Human C4-binding protein: N-terminal amino acid sequence analysis and limited proteolysis by trypsin

Fres isolated blood cells recombined with normal heparinized plasma and then incubated with endotoxin, induced a 100-fold increase in monocyte tissue thromboplastin synthesis. In contrast, recombination of these cells with heat inactivated plasma, cobra venom factor-treated plasma, Ca²⁺-free plasma, or BioRex 70-treated plasma (plasma free of Clq and D) before incubation with endotoxin, failed to induce monocyte synthesis of tissue thromboplastin. These results strongly support the hypothesis that complement is required for endotoxin stimulation of blood monocyte synthesis of tissue thromboplastin.     

Formation of thrombin and its inactivation by antithrombin III following repeated intravenous injections of tissue thromboplastin in animals

A breach in the inactivation of thrombin activity by antithrombin III following numerous repeated intravenous injections of tissue thromboplastin to albino rats was established. Seven injections of tissue thromboplastin to animals (at 30-40 min-interval) caused functional exhaustion of anticoagulation system and increased thrombin blood circulation level.     

Tissue thromboplastin in the human brain

The studies by the scanning electron microscopy have shown that there are small amounts of membrane-like structures in the dry preparation of tissues thromboplastin obtained from the human brain. In water phospholipids of thromboplastin form hexagonal (H11) cylinders. Protein moiety of the tissue factor essentially influences on the phase state of phospholipids and on the dynamic properties of their fatty acid chains' radicals. The interaction of Ca2+ with the tissue thromboplastin does not change the phase state of phospholipids and at the same time rises rigidity of polar parts of phospholipids and their hydrophobic segments in the internal structures.     

The protein component of human brain thromboplastin

The protein component of human brain tissue thromboplastin (factor III) has been purified by deoxycholate (DOC) extraction, ultracentrifugation, gel filtration and finally repeated preparative polyacrylamide gel electrophoresis (PGE) in the presence of sodium dodecylsulphate (SDS). The final preparations gave one band in analytical PGE. Reduced and alkylated protein appeared as a band of molecular weight about 53 000 in SDS-PGE.The protein had a low solubility in aqueous solutions in the absence of detergents. When recombined with an optimal amount of the phospholipid fraction of tissue thromboplastin (fraction B) the procoagulant thromboplastin activity was regained. Neither alone nor after recombination with phospholipid did the protein catalyze the hydrolysis of aminoacyl-β-naphthylamides or casein.     

Hemostatic system function as affected by thromboplastic agents from higher plants

It has been determined that the thromboplastic agents from the inflorescence of the birch Betula pendula Roth, blossoms of the willow Salix daphnoides Vill., seeds of the pea Pisum sativum L. provoke protective reaction of the animal's anticoagulation system, though weaker expressed than the reaction of thromboplastin from brain. The mechanisms of action of thromboplastic agents of plant origin is similar to the mechanism of action of tissue thromboplastin.     

Modification effect of the protein component of tissue thromboplastin (factor III) on its interaction with factors VII and X

The role of protein moiety of tissue thromboplastin during its specific enzymatic modification by papain was studied. Treatment with papain was followed by a decrease of the number of binding sites for factor X on the surface of factor III. The ability of the remaining sites to bind factor X and to form prothrombinase complexes did not change thereby. The specific interaction of thromboplastin with the factors coupled with the external blood coagulation system are based on asymmetric distribution of phospholipids and apoprotein in the cell membrane.     

The calibration of rabbit tissue thromboplastins: experience of the Dutch Reference Laboratory for anticoagulant control

A number of commercial rabbit tissue thromboplastins used in oral anticoagulant control have been calibrated against the first International Reference Preparation for thromboplastins. This was done in a three-stage procedure by one laboratory, each stage representing a different level of thromboplastin comparability. The calibration model recently recommended by ICTH and ICSH was tested. This model proved to be suitable, although a statistically significant aberration was observed for some of the thromboplastins. The bias introduced by using the model in these non-ideal cases was small compared to the overall variation of the International Normalized Ratio, being the universal scale for reporting the prothrombin time during oral anticoagulant control. Batch-to-batch calibration using lyophilized pooled plasmas could be reliably performed for several commercial thromboplastins.     

Free factor Xa is on the main pathway of thrombin generation in clotting plasma

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).     

Antimicrobial peptide buforin I inhibits tissue factor-initiated coagulation

The enhanced extrinsic blood coagulation following septic shock often manifests cardiovascular complications. The upregulated monocytic tissue factor (mTF) was shown to be a primary contributor to the extrinsic hypercoagulation following acute bacterial endotoxin (LPS) infection. A single-stage clotting assay monitors TF-initiated coagulation. We herein demonstrate a novel anticoagulant activity of antimicrobial peptide Buforin I (BF I) in offsetting LPS-induced mTF hypercoagulation in THP-1 cells, which was confirmed in a cell-free in vitro model, showing that BF I effectively blocked rabbit brain thromboplastin (rbTF) procoagulant activity. Upon inclusion of 25 microM BF I into human plasma, the prolonged prothrombin time (PT) was consistent with the depressed TF-initiated coagulation. In a two-stage chromogenic assay monitoring S-2288 hydrolysis, BF I significantly inhibited not only mTF- but also rbTF-catalyzed FVII activation accompanied by the diminished FVIIa formation. The inhibition by BF I of FVII activation accounted for its novel anticoagulant activity in offsetting mTF-initiated hypercoagulation.     

Mechanism of heparin inactivation by thromboplastin (factor III)

Thromboplastin (a commercial one and that obtained from different tissues) is shown to inactivate heparin in proportion to the quantity of thromboplastin or to the heparin:thromboplastin ratio. A degree of inactivation of heparin changes after the modification of protein component of thromboplastin by proteases, however there is no dependence between the protein amount in the preparation and its antiheparin activity. Inactivation of heparin by thromboplastin is stipulated by the formation of associations due to electrostatic interactions between the clusters of amino acid protein residues (which dissociate under physiological conditions as bases) and heparin sulphogroups. It is suggested that factor III circulating in blood flow participates in the creation of hemostatic potential not only as a result of its ability to catalyze thrombinogenesis, but also due to the decrease of the anticoagulant blood activity.     

Acute systemic changes in blood cells, proteins, coagulation, fibrinolysis, and platelet aggregation after frostbite injury in the rabbit

Acute systemic blood changes were measured in New Zealand white rabbits after severe and mild frostbite injury to the foot. There were observed after 72 hr, in the severely frostbitten rabbits, a decrease in erythrocytes, hematocrit, lymphocytes, and albumin, and an increase in total leukocytes, neutrophils, platelets, fibrinogen, and antithrombin III. Mildly frostbitten rabbits showed similar changes except for no changes in the platelets, albumin, and antithrombin III. In severely frostbitten rabbits, after 72 hr, the changes in the plasma coagulation tests were a prolonged partial thromboplastin time, an accelerated prothrombin time, and increased activities of Factors VII, IX, X, and XI. In mildly frostbitten rabbits there were a prolonged partial thromboplastin time and an increased activity of Factor VII. No changes in fibrinolysis were seen in either group of rabbits. Platelet aggregation, studied only in the severely frostbitten rabbits, showed a change only by an increase in the slope of the collagen-induced platelet aggregation. The blood changes observed in the rabbit model are different than those reported in human frostbite cases. No disseminated intravascular coagulation was apparent in the rabbit model after frostbite injury.     

The correlated actions of vitamins C,K, and Q

Three vitamins (C, K, and Q), two of which are unequivocally established and the existence of the third supported by both experimental and clinical evidence, are needed to prevent hemorrhagic states and therefore can be designated the hemostatic vitamins. The coordinated actions of these vitamins can be epitomized by a diagram. Vitamin K is responsible for the synthesis of four basic clotting factors that function in two distinct pathways for the activation of prothrombin to thrombin. Vitamin Q also functions in these pathways. In the intrinsic, it supplies by means of platelets the Q factor that with factors VIII and IX generates intrinsic (plasma) thromboplastin. In the extrinsic pathway, it is related to tissue thromboplastin which has the Q factor as a part of its structure. It appears to be a phospholipid obtained from exogenous sources. Both vitamins C and K have a potential redox mechanism in their structure which can be hypothecated to function in the synthesis and maintenance of mesenchymal tissue.     

Sphingosine inhibits monocyte tissue factor-initiated coagulation by altering factor VII binding

Tissue factor is a lipoprotein, expressed on the surface of cells, which binds coagulation Factor VII or VIIa, leading to activation of Factors X and IX with subsequent fibrin generation. Cellular tissue factor activity is important in pathophysiologic processes such as inflammation and disseminated intravascular coagulation. In this study, the long-chain base sphingosine inhibited coagulation initiated by lipopolysaccharide-stimulated intact human monocytes. Sphingosine (5-100 microM) also profoundly inhibited thromboplastin-initiated coagulation (greater than 90% decrease in thromboplastin activity). This inhibition was dose- and time-dependent. Sphingosine inhibited neither the intrinsic pathway of coagulation nor thrombin generation of fibrin. The sphingosine analogues sphingomyelin, ceramide, or N-acetylsphingosine did not affect thromboplastin activity, suggesting that the polar head of sphingosine was necessary for interaction of the molecule with the coagulation system. Investigation of the biochemical mechanism revealed that sphingosine (5-50 microM), but neither sphingomyelin nor ceramide, inhibited specific binding of radiolabeled Factor VII to lipopolysaccharide-stimulated intact monocytes. The results suggest that sphingosine may regulate monocyte tissue factor-initiated coagulation by modulating Factor VII binding to tissue factor. Sphingosine may represent a new class of inhibitors of hemostasis.     

Interaction of human thrombin I fragment, prethrombin I, and alpha-thrombin with tissue thromboplastin]

The binding of 125I-labeled prothrombin fragment I. prethrombin I and alpha-thrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 of EDTA was studied. The experimental curves plotted in the Scatchard coordinates testify to the presence in thromboplastin of two types of fragment I binding sites: those with a high (Kd = 7.6 x 10(-6) M) and moderate (Kd = 1.3 x 10(-8) M) binding affinity. The parameters of fragment I binding and their changes reproduced, for the most part, the mode of prothrombin binding observed in previous studies. The experimental results provide indirect evidence in favour of a hydrophobic role of Ca(2+)-dependent binding of prothrombin fragment I to thromboplastin. The binding of prethrombin I was nonspecific and Ca(2+)-independent, whereas alpha-thrombin showed a relatively high level of nonspecific electrostatic binding which was competitively inhibited by Ca2+. Thromboplastin proteins interacted (both directly and in a Ca(2+)-independent fashion) with all the prothrombin derivatives under study.     

Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X

We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.     

A family with heterozygous factor X Friuli defect outside Friuli

Three members of the same family were found to have a clotting defect consistent with the diagnosis of heterozygous factor X Friuli disorder. The main features of the defect were a mild prolongation of prothrombin time and partial thromboplastin time, but a normal Stypven-Cephalin clotting time. Factor X activity was 40-50% of normal using tissue thromboplastin, but was perfectly normal using Russell's viper venom and cephalin. Using chromogenic substrate S-2222 the level was 30% of normal. Immunologically, factor X was normal. Bleeding manifestations were mild if any. The hereditary pattern was autosomal. The family comes from an area far away from Friuli and represents the first example of factor X Friuli discovered outside the Friuli.     

Role of adrenergic structures in regulating the release by the intestines of hemocoagulating compounds into the blood stream

Experiments on cats were made to study the capability of adrenaline, tropaphen and propranolol of influencing the intensity of the release of hemocoagulating compounds and anticoagulants from the intestinal vessels and tissues to the bloodstream (perfusate). Adrenaline was found to increase the coagulative activity of the perfusate, provoking an enhanced release into it of thromboplastin, an analogue of plasma factor V and antiheparin compounds and suppressing the release of antithromboplastins. The blockade of the alpha-adrenoreceptors was accompanied by a dramatic increase of antithromboplastins to the intestinal perfusate, whereas the depression of the activity of beta-adrenergic structures by reduction of the release of tissue thromboplastin inhibitors. It is concluded that regulation of the release of antithromboplastins in the intestine is mediated by the structures similar in their characteristics to alpha- and beta-adrenoreceptors.     

Tissue plasminogen activator inhibitor in patients with systemic lupus erythematosus and thrombosis.

OBJECTIVE--To examine the relations among tissue plasminogen activator antigen, plasminogen activator inhibitor, the lupus anticoagulant, and anticardiolipin antibodies in patients with systemic lupus erythematosus. DESIGN--Prospective study of blood samples (a) from selected patients with systemic lupus erythematosus whose disease was and was not complicated by a history of thrombosis or recurrent abortions, or both, and (b) from a series of healthy controls with a similar age and sex distribution. SETTING--University based medical clinic. SUBJECTS--23 Patients with definite systemic lupus erythematosus (American Rheumatism Association criteria), of whom 11 (eight women) aged 26-51 had a history of thrombosis or recurrent abortions, or both, and 12 (10 women) aged 23-53 had no such history. 15 Healthy subjects (10 women) aged 25-58 served as controls. MAIN OUTCOME MEASURES--Tissue plasminogen activator concentrations, plasminogen activator inhibitor activities, detection of the lupus anticoagulant, and values of anticardiolipin antibodies in the two groups of patients and in the patients with a history of thrombosis or abortions compared with controls. Other measurements included concentrations of proteins that are known to change during the acute phase of systemic lupus erythematosus--namely, fibrinogen, C3 and C4, and C reactive protein. RESULTS--Patients with a history of thrombosis or abortions, or both, had significantly higher values of tissue plasminogen activator and plasminogen activator inhibitor than patients with no such history. A significant correlation between tissue plasminogen activator and plasminogen activator inhibitor (r = 0.80) was found only in the patients with a history of complications of their disease. The lupus anticoagulant was detected in six of the 11 patients with a history of thrombosis or abortions when tested by measuring the activated partial thromboplastin time but was found in all 11 patients when tested by measuring the diluted activated partial thromboplastin time. Nine of these 11 patients had raised values of anticardiolipin antibodies. The findings showed no relation to the activity of the disease. CONCLUSIONS--A significant correlation between tissue plasminogen activator concentrations and plasminogen activator inhibitor activities was found only in patients whose systemic lupus erythematosus was complicated by a history of thrombosis or recurrent abortions. The findings show that these patients have raised plasminogen activator inhibitor activities, and the frequent association between these raised activities and the presence of the lupus anticoagulant suggests that the two may be linked.     

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates <Emphasis Type="Italic">in vitro</Emphasis> blood coagulation similarly to thromboplastin on fibroblasts

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.     

Nuclear textural changes preceding endotoxin mediated enhancement of thromboplastin synthesis in human endothelial cells in vitro

Human vascular endothelium plays a major role in hemostatic processes. Human venous endothelial cells (HEC) may promote coagulation by generation of thromboplastin. This tissue factor production is enhanced by bacterial lipopolysaccharide (LPS). However, the mechanisms of this enhancement remain unclear. In order to quantify by image analysis the nuclear modifications induced by LPS on HEC, umbilical cord vein HEC were cultured in vitro with or without E. coli LPS (10 micrograms/ml) for 0 to 6 h. At the end of culture, tissue factor expression was evaluated by the ability of a cellular extract to shorten the coagulation time of human citrated plasma. Simultaneously, the morphology of LPS treated and control HEC was analysed using a SAMBA 200 cell image processor after Feulgen staining. This analysis indicates that LPS treatment induces nuclear modifications as early as 1 h after culture onset, before any tissue factor expression. This activity appears only between 2 and 4 h of culture with LPS. Our data show that image analysis permits the detection of very early nuclear events in HEC and that these events precede the expression of functional properties which may be implicated in thrombotic processes.