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Neutrophils release the serine proteases neutrophil elastase and cathepsin G, which have microbicidal activity and thereby contribute to the innate immune response. Steffen Massberg et al . now show that these neutrophil serine proteases, in association with extracellular nucleosomes, can also promote coagulation and thrombosis within large blood vessels. In a mouse model of systemic bacterial infection, these proteases spurred intravascular coagulation in the microcirculation of the liver, limiting bacterial tissue invasion. These findings point to a role for thrombosis in antimicrobial defense. Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo . The serine proteases and extracellular nucleosomes enhance tissue factor– and factor XII–dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

Abstract Primary hemostasis begins with endothelial injury. VWF, produced by endothelial cells, binds to platelets and links them to subendothelial collagen. Platelet-derived ADP and thromboxane activate non-adhered platelets via their GPIIb/IIIa receptors, allowing these platelets to participate in platelet aggregation. Secondary hemostasis is initiated with the binding of factor VII to extravascular tissue factor (TF). Factors II, VII, IX and X are vitamin K-dependent factors. The role of vitamin K is to assist in the addition of gamma carboxylate groups to glutamic acids in the “GLA” domains of these factors. In vitro the intrinsic pathway is initiated when fresh whole blood is placed in a glass tube. The negative charge of the glass initiates the “contact pathway” where FXII is activated and then FXIa cleaves FIX to FIXa. The extrinsic pathway is triggered when tissue factor, phospholipid and calcium are added to plasma anticoagulated with citrate. In vitro, FVII is activated to FVIIa, and TF-FVIIa preferentially converts FX to FXa activating the common pathway. The prothrombin time is commonly used to monitor warfarin anticoagulant therapy. To correct for differences in reagent and instrument, the international normalized ratio was developed to improve standardization of PT reporting globally. The activated partial thromboplastin time (aPTT) is used to evaluate the intrinsic and common pathways of coagulation. The aPTT is useful clinically as a screening test for inherited and acquired factor deficiencies as well as to monitor unfractionated heparin therapy although the anti-Xa assay is now the preferred measure of the effects of unfractionated heparin. The Clauss assay is the most commonly performed fibrinogen assay and uses diluted plasma where clotting is initiated with a high concentration of reagent thrombin. The mixing study assists in the assessment of an abnormally prolonged PT or aPTT. An equal volume of citrated patient plasma is mixed with normal pooled plasma and the PT or aPTT are repeated on the 1:1 mix. Factor activity assays are most commonly performed as a one-stage assay. The patient’s citrated plasma is diluted and mixed 1-to-1 with a single factor-deficient substrate plasma. A PT or aPTT is performed on the above mix, depending on the factor being tested. Factor inhibitors are antibodies that are most commonly diagnosed in male patients with severe hemophilia A (FVIII deficiency) where they are induced by factor replacement therapy. Factor inhibitors can also appear in the form of spontaneous autoantibodies in both male and female individuals who were previously well. This is an autoimmune condition called “acquired hemophilia.” Most coagulation laboratories can measure the plasma concentration of VWF protein (VWF antigen) by an immunoturbidimetric technique. Testing the functional activity of VWF, utilizes the drug ristocetin. The state of multimerization of VWF is important and is assessed by electrophoresis on agarose gels. Type 2a and 2b VWD are associated with the lack of intermediate- and high molecular weight multimers. The antiphospholipid syndrome (APLS) is an acquired autoimmune phenomenon associated with an increased incidence of both venous and arterial thromboses, as well as fetal loss. Typically, there is a paradoxical prolongation of the aPTT in the absence of any clinical features of bleeding. This is the so-called “lupus anticoagulant (LA) effect.” The laboratory definition of the APLS requires the presence of either a “lupus anticoagulant” or a persistent titer of antiphospholipid antibodies. There are now 2 broad classes of direct-acting oral anticoagulants (DOACs): [1] The oral direct thrombin inhibitors (DTIs) such as dabigatran; and [2] The oral direct factor Xa inhibitors such as rivaroxaban and apixaban. The PT and aPTT are variably affected by the DOACs and are generally unhelpful in monitoring their concentrations. Most importantly, a normal PT or aPTT does NOT exclude the presence of any of the DOACs.

Clotting factor transfusions are vital for people with diseases such as haemophilia. In the 1970s and 1980s, transfusions with pooled plasma led to a devastatingly high number of recipients becoming infected with blood-borne pathogens such as HIV and hepatitis C. This epidemic triggered the development of virus-free factor concentrates through a combination of improved donor selection and screening, effective virucidal technologies, and recombinant protein expression biotechnology. There is now a wide range of recombinant factor concentrates, and an impressive safety record with respect to pathogen transmission. However, remaining therapeutic challenges include the potential threat of transmission of prions and other pathogens, the formation of inhibitory alloantibodies, and the international disparity that exists in product availability due to differences in licensure status as well as prohibitively high costs. In the future, it is likely that bioengineered recombinant proteins that have been modified to enhance pharmacokinetic properties or reduce immunogenicity, or both, will be used increasingly in clinical practice.

A better understanding of the inflammatory, procoagulant, and immunosuppressive aspects of sepsis has contributed to rational therapeutic plans. This review considers optimal management of sepsis from the point of early, goal-directed therapy, lung-protective ventilation, antibiotics, and additional therapies, such as activated protein C, as well as the use of corticosteroids, vasopressin, and intensive insulin. This review considers optimal management of sepsis from the point of early, goal-directed therapy, lung-protective ventilation, antibiotics, and additional therapies, such as activated protein C, as well as the use of corticosteroids, vasopressin, and intensive insulin. A better understanding of the inflammatory, procoagulant, and immunosuppressive aspects of sepsis has contributed to rational therapeutic plans from which several important themes emerge. 1 First, rapid diagnosis (within the first 6 hours) and expeditious treatment are critical, since early, goal-directed therapy can be very effective. 2 Second, multiple approaches are necessary in the treatment of sepsis. 1 Third, it is important to select patients for each given therapy with great care, because the efficacy of treatment — as well as the likelihood and type of adverse results — will vary, depending on the patient. The Spectrum of Sepsis Nomenclature is important when . . .

Vascular injury triggers two intertwined processes, platelet deposition and coagulation, and can lead to the formation of an intravascular clot (thrombus) that may grow to occlude the vessel. Formation of the thrombus involves complex biochemical, biophysical, and biomechanical interactions that are also dynamic and spatially-distributed, and occur on multiple spatial and temporal scales. We previously developed a spatial-temporal mathematical model of these interactions and looked at the interplay between physical factors (flow, transport to the clot, platelet distribution within the blood) and biochemical ones in determining the growth of the clot. Here, we extend this model to include reduction of the advection and diffusion of the coagulation proteins in regions of the clot with high platelet number density. The effect of this reduction, in conjunction with limitations on fluid and platelet transport through dense regions of the clot can be profound. We found that hindered transport leads to the formation of smaller and denser clots compared to the case with no protein hindrance. The limitation on protein transport confines the important activating complexes to small regions in the interior of the thrombus and greatly reduces the supply of substrates to these complexes. Ultimately, this decreases the rate and amount of thrombin production and leads to greatly slowed growth and smaller thrombus size. Our results suggest a possible physical mechanism for limiting thrombus growth.

The protein C anticoagulant pathway serves as a major system for controlling thrombosis, limiting inflammatory responses, and potentially decreasing endothelial cell apoptosis in response to inflammatory cytokines and ischemia. The essential components of the pathway involve thrombin, thrombomodulin, the endothelial cell protein C receptor (EPCR), protein C, and protein S. Thrombomodulin binds thrombin, directly inhibiting its clotting and cell activation potential while at the same time augmenting protein C (and thrombin activatable fibrinolysis inhibitor [TAFI]) activation. Furthermore, thrombin bound to thrombomodulin is inactivated by plasma protease inhibitors > 20 times faster than free thrombin, resulting in increased clearance of thrombin from the circulation. The inhibited thrombin rapidly dissociates from thrombomodulin, regenerating the anticoagulant surface. Thrombomodulin also has direct anti-inflammatory activity, minimizing cytokine formation in the endothelium and decreasing leukocyte-endothelial cell adhesion. EPCR augments protein C activation approximately 20-fold in vivo by binding protein C and presenting it to the thrombin-thrombomodulin activation complex. Activated protein C (APC) retains its ability to bind EPCR, and this complex appears to be involved in some of the cellular signaling mechanisms that down-regulate inflammatory cytokine formation (tumor necrosis factor, interleukin-6). Once APC dissociates from EPCR, it binds to protein S on appropriate cell surfaces where it inactivates factors Va and VIIIa, thereby inhibiting further thrombin generation. Clinical studies reveal that deficiencies of protein C lead to microvascular thrombosis (purpura fulminans). During severe sepsis, a combination of protein C consumption, protein S inactivation, and reduction in activity of the activation complex by oxidation, cytokine-mediated down-regulation, and proteolytic release of the activation components sets in motion conditions that would favor an acquired defect in the protein C pathway, which in turn favors microvascular thrombosis, increased leukocyte adhesion, and increased cytokine formation. APC has been shown clinically to protect patients with severe sepsis. Protein C and thrombomodulin are in early stage clinical trials for this disease, and each has distinct potential advantages and disadvantages relative to APC.

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼ 1% expression of the mouse TF level) and high human TF mice (HTF, ∼ 100% of the mouse TF level) were injected with human rFVIIa (120 µg kg(-1) body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40-50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF's role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.

A mathematical model of the extrinsic or tissue factor (TF) pathway of blood coagulation is formulated and results from a computational study of its behavior are presented. The model takes into account plasma-phase and surface-bound enzymes and zymogens, coagulation inhibitors, and activated and unactivated platelets. It includes both plasma-phase and membrane-phase reactions, and accounts for chemical and cellular transport by flow and diffusion, albeit in a simplified manner by assuming the existence of a thin, well-mixed fluid layer, near the surface, whose thickness depends on flow. There are three main conclusions from these studies. (i) The model system responds in a threshold manner to changes in the availability of particular surface binding sites; an increase in TF binding sites, as would occur with vascular injury, changes the system’s production of thrombin dramatically. (ii) The model suggests that platelets adhering to and covering the subendothelium, rather than chemical inhibitors, may play the dominant role in blocking the activity of the TF:VIIa enzyme complex. This, in turn, suggests that a role of the IXa-tenase pathway for activating factor X to Xa is to continue factor Xa production after platelets have covered the TF:VIIa complexes on the subendothelium. (iii) The model gives a kinetic explanation of the reduced thrombin production in hemophilias A and B.

Historically known for its role in blood coagulation, vitamin K also has been shown to be required for the physiologic activation of numerous proteins that are not involved in hemostasis. Over the last 20 years, vitamin K–dependent proteins have been isolated in bone, cartilage, kidney, atheromatous plaque, and numerous soft issues. Although the precise mechanism of action of many of these proteins remains to be determined, their discovery has proven important from a physiologic point of view.

The coagulation protease thrombin triggers fibrin formation, platelet activation, and other cellular responses at sites of tissue injury. We report a role for PAR1, a protease-activated G protein-coupled receptor for thrombin, in embryonic development. Approximately half of Par1-/-mouse embryos died at midgestation with bleeding from multiple sites. PAR1 is expressed in endothelial cells, and a PAR1 transgene driven by an endothelial-specific promoter prevented death of Par1-/-embryos. Our results suggest that the coagulation cascade and PAR1 modulate endothelial cell function in developing blood vessels and that thrombin's actions on endothelial cells-rather than on platelets, mesenchymal cells, or fibrinogen-contribute to vascular development and hemostasis in the mouse embryo.