Contact phase of coagulation in diabetes mellitus after aspirin administration

The purpose of our study was to investigate the connection between platelets and the contact phase of coagulation. In 17 patients affected by diabetes mellitus we studied the behaviour of prekallikrein, factor XII and factor XI before and after aspirin administration. To evaluate the activity of aspirin we measured platelet production of thromboxane B2 using a radioimmunoassay. In our diabetic patients a hypercoagulable state was confirmed: PK, factor XII and XI levels were significantly higher as compared with normal controls. After aspirin administration a significant decrease of PK levels was found. After 7 days of aspirin administration and 7 days after stopping aspirin a modest but not significant improvement of factor XII and factor XI was observed. In conclusion, we believe that the contact phase of coagulation is another index of the hypercoagulable state in diabetes mellitus. In addition, the decrease of PK obtained by aspirin administration could support the possible connection between the contact phase of coagulation and platelets.     

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI^-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI^-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI^-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

Normal platelet adhesiveness and aggregation in congenital PTA or Hageman factor deficiency

Platelet adhesiveness and aggregation were studied in two patients with congenital factor XI deficiency and in a patient with congenital factor XII deficiency. A normal aggregation pattern was observed in every instance, regardless of the aggregating agent. The same was true for platelet adhesiveness. It is concluded that factor XI and factor XII play no role in platelet aggregation and adhesiveness.     

Activation of the contact pathway of blood coagulation on the circulating microparticles may explain blood plasma coagulation induced by dilution

Recent studies have shown that the contact activation of blood coagulation can be initiated on the surface of circulating microparticles–particles formed as a result of the activation or apoptosis of blood cells or endothelial cells. In the present work, by means of a mathematical model, we investigated the mechanism of the activation of contact pathway of blood plasma coagulation. The model describes membrane-dependent reactions of the activation of factors XII and XI with account of the presence of blood plasma inhibitors. All reactions were described by ordinary differential equations integrated by an implicit multistep method. The current mathematical model is based on our previous model of factor XII activation on the platelet surface. The initial model is modified by the addition of factor XI, kallikrein, and blood plasma inhibitors. We show that the amidolytic activity of the contact pathway factors associated with the microparticles is proportional to the concentration of microparticles. In previous studies, an increase in the overall solution amidolytic activity after the dilution of plasma was observed. Computational analysis of the contact pathway activation in the diluted plasma shows that the increase in the activation appears from the dilution of blood plasma inhibitors. Thus, a well-known experimental phenomenon of the hypercoagulability of plasma after dilution can be explained by an increased activation of the blood plasma coagulation through the contact pathway on the circulating microparticles. In addition, the computational analysis reveals that a rapid stop of the contact pathway activation on the microparticles observed in the experiments could be explained by the rapid depletion of the free activation surface.     

Contact phase of blood coagulation is not activated in edema of high altitude

To examine whether bradykinin generated by the activation of the contact phase of blood coagulation is involved in the pathogenesis of edema occurring after acute exposure to high altitude, 15 mountaineers were examined at 490 m and 1, 3, and 5 days after arrival at 4,559 m. The clotting activity levels of factor XII, factor XI, plasma prekallikrein, and high-molecular-weight kininogen (HMWK) were measured, and plasma kallikrein-induced proteolytic cleavage of HMWK was assessed by ligand blotting by use of radiolabeled factor XI. After an ascent on foot from 1,170 to 4,559 m in 3 days, three subjects developed high-altitude pulmonary edema, and four subjects presented facial edema. There was no evidence for activation of the contact system in any subject as demonstrated by the lack of proteolytic cleavage of HMWK at high altitude. The absence of contact system activation was further supported by stable plasma levels of the individual factors of contact activation. Therefore, we conclude that bradykinin generated by plasma kallikrein-induced cleavage of HMWK is not involved in the pathogenesis of edema due to acute exposure to high altitude.     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Metabolism and toxicity of synthetic analogues of macrocyclic diester pyrrolizidine alkaloids

Seven macrocyclic diesters analogous to hepatotoxic pyrrolizidine alkaloids have been tested in male weanling Wistar rats. The compounds were the succinate (VII), 2,3-dimethylsuccinate (VIII), phthalate (IX), glutarate (X), 2,4-dimethylglutarate (XI), 3,3-dimethylglutarate (XII) and 3,3-pentamethyleneglutarate (XIII) of the synthetic amino dialcohol, synthanecine A. Single doses of these compounds were given i.p. to rats, and liver levels of pyrrolic metabolites were measured 2 h later. For these experiments both normal rats and rats pretreated with the esterase inhibitor tri-orthocresylphosphate (TOCP) were used. In normal rats, low levels of pyrrolic metabolites were formed from compounds VII, IX, X and XI, but these levels were greatly enhanced in rats with inhibited esterase activity. Much higher pyrrole levels were formed from compounds VIII, XII and XIII in normal rats, and esterase inhibition had relatively little effect on their metabolic conversion to pyrroles. This indicated that the last mentioned compounds were relatively resistant to enzymic hydrolysis, whereas VII, IX, X and XI were easily hydrolysed in normal rats, providing an alternative metabolic path which limited their conversion to pyrrolic metabolites. Comparison of results obtained using the 2,4-dimethylglutarate (XI), the 3,3-dimethylglutarate (XII) and the 3,3,-pentamethyleneglutarate (XIII) showed that 3,3-disubstitution but not 2,4-disubstitution in the glutaric acid moiety conferred high resistance to esterase attack. Toxicity tests using four of the compounds confirmed that acute hepatotoxicity was dose related, and associated with the formation of pyrrolic metabolites in the liver. The 3,3-dimethylglutarate (XII) was highly toxic both in normal and in TOCP treated rats, doses of 25-30 mg/kg causing moderate to severe centrilobular necrosis of the liver. In contrast the toxicity of the unsubstituted succinate (VII), glutarate (X) and 2,4-dimethylglutarate (XI) was very low in normal rats but high in rats with inhibited esterase activity. Thus, the glutarate (X) was non-toxic at 200 mg/kg in normal rats, but in TOCP treated rats, in which pyrrolic metabolite formation was enhanced by a factor of 17.5, a 50 mg/kg dose of this compound was severely hepatotoxic. Kidney damage, which was generally limited to the presence of isolated necrotic cells, sometimes accompanied the liver damage caused by these compounds, but acute toxic effects were not observed in any other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of charged groups in factor XI/XIa activity

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.     

Comparative studies of measuring condition of activated partial thromboplastin time and contact factors in experimental animals

For establishing the optimal incubation time (OIT) for measurement of the activated partial thromboplastin time (APTT) in dogs, rabbits, guinea pigs, rats and mice, we determined the shortest clotting time of the plasma from each animal species and compared them with that of human plasma. The OIT for APTT determination was 15 to 30 sec in guinea pigs, rats and mice and 5 to 10 minutes in dogs and rabbits. The mouse APTT (about 30 sec) with the OIT thus determined was similar to human APTT, and relatively longer than APTT in other animal species (10-20 sec). To elucidate the mechanism of the species differences in OIT, we examined the plasma of each animal species for the activity of the contact factors such as factor XII, factor XI, high molecular weight kininogen (HMWK) and prekallikrein (PK) and their effect on the coagulation of contact factor-deficient plasma. The total activity of contact factors was higher in dogs and guinea pigs and lower in rabbits and mice than that in humans. Species difference with the factor XII, Factor XI and HMWK was noted in clotting time but not in OIT. These results suggest that the species difference in OIT for APTT is probably due to difference in activity of the plasma contact factors and in the mode of coagulation for each contact factor.     

IgE-induced blood coagulation alterations in the rabbit: consumption of coagulation factors XII, XI, and IX in vivo.

Intravenous administration of BSA into 3-month-old rabbits producing detectable anti-BSA antibody only of the IgE class of immunoglobulin induced a variety of intravascular blood coagulation alterations observed in the plasma 15 min after antigen challenge included: a) the intravascular consumption of intrinsic blood coagulation factors XII, XI, and IX and possibly the reduction in clottable fibrinogen; b) a significant prolongation of the activated partial thromboplastin time but not the prothrombin time; and c) the production of an inhibitor affecting the last stage of blood coagulation. The observed blood coagulation alterations were not caused by the manipulative procedures utilized, the presence of anti-BSA, IgG or IgM antibody, histamine-induced alterations in the vascular endothelium or the development of hypotensive shock. It is proposed that specific IgE antibody can induce directly or indirectly the activation of intrinsic blood coagulation in vivo.     

Surface-governed molecular regulation of blood coagulation

Among extracellular biological processes the spatial control of blood clotting is a unique phenomenon. Localization in space has very important consequences in both normal and pathological conditions. Under physiological circumstances a clot is formed only in the vicinity of injury, albeit the prerequisites of coagulation are almost completely given in the whole circulation. The local character of blood clotting is secured by the following major conditions: The regulatory signal initiating coagulation-the damaged vascular wall-is itself a surface on which the majority of clotting reactions take place. The first enzyme, factor XII, of the intrinsic coagulation pathway is activated on the collagen fibers exposed in the damaged vascular wall, although the significance of this reaction in respect of the clotting process is ambiguous. On the membrane of platelets adhered to the damaged blood vessel is activated factor XI, too, which is a well-established participant of the intrinsic clotting process. The further consecutive reactions of coagulation are confined to the surface produced by injury, because the enzymes involved contain gamma-carboxyl-glutamyl side chains which are anchored through calcium bridges to the phospholipids of the platelet membrane. The last enzyme of the sequence is thrombin, which is released from the surface. The reactions taking place on the surface form an enzyme cascade, which amplifies the relatively weak triggering signal by several orders of magnitudes. Amplification is ensured not only by the enzyme-substrate relationship of the consecutive reaction partners, but also by spatial confinement, which endows the process with higher efficacy than could be expected on a statistical basis from reactions in solution. It contributes to the efficiency of enzyme cascade that the non-enzymatic regulatory proteins increase the activity of factors IXa and Xa, and thereby the overall process. While the partner of factor IXa, factor VIII, is captured from plasma, factor V, the partner of factor Xa, is derived from the platelets adhered to the damaged surface and orients the binding of factor Xa. The surface localization ensures the protection of the members of clotting system: In the activator complexes found on the surface, the spatial arrangement of clotting factors prevents the inactivation of factors by physiological inhibitors or by proteolytic enzymes and specific antibodies that appear in the circulation in pathological conditions. Platelet factor 4, derived from platelets, binds heparin and thereby markedly decreases the reactivity of antithrombin III, the physiological inhibitor of clotting factors. The above two circumstances are     

A new type of lectin discovered in a fish, flathead (Platycephalus indicus), suggests an alternative functional role for mammalian plasma kallikrein

A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.     

Studies on the demonstration of a factor VIII or IX deficiency by means of partial thromboplastin time]

A report is presented on the performance of the correction of PTT by means of factor VIII and IX deficiency plasma, which may be used at least one year, when preserved in liquid nitrogen. The method allows reliable, qualitative statements to be made about disturbances in the area of the coagulation factors VIII, IX, XI and XII; the small amount of time required for preparing and carrying out these works representing an essential advantage.     

Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling

Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII [Sun, J., and Kaback, H. R. (1997) Biochemistry 36, 11959-11965]. To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label. The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins. Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction. Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR. Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.     

Genetik und Klinik der seltenen autosomal rezessiven hämorrhagischen Diathesen

A complex network of hemostasis proteins maintains the blood flow and integrity of the vascular system. Molecular biology techniques have led to identification and cloning of the corresponding genes, providing the basis for development of various recombinant clotting factor concentrates. Analysis of these genes allowed for phenotype and genotype correlations in patients with hemorrhagic or thromboembolic disorders and analysis of structure and function relationships of the involved proteins. Excepting coagulation factors VIII and X, deficiencies in factors fibrinogen, II, V, VII, X, XI, and XII (except in dysfibrinogenemia) accompanying a tendency to bleed are inherited, autosomally recessive traits and represent 3–5% of all inherited coagulation factor deficiencies. The prevalences for homozygous forms in the general population vary between 1:500,000 for factor VII deficiency and 1:1 million for factor V deficiency.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.     

Factor XI deficiency: implications for management of patients undergoing aesthetic surgery.

We report our experience in patients with an abnormal partial thromboplastin time elevation due to factor XI deficiency (Rosenthal syndrome) who presented for aesthetic surgery consideration. Preoperative evaluation included a thorough history, physical examination, coagulation profile, and hematological consultation. Nine of 10 patients underwent 12 elective aesthetic procedures without undue intraoperative or postoperative bleeding. Based on these findings, we stratified patients as low risk or high risk. Low-risk patients were those with greater than 15 percent factor XI levels, or those with 5 to 14 percent factor XI levels but a history of multiple major surgical procedures without bleeding complications. High-risk patients were those with factor XI levels less than 15 percent, history of bleeding either spontaneously or with surgery, and a family history of bleeding diathesis from factor XI deficiency. Low-risk patients had fresh frozen plasma available for the procedure, whereas high-risk patients received fresh frozen plasma 2 hours before surgery. We conclude that (1) in these patients with abnormally high partial thromboplastin time values and no prior known bleeding disorder, we have identified factor XI deficiency as the prevalent coagulopathy; (2) partial thromboplastin time does not necessarily correlate with factor XI levels; (3) patients can be classified as high or low risk for elective surgery based on factor XI levels and prior surgical or family history; (4) recommendations for perioperative management can be made based on this risk profile; and (5) aesthetic surgery can be performed successfully and safely on patients with factor XI deficiency on a case-by-case basis when appropriate guidelines are enforced.     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.