Physiological Anticoagulants Research Articles

The Role of Leukocytes in Disseminated Intravascular Coagulation Associated with Sepsis

Leukocytes play a pivotal role in the pathogenesis of disseminated intravascular coagulation (DIC) and multiple organ failure associated with sepsis. Cytokines such as tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β) activate monocytes, neutrophils and endothelial cells. TNF-α increases the expression of tissue factor on surfaces of both monocytes and endothelial cells and decreases the anticoagulant potential of endothelial cells, thereby inducing intravascular coagulation. These cytokines also inhibit the fibrinolytic activity by increasing the endothelial production of plasminogen activator inhibitor-1. These cytokines activate neutrophils to release the various inflammatory mediators such as neutrophil elastase and oxygen free radicals, both of which are capable of damaging endothelial cells. Activated neutrophils damage endothelial cells by adhering to endothelial cells through interaction with E-selectin or ICAM-1, endothelial leukocyte adhesion molecules, expression of which are increased by the actions of these cytokines. Both microthrombus formation and the endothelial cell damage could lead to multiple organ failure by inducing microcirculatory disturbances. Among physiological anticoagulants, antithrombin, activated protein C and tissue factor pathway inhibitor exert anti-inflammatory activity by inhibiting leukocyte activation. Gabexate mesilate and nafamostat mesilaste, synthetic anticoagulants, also inhibit leukocyte activation. The reduction of both coagulation abnormalities and inflammatory responses by using these therapeutic agents could be useful in the treatment of DIC associated with sepsis.

Upregulation of the Antithrombotic Protein C Pathway at Birth

Serious thrombotic complications occur in sick neonates, while healthy infants have a very low risk of thrombosis. To better understand the regulation of physiological anticoagulation at birth, components oftheprotein C pathway weremeasured in cordplasma samples from 14 full-term healthy newborns and in samples from 10 adult controls. Although zymogen protein C was significantly reduced in cord plasma (mean ± SEM in cord vs. adult samples 37 ± 1.4% vs. 90 ± 5.5%, p < 0.0001), levels of the active enzyme, activated protein C (APC), were not (119 ± 20% vs. 75 ± 12%, p = 0.0762) Relative to the protein C level, cord plasmas had a 5.2-fold higher APC level (p < 0.01). The A PC increase was partially due to slower inactivation of A PC in cord plasma (half-life for APC 50 min in cord plasma vs. 27 minutes in adult plasma). Increased sensitivity of factor V to inactivation by APC in cord plasma was observed since the activated partial thromboplastin time-based A PC sensitivity ratio was significantly increased for cord vs. adult plasma samples (2.28 ± 0.09 versus 1.97 ± 0.03, p < 0.01). Thus, despite low zymogen protein C, the protein C pathway in newborns seems to be functionally well developed and at an activated stage at birth.

Physiological anticoagulation. Resistance to activated protein C and venous thromboembolism.

Physiological anticoagulation. Resistance to activated protein C and venous thromboembolism.

Coagulation abnormalities in ischaemic optic neuropathy.

The aetiology of non-arteritic ischaemic optic neuropathy (ION) is multifactorial with local anatomical and systemic haemodynamic abnormalities both playing a role. A careful search for treatable vascular disease risk factors is required to allow rational therapy, to optimise the visual prognosis and to allow new insights into pathogenesis. We describe 7 cases in which there was an associated thrombophilic (prothrombotic) state; 4 had deficiencies of the physiological anticoagulants proteins C and S and antithrombin III and 2 had anti-phospholipid antibody (lupus anticoagulant) syndromes. A further patient had reduced levels of the physiological fibrinolytic agent tissue plasminogen activator (t-PA). In 5 patients other risk factors for small vessel occlusive disease were also present, and 4 had recurrent episodes of ION in the same eye. The visual prognosis in these patients may be improved by anticoagulation with warfarin.

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogs

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

Isolation and characterization of thrombomodulin from human placenta.

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.

Impaired fibrin formation in advanced cirrhosis.

The process of fibrin formation was systematically in 25 patients with severe alcoholic cirrhosis. Results of functional tests are reported. A significant lengthening of the thrombin time was found which could not be completely attributed either to hypofibrinogenaemia or to an increase in physiological anticoagulants or to the presence of pathological antithrombins. A defect in fibrin polymerization was seen in the absence of significant levels of antipolymerizing agents. Indirect evidence pointed to an abnormal fibrinogen function. This was mainly suggested by the "polymerization curves" of mixtures of normal and pathological plasmas and the changes in physico-chemical properties of the clot (optical and elastic properties; tensile strength). Altered synthesis in hepatocytes may lead to an "acquired dysfibrinogenaemia" in the late stages of liver cirrhosis, although alteration of a normal fibrinogen molecule after secretion cannot be definitely excluded.

The physiological anticlotting system after acute blood loss

The state of the physiological anticoagulation system was studied according to B. A. Kudryashov during increased blood coagulation. The tolerance of 50 rabbits to the intravenous injection of thromboplastin was determined after an acute blood loss. As revealed, the state of hypercoagulation after blood loss was not provoked by disturbances in the function of the physiological anticoagulation system.

The function of the physiological anticoagulation system in reticuloendothelial block

Trypane blue block of reticuloendothelium in rats provoked some reduction in the protective function of the physiological anticoagulation system. Mortality rate after intravenous administration of thromboplastin (in moderate doses), obtained from the brain tissue of rats, is considerably great in animals with reticuloendothelial block than that in the controls receiving the same thromboplastin doses. The protective reaction of the organism to the thromboplastin administered in the jugular vein is less pronounced during reticuloendothelial block, the rise of the blood fibrinolytic activity in resonse to the administered thromboplastin being less marked in the experimental animals than in the controls. As compared to controls thrombin time of blood subjected to preliminary incubation with protaminsulfate is reduced in the experimental animals, pointing to a lesser discharge or of heparin-like substances in their blood. The restoration of the normal blood coagulation time in the surviving experimental animals was relatively more rapid than in controls.

The reflex pathways of the anticoagulation system.

Further evidence has been obtained that the physiological anticoagulation system in rats is of a reflex-humoral nature. The reflex arc involves the medulla oblongata.