Structural analysis of platelet fragments and extracellular vesicles produced by apheresis platelets during storage.

ABSTRACT: Platelets (PLTs) for transfusion can be stored for up to 7 days at room temperature (RT). The quality of apheresis PLTs decreases over storage time, which affects PLT hemostatic functions. Here, we characterized the membranous particles produced by PLT storage lesion (PSLPs), including degranulated PLTs, PLT ghosts, membrane fragments, and extracellular membrane vesicles (PEVs). The PSLPs generated in apheresis platelet units were analyzed on days 1, 3, 5, and 7 of RT storage. A differential centrifugation and a sucrose density gradient were used to separate PSLP populations. PSLPs were characterized using scanning and transmission electron microscopy (EM), flow cytometry (FC), and nanoparticle tracking analysis (NTA). PSLPs have different morphologies and a broad size distribution; FC and NTA showed that the concentration of small and large PSLPs increases with storage time. The density gradient separated 3 PSLP populations: (1) degranulated PLTs, PLT ghosts, and large PLT fragments; (2) PEVs originated from PLT activation and organelles released by necrotic PLTs; and (3) PEV ghosts. Most PSLPs expressed phosphatidyl serine and induced thrombin generation in the plasma. PSLPs contained extracellular mitochondria and some had the autophagosome marker LC3. PSLPs encompass degranulated PLTs, PLT ghosts, large PLT fragments, large and dense PEVs, and low-density PEV ghosts. The activation-related PSLPs are released, particularly during early stage of storage (days 1-3), and the release of apoptosis- and necrosis-related PSLPs prevails after that. No elevation of LC3- and TOM20-positive PSLPs indicates that the increase of extracellular mitochondria during later-stage storage is not associated with PLT mitophagy.

The effect of factor XIa on thrombin and plasmin generation, clot formation, lysis and density in coagulation factors deficiencies.

INTRODUCTION: Growing evidence supports the importance of factor (F) XI activation for thrombosis and hemostasis as well as inflammation and complement systems. In this study, we evaluated the effect of activated FXI (FXIa) on the detection of factor deficiencies by global hemostasis assays of thrombin generation (TG), plasmin generation (PG), and clot formation and lysis (CFL). MATERIALS AND METHODS: An absorbance and fluorescence microplate assay was used to simultaneously observe TG, PG, and CFL in FV-, FVII-, FVIII-, and FIX-deficient plasmas supplemented with purified factors. Coagulation was initiated with tissue factor with or without FXIa in the presence of tissue plasminogen activator. Thrombin and plasmin peak heights (TPH and PPH), maximal clot density (MCD), times to clotting (CT), thrombin and plasmin peaks (TPT and PPT) and clot lysis (LyT) and a new parameter, clot lifetime (LiT), were evaluated. RESULTS: TG/CFL were elevated by the FXIa at low FV (below 0.1 IU/mL), and at FVIII and FIX above 0.01 IU/mL. FXIa affected PG only at low FV and FVII. At high factor concentrations, FXIa reduced MCD. Thrombin and plasmin substrates had effect on CT, LyT, LiT and MCD parameters. CONCLUSIONS: FXIa reveals new relationships between TG, PG and CFL parameters in factor deficiencies suggesting potential benefits for discrimination of bleeding phenotypes.

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor.

Preclinical evaluation of drugs in animals helps researchers to select potentially informative clinical laboratory markers for human trials. To assess the utility of animal thrombin generation (TG) assay, we studied the sensitivity of animal plasmas to triggers of TG, human Tissue Factor (TF), and Activated Factor XI (FXIa). Pooled human, mouse, rat, guinea pig, rabbit, bovine, sheep, and goat plasmas were used in this study. TF- or FXIa-triggered TG and clotting were measured via fluorescence and optical density, respectively. Thrombin peak height (TPH) and time (TPT), clot time (CT), and fibrin clot density (FCD) were all analyzed. The trigger low and high sensitivity borders (LSB and HSB) for each assay parameter were defined as TF and FXIa concentrations, providing 20 and 80% of the maximal parameter value, unless the baseline (no trigger) value exceeded 20% of the maximal, in which case, LSB was derived from 120% of baseline value. Normal human samples demonstrated lower TPH HSB than most of the animal samples for both TF and FXIa. Animal samples, except mice, demonstrated lower TPT LSB for FXIa versus humans. Most rodent and rabbit samples produced baseline TG in the absence of TG triggers that were consistent with the pre-activation of blood coagulation. FCD was not sensitive to both TF and FXIa in either of the plasmas. Animal plasmas have widely variable sensitivities to human TF and FXIa, which suggests that optimization of trigger concentration is required prior to test use, and this complicates the extrapolation of animal model results to humans.

Sex-dependent balance between thrombin and plasmin generation in the presence of thrombomodulin.

BACKGROUND: Assessing simultaneous generation of thrombin (TG) and plasmin (PG) is an approach to evaluate the balance between coagulation and fibrinolysis with sensitivity to predict endogenous thrombin and plasmin generation. The addition of thrombomodulin (TM), provides the essential component for thrombin activation of protein C and thrombin-activatable fibrinolysis inhibitor. However, the influence of sex on the balance between TG and PG with and without TM addition has not been investigated to date. OBJECTIVES: To investigate the possible sex-based differences in TG and PG in the presence and absence of TM. METHODS: Simultaneous TG and PG were measured in plasma samples obtained from 17 males and 17 females upon tissue factor and tissue plasminogen activator addition. Thrombin- and plasmin-specific fluorogenic substrates Z-Gly-Gly-Arg-AMC and Boc-Glu-Lys-Lys-AMC were used in the study. Thrombin and plasmin peak height (TPH and PPH) and production rate (TPR and PPR) values were determined. To evaluate the balance between TG and PG, the ratios between TPH and PPH (TPH/PPH) and TPR and PPR (TPR/PPR) were calculated. RESULTS AND CONCLUSIONS: TPH between males and females demonstrated significant difference regardless of TM addition. TPR demonstrated differences between males and females only upon TM addition, while PG parameters was not dependent on the sex of the donor. TM significantly lowered TPH/PPH in males, and enhanced TPR/PPR in females. Thus, TPH/PPH and TPR/PPR significantly differed between men and women. Our results indicate that TM may act differently in males and females by shifting the underlying TG/PG balance to fibrinolysis in males and to coagulation in females.

C1-inhibitor influence on platelet activation by thrombin receptors agonists.

INTRODUCTION: Protease activated receptors 1 (PAR1) and 4 (PAR4) agonists are used to study platelet activation. Data on platelet activation are extrapolated across experimental settings. C1-inhibitor (C1INH) is a protease inhibitor present in plasma but not in isolated platelet suspensions. Here we show that C1INH affects platelet activation through PAR1 and PAR4 agonists. METHODS: Platelets were isolated from healthy donor whole blood and then labeled with anti-CD62P and PAC1 antibodies. The platelet suspensions were exposed to PAR1 agonists SFLLRN, TFLLR and TFLLRN; PAR4 agonists AYPGKF and GYPGQV; ADP and thrombin. Flow-cytometric measurements were performed in 5, 10 and 15 min after activation. RESULTS: 0.25 mg/ml C1INH addition made platelets to faster expose CD62P and glycoprotein IIb/IIIa complex after activation with PAR1 agonists. Conversely, C1INH addition led to inhibition of platelet activation with PAR4 agonists and thrombin. Activation with ADP was not affected by C1INH. CONCLUSIONS: Our results suggest that C1INH can modify platelet activation in the presence of synthetic PAR agonists used in platelet research. These observations may be relevant to the development of new methods to assess platelet function.

Interspecies comparison of simultaneous thrombin and plasmin generation.

Animal models of hemostasis are often extrapolated to humans; however, only a few studies have compared coagulation and fibrinolysis across species. Simultaneous thrombin (TG) and plasmin (PG) generation is useful to assessing coagulation and fibrinolysis within the same sample. In this study, we performed simultaneous TG and PG analysis in blood plasma samples from humans and 6 species commonly evaluated in pre-clinical research. TG and PG were investigated in male and female donor platelet-poor plasmas (PPP) obtained from 28 healthy humans, 10 baboons, 12 rhesus monkeys, 20 Yorkshire pigs, 20 Sprague-Dawley rats, 10 New Zealand White rabbits and 14 Hartley guinea pigs. The continuous generation of the 7-amino-4-methylcoumarin (AMC) from substrates specific to thrombin or plasmin was monitored. The thrombin and plasmin concentration peak heights (PH) and production rates (PR) were calculated. TG and PG parameters from baboon and rhesus macaque plasma approximated that of humans. The other species differed significantly from both human and non-human primates. For example, swine and rat plasmas demonstrated similar TG, but swine plasmas did not generate plasmin. TG and PG parameters from Guinea pig samples were extremely low, while rabbit plasmas showed variable PG curves demonstrating one or two peaks with low and high PR values, respectively. Correlations between PH and PR values were significant with the exceptions of human PG, baboon TG, rat TG and Guinea pig PG. These findings are informative to pre-clinical animal species selection and optimization of coagulation and fibrinolysis translational research.

Contradictory to its effects on thrombin, C1-inhibitor reduces plasmin generation in the presence of thrombomodulin.

C1-inhibitor (C1INH) was shown to enhance thrombin generation (TG) in the presence of thrombomodulin (TM) by reducing production of activated protein C. Because C1INH is known to inhibit fibrinolytic system proteases, the objective of this study was to evaluate the effect of moderate (3 IU/ml) and high (16 IU/ml) C1INH concentrations on TG and plasmin generation (PG) in the presence of TM. These concentrations were evaluated based on expected maximum plasma levels following C1INH replacement therapy and recently suggested supraphysiologic dosing. TG and PG were investigated in platelet poor plasmas obtained from 21 healthy donors. An assay designed to monitor the continuous generation of the 7-amino-4-methylcoumarin fluorescence from substrates specific to thrombin or plasmin was used to evaluate the impact of C1INH activity. To characterize the C1INH effects on TG and PG, the thrombin and plasmin concentration peaks and production rates were calculated. TM addition to donor plasma shifted the concentration dependence of C1INH on TG parameters from reduction to enhancement. Conversely, PG parameters were significantly reduced by 16 IU/ml in both the presence and absence of TM. Moderate C1INH concentration (3 IU/ml) reduced TG and PG in the absence of TM but did not significantly affect these parameters in the presence of TM. Finally, 3 IU/ml of C1INH reduced PG more so than TG in the absence of TM. The presented results suggest a mechanism by which C1INH could potentiate thrombosis by inhibition of fibrinolysis.

C1-esterase inhibitor enhances thrombin generation and spatial fibrin clot propagation in the presence of thrombomodulin.

Package inserts for C1-esterase inhibitor (C1INH) products include warnings for an elevated risk of possible thrombosis in certain individuals, referring to thromboembolic events (TEEs) that were reported to occur after C1INH infusions. However, the mechanism(s) that could explain possible development of TEEs due to C1INH remains unknown. In this work, we evaluated plausible impact of C1INH on the protein C (PC) anticoagulant system. We performed thrombin generation (TG) assays (TGA) and analyzed spatial fibrin clot propagation using thrombodynamics in plasma of individual donors after the addition of thrombomodulin (TM) and C1INH. The addition of C1INH was consistent with the plasma concentrations resulting from doses currently approved for the HAE treatment up to ones consistent with off-label use in patients with risk of inflammation. 16 IU/ml of C1INH significantly enhanced thrombin peak (TP) generation in the presence of 12 and 15 nM TM. TG enhancement was observed by the addition of C1INH to make concentrations equal to 2 and 4 IU/ml in some donor plasmas. C1INH addition in the presence of TM enhanced the stop time of spatial clot growth in Thrombodynamics assay. A chromogenic activity assay demonstrated that C1INH inhibited PC activation by thrombin in the presence of TM. Substitution of TM with APC in TGA attenuated the TP enhancing effect of C1INH. The collective results of the present study suggest a concentration dependent C1INH interaction with the PC system. This study introduces a plausible TM-dependent mechanism, that may explain reported TEEs via suppressed production of APC in the presence of C1INH.

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent ß-globin posttranslational modifications, including the irreversible oxidation of ßCys93 and the ubiquitination of ßLys96 and ßLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, ßCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

Thrombodynamics, a new global coagulation test: Measurement of heparin efficiency.

The actual coagulation status may be reliably measured using only highly sensitive global functional tests; however, they are not numerous and all of them have disadvantages. Thrombodynamics (TD), a novel global coagulation test, is sensitive to hypo- and hypercoagulable states. The main properties of this test were investigated, and its capabilities for hemostasis analysis were verified through pharmacodynamic monitoring of the most widely used anticoagulants, heparins. The anticoagulant effects in the plasma of donors (n = 20) and patients after hip replacement (n = 20) spiked with unfractionated heparin or enoxaparin were measured in vitro to eliminate the influence of pharmacokinetic factors. Sensitivity for heparins was compared for activated partial thromboplastin time, thrombin generation tests and TD. TD was shown to reliably characterize the pharmacodynamics of any heparin in the entire range of its prophylactic and therapeutic concentrations. Inter-individual variability for the anticoagulant action of heparins was also calculated using the TD data. This variability did not differ between the investigated groups and did not exceed 12% and 20% for the stationary clot growth rate in the presence of unfractionated heparin and enoxaparin, respectively. That finding was in accordance with the values determined earlier using the thrombin generation test. The study results showed that TD has advantages over the other global methods of coagulation analysis. These advantages are good standardization, high reproducibility, independence of the parameter values from patient age and gender, and a narrower parameter distribution in a normal population. These results indicate that TD is a promising universal assessment method that improves the quality of hemostasis analysis because it more reliably detects deviations from the parameters' reference values.

Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.

BACKGROUND: Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs. METHODS: CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test. RESULTS: SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing-thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a(+) PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. CONCLUSIONS: Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing-thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

Thromboelastography, thrombin generation test and thrombodynamics reveal hypercoagulability in patients with multiple myeloma.

Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism. Therefore, adequate laboratory control of hemostasis and subsequent adjustments of anticoagulant therapy are necessary. We studied hemostasis changes using thromboelastography (TEG), thrombin generation test (TGT) and thrombodynamics (TD) in primary MM patients (PMMpt, n=25) and patients in remission (RMMpt, n=34) during blood stem cell (BSC) mobilization. TD and TEG reveal hypercoagulability in PMMpt (*p<0.05) in relation to healthy volunteers. There was no difference in any of the tests between PMMpt and RMMpt. We detected no heparin effect in 22% of patients one day after the onset of the prophylactic heparin treatment (500 IU/h) during BSC mobilization; tests shifted toward the hypercoagulability in 75% of patients one day after cyclophosphamide (4 g/m2) chemotherapy. Global hemostasis tests were in good agreement with each other, revealed hypercoagulability and heparin "resistance" in patients with MM and may be useful for therapy individualization.

Investigation of the phenotype heterogeneity in severe hemophilia A using thromboelastography, thrombin generation, and thrombodynamics.

BACKGROUND: Hemophilia A (HA) patients with similar factor VIII levels can demonstrate varying bleeding tendencies. In particular, 10-15% of all severe HA patients (FVIII:C<1IUdL(-1)) do not require regular replacement therapy. Modern global coagulation assays can help to detect and study this "mild" bleeding phenotype. Here, we investigated the coagulation status of different bleeding phenotypes using various types of global coagulation assays. MATERIALS AND METHODS: Ten HA patients with severe phenotype and eleven patients with mild phenotypes were included in the study. For each patient, thromboelastography (TE), thrombodynamics (TD), and kaolin- or tissue factor-induced thrombin generation (TG) were measured. TG in platelet-rich plasma (PRP) was investigated using our original modification when the thrombin generation curve showed two peaks, previously shown to depend on platelet activity. We also utilized TG and TD with the addition of thrombomodulin. RESULTS: The second peak amplitude and ETP of PRP TG were the only parameters that were significantly higher in mild bleeders (peak 41.6 ± 3.5 nM, ETP 1966 ± 169 nM*min) than in patients with severe bleeding (peak 28.3 ± 3.3 nM, ETP 1359 ± 130 nM*min). CONCLUSIONS: Our results suggest that severe and mild HA phenotypes could be distiguished by TG assay in PRP suggesting that difference in platelet activity can be involved in the phenotype formation. According to our previous results we can suppose that the mechanism of the phenotypic heterogeneity is linked with TG mediated by PS-expressing platelets.

Antiplatelet agents can promote two-peaked thrombin generation in platelet rich plasma: mechanism and possible applications.

BACKGROUND: Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks. OBJECTIVE: We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve. METHODS: Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers. RESULTS: The addition of the P(2)Y(12) receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E(1) (PGE(1)), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE(1) or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE(1) mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one. CONCLUSIONS: Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types--plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.