Blood coagulation profile of the Asian elephant (Elephas maximus)

The coagulation profile of seven Asian elephants was assessed using human reference plasma as a standard. The plasma values for the majority of the coagulation proteins evaluated, including Factors VII, IX, X, and XI, and antithrombin, were similar to that of human plasma. The average Factor VIII:C value was 1.95 units/ml, approximately twice that of the human value. Human recombinant tissue factor was effective as an activator of the tissue factor-factor VII pathway as measured by the prothrombin time assay. The elephant plasma effectively corrected the clotting defect of human Factor XI-deficient plasma but failed to do so with bovine Factor XI-deficient plasma. However, elephant plasma Factor XII was not readily activated by the commercial activated partial thromboplastin time (APTT) reagent formulated with a soluble activator, and consequently the activity of this protein could not be precisely determined. The average (±SD) APTT result of 65.6 ± 9.2 sec was twice as long as that of the human reference plasma. Despite the presence of relatively high levels of fibrinogen, 4.61 ± 0.49 gm/l, no fibrinolytic activity was detected in any of the elephant plasma samples using a standard fibrin plate assay system. © 1996 Wiley-Liss, Inc.     

Binding of hyaluronic acid to mammalian fibrinogens

We have postulated that the interaction of hyaluronic acid (HA), an extracellular matrix glycosaminoglycan, with fibrin is important during the early stages of wound healing and inflammation (J. Theor. Biol. 119:219; 1986), and have demonstrated the specific binding of 125I-labeled HA to human fibrinogen (J. Biol. Chem. 261:12 586; 1986). To determine whether HA binding is limited to human fibrinogen, we tested the ability of fibrinogens from various mammalian species to bind 125I-HA using a dot-blot assay. Increasing amounts of fibrinogen were adsorbed to nitrocellulose, and incubated with 125I-HA in the presence or absence of a 100-fold excess of nonradiolabeled HA to assess specific binding. In three independent experiments, the amount of 125I-HA bound/mg fibrinogen was determined from the slope derived by linear regression analysis of specifically bound 125I-HA versus protein concentration. A Student's t-test was performed to determine whether the slopes were statistically greater than zero. HA binding was considered statistically significant when P less than 0.05 was obtained by this analysis. Rabbit and dog fibrinogens significantly bound HA in all three trials. Baboon fibrinogen demonstrated significant HA binding in two of three trials. Pig, sheep and goat fibrinogens bound HA significantly in only one of three trials, whereas horse, rat and cow fibrinogens did not bind HA significantly at all. We conclude that fibrinogen from mammalian species other than human can specifically bind HA. The ability of fibrinogen to bind HA appears to correlate with an evolutionary divergence that separated human, baboon, dog, rabbit and rat from cow, pig, horse, goat and sheep.     

Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

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.     

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.     

Naturally Occurring Canine Nephrotic Syndrome is a Potentially Hypercoagulable State

Fourteen dogs with naturally occurring nephrotic syndrome were evaluated for abnormalities in the hemostatic system. Histopathologic diagnoses included 8 dogs with membraneous glomerulonephritis, 1 dog with acute glomerulon-ephritis, 2 dogs with idiopathic glomerulopathy, and 2 dogs with amyloidosis. The coagulation protein assays performed included concentrations of factors V, VII, VIII: C, IX, X, fibrinogen (I), anti-thrombin III, and plasminogen. Thrombocyte counts were also performed. All of these analytes were significantly elevated (P < 0.05) with the exception of ATIΠ which was significantly decreased (P < 0.05). Five of the dogs had histologic evidence and 1 dog had angiographic evidence of thrombosis and thromboembolism. Naturally occurring canine nephrotic syndrome thus represents a potentially hypercoagulable state and may serve as a valuable model in the study of certain components of the human disease.     

Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z

We have recently described a unique trisaccharide linked to a serine residue in the first epidermal growth factor-like domains of bovine blood coagulation factors VII (Ser-52) and IX (Ser-53) (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The sugar chain identified in these clotting factors consists of 1 mol of hexose (glucose (Glc] and 2 mol of pentose (xylose (Xyl]. We report here that human factors VII and IX and protein Z and bovine protein Z also contain such carbohydrate moieties linked to a serine residue at the same position found in bovine factors VII and IX. A glycopeptide derived from each of these proteins was subjected to amino acid sequence and component sugar analyses and fast atom bombardment mass spectrometric analysis. The results indicate that the glycopeptide derived from human factor IX contains 1 mol each of Glc and Xyl. The reducing end of this disaccharide was identified as Glc by analyzing the disaccharide generated by hydrazinolysis. In contrast, human factor VII and protein Z yielded two different glycopeptides which contained Glc and Xyl at molar ratios of 1:1 and 1:2, respectively, suggesting microheterogeneity of these O-linked sugar chains. Bovine protein Z glycopeptide contained 1 mol of Glc and 2 mol of Xyl. These sugar compositions were confirmed by analyses of the intact proteins. In relation to the trisaccharide sugar chain previously discovered in bovine factors VII and IX, these findings indicate the existence of a Xyl2-Glc-Ser and a Xyl-Glc-Ser structure in the first epidermal growth factor-like domains of human factors VII and IX and protein Z in addition to that of bovine protein Z. Whether these carbohydrate moieties contribute to the biological activities of these proteins is unknown.     

Acute-phase protein profile during gestation and diestrous: proposal for an early pregnancy test in bitches

Early pregnancy diagnosis in bitches has special relevance for adequate medical assistance to assure normal gestation, diagnosis of abortion or embryonic resorption, undesirable pregnancy interruption and dog owners wishing to assure medical assistance during parturition and to program participation of females in dog shows. The aim of this study was to verify acute-phase protein profile variation as a consequence of generalized inflammatory reaction due to embryonic endometrial invasion and use alterations as a method for early pregnancy diagnosis. Also the relationship between hormonal status and acute-phase proteins concentrations was assessed. Weekly serum samples were collected from 20 non-pregnant (NP) bitches and 20 pregnant females (P) to determine levels of fibrinogen, haptoglobin, ceruloplasmin, seromucoid, glycoprotein, alpha(2) globulin, progesterone and estradiol-17beta. No correlation was found between the implantation sites formed (number of pups born) and the hepatic stimulus for the acute-phase protein production. The conclusions are that acute-phase proteins can be used as an early pregnancy test for bitches from the 3rd week of gestation (14th-21st day post LH peak) for haptoglobin assay (values above 112.42 mg/dl of HbCN binding capacity), from the 4th to the 6th week (21st-42nd day post LH peak) for ceruloplasmin (values above 12.76+/-5.29 U/l), from the 4th week (21st-28th day post LH peak) for glycoprotein (values above 13.67%) and from the 4th week of gestation (21st-28th day post LH peak) for alpha(2) globulin (values above 0.61 g/dl). Fibrinogen and seromucoid increased in the P group from the 5th to the 6th week, respectively, thus not being suitable as parameters for an early pregnancy diagnosis. Relationship between ceruloplasmin and estradiol-17beta and seromucoid and progesterone were verified. For the acute-phase protein test it is important to verify bitches' healthy condition and to assure the precise mating dates to avoid false-positive and -negative results, respectively.     

Detection of soluble fibrin monomer complexes. Comparison of a haemagglutination assay with the ethanol gelation test

Hypercoagulability and disseminated intravascular coagulation (DIC) are characterized by the presence of circulating fibrin monomer complexes in plasma. In 342 patients with possible DIC fibrin monomers, fibrinogen, Reptilase Time, antithrombin III and other coagulation parameters were determined at frequent intervals. Testing of soluble fibrin monomer complexes was performed using a sensitive and reliable hemagglutation assay with red cells sensitized by fibrin monomers (FM-Test) and the ethanol gelation test (EGT). Method comparison regarding the influence of fibrinogen levels and fibrin degradation products shows that high fibrinogen levels lead to false-positive results with EGT. The same effect is observed for fibrin degradation products and EGT whereas no influence of fibrinogen level and fibrin degradation products on the FM-Test occurs. It is well-known that during DIC AT III level decreases caused by proteolytic activity. In this study it could be shown that fibrin monomer increases parallel to the decrease of AT III. The same effect does not occur due to fibrin degradation products.     

Mast cell restricted mouse and human tryptase·heparin complexes hinder thrombin-induced coagulation of plasma and the generation of fibrin by proteolytically destroying fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.     

The onset of mammary extraction of plasma triglycerides and circulating levels of progesterone during lactogenesis in the cow and goat

In untreated cows and goats, the onset of mammary extraction of plasma triglyceride was sometimes detected several hours before parturition, prior to removal of secretion from the mammary glands by offspring and when circulating progesterone concentrations were low. In cows and goats that had secretion regularly removed from the mammary glands late in gestation, the onset of triglyceride extraction occurred up to several days before parturition, when circulating progesterone concentrations were moderately high (up to 3.9 ng/ml in the cow and 6.2 ng/ml in the goat).     

Comprehensive assessment of the hemostasis system in the participants of the Mars-500 experiment

Hemostasis has been studied in the course of long-term (520 days) isolation in hermetic chamber. Measured parameters included activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (ТT); concentrations of fibrinogen (FBG), plasminogen (PG), Willebrand factor (WF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (TPA), and thrombomodulin (ТМ); activities of the coagulation cascade factors II, V, VII, X, VIII, IX, XI, and XII, antithrombin III (ATIII), protein С (PC), С1-inhibitor (С1), α₂-antiplasmin (АP), TPA and TFPI. The investigation revealed a diversity of changes in plasma FBG concentration, slower blood coagulation in the intrinsic pathway and in final stage, and a relative rise in the activities of ATIII and PC-inhibited factors. The remaining parameters exhibited different trends.     

Citrullinated fibrinogen inhibits thrombin-catalysed fibrin polymerization

Citrullination is the post-translational modification of arginine residues by peptidylarginine deiminases (PADIs). Fibrinogen is one substrate of PADIs under physiological conditions. Fibrinogen is an important factor for blood coagulation and inducing inflammation. The citrullinated form of fibrinogen appears in rheumatoid arthritis synovial tissue together with the production of autoantibodies that target self-peptides containing citrulline. However, whether the function of fibrinogen changes after citrullination remains unclear. We found that citrullinated fibrinogen markedly impairs the function of thrombin-catalysed fibrin polymerization and also inhibits fibrin formation. Increased citrullinated fibrinogen might thus affect the balance between coagulation and fibrinolysis and alter antigenicity under physiological conditions. These data suggest that citrullination of proteins could physiologically change functions and subsequently generate pro-inflammatory conditions and autoimmune reactions.     

Ezrin, a membrane-organizing protein, as a polarization marker of the retinal pigment epithelium in vertebrates

Immunoreactivity for ezrin, a membrane-organizing phosphoprotein that tethers actin microfilaments to cell membrane proteins, was evaluated as a polarization marker in the intraocular neuroepithelial cells of vertebrates, especially in the retinal pigment epithelium (RPE). Six fetal human eyes representing the 14th-28th gestational weeks, 9 normal adult eyes, 12 eyes with intraocular tumors, and 26 eyes from 15 other vertebrate species were analyzed by immunohistochemistry using the avidin-biotinylated peroxidase complex (ABC) method and monoclonal antibody (mAb) 3C12 to ezrin. The apical cytoplasm and microvilli of the human RPE always reacted with mAb 3C12, but the basal cytoplasm was labeled in reactive RPE only. In autopsy eyes and if fixation was delayed, ezrin immunoreactivity in RPE was more diffuse. Developing RPE became gradually immunoreactive from the 14th week of gestation onward. The microvilli of the baboon, pig, raccoon dog, cow, and rat RPE cells were likewise labeled, and their basal cytoplasm was variably immunoreactive as well, but the microvilli of the avian RPE did not react with the antibody used. In all six mammals mentioned, both layers of the ciliary epithelium and the anterior iris epithelium reacted for ezrin, and the posterior epithelium was weakly labeled in pig, cow, and rat eyes. Normal peripheral and reactive human retina, and normal baboon, pig, raccoon dog, cow, rat, black grouse, and jay eyes, showed immunoreaction for ezrin in Müller cells, usually in their microvilli. Ezrin is widely found in RPE and anterior segment neuroepithelia of the mammalian eye, in which it may segregate membrane proteins to specific membrane surfaces, especially to the apical microvilli of the RPE, which intimately interact with outer segments of photoreceptor cells. The ezrin gene on human chromosome 6q25-26 is consequently a candidate gene for causing retinal degenerations.     

Fibrinolytic Enzyme System and Pregnancy

The effect of pregnancy on the components of the fibrinolytic enzyme system was determined by serial observations on 10 healthy women during normal pregnancy, labour, and the puerperium. The plasminogen level was substantially increased in the third trimester; the increase occurred pari passu with a pronounced increase in fibrinogen concentration. After allowing for the expansion of plasma volume in pregnancy a twofold increase in the absolute amounts of circulating fibrinogen and plasminogen was found. In late pregnancy and during labour the level of plasminogen activator was greatly decreased, whereas a normal or increased level was present in the first week of the puerperium. No alteration in the levels of inhibitors of plasminogen activation by urokinase was found during normal pregnancy. The thrombin time and platelet count remained unchanged during pregnancy and labour but the platelet count rose significantly during the first week of the puerperium.The haemostatic mechanism in pregnancy appears to be altered towards an enhanced capacity to form fibrin and a diminished ability to lyse fibrin. These changes may be a physiological development to ensure the integrity of the foetal and maternal circulations and provide rapid and effective haemostasis in the uterus during and after placental separation. Nevertheless, the changes may also establish a vulnerable state for intravascular fibrin deposition.     

Factors V and VII anticoagulant activities in the salivary glands of feeding Dermacentor andersoni ticks

The salivary glands of Dermacentor andersoni ticks possess anticoagulant activities that can alter the clotting time of rabbit whole blood. Salivary gland extracts from female ticks inhibit both the intrinsic and extrinsic coagulation systems, and maximal activities against both pathways occur when the ticks attain about 250 mg feeding weight. These anticoagulants are directed against both coagulation factors V and VII, but they do not affect factors II or X. Despite this salivary anticoagulant activity, heavily tick-infested rabbits suffer no visible alteration of their peripheral blood coagulability and have no detectable circulating fibrin degradation products, suggesting that the ticks do not secrete a factor with fibrinolytic activity.     

Coagulation and fibrinolysis in capybara (Hydrochaeris hydrochaeris), a close relative of the guinea-pig (Cavia porcellus)

Fibrinolytic and coagulation properties of capybara (Hydrochaeris hydrochaeris, LINNAEUS, 1766) plasma were analysed and the results compared to the guinea-pig (Cavia porcellus), a close relative. Capybara fibrinogen was isolated and fibrinolysis of its plasma was carried out in a homologous system and with bovine fibrin. Undiluted plasma did not have fibrinolytic activity on fibrin plates; euglobulins gave a dose-related response. Zymography of capybara and guinea-pig plasma gave the same patterns of activity as human or bovine plasma. Human urokinase (UK) and tissue plasminogen activator (t-PA) produced lysis in capybara fibrin plates. Streptokinase (SK) (500 IU/ml) did not activate capybara or guinea-pig plasma. In this system, human plasma was extensively activated. Coagulation tests for both species of rodent were prolonged. The capybara showed values for prothrombin time (PT) shorter than activated thromboplastin time (APTT). The guinea-pig, as already shown, had longer PT values. Factors X and VII were very low for capybara and guinea-pig when tested using reference curves and diagnostic kits for human plasma. It is suggested that the capybara could be a valuable laboratory animal considering its size and closeness to the guinea-pig, and this could allow for the provision of materials from one single animal when convenient or necessary.     

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.     

Cyclic nucleotides, adenylate cyclase, and cyclic AMP phosphodiesterase in mammary glands from pregnant and lactating mice.

In the mammary glands of mice, levels of cyclic AMP increased during pregnancy and then fell precipitously following parturition. In contrast, levels of cyclic GMP fell during the gestation period and then rose rapidly during the early days of lactation. Adenylate cyclase and cyclic AMP hohsphodiesterase activities were elevated during the pregnancy and lactation periods.     

The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing.