Procoagulant Phosphatidylserine Research Articles

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

BackgroundScott 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. ObjectivesGenetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. MethodsA 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. ResultsWe 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. ConclusionWe 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.

Transducing Force into Procoagulation: PIEZO1-TMEM16F Functional Coupling in Blood Lining Cells

Thrombosis is a major global disease burden that accounts for 1 in 4 deaths worldwide. Hypertension and preeclampsia greatly increase thrombotic risks by imposing high blood pressure and abnormal hemodynamic shear stress on blood-lining cells, namely endothelial cells (ECs) and placental syncytiotrophoblasts (STBs) in pregnant women. Nonetheless, it remains elusive how abnormal mechanical forces under hypertensive conditions promote coagulation. PIEZO1 mechanosensitive ion channel is a major force sensor in a wide variety of cells, including red blood cells (RBCs) and endothelial cells (ECs), where it transduces mechanical forces into chemical signals through its ion permeability to calcium. On the other hand, TMEM16F is a calcium-activated lipid scramblase (CaPLSase) that translocates phosphatidylserine (PS), an important procoagulant factor, to the cell surfaces of blood cells and endothelial cells. We recently discovered that TMEM16F is the sole CaPLSase in placental trophoblasts and RBCs. Our preliminary results show that PIEZO1 and TMEM16F are functionally coupled in RBCs, and calcium entry through PIEZO1 activates TMEM16F, leading to PS exposure. Based on all these, we hypothesize that in the blood lining cells, PIEZO1-TMEM16F coupling is responsible for transducing mechanical forces into PS exposure, a critical procoagulant signal for blood coagulation. By employing immunostaining, patch clamp electrophysiology, and a fluorescence microscopy-based scrambling assay, we show that PIEZO1 is functionally expressed in placental trophoblasts, including STBs. We further demonstrate that PIEZO1 activation, either stimulated by its agonist Yoda1 or by shear stress, induces PS exposure in BeWo human trophoblast cell line and human umbilical vein endothelial cell line (HUVEC) but not the TMEM16F deficient cell lines. In addition, using chromogenic tenase and prothrombinase, and fluorescence fibrin assays, we show that PIEZO1 activation by its agonist, Yoda1, triggered coagulation activities in a TMEM16F-dependent fashion in trophoblasts. Our study thus discovers the functional expression of the PIEZO1 mechanosensitive channel in placental trophoblasts and elucidates a novel force-induced procoagulant mechanism in blood-lining cells. In this mechanism, PIEZO1-TMEM16F coupling transduces mechanical forces into procoagulant PS exposure on the blood lining cells. Enhanced PIEZO1-TMEM16F coupling may contribute to hypercoagulable states in hypertension and preeclampsia, and breaking the coupling is therefore a potential therapeutic strategy to prevent thrombotic risks associated with these diseases.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity exvivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Exvivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Skeletal Muscle Myosin Is Procoagulant By Binding Factor XI Via Its A3 Domain and Enhancing Factor XI Activation By Thrombin

Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI.To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain.Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM.The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop.To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI.In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). [Display omitted] DisclosuresGruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Ultrastructural Alterations of Platelets and Monocytes Associated with Autoantibodies in Systemic Lupus Erythematosus

Introduction. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high titer of autoantibodies, and SLE patients are predisposed to thrombosis. Monocytes and platelets are potentially involved in the pathogenesis of SLE. Monocytes are a source of immune modulators for T and B lymphocytes and they increase the risk of thrombosis through exposing tissue factor on their cell surface. Platelets are activated in SLE and may promote thrombotic events by expressing the procoagulant phosphatidylserine. The aim of this work was to investigate the ultrastructural changes of monocytes and platelets in SLE patients in association with titers of autoantibodies.Methods. SLE patients were included in the study based on the diagnostic criteria of the American College of Rheumatology. Platelets from 5 SLE patients and 3 healthy donors were isolated by gel-filtration on Sepharose 2B from citrated platelet-rich plasma. Monocytes of 6 SLE patients and 3 healthy donors were isolated using density gradient centrifugation of whole citrated blood followed by negative immune-separation. The >96% purity of the isolated monocytes was confirmed using flow cytometry and the viability of the isolated monocytes assessed by exclusion of trypan blue was 95-99%. The ultrastructure of platelets and monocytes from SLE patients and healthy donors was studied with transmission electron microscopy. A total 192 individual platelets and 154 monocytes from SLE patients were analyzed and compared with 60 individual platelets and 22 monocytes from healthy donors.Results . Most platelets from SLE patients and healthy donors had a normal discoid shape without or with 1-2 short filopodia. Intracellular components (α-granules, dense granules, open canalicular system, lysosomes and mitochondria) were well visualized, albeit somewhat less clear in the SLE platelets. The biggest morphological difference between platelets from healthy subjects and SLE patients was revealed in the platelet plasma membrane. All platelets from healthy donors had normal ultrastructure of the plasma membrane, while the membrane of SLE platelets was very often (in 70% of the cells) shaggy and rough and not as smooth as in normal platelets. About 38% of the SLE platelets were fully bordered with a shaggy plasma membrane, while 32% of the platelets had only partially abnormal plasma membrane. Cells from a patient with anti-cardiolipin and anti-beta-2-glypoprotein antibodies had the most striking membrane abnormalities that were found in 100% of platelets analyzed.Monocytes with normal ultrastructure (ruffled cell surface, formation of filopodia, an indented nucleus, and uniform chromatin distribution) comprised 64% of the total in the SLE patients and 95% in the healthy donors. Unlike monocytes from healthy subjects, 19% of monocytes from the SLE patients had ultrastructural signs of apoptosis with a smooth cell surface, electron-dense cytoplasm, mitochondrial swelling, and a spherical nucleus with perinuclear accumulation of condensed chromatin or condensation of chromatin into dense spherical particles. About 17% of monocytes from the SLE patients and 5% from the healthy donors had necrotic features, such as smooth cell surface without or with quite a few filopodia, low electron-density cytoplasm, small electron-dense mitochondria, and spherical pyknotic nucleus with more diffuse euchromatin. The SLE patients with a high titer of anti-dsDNA antibodies (>200 IU/ml) had significantly more apoptotic (25%, p<0.01, χ2-test) and necrotic (20%, p<0.05) monocytes than those of the SLE patients with a normal titer of anti-dsDNA antibodies (<25 IU/ml) (6% and 10%, respectively).Conclusions . In SLE patients, most platelets have abnormal ultrastructure of the plasma membrane (loss of smoothness, shagginess), likely due to deposition of pathological immune complexes. A substantial fraction of monocytes in SLE undergo morphological alternations of apoptotic or necrotic types. Such alterations were more pronounced in patients with an increased level of anti-cardiolipin, anti-beta-2-glypoprotein, and anti-dsDNA autoantibodies. The results confirm the involvement of both platelets and monocytes in the pathogenesis of SLE, including functional and structural alterations associated with increased production of various autoantibodies.The work was supported by the Program for Competitive Growth at Kazan Federal University. DisclosuresNo relevant conflicts of interest to declare.

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis.

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

AbstractArterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

High levels of endothelial and platelet microvesicles in patients with type 1 diabetes irrespective of microvascular complications

IntroductionPatients with type 1 diabetes have high risk of developing microvascular complications, and microangiopathy contributes to premature cardiovascular disease in this population. The role that microvesicles (MVs) may play in the development of microangiopathy in type 1 diabetes remains unclear. Materials and methodsPlasma levels of endothelial MVs (EMVs) and platelet MVs (PMVs) in 130 patients with type 1 diabetes without microangiopathy, 106 patients with microangiopathy and 100 matched healthy controls were analyzed using flow cytometry. MV expression of procoagulant phosphatidylserine (PS) and proinflammatory high mobility group box-1 protein (HMGB1) was also assessed. ResultsPatients with type 1 diabetes had markedly elevated levels of EMVs and PS+ EMVs as well as PMVs and PS+ PMVs compared to healthy controls (p < .001 for all). Furthermore, HMGB1+ EMVs and HMGB1+ PMVs were significantly increased in patients (p < .001 for all). After adjusting for potential confounders, there were no clear differences between patients with or without microvascular complications for any of the MV parameters. ConclusionType 1 diabetes is a prothrombotic and proinflammatory disease state that, regardless of the presence of clinical microangiopathy, is associated with elevated levels of plasma MVs, in particular those of an endothelial origin. We have for the first time demonstrated that patients with type 1 diabetes have higher levels of HMGB1+ MVs. HMGB1 is an alarmin with potent proinflammatory effects that drive endothelial dysfunction, and it would therefore be of interest to further study the role of HMGB1+ MVs in the development of macrovascular complications in type 1 diabetes.

Links Between Complement Activation and Thrombosis

The coagulation and complement systems are central mediators of innate immune defense and aide the peri- and intravascular elimination of invading microorganisms in a process termed immuno-thrombosis. Complement and coagulation proteases are engaged in reciprocal amplifications at different levels of these enzymatic cascades. In addition, activated complement factor (C) 3 specifically stimulates platelets through C3a receptor signaling and thereby amplifies thrombus formation. A non-enzymatic function of complement activation emerged as a crucial mechanism that rapidly alters the function of the extrinsic coagulation activator tissue factor (TF) on monocytes. Activation of the classical complement pathway by therapeutic anti-thymocyte globulin preparations rapidly enhances monocyte TF procoagulant activity. On the one hand, complement activation-associated thiol-disulfide exchange supports protein disulfide isomerase-dependent conformational changes in TF. Insertion of the C5b-C7 complex supports these conformational changes in TF by exposing procoagulant phosphatidylserine (PS). Furthermore, these dual roles of complement activation play a central role in venous thrombosis. C3, but not C5-deficient mice are protected from platelet deposition. In contrast, C5 knock-out mice show diminished PS exposure on leukocytes and lack TF-dependent coagulation activation and fibrin deposition at the flow restricted venous vessel wall. Antiphospholipid antibodies (aPL) also cause TF- and complement-dependent thrombosis. Whereas C3-dependent thiol-disulfide exchange is again required for converting TF to a procoagulant form, aPL do not rely on downstream complement components for the exposure of PS and induce pathological thrombosis in C5-deficient mice. Our recent data show that aPL dissociate a cell surface-localized, inhibited complex with monocyte-expressed TF pathway inhibitor (TFPI). In addition to promoting TF procoagulant activation, complement-dependent thiol-disulfide exchange emerged as an additional central mechanism that allows for aPL internalization and endosomal signaling involving integrin- and TF cytoplasmic domain-dependent translocation of the NADPH oxidase to the endosome. A monoclonal antibody that traps TF with low procoagulant activity on the surface prevents TF endosomal trafficking, aPL signaling and aPL-induced pregnancy loss. These complement-dependent effects require assistance by thrombin-PAR1/PAR2 heterodimer signaling initiated by FXa dissociated from the inhibited TF complex. Monocyte TFPI-dysfunctional mice are protected from aPL pathogenic signaling in vitro and from aPL-induced thrombosis, but form thrombi normally in other experimental thrombosis models. These data show that formation of an inhibited TF cell surface complex specifically primes monocytes for thrombosis and pathogenic aPL signaling. Thus, the evolutionary conserved crosstalk of complement and coagulation cascades not only plays crucial roles in thrombosis, but also regulates thrombo-inflammatory signaling in autoimmune disease. Disclosures Ruf: MeruVasimmune: Equity Ownership; Iconic Therapeutics: Consultancy.

Protein Cysteine Sulfenylation By CD36-Dependent Reactive Oxygen Species Signaling Promotes Platelet Activation

Arterial thrombosis in the setting of dyslipidemia produces clinically significant events, including myocardial infarction and stroke. Oxidized lipids in circulating lipoproteins (oxLDL) are a risk factor for atherothrombosis in dyslipidemia and are recognized by platelet scavenger receptor CD36. OxLDL binding to CD36 promotes platelet activation and thrombosis by generating intracellular reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). Downstream signaling events initiated by ROS in this setting are largely unknown. We hypothesize that H2O2 generated by CD36 signaling promotes oxidative cysteine modification of cellular regulators of arterial thrombosis. Platelets isolated from healthy human donors were stimulated with oxLDL and H2O2 levels were measured by HPLC fluorescence quantification of 7-hydroxycoumarin generated by H2O2-specific oxidation of the coumarin boronic acid probe. OxLDL induced time- and concentration-dependent H2O2 formation (up to 80 ±13% greater than unstimulated platelets), while control "native" LDL showed negligible H2O2 formation. Pre-treatment of platelets with a CD36-blocking antibody or with PEG-catalase, an enzyme that degrades H2O2, normalized H2O2 formation by oxLDL to levels observed in unstimulated platelets. To mimic pathophysiologic conditions platelets were sensitized with oxLDL before stimulating with classic activators: adenosine diphosphate (ADP) and collagen-related peptide (CRP). Sensitization synergistically increased H2O2 formation through collagen receptors, but not ADP receptors. Since H2O2 can induce transient protein cysteine sulfenylation as a posttranslational oxidative modification, we assayed platelet cysteine sulfenylation using an alkyne-containing benzothiazine-based probe, BTD. BTD was loaded into platelets prior to exposure to oxLDL and detected by click-chemistry with biotin-PEG-azide followed by detection by western blot with streptavidin. OxLDL, but not LDL, induced two-fold increase in sulfenylation in the platelet proteome within 15 minutes. Src family kinases (SFK) are known to be recruited to and activated by CD36 in a ligand-dependent manner and SFK were also previously shown to be sulfenylated by H2O2 at Cys185 and Cys277, maintaining the kinase in an activated state. To determine if SFK cysteines are oxidatively modified by CD36 signaling, lysates from BTD-loaded, oxLDL-stimulated platelets were biotinylated as above, immunoprecipitated with anti-Src antibody, and then analyzed by immunoblot to detect BTD incorporation. SFK were sulfenylated in a time-dependent manner and this was blocked by a CD36 blocking monoclonal antibody or by treatment with PEG-catalase. We then showed by immunoblot that SFK cysteine sulfenylation by CD36/H2O2 was associated with phosphorylation at Y416, a signature activation motif in the kinase domain. OxLDL induced Src Y416 phosphorylation was prevented by PEG-catalase. Platelet aggregometry was used to determine the functional impact of protein sulfenylation and revealed that oxLDL-induced platelet aggregation was inhibited in a concentration-dependent manner (IC50 2.02 mM) by modifying sites of sulfenylation with BTD. BTD had no impact on aggregation induced by low- or high-concentration of ADP or CRP, suggesting these physiologic activators alone do not generate sufficient H2O2 to promote cysteine sulfenylation. OxLDL/CD36 induced procoagulant phosphatidylserine externalization, assessed by flow cytometry using fluorophore-tagged annexin V, was also prevented by BTD. In conclusion, our studies show that platelet CD36 signaling in response to oxLDL induces intracellular H2O2 generation which in turn induces cysteine sulfenylation of Src family kinases to promote platelet activation. Cysteine sulfenylation by CD36 could potentially be targeted to reduce the risk for clinically significant thrombotic events while maintaining hemostasis. Disclosures No relevant conflicts of interest to declare.

Increased Phosphatidylserine-Exposing Microparticles and Their Originating Cells Are Associated with the Coagulation Process in Patients with Oral Squamous Cell Carcinoma

Introduction:Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck area, and the incidence remains high.Despite advances in diagnosis and treatment, the poor prognosis of OSCC is characterized by a high rate of local recurrence and the overall five-year survival rate remains at approximately 50%. Therefore, the mechanisms underlying the development of OSCC still need to be clarified. Patients with cancer tend to develop a hypercoagulable state which predisposes them to thromboembolic events. Cancer increases the risk of venous thrombosis several fold with varying degree of relative risks (range 4-7). A recent study has reported that microparticles (MPs) increased procoagulant activity (PCA) in OSCC. MPs are small membrane vesicles of 0.1-1 µm containing negatively charged, procoagulant phosphatidylserine (PS), which plays an important role in thrombosis. The definitive role of PS in the hypercoagulable state in patients with OSCC remains unclear. Our objectives were to measure the PS exposure on MPs, blood cells, and endothelium, and to evaluate their PCA in different stages of OSCC.Methods: OSCC patients (n = 57) and healthy controls (n = 26) were included in our study. Blood samples were obtained from controls and OSCC patients within 1 day before surgery and 2-week after surgery. Human umbilical vein endothelial cells (HUVECs) were incubated in growth media containing 20% of pool serum obtained from either OSCC patients or healthy donors at room temperature for 24 h, respectively. Exposed PS was analyzed with flow cytometry and confocal microscopy. Lactadherin was used to quantify PS exposure on MPs and their original cells. PCA of MPs and these cells was evaluated using clotting time, purified coagulation complex, and fibrin formation assays. Meanwhile, the inflammation-related cytokines were detected by enzyme-linked immunosorbent assay.Results: Using flow cytometry, plasma levels of PS+ blood cells and MPs in OSCC patients with stage III/IV were significantly higher than those in stage I/II patients or healthy controls (all P < 0.05). However, we only found a significant difference between stage I or II and controls (P < 0.05) in total PS+ MPs and PMPs. Similarly, we found that the endothelial cells (ECs) treated with OSCC serum in vitro exposed more PS than those with healthy serum. Moreover, in OSCC patients with stage IV, MPs primarily originated from platelets (53.9 ± 3.2%) followed by leukocytes (21.8 ± 2.1%, including MPs from neutrophils, monocytes and lymphocytes), erythrocytes (6.3 ± 0.6%) and ECs (6.9 ± 0.8%). Additionally, PS+ blood cells, MPs and OSCC serum-cultured ECs markedly promoted shortened coagulation time and significantly increased FXa/thrombin/fibrin generation in stage IV or III OSCC patients compared with controls (all P < 0.05). Interestingly, confocal imaging of MPs or OSCC serum-treated ECs showed binding sites for FVa and FXa to form prothrombinase. Furthermore, blockade of PS on MPs/blood cells/ECs with lactadherin inhibited PCA by approximately 80%. Most importantly, we found treatment with radical resection significantly reduced the amount of PS+ blood cells, ECs and MPs, and prolonged the clotting times of those cells and MPs compared with presurgery patients. Lastly, the correlation analysis revealed that the plasma levels of interleukin 6, interleukin 8 and tumor necrosis factor α were positively correlated with the levels of total PS+ MPs and PS+ platelets in OSCC patients.Conclusions: Our results suggested that PS+ blood cells and MPs play a prominent role in inducing the hypercoagulable and prothrombotic state especially in advanced OSCC. Interestingly, we found treatment with radical resection could attenuate this effect. Moreover, the released inflammatory cytokines may contribute to PS exposure on platelets and MPs and the increased procoagulant activity in patients with OSCC. Notably, our findings also show that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Therefore, directly targeting FXa and prothrombinase complexes might decrease thrombotic risk OSCC patients. This study shows that future research should focus on the application of PS inhibitors as a novel therapeutic strategy in OSCC patients when coagulation is abnormally enhanced. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombin-Generating Capacity of Microvesicles (MVs) in Patients with Immune Thrombocytopenia (ITP) before and during Treatment with Thrombopoietin-Receptor Agonists (TPO-RA)

Introduction:ITP is an acquired autoimmune disorder characterized by isolated thrombocytopenia and an increased risk of bleeding. Paradoxically, ITP is also associated with an increased risk of thrombosis, which may be exacerbated with TPO-RA-treatment. The underlying mechanism(s) involved in the development of thrombosis in ITP and especially in TPO-RA-treated ITP-patients remain poorly understood.MVs released from activated/apoptotic cells are procoagulant due to the presence of tissue factor (TF) and phospholipids such as phosphatidylserine (PS) on their membrane. MVs have been shown to be increased in ITP-patients but the prothrombotic role of these MVs before and after treatment with TPO-RA is unclear. We measured MV-associated thrombin generation, PS-dependent thrombin generation in plasma, TF-activity and PS-activity in plasma of ITP-patients vs controls and investigated the effect of TPO-RA on these measurements.Methods:In 15 controls and 11 ITP patients, before TPO-RA and 2 and 6 weeks after the initiation of TPO-RA, citrated plasma was prepared (2000gx20 min) and immediately frozen. After thawing of plasma, MVs were isolated by centrifugation (17,000gx30 min), the supernatant removed and the remaining MV-pellet washed twice. Isolated plasma-derived MVs were added to pooled normal plasma (PNP), and to obtain measureable thrombin generation, antibodies against tissue factor pathway inhibitor were also added to the PNP. The ability of MVs to generate thrombin was measured by the calibrated automated thrombogram method and the thrombin generation parameters lag time (LT), peak, endogenous thrombin potential (ETP), time to peak (ttPeak) and velocity index (VI) were calculated by the Thrombinoscope software.To estimate the contribution of procoagulant PS, PS-activity in plasma (PS equivalents) was measured with the Zymuphen MP-activity assay. In addition, thrombin generation was measured directly in plasma where only TF (1pM), but not PS, had been added (PS-dependent thrombin generation). TF-activity in plasma was measured with the Zymuphen MP-TF-activity assay.Friedman test with Dunn's multiple comparisons was used to compare measurements in ITP-patients before and after TPO-RA-treatment. Kruskal-Wallis test was used to compare measurements in ITP-patients and controls.Results:Median age of ITP-patients and controls: 53 and 50 years. Eight (73%) were on romiplostim and three (27%) were on eltrombopag. Median values (IQR) for all measurements before, 2 weeks and 6 weeks on TPO-RA-treatment and in controls are shown in the table.ITP-patients before treatment with TPO-RAs vs controls: No significant difference was found in MV-associated thrombin generation, PS-activity or TF-activity in plasma. There was a trend towards a higher peak, VI and ETP and lower ttPeak in PS-dependent thrombin generation in plasma of ITP-patients vs controls; lack of statistical significance may be due to the small sample size.TPO-RA-treated ITP-patients (2 and 6 weeks) vs controls: MV-associated thrombin generation: significant changes after 2 weeks (shorter LT/ttPeak (p=0.03/p=0.03), higher peak/VI (p=0.04/p=0.04)). PS-dependent thrombin generation in plasma: significant changes after 2 weeks (shorter ttPeak (p=0.007), higher peak/VI (p=0.003/p=0.001)), and after 6 weeks (shorter ttPeak (p=0.002), higher peak/ETP/VI (p=0.004/p=0.02/p=0.002)). PS-activity in plasma: significant changes after 2 and 6 weeks (p=0.006/p=0.02). TF-activity in plasma: no significant changes.ITP-patients before vs after TPO-RA-treatment (2 and 6 weeks): Only MV-associated thrombin generation was significantly increased after 2 weeks (higher peak/VI (p=0.03/p=0.04)).Conclusions: Compared with controls, treatment with TPO-RAs increases PS-dependent thrombin generation and PS-activity in plasma that is partly accompanied by an increase in MV-associated thrombin generation, but not with an increase in TF-activity in plasma. Similarly, we find a trend of increase in PS-dependent thrombin generation in ITP vs controls. This suggests that PS-positive MVs, most likely released from activated/apoptotic platelets, may contribute to the pre-existing increased thrombotic risk present in at least some of the patients with ITP, and may be used as a potential marker to estimate the risk of future thromboembolic events in TPO-RA-treated ITP-patients. [Display omitted] DisclosuresGhanima:Roche, Amgen, Novartis, Bayer, BMS: Other: Personal Fees, Research Funding; GlaxoSmithKline and Pfizer: Other: Personal Fees. Bussel:Prophylix: Consultancy, Research Funding; Protalex: Consultancy; Uptodate: Honoraria; Novartis: Consultancy, Research Funding; Amgen Inc.: Consultancy, Research Funding; Momenta: Consultancy; Rigel: Consultancy, Research Funding.

Platelet CD36 signaling through ERK5 promotes caspase-dependent procoagulant activity and fibrin deposition in vivo

AbstractDyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction–induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI–mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet–fed conditions comparable to that seen in chow diet–fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.

Simvastatin reverses multiple myeloma serum-induced prothrombotic phenotype in endothelial cells via ERK 1/2 signalling pathway.

: The introduction of new agents in multiple myeloma therapy has increased the overall response rate and improved clinical outcomes, but the increased risk of thrombotic complications impairs the quality of life of patient and the optimal thromboprophylaxis remains unknown. Increasing evidence has shown that statins can prevent venous thrombosis. Hence, we investigated the effects of simvastatin on multiple myeloma serum-related haemostatic imbalance in endothelial cells in vitro. The effects of simvastatin on procoagulant and anticoagulant protein expression were assessed on mixed multiple myeloma serum-treated human umbilical vein endothelial cells (HUVECs). The activity of these proteins was measured by thrombin generation and protein C activation assay. Then, the effects of extracellular signal-regulated kinase (ERK) 1/2 on endothelial activation were assessed by western blot and inhibition assay. We found that simvastatin inhibited multiple myeloma serum-induced expression of procoagulant protein tissue factor and phosphatidylserine and generation of thrombin on HUVECs. In contrast, simvastatin increased multiple myeloma serum-inhibited expression of anticoagulant protein endothelial protein C receptor and activation of protein C on HUVECs. Moreover, simvastatin reversed the multiple myeloma serum-induced prothrombotic phenotype, at least in part, via the inhibition of ERK 1/2 activation in endothelial cells. This study supports the beneficial effects of simvastatin on multiple myeloma serum-induced endothelial haemostatic imbalance, which suggests that simvastatin may serve as a new and appropriate antithrombotic approach for multiple myeloma patients.

Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

BackgroundThe release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response.MethodsIsolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation.ResultsCell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y12 and αIIbβ3 receptors.ConclusionsIn vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.

Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies

AbstractThe complement and coagulation cascades interact at multiple levels in thrombosis and inflammatory diseases. In venous thrombosis, complement factor 3 (C3) is crucial for platelet and tissue factor (TF) procoagulant activation dependent on protein disulfide isomerase (PDI). Furthermore, C5 selectively contributes to the exposure of leukocyte procoagulant phosphatidylserine (PS), which is a prerequisite for rapid activation of monocyte TF and fibrin formation in thrombosis. Here, we show that monoclonal cofactor-independent antiphospholipid antibodies (aPLs) rapidly activate TF on myelomonocytic cells. TF activation is blocked by PDI inhibitor and an anti-TF antibody interfering with PDI binding to TF, and requires C3 but unexpectedly not C5. Other prothrombotic, complement-fixing antibodies, for example, antithymocyte globulin, typically induce TF activation dependent on C5b-7–mediated PS exposure on the outer membrane of monocytes. We show that aPLs directly induce procoagulant PS exposure independent of C5. Accordingly, mice deficient in C3, but not mice deficient in C5, are protected from in vivo thrombus formation induced by cofactor-independent aPLs. Only immunoglobulin G (IgG) fractions with cofactor-independent anticardiolipin reactivity from patients with antiphospholipid syndrome (APS) induce complement-independent monocyte PS exposure and PDI-dependent TF activation. Neither a human monoclonal aPL directed against β2-glycoprotein I (β2GPI) nor patient IgG with selective reactivity to β2GPI rapidly activated monocyte TF. These results indicate that inhibitors of PDI and TF, but not necessarily clinically available drugs targeting C5, have therapeutic benefit in preventing thrombosis associated with APS caused by pathogenic aPLs primarily reactive with lipid, independent of β2GPI.

Platelet subpopulations remain despite strong dual agonist stimulation and can be characterised using a novel six-colour flow cytometry protocol

It is recognised that platelets respond differently to activation, where a subpopulation of platelets adopt a procoagulant phenotype while others are aggregatory. However, it has not been thoroughly tested whether these subpopulations will remain in maximally activated samples, or if they are merely a result of different platelet sensitivities to agonist activation. Here platelets were activated with gradually increasing concentrations of thrombin and/or the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Platelet activation was investigated using a novel six-colour flow cytometry protocol evaluating exposure of phosphatidylserine, active conformation of the fibrinogen receptor αIIbβ3, α-granule and lysosomal release (P-selectin and LAMP-1 exposure), mitochondrial membrane integrity and platelet fragmentation. Upon activation by CRP-XL or thrombin+CRP-XL, platelets formed three differently sized subpopulations. Normal-sized platelets showed high exposure of aggregatory active αIIbβ3 and intact mitochondria, while the smaller platelets and platelet fragments showed high exposure of procoagulant phosphatidylserine. The distribution of platelets between the differently sized subpopulations remained stable despite high agonist concentrations. All three were still present after 30 and 60 min of activation, showing that all platelets will not have the same characteristics even after maximal stimulation. This suggests that platelet subpopulations with distinct activation patterns exist within the total platelet population.

Auranofin, a thioredoxin reductase inhibitor, causes platelet death through calcium overload

Platelets are central to normal hemostasis and must be tightly controlled to prevent thrombosis. However, drug treatments that also affect platelets could lead to unwanted side effects on hemostasis or thrombosis. In this study, the effect of auranofin on platelets was tested. Auranofin, a gold-based thioredoxin reductase (TRXR) inhibitor, has been previously used in arthritis. Recently, auranofin and other inhibitors of the thioredoxin system have been proposed as novel anti-cancer therapies. TRXR is an important part of the antioxidant defenses in many cells that maintain intracellular proteins in their reduced state.TRXR activity in platelets could be completely inhibited by auranofin. Auranofin-treated platelets showed several features of cell death, including the inability to aggregate in response to thrombin, leakage of cytosolic lactate dehydrogenase, and surface exposure of procoagulant phosphatidylserine. Auranofin increased platelet reactive oxygen species production and intracellular calcium concentration. DTT, a sulfydyl reducing agent, and BAPTA-AM, which chelates intracellular calcium, prevented auranofin-induced phosphatidylserine exposure. These data suggest that TRXR is an important part of the platelet antioxidant defense. TRXR inhibition by auranofin triggers oxidative stress and disrupts intracellular calcium homeostasis, leading to platelet necrosis. The use of auranofin or other TRXR inhibitors could therefore lead to unwanted side effects.

Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking.

Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood. Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation. These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.