Prothrombinase Activity Research Articles

Glycoside Residues on Platelet's Surface Regulate Platelet Function, Apoptosis and Binding of Coagulation Complexes in Patients with Immune Thrombocytopaenia

Introduction: Platelet surface glycoproteins (GPs) are highly glycosylated and are key elements for platelet function since most of them constitute receptors for adhesion ligands. However, exact role of their glycan composition is not clear. Under normal conditions, platelets contain sialic acid in the carbohydrate side chains of their GPs, and it has been described that alterations in the degree of their sialinization can affect the clearance of platelets. This mechanism has been proposed as involved in etiopathogenesis of immune thrombocytopaenia (ITP), mainly in those patients who do not respond to treatments. Thus, after the loss of sialic acid, there would be a greater exposure of galactose and of N-acetyl-glucosamine residues on the surface of circulating platelets to hepatic Ashwell-Morell receptors, which could induce their phagocytosis and platelet clearance. On the other hand, procoagulant platelets, defined as the platelet subpopulation that binds functional prothrombinase, exposed on their surface increased levels of P-selectin and GPIb, two glycan rich GPs. So, it is tempting to speculate that changes in glycan residues on platelet surface may induce changes in their function. Aim: We aimed to assess in ITP patients whether changes in platelet glycosylation, mainly the loss of sialic acid, may condition platelet function, apoptosis and binding of prothrombinase complex. Methods: This is an observational, prospective and transversal study approved by Ethics Committee from La Paz University Hospital. One hundred and eight patients with chronic primary ITP (68 with a platelet count ≥30x103 platelets/µL and 40 with a platelet count <30x103 platelets/µL) and 132 healthy controls were included after signing the informed consent. Platelet activation markers were determined in platelet rich plasma; whereas platelet glycosylation, binding of prothrombinase, annexin V and caspase's activities were assayed in washed platelets. Samples were analyzed by flow cytometry. Table 1 shows lectins tested and their sugar-binding specificity. Data were analyzed with GraphPad Prism 6.0 software. Results: Platelets from ITP patients with a platelet count <30x103/µL exposed less sialic acid in correspondence to an enhanced binding of lectins to non-sialylated residues. Moreover, levels of α1,6-Fucose, a glycan residue which could directly regulate antibody-dependent cellular cytotoxicity, and of α-Mannose, which could be recognized by the mannose binding lectin and activate complement pathway, were increased in platelets from these ITP patients. In accordance, sialic acid loss and consequent platelet surface exposure of other glycoside residues were inversely related to platelet count and ability to be activated (Table 1). These differences in glycosylation observed in ITP patients with a platelet count <30x103/µL were accompanied by a less ability of platelets to be activated (Figure 1), an increased exposure of phosphatidylserine and higher caspase activites (Figure 2). Moreover, increased exposure of phosphatidylserine and of N-acetyl-glucosamine residues (measured through the binding of WGA) enhanced binding of prothrombinase complex (Figure 3). Conclusion: Changes in glycoside composition of GPs on platelet's surface impaired their functional capacity, increases their apoptosis and modifies conditions for the binding of coagulation proteins. These modifications in platelet's glycoside residues seem to be related to severity of ITP. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772) and and Platelet Disorder Support Association. EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Butta: Grifols: Research Funding; Novartis: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; SOBI: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Speakers Bureau. Alvarez Román:Grifols: Research Funding; Bayer: Consultancy; Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau. Martín:SOBI: Research Funding; Pfizer: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau. Rivas Pollmar:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Justo Sanz:Takeda: Current Employment. García Barcenilla:NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Pfizer,: Speakers Bureau; Roche: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Speakers Bureau. Canales:Celgene: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Sandoz: Honoraria; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding.

The effect of pathogen inactivation on cryoprecipitate: a functional and quantitative evaluation.

As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.

An exploratory study of the effects of strenuous exercise on markers of coagulation activation, circulating microparticles, and inflammation in sickle cell trait.

This exploratory study evaluated the effect of intense exercise on biomarkers of inflammation and coagulation activation in subjects with and without sickle cell trait (SCT). Fifteen healthy African American men (18‐35 years, 5 SCT, 10 control) completed a strenuous exercise protocol. Microparticle‐associated prothrombinase and tissue factor activities, as well as soluble VCAM, total white cell and monocyte count increased transiently in all subjects following exercise. In the SCT group, exercise resulted in increased d‐dimer, erythrocyte phosphatidylserine exposure, as well as increased circulating erythrocyte‐ and endothelial‐derived microparticle numbers. These alterations could contribute to exercise‐related complications in people with SCT.

Influence of Different Antithrombotic Regimens on Platelet-Mediated Thrombin Generation in Patients with Left Ventricular Assist Devices.

We characterized the biologic background of prothrombotic platelet function in the setting of durable left ventricular assist devices (LVADs) evaluating the role of different antithrombotic regimens. Platelet-mediated thrombin generation was quantified using the Platelet Activity State (PAS) Assay and the Thrombin Generation Test (TGT) in 78 patients implanted with the HeartMate II (n = 10, 13%), the HeartMate 3 (HM3) (n = 30, 38%), or the HVAD (n = 38, 49%) and managed with oral anticoagulation plus aspirin (n = 46, 59%) or anticoagulation alone (n = 32, 41%). Coagulation parameters (platelet count, International Normalized Ratio (INR), activated Partial Thromboplastin Time, Fibrinogen and D-Dimer levels) and hemolysis (lactate dehydrogenase levels [LDH]) were also recorded to comprehensively characterize the hemostatic profile in the two groups. In patients without aspirin, the PAS assay revealed low-intensity increase in platelet prothrombinase activity (1.11-fold, p = 0.03). Similarly the TGT revealed moderate higher platelet reactivity when compared with patients receiving aspirin, consistent with reduction in lag time (0.87-fold, p < 0.001), increase in peak of thrombin generation (1.5-fold, p = 0.002) and thrombin generation rate (2-fold, p = 0.02), but comparable endogenous thrombin potential (p = 0.50). Coagulation parameters and LDH were comparable in the two groups (p > 0.05). Moreover, no differences were noted in platelet prothrombinase activity of patients implanted with the HM3 or HVAD. Our results suggest that, in the setting of durable LVADs, aspirin minimally modulates the biochemical pathway of platelet-mediated thrombin generation. Accordingly, re-evaluation of current antithrombotic management criteria in patients stratified according to bleeding/thromboembolic risk might be safe and beneficial to prevent adverse events.

Serum amyloid A4 is a procoagulant apolipoprotein that it is elevated in venous thrombosis patients

BackgroundSerum amyloid A4 (SAA4) is an apolipoprotein that is in the SAA family and it is constitutively translated. Previously, acute‐phase SAA1 and SAA2 levels were associated with venous thromboembolism (VTE). ObjectiveWe investigated the association of plasma SAA4 with VTE and the role of SAA4 in coagulation. Patients and MethodsThe association of SAA4 with VTE in a case‐control study of adult VTE subjects (N = 113 each group) and the effects of recombinant SAA4 on plasma blood coagulation assays and prothrombin activation initiated by factor Xa were evaluated. ResultsPlasma SAA4 levels in VTE subjects were higher vs. controls (48.1 vs. 38.4 µg/mL; P < .001). Elevated plasma SAA4 level (above the 90th percentile of controls) was associated with increased VTE occurrence (odds ratio, 3.8; 95% confidence interval, 1.8‐8.0). This association remained significant after the adjustment for acute‐phase SAA level, suggesting that SAA4 associated with VTE is independent of acute‐phase SAA. Two isoforms of SAA4, that is, glycosylated and nonglycosylated SAA4 isoforms, were each higher in VTE patients. When recombinant SAA4 was added to plasma, it shortened factor Xa‐1‐stage clotting times, showing that it enhances clotting in plasma. In reaction mixtures containing purified factors Xa and Va and prothrombin, recombinant SAA4 increased prothrombin activation, showing that it enhances prothrombinase activity. ConclusionElevated plasma constitutive SAA4 levels were linked to VTE in adults, and SAA4 can enhance thrombin generation in plasma. Our data highlight a previously unknown procoagulant activity of SAA4 that appears to be related to risk of venous thrombotic events.

Phagocytosis by endothelial cells inhibits procoagulant activity of platelets of essential thrombocythemia in vitro

BackgroundEssential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). MethodsPhosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. ResultsThe PS exposure on platelets in ET patients is higher than that in healthy controls. The PS‐exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time‐dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin αvβ3 on ECs, and P‐selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS‐exposed platelets were decreased after incubation with ECs. ConclusionOur results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.

Cardiac Myosin Acts Is a Potent Procoagulant in Vitro and In Vivo

Thrombin generation and fibrin formation can cause occlusive thrombosis and myocardial infarction is caused by occlusive thrombi. Exposure and release of cardiac myosin (CM) are linked to myocardial infarction, but CM has not been accorded any thrombotic functional significance. Skeletal muscle myosin (SkM), which is structurally similar to CM, was previously shown to exert procoagulant activities (Deguchi H et al, Blood. 2016;128:1870), leading us to undertake new studies of the in vitro and in vivo procoagulant activities of CM. First, the setting of hemophilia A with its remarkable bleeding risk was used to evaluate the procoagulant properties of CM. In studies of human hemophilia plasma and of murine acquired hemophilia A plasma, CM was added to these plasmas and tissue factor (TF)-induced thrombin generation assays were performed. Plasmas included human hemophilia A plasma and C57BL/6J mouse plasma with anti-FVIII antibody (GMA-8015; 5 microgram/mL final). CM showed strong procoagulant effects in human hemophilia A plasma, which is naturally deficient in factor VIII (&amp;lt;1% FVIII). The addition of only CM (12.5-200 nM) greatly increased thrombin generation in a manner comparable to addition of only recombinant FVIII. In the wild-type C57BL/6J mouse plasma, anti-FVIII antibody greatly reduced TF-induced thrombin generation, as reported. When CM (12.5-200 nM) was added to mouse plasma containing anti-FVIII antibodies, TF-induced thrombin generation was concentration-dependently restored. To study the in vivo hemostatic ability of SkM, an acquired hemophilia A mouse model was employed. Intravenous injection of anti-FVIII antibody (GMA-8015; 0.25 mg/kg) or control vehicle was given retro-orbitally to wild type C57BL/6J mice at 2 hours prior to tail cutting. The distal portion of the tail was surgically removed at 1.5 mm tail diameter to induce moderate bleeding. Tails were immersed in 50 mL of saline at 37 degrees. Total blood loss was measured as the blood volume collected during 20 min normalized for mouse weight (microL/g). Mice given only anti-FVIII antibody had more blood loss (median = 6.7 microL/g) compared to control mice (median &amp;lt; 2 microL/g) (Figure). In this mouse model receiving anti-FVIII antibody, CM (5.4 mg/kg) injected at 15 min prior to tail cutting significantly reduced the median blood loss from 6.7 to 2.0 and 3.2 microL/g, respectively (p &amp;lt; 0.001 for each myosin) (Figure). Thus, these studies provide in vivo proof of concept that both CM and SkM can reduce bleeding and are procoagulant in vivo. Second, studies of the effects of CM on thrombogenesis ex vivo using fresh human flowing blood showed that perfusion of blood over CM-coated surfaces at 300 s-1 shear rate caused extensive fibrin deposition. Addition of CM to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of CM's thrombogenicity. Further studies showed that CM enhanced thrombin generation in platelet rich plasma and platelet poor plasma, indicating that CM promotes thrombin generation in plasma primarily independently of platelets. To address the mechanistic insights for CM's procoagulant activity, purified coagulation factors were employed. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, CM greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for CM's prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. Binding studies showed that CM directly binds factor Xa. In summary, here we show that CM is procoagulant due to its ability to bind factor Xa and strongly promote thrombin generation. In summary, CM acts as procoagulant by its ability to bind factor Xa and strongly promote thrombin generation both in vivo an in vitro. These provocative findings raise many questions about whether and how the protective pro-hemostatic properties or the pathogenic prothrombotic properties of CM contribute to pathophysiology in the coronary circulation. This discovery raises many questions about CM and coronary pathophysiology, and future CM research may enable novel translations of new knowledge regarding CM's procoagulant activities for coronary health and disease. Figure Disclosures Mosnier: The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

Prothrombin Complex Concentrates (PCCs) Have Limited Effect on TF-Initiated Thrombin Generation in FXa Inhibitor-Anticoagulated Plasma: In Vitro Comparison between Direct Reversal By Andexanet Alfa and “Work Around” By PCCs

Introduction: Direct oral FXa inhibitors (DOACs, e.g., apixaban [Apix] and rivaroxaban [Riva]) exert their anticoagulant activity by specifically targeting FXa with subnanomolar affinities. Reversal of DOAC-induced anticoagulation can be achieved by immediate and effective sequestration of the inhibitors to relieve inhibition of FXa activity and restore normal thrombin generation (TG). Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for patients treated with Apix or Riva, when reversal of anticoagulation is needed due to life-threatening bleeding. Factor replacement therapies (e.g., 4 factor-PCCs [4F-PCCs]) have demonstrated clinical benefit for vitamin K antagonist-anticoagulated patients by replacing factors II, VII, IX, and X rendered deficient by the anticoagulant. In contrast, the effectiveness and clinical benefit of 4F-PCCs are unclear for reversing DOACs because PCCs do not affect anti-FXa activity. In the present study, we evaluated the effect of individual coagulation factors (FIX, FX, FII), as well as 4F-PCCs on TF-initiated TG (TF-TG) in human platelet poor plasma (PPP), with or without Apix or Riva, and compared it with AnXa under the same conditions. Methods: TF-TG was measured using a calibrated automated thrombogram (CAT, 5 pM TF, Diagnostica Stago). Purified plasma proteins (FIX, FX, FII), PPP, and a commercially available 4F-PCC (Kcentra/Beriplex) were used. 4F-PCC was used with or without pretreatment with the heparinase cup (Haemonetics Corporation) at room temperature for 30 minutes. TF-TG was measured in PPP spiked with purified plasma proteins, 4F-PCC (0-1.0 IU/mL), 4F-PCC + Riva or Apix (0-250 ng/mL), or PPP with AnXa (0-4 μM) + Riva or Apix (0-2000 ng/mL). For comparison, reversal of warfarin anticoagulation by 4F-PCC was performed under similar conditions using individual plasma from warfarin-treated patients (INR = 1.5-6.9) spiked with 4F-PCC (0-1.0 IU/mL). Five CAT parameters were collected, and the endogenous thrombin potential (ETP) and Peak thrombin (Peak) were used for the comparisons. Results: In normal PPP, addition of FX (up to 1.0 IU/mL) had minimal effect on TF-TG parameters, whereas FIX (1.0 IU/mL) mainly increased Peak (~60%). In contrast, FII (1.0 IU/mL) increased both the ETP (~2x) and Peak (~50%). These observations are consistent with the relative plasma concentration and affinity of these coagulation factors as the substrate for the respective enzyme complexes, demonstrating the predominant role of prothrombinase activity in the TF-TG. Addition of 4F-PCC (1.0 IU/mL, equivalent to the therapeutic dose of 50 IU/kg) to PPP caused similar increases of ETP (~2.4x) and Peak (~40%). However, individual plasma protein (FIX, FX, FII) or 4F-PCC up to 1.0 IU/mL was unable to restore normal ETP or Peak in the presence of Apix or Riva (75-250 ng/mL). ETP and Peak increased only when the inhibitor concentrations were sufficiently low (&amp;lt;75 ng/mL), leading to less inhibition of FXa and increased FIIa formation, likely due to an increased FII level from 4F-PCC addition. Similar results were obtained with or without preincubation of 4F-PCC with heparinase prior to spiking in plasma. In contrast, AnXa (0-4.0 μM) dose-dependently reversed Apix- and Riva- (up to 2000 ng/mL) induced inhibition of TF-TG. In the same TF-TG assay, 4F-PCC dose-dependently reversed warfarin anticoagulation in warfarin-treated patients' plasma spiked with 4F-PCC. Conclusions: Specific reversal agents are effective for restoration of TF-TG by either direct sequestration of the FXa inhibitor and restoration of FXa activity (in the case of AnXa with FXa inhibitors) or by replacement of inactive factors due to warfarin treatment (in the case of 4F-PCCs). Addition of coagulation factors or use of 4F-PCCs had limited effect on restoration of normal TG in the presence of a FXa inhibitor. These data demonstrate that AnXa can normalize TF-TG over a wide range of FXa inhibitor concentrations (0-2000 ng/mL), in particular over the range observed in bleeding patients in the ANNEXA-4 study, where 77% of bleeding patients had inhibitor levels ≥75 ng/mL at baseline. In contrast, 4F-PCCs were only able to restore normal TF-TG at low inhibitor levels (&amp;lt;75 ng/mL). These in vitro data are consistent with ANNEXA-4, where AnXa demonstrated &amp;gt;90% immediate reversal of anti-FXa activity and &amp;gt;80% hemostatic efficacy with a wide range of FXa inhibitor levels and multiple bleed types. Disclosures Lu: Portola Pharmaceuticals: Employment, Equity Ownership. Lin:Portola Pharmaceuticals, Inc: Employment, Equity Ownership. Bui:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Curnutte:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals: Employment, Equity Ownership.

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα.

Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X.

The Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.

P2Y12 Receptor Antagonists: Do They Really Inhibit Shear-Mediated Platelet Activation within MCS?

Purpose Shear-mediated platelet activation (SMPA) by supra-physiological blood flow found within MCS is a main driver and contributor of device-related thrombosis. Thus, antiplatelet drugs targeting the main pathways of platelet activation are routinely prescribed. We have shown, that aspirin and GP IIbIIIa blockers have limited ability in mitigating SMPA, and do NOT protect platelets from integrin shedding and microparticle (MP) generation. In contrast, ticagrelor, a direct P2Y12 purinergic receptor antagonist, drastically inhibits platelet prothrombinase activity induced by high shear. The aim of the study was to define the role of the P2Y12 pathway in SMPA and evaluate the effect of P2Y12 antagonists on platelet procoagulant activity and MP generation induced by high shear stress. Methods The following P2Y12 antagonists were used: clopidogrel active metabolite (10 µM), cangrelor (1 µM) and ticagrelor (1 µM). After 10 min incubation with the agent, human platelets were exposed to continuous high shear stress (70 dyn/cm2, 10 min) in a hemodynamic shearing device. Platelet procoagulant activity was assessed using the annexin V binding assay, detecting procoagulant surface exposure, and platelet activity state (PAS) assay measuring thrombin generation. MPs were quantified by flow cytometry as CD41+/AnV+ particles distinguished from platelets by their FS/SS. Results Ticagrelor and cangrelor showed similar inhibitory effect, consistently decreasing both platelet procoagulant activity and microparticle generation induced by shear stress. The number of AnV+ platelets, AnV+ MPs, and PAS levels were decreased by 26%, 27%, and 30%, respectively, as compared with non-treated controls subjected to shear. Clopidogrel active metabolite, known for its low affinity to the targeted receptor, reduced PAS by 15%, while annexin V binding was diminished in more than 10 times for platelets and 2 times for MPs, indicating a significant reduction of platelet procoagulant surface exposure but limited inhibition of thrombin generation. Conclusion P2Y12 purinergic receptor antagonists inhibit shear-mediated platelet procoagulant surface formation and microparticle generation, both contributors in overall platelet prothrombotic activity induced by high shear stress within MCS. Our results suggest that P2Y12 pathway is involved in the regulation of SMPA.

Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support.

We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support. Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support. Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001). We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.

Top New Drug Approvals of 2018

Top New Drug Approvals of 2018

Phospholipid-Independent Activity of Fviiia Mimetic Bispecific Antibodies in Plasma

Introduction: An important coagulation regulatory mechanism is localization of clotting complexes to exposed phosphatidylserine (PS) on cell surfaces. All components of the intrinsic tenase complex (factor (F)VIIIa, FIXa, and FX) bind to PS. FVIIIa mimetic bispecific antibodies are drugs in development for hemophilia A that aim to mimic the cofactor function of FVIIIa by bringing together FIXa and FX to generate FXa. However, these antibodies differ from FVIII in many ways including no requirement of activation and a lack of direct PS binding. Emicizumab is a bispecific antibody currently on the market for hemophilia A patients with inhibitors. It binds to factor FIX, FIXa, FX, and FXa with micromolar affinities in solution. Previously, we have shown that in-house preparations of sequence-identical emicizumab (SI-Emi) showed similar weak affinities to its antigens and similar in vitro activity to published emicizumab results by one-stage clotting, chromogenic FXa generation, and thrombin generation. However, in chromogenic FXa generation using antibody concentrations in the range of the mean steady state plasma concentration of patients on emicizumab prophylaxis [~360 nM, (Oldenburg, et al., NEJM 2017)], SI-Emi maintained 28% of its activity even in the absence of PS-containing phospholipid vesicles. Another FVIIIa mimetic antibody, BS-027125, was discovered by our group and binds with low nanomolar affinity to FIX, FIXa and FX, with no detectable binding to FXa. In one-stage clotting, BS-027125 achieved clot times similar to physiological levels of FVIII, but had poor activity in thrombin generation at these concentrations. Furthermore, it too maintained small amounts of phospholipid-independent activity in chromogenic FXa generation. Given the artificial nature of the chromogenic FXa generation assay, and that activity of prothrombinase is PS-dependent thereby precluding omission of phospholipids from thrombin generation assays, we developed an assay to detect FXa generation in a plasma milieu by FVIIIa mimetic antibodies or FVIII with and without phospholipid vesicles.Methods: FVIIIa mimetic antibodies or recombinant FVIII (rFVIII) were incubated with thrombin for 5 minutes, quenched with hirudin, then spiked into platelet-free congenital hemophilia A plasma treated with additional hirudin. FXIa (to generate FIXa in situ) with and without PC:PE:PS (40:40:20 molar ratio) phospholipid vesicles was added and reactions were triggered with a solution of CaCl2 and fluorogenic FXa substrate (Mes-D-LGR-ANSN(C2H5)2). Substrate cleavage was monitored kinetically on a fluorescent plate reader. Substrate cleavage by FXIa could not be detected, yet another unknown plasma peptidase did cleave substrate at a constant low rate that was background subtracted.Results: In the absence of phospholipid vesicles, SI-Emi maintained 51±3.7% of its FXa generation activity at all concentrations tested (3.8±0.4 versus 8.0±1.1 RFU/min at 333 nM). BS-027125 showed very low activity (0.43±0.12 RFU/min at 50 nM) in the presence of phospholipid vesicles, however, in the absence of phospholipid vesicles, BS-027125 activity was not detectable above baseline. Nearly all rFVIII activity (>99%) was lost in the absence of phospholipid vesicles (0.14±0.04 versus 15.1±1.8 RFU/min at 0.3 IU/mL). Addition of annexin V was sufficient to block all rFVIII activity in the presence or absence of phospholipid vesicles, but could not block SI-Emi activity. Furthermore, addition of rivaroxaban, a direct FXa inhibitor, confirmed that detection of substrate cleavage was due to FXa activity.Conclusions: In the absence of phosphatidylserine-containing phospholipid vesicles, SI-Emi promoted the generation of FXa in plasma triggered with FXIa. The activity of BS-027125 was too low in this assay to clearly determine its phospholipid-independent activity. These results suggest SI-Emi has mis-regulated (PS-independent) procoagulant activity due to a lack of phospholipid localization of the antibody-FIXa-FX complex. Given the weak affinity of SI-Emi for its antigens, the exact mechanism enabling this activity is unclear. Further study of this phenomenon and its relevance to overall thrombin generation and in vivo activity are needed. DisclosuresAleman:Bioverativ, a Sanofi company: Employment. Jindal:Bioverativ, a Sanofi company: Employment. Leksa:Bioverativ a Sanofi company: Employment. Peters:Bioverativ a Sanofi company: Employment, Equity Ownership. Salas:Bioverativ a Sanofi company: Employment, Equity Ownership.

A Plasma-Based Assay to Measure the Susceptibility of Factor V(a) to Inhibition By TFPIα

Background: Coagulation factor V (FV) is the precursor of activated FV (FVa), which assembles with factor Xa (FXa) on phospholipid surfaces to form the prothrombinase complex, accelerating prothrombin activation >1000-fold. FV is activated by FXa or thrombin via limited proteolysis at Arg709, Arg1018 and Arg1545. These cleavages progressively expose the FXa-binding site, generating activation intermediates with increasing affinities for FXa. Recently, it has been shown that tissue factor pathway inhibitor α (TFPIα) inhibits both the activation of FV (by interfering with cleavage at Arg1545) and the ability of partially activated FV(a) species to enhance prothrombin activation. These effects are mediated by interactions of the C-terminus of TFPIα with an acidic region in the B-domain of FV as well as with the FV(a) heavy chain. The pathophysiological relevance of these novel anticoagulant activities of TFPIα is still unexplored, but evidence has been provided that prothrombinase complexes assembled with FV(a) Leiden are less susceptible to TFPIα inhibition. Moreover, FV splicing variants (FV-short) with increased affinity for TFPIα have recently been discovered in two unrelated families (from East Texas and Amsterdam, respectively) with bleeding tendencies.Rationale and Aim: The FV present in plasma from different individuals may differ in its sensitivity to inhibition by TFPIα, with potential implications for the risk of venous thrombosis or bleeding. Therefore, the aim of this study was to develop a plasma-based assay that measures the susceptibility of FV(a) to TFPIα inhibition.Methods: FV in 1/1000 diluted plasma was activated for 3 minutes with a suboptimal FXa concentration on 20/60/20 DOPS/DOPC/DOPE lipids in the presence or absence of a peptide mimicking the C-terminus of TFPIα (TFPIα C-term). Purified prothrombin and a chromogenic substrate for thrombin were then added, and the activity of the prothrombinase complex was monitored continuously up to 30 minutes. The parabolic absorbance curves were fitted to second-order polynomial equations and the rate of prothrombin activation was calculated from the coefficient of the x2-term. The assay outcome was expressed as residual prothrombinase ratio (RP-ratio), defined as the ratio between the rates of prothrombin activation obtained in the presence and absence of TFPIα C-term. The assay was validated using plasma from 4 FV Leiden homozygotes and 4 normal controls. In addition, we tested plasma from 3 members of the FV Amsterdam family (2 carriers of the mutation up-regulating FV-short Amsterdam and 1 non-carrier).Results: The rate of prothrombin activation in the absence of peptide was a function of plasma FV level and pre-incubation time, and was inhibited by TFPIα C-term in a dose-dependent manner. A pre-incubation time of 3 minutes and a peptide concentration of 100 nM, yielding an RP-ratio of 0.30 in normal pooled plasma, were chosen. The RP-ratio was independent of the plasma FV level in the 75-150% range. Moreover, control experiments indicated that, at this high dilution, the plasma background did not influence the assay outcome. The intra- and inter-assay coefficients of variation of the RP-ratio were 5.4% and 12%, respectively. FV Leiden homozygotes had higher RP-ratios than normal controls (0.45 ± 0.04 vs. 0.30 ± 0.03, p=0.002), indicating resistance to inhibition by TFPIα C-term. Differently, the 2 carriers of the FV Asterdam mutation, who express high levels of FV-short Amsterdam, had markedly reduced RP-ratios (0.18 and 0.16 vs. 0.29 in the non-carrier), as expected from the high affinity of FV-short Amsterdam for TFPIα.Conclusions: We have developed and validated an assay that measures the susceptibility of plasma FV(a) to inhibition by TFPIα. This assay can be used to test whether TFPIα-mediated inhibition of FV activation and prothrombinase activity differs for (genetically) different FV variants and whether it correlates with the risk of thrombosis or bleeding.Supported by grant 2014-1 from the Dutch Thrombosis Foundation. DisclosuresNo relevant conflicts of interest to declare.

Necrotic but Not Apoptotic Platelets Are Functionally Procoagulant

There are numerous modes of cell death in mammalian cells. In platelets the primary identified modes are apoptosis and necrosis. Apoptosis is a form of programmed cell death, and in platelets it is considered to be important in their clearance. Besides pyknosis (cell shrinkage) and engulfment by resident phagocytes in vivo, a characteristic feature of apoptosis in nucleated cells is retraction of pseudopodia. However, in platelets, this does not occur. Instead, distinct from that which occurs during activation, lamelopodia and filopodia are not observed in apoptotic platelets suggesting that they have a limited potential to actively participate in coagulation. Necrosis, either regulated or pathologic, is characterized by cytoplasmic swelling (oncosis), swelling of cytoplasmic organelles, and, in later stages, rupture of the plasma membrane. Additionally, secondary necrosis can occur as a consequence of the activation of apoptotic pathways. Apoptosis is an ATP requiring process. In instances when an apoptotic signal was initiated and insufficient ATP is present for the completion of apoptosis, this ATP-depleted cell will undergo death with morphologic features of necrosis. Procoagulant platelets are those platelets that are capable of supporting the assembly of functional tenase and prothrombinase complexes. Due to the increased complexity of experiments demonstrating functional activity, the ability to support procoagulant activity has been presumed in subpopulations of platelets with variable degrees of phosphatidylserine exposure. Here we investigated the procoagulant function and the extent of PSer exposure in platelets undergoing different modes of cell death.Washed human platelets were stimulated with the BH-3 mimetic ABT-737 (100 nM) (apoptotic initiator) or convulxin (250 ng/mL, CVX) (initiator of regulated necrosis). The extent of PSer externalization was estimated by cytometric evaluation of annexin V (AnnV) binding, and functional activity was assessed using tissue factor-initiated thrombinoscopy. Upon stimulation two distinct subpopulations of PSer exposing platelets with “low” and “high” AnnV binding could be distinguished. In CVX-stimulated platelets only a single subpopulation (“high”) of PSer exposing platelets could be distinguished. But in platelets stimulated with the apoptotic initiator ABT-737 both “low” and “high” subpopulations of AnnV binding platelets could readily be discerned. To assess the procoagulant functionality of these various modes of cell death ABT-737 (generating ~38% “high” and ~46% “low” AnnV positive platelets) and CVX (~33% “high” AnnV) stimulated platelets were compared by thrombinoscopy. Relative to unstimulated platelets, inclusion of CVX-stimulated platelets within the reaction significantly shortened both the lag time (~5 to ~3 min (p<0.05)) and time to peak thrombin (~13 to ~8 min (p<0.05)). These shortened response times were accompanied by a ~2-fold increase in peak thrombin (p<0.05). In contrast to the potentiating effects of CVX-stimulation, ABT-737 stimulation had no effect on measures of thrombin; this despite having equivalent (when assessing only high) or even a greater percentage of AnnV binding (when assessing low and high).These results are the first to differentiate the mode of platelet cell death and its corresponding effects on PSer exposure and a functional measure of procoagulant function. Apoptotic and necrotic platelets are not only distinguished by their molecular mechanism of initiation, but also by their functional ability to support coagulation. Only platelets undergoing cell death by necrotic pattern were functionally procoagulant as measured by direct prothrombinase activity. Advancement in understanding the principles of cell death in procoagulant platelet formation might be beneficial for the determination and guidance of novel pharmacological strategy targeted for the treatment of procoagulant related hematopathologies. DisclosuresJobe:CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.

Histones Induce the Release of Extracellular Hemoglobin and Red Blood Cell-Derived Microvesicles with Procoagulant Activity

BackgroundHistones are nuclear proteins that can be released extracellularly following cell death or certain mechanisms of cell activation. Infusion of high doses of histones into mice causes lethality. Circulating levels of extracellular histones are elevated in several disease states associated with inflammation and thrombosis. The mechanisms by which extracellular histones induce morbidity and mortality are not fully understood. Histones may activate or be toxic to several cell types in vitro. Most of these activities have been shown to be dependent on histone signaling through toll-like receptors (TLRs). Red blood cells (RBC) do not express TLRs. However, previous studies have reported that histones promote RBC aggregation, increase shear-dependent RBC fragility, reduce deformability under flow and cause hemolytic anemia in mice. Histones also induce calcium influx and promote PS expression on RBC membranes thereby supporting the prothrombinase activity and shortening clotting times in plasma. Histones may insert into cell membranes (independently of TLRs), and form pore-like structures similar to ionophore. The role of this process in histone-induced RBC toxicity has not been fully investigated.ObjectiveWe assessed the ability of individual histones to promote the release of extracellular hemoglobin (Ex-Hb) and RBC-derived microvesicles (RBC-MVs), as well as RBC-MVs' ability to initiate or propagate thrombin generation (TG) in MV-free plasma.MethodsRBCs were isolated from citrated whole blood from healthy volunteers, and were re-suspended in buffer with or without 10uM A23187 (calcium ionophore), recombinant histones H2A, H2B, H3 or H4 for 1 hour at 37°C.To assess histone binding, RBCs were washed and dual-stained with fluorophore-conjugated anti-glycophorin A (GPA) and anti-panhistone antibodies. Stained RBCs were analyzed using imaging flow cytometry, and were considered positive for histone binding if the fluorescence intensity was > 95th percentile of RBCs processed in the absence of histones.RBCs were removed by centrifugation at 2,500g for 15 minutes x2. RBC-MVs present in the supernatant were pelleted by centrifugation at 20,000g for 30 min. Isolated RBC-MVs were then re-suspended in buffer to assess PS exposure using the microparticle activity assay (Hyphen Biomed). RBC-MVs were also re-suspended in re-calcified MV-free plasma supplemented with 4uM 41%PC/44%PE/15%PS lipid vesicles to assess their ability to initiate TG by monitoring the activity of thrombin on a fluorogenic substrate over time. To assess their ability to propagate TG, coagulation was initiated by 1pM tissue factor in MV-free plasma containing histone-induced RBC-MVs without addition of synthetic lipid vesicles. In this condition, phospholipids present on the surface of histone-induced RBC-MVs are the limiting factor for TG.The supernatant collected after pelleting RBC-MVs was used to measure Ex-Hb by absorbance at 540 nm.ResultsThe percentage of RBCs positive for histone binding was 21.6%, 2.3%, 76.9% and 98.1% for H2A, H2B, H3 and H4, respectively. Representative images of RBC staining for GPA and histone H4 is shown in Figure 1A.All histone types induced the release of Ex-Hb (Fig. 1B) and RBC-MVs that possess PS activity (Fig. 1C) in a dose-dependent manner, with the highest PS activity observed with H2A. RBC-MVs released by H2A or H2B clearly initiated TG in MV-free normal pool plasma (Fig.1D). They also significantly enhanced TF-initiated TG in the absence of additional lipid vesicles. The release of Ex-Hb and procoagulant RBC-MVs induced by histones were re-capitulated by ionophore to a greater degree, as expected (Fig.1B-E).ConclusionsAll 4 types of core histones bind to RBCs and induce the release of Ex-Hb. Our findings suggest that despite their lower binding to RBCs, histones H2A and H2B may have a greater ability to induce the release of procoagulant RBC-MVs that can initiate and propagate TG in plasma. The reason for the differential toxicity of histone types on RBCs and the discrepancy between the relative effect of histone-induced RBC-MVs on PS equivalents and TG are unknown. Histone-induced RBC toxicity appears to mimic calcium ionophore, supporting the idea that histones may insert and form functional pores in cell membranes. The contribution of Ex-Hb and procoagulant RBC-MVs to the morbidity and mortality of extracellular histones in vivo requires further studies. [Display omitted] DisclosuresMonroe:Novo Nordisk A/S: Honoraria, Research Funding.

Abstract 148: Cardiac Myosin Promotes Thrombin Generation and Attenuates Tissue Plasminogen Activator-induced Plasma Clot Lysis

Recently we discovered that skeletal muscle myosin, which is in the same family as cardiac myosin, exerts prothrombotic effects by binding factor Xa and enhancing prothrombin activation in the presence of factor Va. Thus, we tested the influence of cardiac myosin on thrombus formation and fibrinolysis. Studies of the effects of cardiac myosin on thrombogenesis ex vivo using fresh human flowing blood showed that perfusion of blood over cardiac myosin-coated surfaces at 300 s -1 shear rate caused extensive fibrin deposition. Addition of cardiac myosin to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of myosin’s thrombogenicity. Further studies showed that cardiac myosin enhanced thrombin generation in whole blood, platelet rich plasma and platelet poor plasma, indicating that myosin promotes thrombin generation in plasma primarily independently of platelets or other blood cell components. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, cardiac myosin greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for cardiac myosin’s prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. In studies of tissue plasminogen activator (tPA)-induced plasma clot lysis, increasing concentrations of cardiac myosin attenuated tPA-mediated clot lysis. The ability of cardiac myosin to inhibit tPA-induced plasma clot lysis was ablated in the presence of the carboxypeptidase inhibitor from potatoes, an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI). Clot lysis assays using TAFI-deficient plasma confirmed the requirement for TAFI for the antifibrinolytic action of cardiac myosin. We hypothesize that cardiac myosin-dependent thrombin generation increases TAFI activation and subsequent inhibition of clot lysis. In summary, here we show that cardiac myosin is both procoagulant and anti-fibrinolytic due to its ability to bind factor Xa and strongly promote thrombin generation. This raises new questions about potential procoagulant functions for cardiac myosin in coronary health and disease.

Abstract 520: Skeletal Muscle Myosin Supports the Anticoagulant Functions of Activated Protein C and Protein S but not Protein C Activation by Thrombin:Thrombomodulin

Skeletal muscle myosin was reported by Deguchi et al to promote thrombus formation ex vivo for flowing human whole blood, to support prothrombinase activity in plasma and to promote prothrombin activation by factors (F) Xa and Va in purified systems. Skeletal muscle myosin binds FXa and FVa and effectively replaces the procoagulant activity of phospholipid vesicles. We therefore assessed whether myosin might replace phospholipids in support of the anticoagulant function of activated protein C (APC) and its cofactor protein S (ProS) for inactivation of purified FVa. When purified component prothrombinase assays were used to monitor FVa inactivation by APC, FVa activity was significantly reduced by APC in the presence of myosin compared to controls, and FVa inactivation in the presence of 40 nM myosin over 25 min was much greater than that in the presence of 40 nM phospholipids and similar to the rate of FVa inactivation in the presence of 25 μM phospholipids (see Figure). Inhibition of the APC-enhancing effects of myosin were confirmed to be specific for myosin because the anti-myosin inhibitor, myoVin-1, inhibited myosin’s effects. ProS at ≥ 25 nM enhanced the myosin-stimulated inactivation of FVa by APC. The ProS anticoagulant enhancement effect was most obvious at lower APC concentrations, as expected. Immunoblots showed that myosin enhanced Arg506 cleavage in FVa by APC alone; but notably in the presence of ProS, myosin enhanced both Arg506 and Arg306 cleavages in FVa. Myosin did not enhance activation of protein C by thrombin in the presence or absence of thrombomodulin. Thus, in purified reaction mixtures, skeletal muscle myosin remarkably can support both procoagulant thrombin generation, as previously reported, and compensatory anticoagulant mechanisms in the presence of APC and ProS, as shown here. Further studies are needed to determine the molecular basis for these reactions and to assess the physiologic and pathologic relevance of these reaction mechanisms.

Microparticles formed during storage of red blood cell units support thrombin generation.

Intact red blood cells (RBCs) appear to support thrombin generation in in vitro models of blood coagulation. During storage of RBC units, biochemical, structural, and physiological changes occur including alterations to RBC membranes and release of microparticles, which are collectively known as storage lesion. The clinical consequences of microparticle formation in RBC units are unclear. This study was performed to assess thrombin generation via the prothrombinase complex by washed RBCs and RBC-derived microparticles as a function of RBC unit age. Well-characterized kinetic and flow cytometric assays were used to quantify and characterize microparticles isolated from leukocyte-reduced RBC units during storage for 42 days under standard blood banking conditions. Stored RBCs exhibited known features of storage lesion including decreasing pH, cell lysis, and release of microparticles demonstrated by scanning electron microscopy. The rate of thrombin formation by RBC units linearly increased during storage, with the microparticle fraction accounting for approximately 70% of the prothrombinase activity after 35 days. High-resolution flow cytometric analyses of microparticle isolates identified phosphatidylserine-positive RBC-derived microparticles; however, their numbers over time did not correlate with thrombin formation in that fraction. Red blood cell-derived microparticles capable of supporting prothrombinase function accumulate during storage, suggesting an increased potential of transfused units as they age to interact in unplanned ways with ongoing hemostatic processes in injured individuals, especially given the standard blood bank practice of using the oldest units available.