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Background: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms., Methods: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results., Results: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography., Conclusion and Significance: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Nonleukoreduced units of red blood cells (RBCs) contain activated platelets (PLTs) that interact with white blood cells (WBCs) and may promote inflammation and thrombosis in the recipient. The aim of this study was to characterize PLT-WBC interactions (PLT-WBC aggregates [PLAs]), WBC apoptosis, WBC death, and the development of procoagulant activity in RBCs during storage., Study Design and Methods: RBCs were prepared from volunteer donor blood and stored. Samples were analyzed with flow cytometry between Days 1 and 15 to measure PLT-monocyte aggregate (PMA) and PLT-neutrophil aggregate (PNA) formation, WBC apoptosis (annexin V binding), and cell death (binding of 7-aminoactinomycin D). Procoagulant activity in the supernatant of four RBC preparations was assessed between Days 1 and 39 using a clotting assay with and without the addition of an inhibitory anti-tissue factor (TF) antibody, αTF-5., Results: PLA formation was extensive and maximal on Day 3 of storage (PNA, 23 ± 13%; PMA, 93 ± 4%; n = 6). Apoptosis was progressive throughout storage, with 95 ± 4% of neutrophils and 73 ± 19% of monocytes binding annexin V on Day 15. Cell death became measurable after apoptosis. Procoagulant activity was observed in all RBCs but with varying temporal patterns. It was partially TF dependent and removed with high-speed centrifugation, suggestive of an association with microparticles., Conclusion: The activation of PLTs during the storage of RBCs induces PLA formation that precedes WBC apoptosis and death. Procoagulant activity, likely associated with microparticles derived from apoptotic WBCs, may contribute to adverse effects of stored, nonleukoreduced RBCs., (© 2010 American Association of Blood Banks.)

Our current understanding of hemostasis and the roles of coagulation and platelets has evolved in recent years from the old "coagulation cascade" and "platelet pathway" models to an inter-related, cell-based model that more accurately represents in-vivo processes and better reflects clinical observations. The new model makes it apparent that thrombin is a key effector and modulator of hemostasis, and that activation of coagulation and platelets cannot be viewed as separate processes. Thrombin is a potent platelet agonist, and recent data indicate that thrombin may be the most critical activator of tissue-factor-induced thrombosis. Minute quantities of thrombin generated at the site of plaque disruption or arterial injury activates platelets and amplifies its own production, resulting in an explosive burst of thrombin production after clot formation. Thrombin-induced activation of platelets and other cells may contribute to inflammatory processes that are increasingly recognized as playing an important role in acute coronary syndromes (ACS) and coronary interventions. Along with a clearer understanding of the biology of thrombosis and hemostasis, better methods for studying the mechanisms of coagulation and platelet activation and the interaction of these processes in vivo have provided a new understanding of the method by which antithrombotic agents work. The physiological basis of thrombosis and hemostasis has important implications for treatment strategies in ACS, and for outcomes of percutaneous coronary interventions (PCI). With this increased understanding, the limitations of heparin and the advantages of the direct thrombin inhibitor bivalirudin (Angiomax, The Medicines Company) become apparent--providing a mechanistic rationale for the clinical benefit recently demonstrated in the Randomized Evaluation of PCI Linking Angiomax to Reduced Clinical Events (REPLACE-2) PCI trial.