Regulation of inflammatory responses by natural anticoagulants

Summary: Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) are critically involved in activation of the coagulation system in sepsis, leading to disseminated intravascular coagulation (DIC). Natural anticoagulants such as antithrombin (AT) and activated protein C (APC) regulate the coagulation system by inhibiting thrombin generation. In addition to these anticoagulant effects, both AT and APC have been shown to attenuate inflammatory responses induced by various noxious stimuli in rats such as lipopolysaccharide (LPS) challenge. AT promotes the endothelial release of prostacyclin, a potent anti-inflammatory prostaglandin that inhibits the monocytic production of TNF-α, by interacting with cell-surface heparin-like substances. APC directly inhibits the production of TNF-α by inhibiting the activation of both nuclear factor κB (NFκB) and activator protein-1 in monocytes stimulated with LPS. Thrombomodulin, an endothelial membranous integral protein that binds thrombin, exerts anti-inflammatory effects by generating APC. Furthermore, tissue factor pathway inhibitor, a natural anticoagulant for the extrinsic pathway of the coagulation system, also attenuates LPS-induced inflammatory responses in rats by inhibiting TNF-α production by monocytes. These findings strongly suggest that natural anticoagulants could regulate inflammatory responses as well as the coagulation system in rats by inhibiting the monocytic production of TNF-α. Such anti-inflammatory properties of natural anticoagulants are potentially important for their replacement in patients with sepsis who frequently develop DIC and organ failure as inflammatory responses.     

Thrombin generation measurement in factor VII-depleted plasmas compared to inherited factor VII-deficient plasmas

Activated factor VII (FVIIa)/tissue factor enzyme complex is the initiator of the coagulation cascade in vivo. FVIIa is of particular interest because it has been found to induce haemostasis in various bleeding disorders. In order to evaluate the FVII threshold that is required to initiate the clotting cascade, we measured thrombin generation in FVII-depleted plasmas spiked with increasing amounts of normal pooled plasma and in inherited FVII-deficient plasmas. According to the literature, only trace amounts of FVII are sufficient to initiate blood coagulation in vitro. By contrast, results on inherited FVII-deficient plasmas showed a wide variety of the amounts of thrombin generated in plasmas with the same FVII coagulant activity levels. This suggests that the threshold of FVII required to initiate haemostasis in vivo depends on one or more, hitherto unknown, plasmatic or cellular factors.     

Ancylostoma caninum anticoagulant peptide-5: immunolocalization and in vitro neutralization of a major hookworm anti-thrombotic

Hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia in the developing world. Recently two major anticoagulant serine protease inhibitors have been identified and cloned from adult Ancylostoma caninum hookworms. One of these, A. caninum anticoagulant peptide 5 (AcAP5), is a potent and specific inhibitor of human coagulation factor Xa. A polyclonal IgG has been purified from rabbits immunized with recombinant AcAP5 using affinity chromatography. Using immunohistochemistry, the polyclonal alpha-rAcAP5 IgG localized to the cephalic or amphidial glands, confirming previous biochemical studies that had identified this secretory gland as the primary source of anticoagulant activity in the adult worm. This polyclonal IgG also neutralized the inhibitory activity of recombinant and native AcAP using a single stage chromogenic assay of coagulation factor Xa activity. In addition, the polyclonal IgG also neutralized the anticoagulant activity of native and recombinant AcAP5 as measured by the activated partial thromboplastin time clotting assay. Importantly, this neutralizing activity is species specific, as the polyclonal IgG failed to neutralize the anticoagulant activity of A. ceylanicum. Taken together, these data suggest that the hookworm anticoagulant AcAP5 represents a viable target for future immunization strategies aimed at inhibiting the ability of the adult hookworm to feed on blood in vivo.     

Effect of gaboon viper (Bitis gabonica) venom on blood coagulation, platelets, and the fibrinolytic enzyme system

The action of the venom of the gaboon viper (Bitis gabonica) on blood coagulation, platelets, and the fibrinolytic enzyme system was studied. The results confirm that the venom of Bitis gabonica has a marked anticoagulant action in vitro. The venom appears to impair clot formation by a direct proteolytic action on fibrinogen, releasing soluble breakdown products.     

Protein C, protein S

Protein C is a potent inhibitor of blood coagulation, and, in addition, appears to be a profibrinolytic agent. In a first step, protein C must be converted to a serine protease. This activation is catalyzed by a complex formed between thrombin and thrombomodulin, an endothelial cell surface protein. Activated protein C exhibits its anticoagulant activity through the proteolytic inactivation of two blood coagulation cofactors, factors Va and VIIIa. This reaction requires phospholipids, originating from platelets or endothelial cells, and a cofactor protein, protein S. Protein S enhances the binding of activated protein C to phospholipids. In addition, activated protein C stimulates fibrinolysis, through the inactivation of the tissue plasminogen activator (tPA) inhibitor. An isolated constitutional, quantitative or qualitative, protein C or protein S deficiency increases the risk of thrombosis, the clinical features are different in the rare cases of homozygous protein C deficiency (neonatal purpura fulminans) or in the heterozygous patients (recurrent venous thrombosis in young adults). Acquired deficiency in protein C and S had been observed in liver disease, during vitamin K antagonists or L-Asparaginase treatment, and in disseminated intravascular coagulation.     

Extrinsic coagulation blockade attenuates lung injury and proinflammatory cytokine release after intratracheal lipopolysaccharide

Initiation of coagulation by tissue factor (TF) is a potentially powerful regulator of local inflammatory responses. We hypothesized that blockade of TF-factor VIIa (FVIIa) complex would decrease lung inflammation and proinflammatory cytokine release after tracheal instillation of Escherichia coli lipopolysaccharide (LPS 0111:B4). At the time of injury, rats received one dose of site-inactivated FVIIa (FFR-FVIIa) or saline intravenously. At 0, 6,12, 24, and 48 h after injury, lungs were examined for histologic changes and bronchoalveolar lavage (BAL) was performed to assess protein, lactate dehydrogenase (LDH) activity, cell counts, and cytokine levels. LPS-injured rats treated with FFR-FVIIa showed decreased intra-alveolar inflammation and fibrin deposition by light microscopy compared with untreated rats. This was accompanied by decreased protein leakage (P < 0.0001), LDH activity (P < 0.0001), and local elaboration of interleukin (IL)-1beta, IL-6, and IL-10 (all P < 0.0001), but not tumor necrosis factor (TNF)-alpha. Protection was associated with reduction of TF mRNA expression in whole lung, but not with changes in nuclear translocation of nuclear factor (NF)-kappaB. FFR-FVIIa given 6 h after LPS afforded equivalent lung protection. Therefore, blockade of TF-FVIIa complex protects the lung from injury by LPS in part by reducing local expression of proinflammatory cytokines and may offer promise for therapy of acute lung injury.     

Tissue factor pathway inhibitor: structure, biology and involvement in disease

Tissue factor (TF)-initiated coagulation plays a significant role in the pathophysiology of many diseases, including cancer and inflammation. Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor, which modulates initiations of coagulation induced by TF. In a factor (F) Xa-dependent feedback system, TFPI binds directly and inhibits the TF-FVII/FVIIa complex. Normally, TFPI exists in plasma both as a full-length molecule and as variably carboxy-terminal truncated forms. TFPI also circulates in complex with plasma lipoproteins. The levels and the dual inhibitor effect of TFPI on FXa and TF-FVII/FVIIa complex offers insight into the mechanisms of various pathological conditions triggered by TF. The use of selective pharmacological inhibitors has become an indispensable tool in experimental haemostasis and thrombosis research. In vivo administration of recombinant TFPI (rTFPI) in an experimental animal model prevents thrombosis (and re-thrombosis after thrombolysis), reduces mortality from E. coli-induced-septic shock, prevents fibrin deposition on subendothelial human matrix and protects against disseminated intravascular coagulation (DIC). Thus, TFPI may play an important role in modulating TF-induced thrombogenesis and it may also provide a unique therapeutic approach for prophylaxis and/or treatment of various diseases. In this review, we consider structural and biochemical aspects of the TFPI molecule and detail its inhibitory mechanisms and therapeutic implications in various disease conditions.     

Circulating heparan sulfate anticoagulant in a patient with a fatal bleeding disorder

We have identified a circulating, heparin-like anticoagulant in a patient with multiple myeloma (IgG4 lambda) who had serious clinically evident bleeding that contributed to his death. Purification of the patient's circulating coagulation inhibitor was accomplished by ammonium sulfate concentration, anion exchange chromatography, and affinity chromatography on protamine sulfate. Analysis of the purified inhibitor showed that it was a proteoglycan that comigrated with heparan sulfate on lithium acetate-agarose-gel electrophoresis and that it contained 39 per cent L-iduronic acid. Control samples of heparan sulfate and heparin contained 29 and 68 per cent L-iduronic acid, respectively. Functional coagulation studies revealed that the purified inhibitor had cofactor activity with antithrombin III that could be abolished by prior incubation with protamine sulfate or platelet factor 4. Recognition of the existence of this or of other similar inhibitors in bleeding patients is important because of the potential for treatment with agents such as protamine sulfate and platelet factor 4, which neutralize the anticoagulant effects of proteoglycans.     

Stimulation of Naïve Monocytes and PBMCs with Coagulation Proteases Results in Thrombin‐Mediated and PAR‐1‐Dependent Cytokine Release and Cell Proliferation in PBMCs Only

Protease‐activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain. Coagulation proteases, such as FVIIa, the binary TF‐FVIIa complex, free FXa, the ternary TF‐FVIIa‐FXa complex and thrombin, are able to stimulate PARs. Whereas the role of PARs on platelets is well known, their function in naïve monocytes and peripheral blood mononuclear cells (PBMCs) is largely unknown. This is of interest because PAR‐mediated interactions of coagulation proteases with monocytes and PBMCs in diseases with an increased activation of coagulation may promote inflammation. To evaluate PAR‐mediated inflammatory reactions in naïve monocytes and PBMCs stimulated with coagulation proteases. For this, PAR expression at protein and RNA level on naïve monocytes and PBMCs was evaluated with flow cytometry and RT‐PCR. In addition, cytokine release (IL‐1β, IL‐6, IL‐8, IL‐10, TNF‐α) in stimulated naïve and PBMC cell cultures was determined. In this study, it is demonstrated that naïve monocytes express all four PARs at the mRNA level, and PAR‐1, ‐3 and ‐4 at the protein level. Stimulation of naïve monocytes with coagulation proteases did not result in alterations in PAR expression or in the induction of inflammation involved cytokines like interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8, interleukin‐10 or tumour necrosis factor‐α. In contrast, stimulation of PBMCs with coagulation proteases resulted in thrombin‐mediated induction of IL‐1β and IL‐6 cytokine production and PBMC cell proliferation in a PAR‐1‐dependent manner. These data demonstrate that naïve monocytes are not triggered by coagulation proteases, whereas thrombin is able to elicit pro‐inflammatory events in a PAR‐1‐dependent manner in PBMCs.     

抗重组人组织因子途径抑制物1单抗抑制rhTFPI-1的抗凝作用

目的研制抗重组人组织因子途径抑制物1单克隆抗体(rhTFPI-1 M cAb),了解其对rhTFPI-1抗凝活性的抑制作用。方法将分泌rhTFPI-1 M cAb的杂交瘤细胞注入经降植烷预处理的Balb/C小鼠腹腔中,诱生腹水。腹水先经硫酸铵沉淀处理,再用rprote in A亲和层析柱进一步纯化。对纯化后的单克隆抗体进行SDS-PAGE检测和W estern b lot分析。并用发色底物法测定rhTFPI-1 M cAb对rhTFPI-1的活性抑制。结果纯化后的抗rhTFPI-1 M cAb经SDS-PAGE检测,其纯度为98%;W estern b lot分析显示其能特异识别rhTFPI-1抗原。发色底物法检测结果表明,其能有效阻断rhTFPI-1的抗FVIIa/TF活性,M cAb浓度为8 mg/L时FVIIa/TF剩余活性达到86%;但对rhTFPI-1的抗FXa活性作用不明显,M cAb浓度为80 mg/L时FXa的剩余活性为48.4%。结论rhTFPI-1 M cAb可明显抑制rhT-FPI-1活性,阻碍rhTFPI-1对TF/FVIIa的抑制活性。纯化后的rhTFPI-1 M cAb的各项鉴定指标均较为理想,rprote inA亲和层析柱纯化方法简便易行值得推广。     

The Taipan snake venom time: a new test for lupus anticoagulant.

AIMS--To develop a specific test for lupus anticoagulant activity with reduced sensitivity to coagulation factor deficiency that would be suitable for analysis of plasmas from patients receiving oral anticoagulants. METHODS--A coagulation test based on the Taipan snake venom time (TSVT) with a platelet neutralisation procedure (PNP) was developed and compared with dilute Russell's viper venom time (DRVVT). The TSVT was used to test plasmas from patients receiving oral anticoagulant or heparin with mild liver dysfunction and with documented lupus anticoagulant. RESULTS--The optimised conditions for the TSVT were established and a reference range was determined in normal healthy subjects. Results were considered positive for lupus anticoagulant if the ratio was > or = 1.1 and was reduced by > or = 10% or to < 1.1 in the PNP. In 43 samples from patients receiving oral anticoagulants there was no correlation between level of anticoagulation and TSVT, and only seven samples had increased TSVTs. Of these, five corrected on mixing with normal plasma and two gave equivocal results. The patients with mild liver dysfunction all had normal TSVTs. The TSVT in plasmas from patients receiving heparin correlated with the heparin concentrations (as measured by the APTT, r2 = 0.81). Some anticoagulated plasmas showed correction in the PNP and were regarded as false positive. Fourteen of 17 patients known to have lupus anticoagulant (on the basis of DRVVT results) were also positive by the TSVT; two of the remaining three were borderline and one was negative. CONCLUSIONS--The TSVT showed satisfactory intra-assay precision and reasonable sensitivity to lupus anticoagulant, compared with the DRVVT. The TSVT was influenced by the presence of heparin but was not sensitive to the effects of oral anticoagulant. Like other lupus anticoagulant tests, it does not seem to have a 100% detection rate, but this may be due to the presence of lupus anticoagulant subtypes with distinct activities or the requirement of cofactors other than prothrombin or beta 2 glycoprotein-I.     

Effects of three cobra venoms on blood coagulation, platelet aggregation, and fibrinolysis

The effects of the venoms of Naja melanoleuca, Naja nigricollis, and Ophiophagus hannah on blood coagulation, platelet aggregation, and fibrinolysis were studied in vitro. All three venoms were shown to be anticoagulant. This action appeared to be due to an effect on both the extrinsic and blood thromboplastin mechanisms. Platelet aggregation in Chandler's tubes and adenosine diphosphate reactivity were inhibited by the three venoms, although in the case of Ophiophagus hannah venom they were inhibited only with intermediate concentrations. The three venoms possessed proteolytic properties, but when incorporated into purified caseinolytic systems and euglobulin clot lysis systems inhibition of plasmin activity was observed.     

The role of endothelium in the homeostatic balance of haemostasis

As the cells forming the luminal vascular surface, endothelial cells are strategically positioned to play an important role in the regulation of coagulation. They cannot be regarded as an inert surface lining the vessel wall since they possess multiple activities. Anticoagulant properties include provision of a cell surface with heparin-like molecules (which can serve as binding sites for antithrombin III), synthesis of thrombomodulin (which alters the substrate specificity of thrombin), maintenance of a low level of tissue factor and generation of prostacyclin. In addition to these anticoagulant properties, endothelial cells can play a role in procoagulant reactions. Studies which have examined mechanisms underlying the localization of thrombotic processes have suggested the possible involvement of endothelial cells in procoagulant events. Endothelial cells have been found to propagate factor X and prothrombin activation once factor IXa and factor Xa have been formed. Factors regulating the balance of plasminogen activator and inhibitor synthesis are also under study. Perturbation of endothelial cells with induction of tissue factor and production of platelet-activating factor and thromboxane provides a model of the thrombotic state in which endothelium can promote coagulation. The multiple properties of endothelial cells indicate that a continuous blood flow in an unperturbed region of the vessel wall results from a complex interplay of anticoagulant and procoagulant activities. In a perturbed region, endothelial cells might initiate coagulation and the thrombin formed on the surface of endothelial cells might then lead to recruitment of platelets.     

Coagulation under flow: the influence of flow-mediated transport on the initiation and inhibition of coagulation

A mathematical model of intravascular coagulation is presented; it encompasses the biochemistry of the tissue factor pathway, platelet activation and deposition on the subendothelium, and flow- and diffusion-mediated transport of coagulation proteins and platelets. Simulation experiments carried out with the model indicate the predominant role played by the physical processes of platelet deposition and flow-mediated removal of enzymes in inhibiting coagulation in the vicinity of vascular injury. Sufficiently rapid production of factors IXa and Xa by the TF:VIIa complex can overcome this inhibition and lead to formation of significant amounts of the tenase complex on the surface of activated platelets and, as a consequence, to substantial thrombin production. Chemical inhibitors are seen to play almost no (TFPI) or little (AT-III and APC) role in determining whether substantial thrombin production will occur. The role of APC is limited by the necessity for diffusion of thrombin from the site of injury to nearby endothelial cells to form the thrombomodulin-thrombin complex and for diffusion in the reverse direction of the APC made by this complex. TFPI plays an insignificant part in inhibiting the TF:VIIa complex under the conditions studied whether its action involves sequential binding of TFPI to Xa and then TFPI:Xa to TF:VIIa, or direct binding of TFPI to Xa already bound to the TF:VIIa complex.     

The role of complex formation and epidermal growth factor-like domains in the regulation of blood coagulation by the thrombomodulin-protein C system

The explosive nature of the coagulation cascade led many scientists to investigate how it is regulated. Proteinase inhibitors such as antithrombin III inhibit active proteases of the coagulation cascade. Cofactors such as factor Va and factor VIIIa are proteolytically inactivated by activated protein C. Protein C is activated by the thrombin-thrombomodulin complex on the endothelial cell surface. Thus, the independent actions of the proteinase inhibitor system and the thrombomodulin-protein C system complement each other to maintain regulation of blood coagulation. The thrombin binding site of thrombomodulin was identified to be the fifth and sixth repeats of the epidermal growth factor-like domain. The same binding template contains sufficient information to block the functions of thrombin as a procoagulant. However, additional repeats are required for the activation of protein C. Thrombomodulin is the first example which illustrates that the epidermal growth factor-like domain functions as a binding template for thrombin and as a switch to turn off the procoagulant activity of thrombin as well as to trigger the protein C anticoagulant pathway. Epidermal growth factor-like structures are found in many of the coagulation factors. Complex formation is a repeated theme not only in the blood coagulation cascade but also in the thrombomodulin-protein C anticoagulant pathway.     

Effects of the venom of the rhinoceros horned viper (Bitis nasicornis) on blood coagulation, platelet aggregation, and fibrinolysis

The venom of the rhinoceros horned viper (Bitis nasicornis) has been studied in vitro and has been shown to be anticoagulant. This action appeared to be due to an effect on both the extrinsic and intrinsic blood thromboplastin mechanisms. The venom was also proteolytic and in purified caseinolytic systems activated plasminogen, enhanced the activation of plasminogen by streptokinase, and potentiated the action of plasmin. In the euglobulin clot lysis system high concentrations of venom produced inhibition. The crude venom increased platelet adhesiveness but in high concentrations delayed the snowstorm effect in the Chandler's tube system and inhibited platelet adenosine diphosphate reactivity. Passage through carboxymethylcellulose yielded six fractions. One possessed anticoagulant activity, inhibited plasmin, and increased the optical density of platelet-rich plasma. The other five fractions shortened the plasma recalcification time but had no effect on plasmin activity. Four fractions aggregated platelets and enhanced platelet adenosine diphosphate reactivity.     

Anticoagulant activity of enzymatically synthesized amylose derivatives containing carboxy or sulfonate groups

Heparin is an extracellular matrix polysaccharide. It is widely employed as an anticoagulant and can be used to form an anticoagulant surface on various medical devices such as renal dialysis devices to prevent thrombosis. However, heparin may cause hemorrhage and thrombocytopenia. Moreover, commercially available heparin may be contaminated with viruses and allergens of animal origin, as it is derived mainly from porcine or bovine tissue. To avoid these problems, we prepared succinated and sulfonated enzymatically synthesized amylose (SucESA and SulfESA, respectively) and assessed their anticoagulant activity. SucESA and SulfESA inhibited factor Xa activity in normal human plasma to an equal extent. However, SucESA strongly inhibited thrombin activity, whereas SulfESA only inhibited it slightly. These results suggest that SucESA inhibits the activities of both factor Xa (or its upstream coagulation factors) and thrombin and that SulfESA inhibits only factor Xa activity (or that of its upstream coagulation factors). SucESA and SulfESA with a high degree of substitution strongly inhibited factor Xa and thrombin activity compared with those of the derivatives with a low degree of substitution, even when present in high concentrations. This suggests that the density of the anion group determines the degree of inhibition of factor Xa and thrombin activity. SucESA, which has a high molecular weight, inhibited thrombin activity to a greater degree than low molecular weight SucESA. Because SucESA and SulfESA inhibited both purified factor Xa and thrombin irrespective of the presence of antithrombin, it is suggested that SucESA and SulfESA inhibit via direct action with both enzymes.     

Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa.     

General mechanisms of coagulation and their physiological inhibition. I. General mechanisms of blood coagulation

Blood coagulation results from a sequence of enzymatic reactions, which involve 12 plasma proteins (coagulation factors), platelets, a tissue lipoprotein (tissue factor), vascular components and calcium ions. These reactions are induced by vascular lesions, and are promoted by blood contact with sub-endothelial structures and by the release of tissue factor into the circulation; they result in the formation of an hemostatic plug constituted of platelets and fibrin. The enzymatic reactions consist of proteolytic reactions, in which zymogens are converted to proteinases. These reactions are accelerated by both proteins-proteins interactions and by proteins-membrane surface (vascular wall, platelets) interactions, responsible for the amplification of the activation process, and its localization at the site of injury. The coagulation process is regulated by positive and negative feed-backs and by physiological inhibitors.     

Activation of coagulation by administration of recombinant factor VIIa elicits interleukin 6 (IL-6) and IL-8 release in healthy human subjects

The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa.