging modalities towards a complete understanding of platelet roles in thrombus formation.

Coagulopathy commonly arises in patients following traumatic injury and is a major clinical burden [1]. Trauma-induced coagulopathy (TIC) is a multi-factorial condition associated with increased thrombin generation, coagulation factor consumption and activation of fibrinolytic pathways, leading to increased generation of plasmin, fibrin and fibrin degradation products, including D-dimers [1-3]. TIC also affects platelet function, where trauma patients can present with a hypofunctional platelet phenotype even with normal platelet count, which predicts mortality and poor patient outcome [4-7]. This article is protected by copyright. All rights reserved.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

Despite advances in design and materials, as well as pharmacological prophylaxis, hemostatic complications continue to plague device recipients. Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. We examined blood samples from patients with heart failure or in receipt of high fluid shear mechanocirculatory support to assess whether loss of platelet receptors occurs in vivo , and the relationship with acquired von Willebrand syndrome (AVWS) and changes in inflammatory cytokines. Platelet counts, levels of inflammatory cytokines IL-1β, IL-6, TNFα, MCP-1, IL-17A and IFNγ, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 13 continuous flow VAD (CF-VAD), 14 ECMO, and 24 heart failure patients. Levels of platelet receptors were measured by flow cytometry or ELISA. The loss of high molecular weight VWF multimers was observed in 12 of 13 CF-VAD and 7 of 14 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Significant differences in levels of inflammatory cytokines monocyte-chemoattractant protein and tissue necrosis factor-a in a subset of patients with decompensated HF. These data link AVWS and increased platelet receptor shedding in patients with CFVADs or ECMO. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CFVAD and ECMO patients may be linked with extent of inflammation and may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.

The ability to upregulate and downregulate surface-exposed proteins and receptors is a powerful process that allows a cell to instantly respond to its microenvironment. In particular, mobile cells in the bloodstream must rapidly react to conditions where infection or inflammation are detected, and become proadhesive, phagocytic, and/or procoagulant. Platelets are one such blood cell that must rapidly acquire and manage proadhesive and procoagulant properties in order to execute their primary function in hemostasis. The regulation of platelet membrane properties is achieved via several mechanisms, one of which involves the controlled metalloproteolytic release of adhesion receptors and other proteins from the platelet surface. Proteolysis effectively lowers receptor density and reduces the reactivity of platelets, and is a mechanism to control robust platelet activation. Recent research has also established clear links between levels of platelet receptors and platelet lifespan. In this review, we will discuss the current knowledge of metalloproteolytic receptor regulation in the vasculature with emphasis on the platelet receptor system to highlight how receptor density can influence both platelet function and platelet survival.

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)–FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.

C-type lectin-like receptor 2 (CLEC-2) is a glycoprotein expressed on the surface of platelets that induces platelet activation including aggregation. Binding of the endogenous ligand podoplanin or the exogenous ligand rhodocytin to CLEC-2 induces tyrosine phosphorylation of the hemITAM motif expressed in its cytoplasmic tail. This hemITAM phosphorylation induces subsequent downstream signalling leading to calcium mobilisation and platelet activation. Although CLEC-2 stimulation induces powerful platelet activation, the interaction of CLEC-2 with podoplanin is not crucial for haemostasis. During development, CLEC-2 interacts with podoplanin on lymphatic endothelial cells (LECs) and is crucial for blood/lymphatic vessels separation, as well as development of the cerebrovasculature. Moreover, CLEC-2 critically maintains the integrity of high-endothelial venules within lymph nodes post-development through its interaction with podoplanin on fibroblastic reticular cells.

Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal haematopoietic stem cell disorder characterised by haemolytic anaemia, thrombosis and bone marrow failure. Thrombosis is considered a major cause for high morbidity and mortality in PNH patients (Hillmen P et al N Eng J Med 1995). However, the underlying mechanisms of thrombosis in PNH are still poorly understood. Various factors including defective interactions between the complement system, platelets and coagulation systems have been proposed (Peacock-Young B et al Haematologica 2017). Platelet function and clot formation in this disorder have not been comprehensively evaluated. Here we describe standard and novel techniques to evaluate platelet function in blood from PNH patients, to elucidate the underlying pro-thrombotic mechanisms. Methods Whole blood was collected from five PNH patients and compared to same-day healthy donor (HD) controls. The samples were collected at trough for those patients who were on eculizumab (Complement C5 inhibitor used in the treatment of PNH). Surface levels of platelet adhesion proteins and activation markers P-selectin and phosphatidylserine (PS) were assessed using flow cytometry. Plasma soluble GPVI (a platelet-specific activation marker) and cytokine levels were measured by ELISA. Clot formation was assessed by viscoelastic testing (ROTEM). Adhesion of platelets to collagen under flow in a whole blood assay was evaluated by Digital Holographic Microscopy (DHM) and thrombus height, surface area and volume quantified using custom-built MatLab-based software. Results Clinical characteristics of PNH patients were highly variable: two patients had history of thrombosis, three patients were on eculizumab, four patients were thrombocytopenic ( Conclusion Analysis of these PNH patients with highly variable clinical characteristics did not identify a unifying platelet lesion. DHM could detect and quantify parameters of small thrombi in real time, in PNH patient samples and these data were consistent with enhanced thrombogenic potential in PNH patients. Mechanisms beyond platelet activation that contribute to increased thrombosis in these patients and the impact of eculizumab therapy on thrombotic propensity need to be explored further. Disclosures D'Rozario: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The authors received funding from the National Health and Medical Research Council of Australia and the Australian Research Council.