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Summary Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII-activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Infusion of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, which lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. (Blood. 2011;118(7):1952-1961) IntroductionHistones are cationic proteins that associate with DNA in nucleosomes and are involved in chromatin remodeling and regulation of gene transcription. Despite their physiologic nuclear localization, nucleosomes have been found in the circulation of both healthy subjects and patients, where they can be released from dying cells 1 or actively secreted by activated inflammatory cells (neutrophils, basophils, and mast cells) in the form of "extracellular traps," complex structures of DNA strands, histones, and cell-specific granule proteins. 2,3 High blood levels of nucleosomes have been detected in several inflammatory, ischemic, autoimmune, and neoplastic diseases 4 ; in some cases, a correlation with disease severity has been found. 5 Whether extracellular nucleosomes are merely bystanders or active mediators in disease and which role histones play are important emerging questions. Histones are known to possess cytotoxic properties against both microorganisms 6 and eukaryotic cells. 7 Xu et al 8 reported that extracellular histones behave as late mediators of cell damage and organ dysfunction during the hyperinflammatory reaction that characterizes sepsis, as shown by the efficacy of a neutralizing antibody against histone H4 in reducing mortality in several experimental models of murine sepsis. Moreover, direct injection of histones into mice resulted in death with pathologic lesions suggestive of a massive prothrombotic response similar to that found in sepsis, including diffuse microvascular thrombosis, fibrin and platelet deposition in the lung alveoli, and intra-alveolar hemorrhage. Fuchs et al 9 rec...

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti–β2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer’s threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing.factor V ͉ platelets ͉ tissue factor pathway inhibitor ͉ acidocalcisomes ͉ dense granules P olyphosphate (polyP) is widely distributed in biology, being found in bacteria, fungi, plants, and animals (1). The biologic functions of polyP have been studied most extensively in prokaryotes and unicellular eukaryotes, in which high levels of polyP accumulate in acidic organelles known as acidocalcisomes. PolyP in these organisms can reach chain lengths of several hundred phosphate units. In unicellular organisms, polyP has been shown to play essential roles in stress responses and virulence (1, 2), although its function in higher eukaryotes, including man, has not been extensively investigated. Recently, we reported that dense granules of human platelets strongly resemble acidocalcisomes and contain millimolar levels of polyP (with chain lengths of Ϸ70-75 phosphate units), making acidocalcisomes the only known class of organelle conserved during evolution from bacteria to humans (3). Human platelets each have three to eight dense granules (also called ␦ granules), a type of secretory granule that contains serotonin, ADP, ATP, and PP i in addition to polyP. Patients with dense granule defects exhibit bleeding diatheses, underscoring the role of these secretory granules in hemostasis (4). Platelets contain Ϸ0.74 nmol polyP per 10 8 platelets, which is secreted after stimulation by platelet agonists such as thrombin (3). Therefore, released polyP could readily attain a concentration of 3 M in whole blood, and this could be far higher in platelet-rich thrombi. (Concentrations of polyP are expressed in this paper as phosphate monomer.)The importance of platelets in hemostasis suggested to us that polyP may play an important role in the blood clotting system. In this study, we found that polyP of the size released from activated platelets has a strongly net procoagulant effect, which is exerted at several levels in the clotting͞fibrinolysis system: PolyP activate...