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Introduction: Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease., Materials and Methods: We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined., Results: Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function., Conclusions: Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

We have found that synthetic peptides derived from the two epidermal growth factor-like domains of factor VII are inhibitors of tissue factor dependent factor X activation. Inhibition was most pronounced for a constrained sequence of amino acids corresponding to positions 91-102 of factor VII, Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gin-Tyr-Cys. The biological activity appeared to be localized to the tripeptide 'motif', Glu-Gln-Tyr, within the larger sequence. The cyclic peptide was also an inhibitor of tissue factor induced coagulation of plasma, using lipidated tissue factor or tissue factor expressed on the surface of living cells. However, it did not interfere with intrinsic coagulation. Inhibition of factor X activation was dose-dependent with an IC50 value of 350 microM. Kinetic analyses revealed non-competitive inhibition with respect to factor X and suggested that the peptide sequence interferes with the factor VII/tissue factor/factor X complex formation and function. A pentapeptide analog of the putative pharmacophore was also a dose-dependent inhibitor of factor X activation with an IC50 value of 560 microM, but the tripeptide, Glu-Gin-Tyr, alone was without effect. Our results suggest a direct role for the second epidermal growth factor-like domain of factor VII, and in particular its loop I, in the formation and function of the factor VII/tissue factor/factor X complex.

After a bite by the aglyphous red-necked keelback snake Rhabdophis subminiatus a complete defibrinogenation syndrome with severe hemorrhagic diathesis developed in a 25-year-old man. In vitro studies showed that the venom gland extract of the snake contains a very active prothrombin (Factor II) activator. The thrombin generated is inhibited neither by antithrombin III nor the antithrombin-III-heparin complex. The venom gland extract stimulated also the tissue plasminogen activator; however, it did not cause direct activation of plasminogen, protein C, Factor X or direct degradation of fibrinogen.

Tissue factor activity expressed by Hela cells cultured in 96-well plates has been quantitated in situ using a continuous spectrophotometric assay. Following the assay, cells assayed without physical disruption remained as viable as cells not subjected to the assay. Very little (or no) tissue factor was expressed in nondisrupted cells relative to that available in cells disrupted by freeze-thawing and sonication. Total tissue factor activity (that available in disrupted cells) decreased not as a simple function of time after subculturing, but was inversely related to cell density.

The major plasma inhibitor of factor Xa is thought to be anti-thrombin III (At III). However, adsorption of plasma by aluminium hydroxide (A1(OH)3) increases its rate of neutralisation 7-8 fold, and this 'fast-acting' anti-Xa activity has been shown to be independent of At III. Gel filtration of plasma indicated that the anti-Xa activity after A1(OH)3 adsorption was located largely in the high molecular weight (greater than 200,000) fractions, which contain most of the plasma lipoproteins. Purified lipoproteins of very low-density (VLDL), low-density (LDL) and high density (HDL) were prepared by ultracentrifugation and their anti-Xa activities measured before and after adsorption by A1(OH)3. Both LDL and HDL had significant anti-Xa activities by clotting and amidolytic assays. A1(OH)3 adsorption of LDL and HDL gave a marked increase in anti-Xa clotting activity and a decrease in amidolytic activity. Incubation of the adsorbed lipoproteins with phospholipase enzymes destroyed the anti-Xa activity, and prior incubation of Factor Xa with Ca++ and phospholipid protected it against inactivation, indicating that the anti-Xa activity of the adsorbed lipoproteins is mediated via binding of Xa to phospholipid in the lipoproteins. These results indicate that lipoproteins, especially LDL and HDL, are responsible for the increased anti-Xa activity of plasma after A1(OH)3 adsorption. These lipoproteins appear to contain high affinity phospholipid binding sites for Xa which are revealed by A1(OH)3 adsorption.

Inhibition of the procoagulant activity of a tissue factor-Factor VII (TF-FVII) complex by Al(OH)3-adsorbed plasma (AP) was found to require the presence of Factor Xa (FXa). Inhibitory activity seems to be generated through the interaction of FXa with a component in AP rather than with the TF-FVII complex. Quantitation of inhibitor activity was carried out using an amidolytic assay for TF-FVII activity. Incubation of AP with various antisera demonstrated that the inhibition was mainly associated with the presence of apolipoprotein B (apo B) rather than alpha 2-macroglobulin or antithrombin III. Purified lipoprotein-rich fractions prepared from AP, using density gradient ultracentrifugation, all contained some inhibitory activity. Incubation with anti-apo B greatly reduced the inhibitor in the very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-rich fractions but had essentially no effect on inhibition by the high density lipoprotein (HDL) fraction, which was rich in apo A. The inhibitory activity of AP was 60% that of normal plasma and this correlated well with the relative apo A and apo B concentrations. It is proposed that inhibition requires the interaction of FXa with plasma lipoproteins or associated components and that the product of this interaction is then able to bind to and inhibit the TF-FVII complex.

Normal plasma exposed to an insolubilized omega-NH2-hexyl-agarose derivative of PGE1 inhibits ADP-induced platelet aggregation. To define the plasma macromolecule responsible, we tested plasma congenitally deficient in coagulation factors for this property. Only plasma deficient in factor X failed to inhibit platelet aggregation under these conditions. The inhibition could be restored by reconstituting the defective plasma with bovine or human factor X. Purified factor X or Xa exposed to insolubilized PGE1 also inhibited ADP-induced platelet aggregation. Factor Xa could reverse suggesting that factor Xa was the responsible component. This conclusion was further supported by the ability of heparin to reverse the inhibitory effect of altered factor Xa when the anticoagulant was added simultaneously to platelets. The results were not due to the release of PGE1 from the insoluble derivative. Factor Xa may have a heretofore unrecognized effect on modulating platelet functions.

Infusion of dogs with thrombin (0.5 U/kg/min) for 90 minutes significantly increased percent survival following infusion of endotoxin (0.06 mg/kg/min) for 30 minutes. Nine of fourteen dogs infused with thrombin survived seven days (permanent survivors), whereas thirteen of fourteen dogs infused with saline died within 36 hours. Those dogs which survived responded immediately to endotoxin with enhanced anticoagulant and fibrinolytic activity as measured by the Xa one-stage and fibrin degradation product assays, respectively. Those dogs receiving saline instead of thrombin did not respond with anticoagulant or fibrinolytic activity until the end of the study. We concluded that thrombin in the correct amounts protected dogs from endotoxin and that this protection was associated with an early anticoagulant and fibrinolytic response to endotoxin infusion.

An abnormal prothrombin has been detected in a 23 yr-old healthy female and her mother. Both patients appeared to be heterozygous for the abnormality, plasma prothrombin being 50% of normal using the usual one stage assay, but normal when measured either by using Echis carinatus venom or by immunoassay. No abnormality in the immunoelectrophoretic pattern was observed. Prothrombin isolation on DEAE Sephadex failed to separate the abnormal population (prothrombin Clamart) from the normal one. The rates of prothrombin activation by factor Xa, in the presence or absence of phospholipids and/or factor Va, were determined by measuring the production of both clotting and amidolytic activities. The thrombin generation rate from prothrombin isolated from the propositus plasma was 50% slower than normal whatever the method of measurement and the composition of the activation mixture. Analysis of the final activation products by SDS polyacrylamide gel electrophoresis revealed that equal amounts of prethrombin 2 and thrombin had been formed. Prethrombin 2 Clamart was shown to be resistant to proteolysis upon further incubation with factor Xa, whereas it was readily converted to thrombin by Echis carinatus venom. Prothrombin Clamart appears to be characterized by an impairment of Arg 320-IIe cleavage by factor Xa.

To determine the antagonization of anticoagulant and lipolytic effects of a low molecular weight [LMW] heparin preparation protamine chloride was given intravenously after i.v. injection of LMW or normal heparin. The effects of normal heparin on factor Xa, thrombin, aPTT, lipoprotein [LPL] and hepatic triglyceride lipase [HTGL] activities were neutralized immediately by i.v. protamine. The inhibition of thrombin and aPTT by LMW heparin were also abolished, whereas the effects on LPL and HTGL were counteracted to 80% and on factor Xa only to 40% by i.v. protamine chloride. No rebound of the anticoagulant or lipolytic effect was detected. It is assumed that haemorrhagic complication during therapy can be antagonized by protamine chloride. The incomplete inhibitory effect of protamine chloride on LPL, HTGL and factor Xa activities of LMW heparin indicate that protamine chloride requires more than 14 saccharide units in the heparin molecule for interaction.

In this report, we describe the anticoagulant and antithrombotic properties of a peptide (residues 1-44) derived from the amino-terminus of the bovine Factor X light chain by limited proteolysis with chymotrypsin, and subsequently purified by QAE-Sephadex chromatography. The effect of Factor X gla-peptide on the activation of human 3H-Factors IX and X was studied using radiometric assays and purified coagulation factors. Factor VIIa-tissue factor catalyzed activation of Factors IX and X was half-maximally inhibited by Factor X gla-peptide at concentrations of 0.8 microM and 0.2 microM, respectively. Factor IXa-VIII catalyzed Factor X activation was half-maximally inhibited at a gla-peptide concentration of 0.5 microM. In addition, thrombin formation by platelets incubated with Factor Xa and prothrombin could be similarly blocked by gla-peptide. Studies with bovine aortic endothelial cells indicated that the Factor X gla-peptide blocked in parallel Factor X binding and activation on the cell surface. Decarboxylation of the peptide by acid heat treatment destroyed its anticoagulant activity. The in vivo anticoagulant potential of native gla-peptide was demonstrated by a rapid prolongation of the PT and APTT following intravenous infusion into a rabbit. In addition, gla-peptide prevented thrombus formation in response to Factors IXa and Xa, but not thrombin, in a Wessler venous stasis model.