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Background: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism., Objectives: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets., Methods: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied., Results: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold., Conclusion: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tissue factor (TF) is the most important trigger for the extrinsic coagulation pathway. TF, earlier denoted as thromboplastin, has always been a mystery since its discovery due to its abundant presence in most human tissues but not blood. The latter has been extensively studied in a vast quest for possible sources of blood-borne TF yielding many conflicting findings and confusing conclusions regarding the presence of TF mRNA, protein or functional procoagulant activity in virtually all blood cells. Platelets, in particular, have been heavily scrutinized by investigators eager to demonstrate expression of TF. However, some investigators including our own groups have not found evidence for TF in platelets. This article discusses notable reports and possible reasons for erroneous detection of platelet TF antigen and activity including artificially hyper-stimulated platelets, suboptimal purity of cell preparations, flaws in study design and/or choice of reagents.

Background: The controversy about the expression of tissue factor (TF) in platelet after de novo synthesis prevail despite many groups recognize that platelet isolation, assays and reagents, particularly non-specific antibodies, may account for the diversity. In this study the potential of TF expression was evaluated using immune-purified human platelets and employing a very sensitive and highly specific TF activity assay., Methods: Isolated platelets in plasma anti-coagulated with Fragmin were subjected to stimulation by LPS plus PMA, IgG antibody or TRAP and tested for TF activity., Results: Platelets stimulated with LPS plus PMA for 4 hours expressed trace amounts of TF like activity (PCA), not inhibited by anti-TF antibody (0.2±0.1 mU/ml blood). Platelets, not immune-adsorbed to remove monocytes, showed significant TF activity (2.0±0.9 mU/ml blood) that was nearly abolished by anti-TF antibody. IgG antibody from patient with lupus anticoagulant failed to enhance the trace amount of PCA as compared to the control in contrast to high TF activity induced in monocytes (0.4±0.1 mU/ml blood versus 27.5±10.5 mU/10(6) cells) showing that activation of complement is not mediating TF expression. Platelet subjected to TRAP activation for 10 min possessed only trace amounts of PCA that was not inhibited by anti-TF antibody and slightly enhanced by anti-TFPI antibody., Conclusions: It is concluded that platelets free of monocytes do not express TF activity when stimulated by LPS or activated complement factors, implying no role for Toll like receptor (TLR4) as suggested recently. There is no evidence of TF activity associated with platelets as a result of rapid and dynamic process., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The transmembrane glycoprotein TF (tissue factor) plays an essential role in haemostasis as the principal initiator of blood coagulation. In this paper, we describe how the circulating blood cells--monocytes, platelets, neutrophils and their microparticles--co-operate in regulating the expression, availability and activity of monocyte-derived TF.

The present study was performed to explore differences in the expression of P-selectin and IL-8 production of blood cells in healthy individuals with large size platelets (MI-families) as compared to people having normal size platelets. A positive correlation between LPS-induced IL-8 production per platelet in whole blood and mean platelet volume (MPV) was found in the large platelet group (R=0.74, P<0.05). When the large and normal groups were combined the correlation was nearly, but not quite significant (R=0.46, P<0.06). There was also a positive correlation between sP-selectin and MPV (R=0.42, P<0.05). Furthermore, IL-8 in serum was positively correlated to sP-selectin in serum (R=0.68, P<0.005). sP-selectin baseline values in citrated plasma correlated significantly with values found in serum (R=0.72, P<0.0005), indicating that sP-selectin in blood originates from the platelets rather than from endothelial cells. Significant correlations were also found in both groups between P-selectin and CD40L (R=0.44, P<0.05) and P-selectin and RANTES (R=0.44, P<0.05). A significant correlation was also found between PDGF and RANTES (R=0.44, P<0.05). Our results suggest that larger platelets enhance the production of IL-8 more than normally sized platelets. This phenomenon is probably mediated through P-selectin exposed on platelets.

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide (LPS)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.