Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

Essentials

• Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF).
• Salivary EVs expose CD24, a ligand of P‐selectin.
• CD24 and coagulant TF co‐localize on salivary EVs.
• TF⁺/CD24⁺ salivary EVs bind to activated platelets and trigger coagulation.

Summary

Background

Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF‐exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell‐derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P‐selectin glycoprotein ligand‐1 (PSGL‐1) and P‐selectin.

Objectives

We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P‐selectin.

Methods

We investigated the presence of two ligands of P‐selectin on salivary EVs, PSGL‐1 and CD24.

Results

Salivary EVs expose CD24 but PSGL‐1 was not detected. Immune depletion of CD24‐exposing EVs completely abolished the TF‐dependent coagulant activity of cell‐free saliva, showing that coagulant TF and CD24 co‐localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24.

Conclusions

A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P‐selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.     

Interleukin-6 release and the acute-phase reaction in patients with acute myocardial infarction: a pilot study.

We investigated the potential role of interleukin-6 as a mediator of the acute-phase reaction (APR) in patients with acute myocardial infarction. Of the six patients studied, five demonstrated increased plasma interleukin-6 levels. Interleukin-6 levels began to increase at 14 hours (mean; range = 8 to 20 hours) after the initial complaints and reached maximal levels of 28 to 250 U/mL (normal values less than 10 U/mL) after 36 hours (mean; range = 24 to 52 hours). No correlation was seen between the size of the interfaction as indicated by creatine kinase MB assays and the extent of the interleukin-6 increases (r = 0.44; p = 0.38). As an indicator of the APR, plasma C-reactive protein (CRP) levels were measured. CRP levels began to increase after 16 hours (mean; range = 8 to 24 hours) and reached maximum levels of 56 to 322 mg/L (normal values less than 3 mg/L) after 65 hours (mean; range = 48 to 92 hours). The increase of the interleukin-6 level preceded the increase of the CRP level in three patients and was simultaneous in two patients. Maximal interleukin-6 levels correlated significantly with maximal CRP levels (r = 0.96; p = 0.002). Thus these findings indicate that interleukin-6 is an important endogenous mediator for the APR in patients with acute myocardial infarction.     

Subcellular distribution and phosphorylation of vinculin isoforms in human blood platelets

In this study we investigated human blood platelet vinculin microheterogeneity, the subcellular localization and phosphorylation of the different isoforms before and after platelet stimulation. At least 5 vinculin isoforms could be detected, as well as meta-vinculin. These isoforms did not demonstrate a specific subcellular localization, i.e. their relative content was similar in cytoskeleton, membrane skeleton and cytosol. Upon platelet stimulation with thrombin a small increase in alpha-vinculin was noted in all platelet subfractions. The cytoskeleton of non-stimulated platelets contained a minor quantity of vinculin. Upon thrombin stimulation of the platelets the cytoskeletal vinculin content increased significantly; previously we already reported a maximal 10% incorporation of the total platelet vinculin content into the cytoskeleton upon stimulation. A phosphorylation of a minor vinculin-isoform, i.e. at the alpha'/alpha location was mainly detected in the cytoskeleton. This phosphorylation was observable in the non-stimulated platelet cytoskeletal vinculin. These findings argue against a regulatory role for vinculin phosphorylation in the uptake of the main isoforms of this protein in the platelet cytoskeleton upon thrombin stimulation. The function of the phosphorylated cytoskeletal vinculin remains to be established.     

Activated complement components and complement activator molecules on the surface of cell-derived microparticles in patients with rheumatoid arthritis and healthy individuals

OBJECTIVES: In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell-derived microparticles of RA patients and healthy individuals. METHODS: Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex- and age-matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). RESULTS: Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. CONCLUSIONS: This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid.     

Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation

We determined the numbers, cellular origin and thrombin-generating properties of microparticles in healthy individuals (n = 15). Microparticles, isolated from fresh blood samples and identified by flow cytometry, originated from platelets [237 x 10(6)/L (median; range 116-565)], erythrocytes (28 x 10(6)/L; 13-46), granulocytes (46 x 10(6)/L; 16-94) and endothelial cells (64 x 10(6)/L; 16-136). They bound annexin V, indicating surface exposure of phosphatidylserine, and supported coagulation in vitro. Interestingly, coagulation occurred via tissue factor (TF)-independent pathways, because antibodies against TF or factor (F)VII were ineffective. In contrast, in our in vitro experiments coagulation was partially inhibited by antibodies against FXII (12%, p = 0.006), FXI (36%, p <0.001), FIX (28%, p <0.001) or FVIII (32%, p <0.001). Both the number of annexin V-positive microparticles present in plasma and the thrombin-generating capacity inversely correlated to the plasma concentrations of thrombin-antithrombin complex (r = -0.49, p = 0.072 and r = -0.77, p = 0.001, respectively), but did not correlate to prothrombin fragment F1+2 (r = -0.002, p = 0.99). The inverse correlations between the number of microparticles and their thrombin-forming capacity and the levels of thrombin-antithrombin complex in plasma may indicate that microparticles present in the circulation of healthy individuals have an anticoagulant function by promoting the generation of low amounts of thrombin that activate protein C. We conclude that microparticles in blood from healthy individuals support thrombin generation via TF- and FVII-independent pathways, and which may have an anticoagulant function.     

Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms

AIM: We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. METHODS: MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. RESULTS: Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = -0.51, p = 0.013, and r = -0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r= -0.62, p = 0.013). CONCLUSIONS: In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.     

Two novel and one recurrent missense mutation in the factor XIII A gene in two Dutch patients with factor XIII deficiency

Congenital factor XIII (FXIII) deficiency is a rare autosomal recessive disorder, usually attributed to a defect in the FXIII A subunit, whose genetic basis has been studied in a number of cases. We describe here the genetic variations found in two unrelated patients with FXIII deficiency. Both patients, under prophylactic substitution with FXIII concentrate, showed low plasma FXIII A subunit antigen levels with undetectable A subunit antigen in the platelets and normal plasma B antigen levels, which indicate that the defects are present in the A subunit of the molecule. Both probands were heterozygous for a previously reported G-->A transversion in exon 8 of the FXIII A subunit gene (Arg326Gln substitution). Proband 1 was also heterozygous for a novel G-->T transversion in exon 7, which predicts a Val316Phe substitution. Two of her sons were heterozygous for this mutation and showed low FXIII activity and FXIII A subunit antigen levels. Val316 is a well-conserved amino acid among the transglutaminase family, located within the core domain, close to the Cys314 member of the catalytic triad. Proband 2 had a unique 2-bp (TT) insertion in one of the alleles within or adjacent to the -7 to -20 T tail of intron A. This insertion was not found in 50 healthy individuals, which supports this being the second mutation in this patient.