Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome.

BACKGROUND: Scott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized. OBJECTIVES: Genetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. METHODS: A targeted gene panel of known bleeding and platelet genes was used to identify possible genetic variants. Platelet phenotyping, flow adhesion, flow cytometry, whole blood and platelet-rich plasma thrombin generation, and specialized extracellular vesicle measurements were performed. RESULTS: We detected a novel homozygous frameshift variant, c.1943del (p.Arg648Hisfs∗23), in ANO6 encoding Anoctamin 6, in a patient with a bleeding history but interestingly with normal ANO6 expression. Phenotyping of the patient's platelets confirmed the absence of PS expression and procoagulant activity but also revealed other defects including reduced platelet δ granules, reduced ristocetin-mediated aggregation and secretion, and reduced P-selectin expression after stimulation. PS was absent on spread platelets, and thrombi formed over collagen at 1500/s. Reduced thrombin generation was observed in platelet-rich plasma and confirmed in whole blood using a new thrombin generation assay. CONCLUSION: We present a comprehensive report of a patient with Scott syndrome with a novel frameshift variant in AN06, which is associated with no platelet PS exposure and markedly reduced thrombin generation in whole blood, explaining the significant bleeding phenotype observed.

The Ways of the Virus: Interactions of Platelets and Red Blood Cells with SARS-CoV-2, and Their Potential Pathophysiological Significance in COVID-19.

The hematological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important in COVID-19 pathophysiology. However, the interactions of SARS-CoV-2 with platelets and red blood cells are still poorly understood. There are conflicting data regarding the mechanisms and significance of these interactions. The aim of this review is to put together available data and discuss hypotheses, the known and suspected effects of the virus on these blood cells, their pathophysiological and diagnostic significance, and the potential role of platelets and red blood cells in the virus's transport, propagation, and clearance by the immune system. We pay particular attention to the mutual activation of platelets, the immune system, the endothelium, and blood coagulation and how this changes with the evolution of SARS-CoV-2. There is now convincing evidence that platelets, along with platelet and erythroid precursors (but not mature erythrocytes), are frequently infected by SARS-CoV-2 and functionally changed. The mechanisms of infection of these cells and their role are not yet entirely clear. Still, the changes in platelets and red blood cells in COVID-19 are significantly associated with disease severity and are likely to have prognostic and pathophysiological significance in the development of thrombotic and pulmonary complications.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

Platelet Subtypes in Inflammatory Settings.

In addition to their essential role in hemostasis and thrombosis, platelets also modulate inflammatory reactions and immune responses. This is achieved by specialized surface receptors as well as secretory products including inflammatory mediators and cytokines. Platelets can support and facilitate the recruitment of leukocytes into inflamed tissue. The various properties of platelet function make it less surprising that circulating platelets are different within one individual. Platelets have different physical properties leading to distinct subtypes of platelets based either on their function (procoagulant, aggregatory, secretory) or their age (reticulated/immature, non-reticulated/mature). To understand the significance of platelet phenotypic variation, qualitatively distinguishable platelet phenotypes should be studied in a variety of physiological and pathological circumstances. The advancement in proteomics instrumentation and tools (such as mass spectrometry-driven approaches) improved the ability to perform studies beyond that of foundational work. Despite the wealth of knowledge around molecular processes in platelets, knowledge gaps in understanding platelet phenotypes in health and disease exist. In this review, we report an overview of the role of platelet subpopulations in inflammation and a selection of tools for investigating the role of platelet subpopulations in inflammation.

Immune-Mediated Platelet Activation in COVID-19 and Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Both qualitative and quantitative platelet abnormalities are common in patients with coronavirus disease 2019 (COVID-19) and they correlate with clinical severity and mortality. Activated platelets contribute to the prothrombotic state in COVID-19 patients. Several groups have shown immune-mediated activation of platelets in critically ill COVID-19 patients. Vaccine-induced immune thrombotic thrombocytopenia is an autoimmune condition characterized by thrombocytopenia and life-threatening thrombotic events in the arterial and venous circulation. Although the initial trigger has yet to be determined, activation of platelets by immune complexes through Fc gamma RIIA results in platelet consumption and thrombosis. A better understanding of platelet activation in COVID-19 as well as in vaccine-induced thrombotic complications will have therapeutic implications. In this review, we focused on the role of immune-mediated platelet activation in thrombotic complications during COVID-19 infection and vaccine-induced immune thrombotic thrombocytopenia.

Mechanisms Underlying Dichotomous Procoagulant COAT Platelet Generation-A Conceptual Review Summarizing Current Knowledge.

Procoagulant platelets are a subtype of activated platelets that sustains thrombin generation in order to consolidate the clot and stop bleeding. This aspect of platelet activation is gaining more and more recognition and interest. In fact, next to aggregating platelets, procoagulant platelets are key regulators of thrombus formation. Imbalance of both subpopulations can lead to undesired thrombotic or bleeding events. COAT platelets derive from a common pro-aggregatory phenotype in cells capable of accumulating enough cytosolic calcium to trigger specific pathways that mediate the loss of their aggregating properties and the development of new adhesive and procoagulant characteristics. Complex cascades of signaling events are involved and this may explain why an inter-individual variability exists in procoagulant potential. Nowadays, we know the key agonists and mediators underlying the generation of a procoagulant platelet response. However, we still lack insight into the actual mechanisms controlling this dichotomous pattern (i.e., procoagulant versus aggregating phenotype). In this review, we describe the phenotypic characteristics of procoagulant COAT platelets, we detail the current knowledge on the mechanisms of the procoagulant response, and discuss possible drivers of this dichotomous diversification, in particular addressing the impact of the platelet environment during in vivo thrombus formation.

Measurement of procoagulant platelets provides mechanistic insight and diagnostic potential in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse drug reaction associated with high rates of thrombosis-related morbidity and mortality caused by FcγRIIa-activating pathogenic antibodies to PF4-heparin. Procoagulant platelets are a platelet subset that promote thrombin generation, are clinically relevant in prothrombotic diseases, and are formed when platelet G-protein-coupled receptor (GPCR) and ITAM-linked receptors are co-stimulated. OBJECTIVES: We examined the procoagulant platelet response of healthy donors to platelet agonists in the presence of HIT plasma and determined the contribution of FcγRIIa. PATIENTS/METHODS: Our previously established flow cytometry-based procoagulant platelet assay was modified to incorporate plasma samples, performed using FcγRIIa-responsive donor platelets. Plasma samples were serotonin-release assay-confirmed HIT (HIT+), or negative on HIT screening. RESULTS: In response to GPCR stimulation, only HIT+ plasma produced a heparin-dependent sensitization that required active FcγRIIa. As a potential diagnostic tool, the procoagulant platelet assay achieved 98% accuracy in identifying clinically verified HIT when performed blinded to the diagnoses of a validation cohort. Samples inducing a higher procoagulant platelet response were more likely from patients with thrombotic complications. Thrombin stimulation markedly increased the procoagulant platelet response with HIT+ plasma that was heparin independent and only partially reversed by FcγRIIa blockade, possibly reflecting ongoing thrombotic risk after heparin cessation. CONCLUSIONS: We demonstrate that HIT plasma together with platelet agonists increased the procoagulant platelet proportions, which may contribute to thrombotic risk in HIT. Targeting procoagulant platelet activation may represent a novel treatment strategy. This assay may be a rapid, clinically relevant functional assay for accurately detecting pathological HIT antibodies.

Platelet procoagulant potential is reduced in platelet concentrates ex vivo but appears restored following transfusion.

BACKGROUND: The procoagulant profile of platelet concentrates (PCs) following transfusion has been difficult to evaluate due to lack of specific markers. This study aimed to characterize procoagulant platelets in PCs and the effect of transfusion. STUDY DESIGN AND METHODS: Buffy coat-derived PCs from 12 donors were pooled, split, then stored conventionally, cold (2-6°C) or cryopreserved (-80°C). Procoagulant platelet profiles were assessed by flow cytometry (GSAO+ /P-selectin+ ), lactadherin-binding, and calibrated automated thrombogram, during storage, unstimulated, or after thrombin and collagen stimulation and compared with blood from healthy volunteers. Platelet activation (P-selectin) and procoagulant platelet formation potential were measured (flow cytometry) in patients receiving clinically indicated conventional PC transfusion. RESULTS: Independent of significant increases with storage, procoagulant platelet proportions with and without agonist stimulation were significantly blunted in conventionally stored PCs (stimulated day 5 conventional PC 4.2 ± 1.3%, healthy volunteer blood 11.1 ± 2.9%; p < .0001). Cryopreserved PCs contained the highest proportion of procoagulant platelets (unstimulated: cryopreserved 25.6 ± 1.8% vs. day 5 conventional 0.5 ± 0.1% vs. day 14 cold-stored 5.8 ± 1.0%, p < .0001), but demonstrated minimal increase with agonist. Transfusion of PCs was associated with an increase in procoagulant platelets (2.2 ± 1.4% vs. 0.6 ± 0.2%; p = .004) and reversal of the blunted agonist response (15.8 ± 5.9% vs. 4.0 ± 1.6%; p < .0001). Procoagulant responses post-transfusion were significantly higher than healthy controls, suggesting a priming effect. The P-selectin agonist response was not restored upon transfusion (79.4 ± 13.9% vs. 82.0 ± 2.5%). CONCLUSION: Storage blunts the procoagulant platelet response to agonist stimulation in PCs. Despite this, conventionally stored PCs have high procoagulant potential following transfusion, with a discordant, persistent reduction in P-selectin response.

Procoagulant platelets: Generation, characteristics, and therapeutic target.

Platelets play a pivotal role in hemostasis. Activated platelets are classified into two groups, according to their agonist response: aggregating and procoagulant platelets. Aggregating platelets consist of activated integrin αIIbß3 and stretch out pseudopods to further attract platelets to the site of injury by connecting with fibrinogen. They mainly gather in the core of the thrombus and perform a secretory function, such as releasing adenosine diphosphate (ADP). Procoagulant platelets promote the formation of thrombin and fibrin by interacting with coagulation factors and can thus be considered as the connector between primary and secondary hemostasis. In addition to their functions in blood coagulation, procoagulant platelets play a proinflammatory role by releasing platelet microparticles and inorganic polyphosphate. Considering these important functions of procoagulant platelets, this subpopulation warrants detailed study to analyze their potential in preventing human diseases. This review summarizes the generation and important characteristics of procoagulant platelets, as well as their potential for preventing the adverse effects associated with current antiplatelet therapies.

Bisphenol AF elevates procoagulant platelets by inducing necroptosis via RIPK1-inflammasome axis.

Bisphenol AF, an analogue of Bisphenol A, is an important raw material used in the production of plastic and rubber substances like plastic bottles and containers, toys, and medical supplies. Increased contamination of air, water, dust, and food with BPA/BPAF, poses an enormous threat to humans, globally. BPAF/BPA are endocrine-disrupting chemicals that mimic estrogen hormone, thus increasing the risks of various metabolic and chronic disorders. Exposure of human blood cells to BPA/BPAF induces oxidative stress and genotoxicity. However, its effects on platelets, which play central roles in hemostasis and thrombosis, are not well-documented. In this study, we demonstrate that BPAF induces RIPK1-inflammasome axis-mediated necroptosis in platelets, increasing procoagulant platelet levels in vivo and in vitro. We also show that BPAF-induced rise in procoagulant platelets worsens pulmonary thromboembolism in vivo. The elevated procoagulant platelets are shown to increase platelet-neutrophil/monocyte aggregates that mediate pathogenesis of CVD, thrombosis, and chronic inflammatory diseases. Our results demonstrate the toxic effects of BPAF on platelets and how it propagates the clinical complications by elevating procoagulant platelet numbers. Altogether, our study sends a cautionary message against extensive use of BPAF in the plastic and rubber industries, resulting in frequent human exposure to it, thus endangering platelet functions.

Thrombocytopathies: Not Just Aggregation Defects-The Clinical Relevance of Procoagulant Platelets.

Platelets are active key players in haemostasis. Qualitative platelet dysfunctions result in thrombocytopathies variously characterized by defects of their adhesive and procoagulant activation endpoints. In this review, we summarize the traditional platelet defects in adhesion, secretion, and aggregation. In addition, we review the current knowledge about procoagulant platelets, focusing on their role in bleeding or thrombotic pathologies and their pharmaceutical modulation. Procoagulant activity is an important feature of platelet activation, which should be specifically evaluated during the investigation of a suspected thrombocytopathy.

Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia.

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

Improving long circulation and procoagulant platelet targeting by engineering of hirudin prodrug.

To reduce systemic bleeding risks during anticoagulant treatment, a new concept named "precise anticoagulation" was proposed to localize the effects of anticoagulants via the targeted delivery of prodrugs to the coagulation site. In this study, the fusion protein Annexin V-hirudin 3-ABD (hAvHA) was constructed to achieve the prolonged circulation and targeted delivery of hirudin to coagulation sites. hAvHA was inactive as a prodrug, and it could bind to albumin during circulation. The drug was quickly activated via factor Xa-mediated cleavage once coagulation occurred, and hirudin was efficiently released to exert antithrombin activity in vitro. The hAvHA protein could be activated in mouse blood and exert significant anticoagulation effects. The results of FITC labeling illustrated that hAvHA bound to procoagulant platelets, suggesting the Annexin V modification permits targeted delivery to sites of thrombosis. hAvHA bound to albumin in vitro with an equilibrium dissociation constant of 8 pM, suggesting the ABD modification permitted prolonged circulation in vivo. Moreover, the bleeding time was much shorter in hAvHA-treated mice than in hirudin-treated mice. Therefore, our results suggested that that hAvHA is a potential and promising anticoagulant in vivo.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Thrombin Generation and Cancer: Contributors and Consequences.

The high occurrence of cancer-associated thrombosis is associated with elevated thrombin generation. Tumour cells increase the potential for thrombin generation both directly, through the expression and release of procoagulant factors, and indirectly, through signals that activate other cell types (including platelets, leukocytes and erythrocytes). Furthermore, cancer treatments can worsen these effects. Coagulation factors, including tissue factor, and inhibitors of coagulation are altered and extracellular vesicles (EVs), which can promote and support thrombin generation, are released by tumour and other cells. Some phosphatidylserine-expressing platelet subsets and platelet-derived EVs provide the surface required for the assembly of coagulation factors essential for thrombin generation in vivo. This review will explore the causes of increased thrombin production in cancer, and the availability and utility of tests and biomarkers. Increased thrombin production not only increases blood coagulation, but also promotes tumour growth and metastasis and as a consequence, thrombin and its contributors present opportunities for treatment of cancer-associated thrombosis and cancer itself.

Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response.

Essentials Procoagulant platelets can be detected using GSAO in human whole blood. Stable coronary artery disease is associated with a heightened procoagulant platelet response. Agonist-induced procoagulant platelet response is not inhibited by aspirin alone. Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel. SUMMARY: Background Procoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay. Objectives We developed a novel assay, utilizing the cell death marker, GSAO [(4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P-selectin, to identify procoagulant platelets in whole blood by flow cytometry. Patients/Methods Using this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography. Results In patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α2b ß3 integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel. Conclusions We report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P-selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD.

Assessment of Platelet Function in Whole Blood by Flow Cytometry.

Evaluation of platelet function is important for understanding the physiology of hemostasis and thrombosis and is utilized in clinical practice to diagnose inherited and acquired platelet bleeding disorders. Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large number of platelets in whole blood and offers many advantages over other traditional methods. Attention to pre-analytical factors is required to ensure biologically valid and robust results.

Flow Cytometry Protocols for Assessment of Platelet Function in Whole Blood.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.