Deficient Human Plasma Research Articles

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Purification and characterization of a thrombin-like enzyme isolated from Vipera lebetina venom: its interaction with platelet receptor.

: Snake venoms contain various molecules that can be used as tools in the diagnosis and in the treatment of hemostatic disorders. This study reports the isolation and functional characterization of a new thrombin-like enzyme and its role in the modulation of platelet aggregation and coagulation. The molecule was purified by gel filtration, anion exchange chromatography and reverse-phase-HPLC on C8 column; its molecular weight was determined. Natural and synthetic substrates were used to evaluate its enzymatic activities. The fibrinogenolytic activity was tested electrophoretically and by reverse-phase-HPLC on C18 column. Otherwise, the effect on blood coagulation and deficient plasma factors were also evaluated. The mechanism by which a thrombin-like enzyme VLCV (thrombin-like enzyme)-induced platelet aggregation was explored in presence of ticlopidin, clopidogrel and aspirin. VLCV (45 kDa) isolated from Vipera lebetina as a thrombin-like enzyme seems to be able to modulate platelet function. This enzyme showed an amidolytic activity by hydrolyzing the chromogenic-specific substrate of thrombin and the α-chain of fibrinogen. It is also able to clot human plasma and the deficient human plasma in factor X, suggesting that it is involved in the intrinsic and common pathways. The aggregating effect of VLCV is more sensitive to ticlopidine than to the clopidogrel suggesting the involvement of ADP/P2Y12/PI3K pathway. VLCV seems to be able to promote human platelet aggregation suggesting an interaction between P2Y12 and PAR1. Due to its ability to replace the missing factor X and its proaggregating activity, VLCV could be used as molecular tool to better understand the hemostasis mechanism.

The In Vitro Effects of Pentamidine Isethionate on Coagulation and Fibrinolysis.

Pentamidine is bis-oxybenzamidine-based antiprotozoal drug. The parenteral use of pentamidine appears to affect the processes of blood coagulation and/or fibrinolysis resulting in rare but potentially life-threatening blood clot formation. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. To investigate the potential underlying molecular mechanism(s) of pentamidine’s effects on coagulation and fibrinolysis, we studied its effects on clotting times in normal and deficient human plasmas. Using normal plasma, pentamidine isethionate doubled the activated partial thromboplastin time at 27.5 µM, doubled the prothrombin time at 45.7 µM, and weakly doubled the thrombin time at 158.17 µM. Using plasmas deficient of factors VIIa, IXa, XIa, or XIIa, the concentrations to double the activated partial thromboplastin time were similar to that obtained using normal plasma. Pentamidine also inhibited plasmin-mediated clot lysis with half-maximal inhibitory concentration (IC50) value of ~3.6 μM. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4 µM and 8.4 µM, respectively. Interestingly, it did not significantly inhibit thrombin, factor XIa, factor XIIIa, neutrophil elastase, or chymotrypsin at the highest concentrations tested. Michaelis-Menten kinetics and molecular modeling studies revealed that pentamidine inhibits factor Xa and plasmin in a competitive fashion. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on coagulation and fibrinolysis via the disruption of the proteolytic activity of factor Xa and plasmin.

Polyguluronate sulfate, polymannuronate sulfate, and their oligosaccharides have antithrombin III- and heparin cofactor II-independent anticoagulant activity

Cardiovascular disease is the leading causes of death. However, the complications can be treated with heparin and heparinoids, such as heparin pentasaccharide Fondaparinux, dermatan sulfate, and PSS made from alginate extracted from brown seaweeds by chemical sulfation. Alginate is composed of a linear backbone of polymannuronate (PM), polyguluronate (PG), and alternate residues of mannuronic acid and guluronic acid. It is unknown if heparin and sulfated PG (PGS)/PM (PMS) have the same or different anticoagulant molecular targets. In the current study, the anticoagulant activities of PGS, PMS, and their oligosaccharides were directly compared to that of heparin, Fondaparinux, and dermatan sulfate by the activated partial thrombinplastin time (aPTT) assay using normal, antithrombin III (ATIII)-deficient, heparin co-factor II (HCII)-deficient, and ATIII- and HCII-double deficient human plasmas. Our results showed that PGS, PMS, and their oligosaccharides had better anticoagulant activity than that of Fondaparinux in all four human plasmas tested. As expected, heparin was the best anticoagulant in normal plasma. Moreover, PGS, PGS6, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than dermatan sulfate in HCII-deficient plasma. Most strikingly, PGS, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than that of heparin in ATIII- and HCII-double deficient human plasma. The results revealed for the first time that sulfated alginate had ATIII- and HCII-independent anticoagulant activities. Therefore, developing PGS and PMS-based anticoagulants might require to discover their major molecular targets and to develop target-specific anticoagulant assays.

The interaction between fibrinogen and zymogen FXIII-A2B2 is mediated by fibrinogen residues γ390-396 and the FXIII-B subunits

AbstractCoagulation transglutaminase factor XIII (FXIII) exists in circulation as heterotetrameric proenzyme FXIII-A2B2. Effectively all FXIII-A2B2 circulates bound to fibrinogen, and excess FXIII-B2 circulates in plasma. The motifs that mediate interaction of FXIII-A2B2 with fibrinogen have been elusive. We recently detected reduced binding of FXIII-A2B2 to murine fibrinogen that has γ-chain residues 390-396 mutated to alanines (Fibγ390-396A). Here, we evaluated binding features using human components, including recombinant fibrinogen variants, FXIII-A2B2, and isolated FXIII-A2 and -B2 homodimers. FXIII-A2B2 coprecipitated with wild-type (γA/γA), alternatively-spliced (γ′/γ′), and αC-truncated (Aα251) fibrinogens, whereas coprecipitation with human Fibγ390-396A was reduced by 75% (P < .0001). Surface plasmon resonance showed γA/γA, γ′/γ′, and Aα251 fibrinogens bound FXIII-A2B2 with high affinity (nanomolar); however, Fibγ390-396A did not bind FXIII-A2B2. These data indicate fibrinogen residues γ390-396 comprise the major binding motif for FXIII-A2B2. Compared with γA/γA clots, FXIII-A2B2 activation peptide release was 2.7-fold slower in Fibγ390-396A clots (P < .02). Conversely, activation of recombinant FXIII-A2 (lacking FXIII-B2) was similar in γA/γA and Fibγ390-396A clots, suggesting fibrinogen residues γ390-396 accelerate FXIII-A2B2 activation in a FXIII-B2–dependent mechanism. Recombinant FXIII-B2 bound γA/γA, γ′/γ′, and Aα251 with similar affinities as FXIII-A2B2, but did not bind or coprecipitate with Fibγ390-396A. FXIII-B2 also coprecipitated with fibrinogen from FXIII-A–deficient mouse and human plasmas. Collectively, these data indicate that FXIII-A2B2 binds fibrinogen residues γ390-396 via the B subunits, and that excess plasma FXIII-B2 is not free, but rather circulates bound to fibrinogen. These findings provide insight into assembly of the fibrinogen/FXIII-A2B2 complex in both physiologic and therapeutic situations.

Isolated biomolecules of pharmacological interest in hemostasis from Cerastes cerastes venom

Biomolecules from Cerastes cerastes venom have been purified and characterized. Two phospholipases isolated from Cerastes cerastes venom share 51% of homology. CC2-PLA2 exhibits antiplatelet activity that blocks coagulation. CCSV-MPase, a non-hemorrhagic Zn2+-metalloproteinase, significantly reduced the plasmatic fibrinogen level and hydrolyzes only its Bβ chain. Serine proteinases such as RP34, afaâcytin and CC3-SPase hydrolyze the fibrinogen and are respectively α, αβ and αβ fibrinogenases. In deficient human plasma, afaâcytin replaces the missing factors VIII and IX, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. RP34 proteinase also had no effect on both human and rabbit blood platelet aggregation. CC3-SPase revealed a pro-coagulant activity. However, the insolubility of the obtained clot indicates that CC3-SPase does not activate factor XIII. In addition, CC3-SPase clotting activity was carried out with human plasmas from volunteer patients deficient in clotting factors. Results showed that CC3-SPase shortens clotting time of plasma deficient in factors II and VII but with weaker clotting than normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma; suggesting that CC3-SPase is able to replace both factors IIa and VII in the coagulation cascade and thus could be involved in the blood clotting process via an extrinsic pathway. These results imply that CC3-SPase and afaâcytin could repair hemostatic abnormalities and may replace some factors missing in pathological deficiency. Afaâcytin also exhibits α fibrinase property similar to a plasmin-like proteinase. Despite its thrombin-like characteristics, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin might have potential clinical applications.

DNA Aptamers for Human Thrombin with High Anticoagulant Activity Demonstrate Target- and Species-Specificity

G-quadruplex-based DNA aptamers for human thrombin represent promising pharmaceuticals having high anticoagulant activity, rapid clearance from the bloodstream, and availability of DNA antidote. High specificity of aptamers minimizes side-effects but at the same time leads to difficulties when choosing animal model for preclinical trials. Well described minimal G-quadruplex 15-TBA, elongated with hinge and duplex regions 31-TBA, and developed recently modular aptamer RA-36 were investigated thoroughly and compared in this paper. To estimate the species-specificity of aptamers the inhibition constants and types were determined for human fibrinogen hydrolysis with human, bovine, porcine, rabbit, rat, and mouse thrombins using turbidimetric assay. Coagulation tests on human fibrinogen, factor II deficient human plasma and animal plasmas were conducted to verify turbidimetric data and reveal either the concentration effects or the influence of other plasma components. And finally, a set of tests on blood clotting cascade was performed to investigate target-specificity of RA-36 aptamer.

The Hag-protease-II is a fibrin(ogen)ase from Hippasa agelenoides spider venom gland extract: Purification, characterization and its role in hemostasis

The current study describes the biochemical, biophysical and pharmacological properties of Hag-protease-II from Hippasa agelenoides spider venom gland extract. The Hag-protease-II was purified to homogeneity using gel filtration and ion-exchange chromatography. The molecular mass was found to be 28.749 kDa by MALDI-TOF mass spectrometry. PMSF abolished the activity while EDTA, EGTA, IAA and 1, 10-phenanthrolene did not. Hag-protease-II hydrolyzed casein, fibrinogen and fibrin, however it did not hydrolyze gelatin, fibronectin and collagen types- I and IV. It was non-lethal and devoid of hemorrhagic, myotoxic and edema forming activities. It dose dependently reduced re-calcification time of citrated human plasma. Strikingly; the Hag-protease-II coagulated the factor X deficient congenital human plasma. It hydrolyzed Bβ-chain but, did not degrade Aα- and γ-chains of fibrinogen while, it hydrolyzed α-polymer and α-chain but not the β-chain and γ–γ dimers of partially cross-linked fibrin clot. The Hag-protease-II induced aggregation of human platelets in PRP dose dependently, however it did not interfere in collagen induced aggregation of PRP and washed human platelets.

Purification and characterization of ‘Trimarin’ a hemorrhagic metalloprotease with factor Xa-like Activity, from Trimeresurus malabaricus snake venom

In the present study, we describe the purification and characterization of a metalloprotease ‘trimarin’ from Trimeresurus malabaricus snake venom. Trimarin is a single-chain basic protein, with a molecular mass of 29.6 kDa. Trimarin showed proteolytic activity towards casein and fibrinogen, which was irreversibly inhibited by EDTA and 1,10-phenanthroline. The metal ion associated with trimarin was found to be Zn 2+. Trimarin exhibited pharmacological activities including hemorrhage, myotoxicity, procoagulant and factor Xa-like activities. The hemorrhage and myotoxicity correlated with degradation of extracellular protein components type-IV collagen and fibronectin. Myotoxicity due to muscle tissue necrosis was substantiated with increased serum CK activity. Trimarin showed procoagulant activity with reduced re-calcification time of citrated human plasma. Trimarin shortened the activated partial thromboplastin time (aPTT) and prothrombin time (PT), suggesting its involvement in common pathway of blood coagulation. Trimarin coagulated the citrated human plasma in the absence of CaCl 2, but it was lacking thrombin like activity as it did not clot the purified fibrinogen. Remarkably, the enzyme clotted the factor X deficient human plasma, suggesting that trimarin has factor Xa-like activity. Thus, trimarin may play a key role in the pathophysiological conditions that occur during T. malabaricus envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130-138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl(2) but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl(2) and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aalpha chain and then the Bbeta-chain, but not the gamma-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (alpha-polymer, alpha-chain, beta-chain, and gamma-gamma dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.

Differential Effects of TAK-442, a Factor Xa Inhibitor, and Ximelagatran, a Thrombin Inhibitor, on Bleeding: Possible Role of Differences in Effects on Factor V-Mediated Feedback on Blood Coagulation Cascade

Thrombin generation serves to amplify the coagulation cascade via positive feedback activation of factor V (FV) and factor VIII. We hypothesized that factor Xa (FXa) inhibitors, unlike thrombin inhibitors, would not block the feedback activation of the coagulation cascade but would have a favorable anticoagulating profile—sufficient to prevent thrombus formation, yet not interfere with hemostatic plug formation. TAK-442 is a newly synthesized, selective FXa inhibitor that strongly inhibits FXa (with a Ki value of 1.8 nM), and displays more than 440x selectivity toward FXa than other serine proteases. In the present study, we compared the effects of TAK-442 versus ximelagatran on FV-mediated positive feedback in vitro, and on their antithrombotic and hemorrhagic effects in a rat model of venous thrombosis.In vitro, TAK-442 gradually inhibited thrombin generation and prolonged prothrombin time (PT) in a dose-dependent manner, while melagatran, an active form of ximelagatran, exhibited a steeper effect at higher doses tested. The PT prolonging potency was increased in FV–deficient human plasma, with CT2 values (the concentration that causes 2 times prolongation of clotting times) of 120 nM for TAK-442 and 32 nM for melagatran, compared with 500 nM and 360 nM for TAK-442 and melagatran, respectively in normal plasma. In the rat model of venous thrombosis, TAK-442 (10 mg/kg, po) prevented thrombus formation by 55% and prolonged PT by 1.3 times of control values; a similar effect was observed in ximelagatran-treated (3 mg/kg, po) animals, with 59% inhibition of thrombus formation and 1.2 times prolongation of PT. TAK-442 at 100 mg/kg, prolonged PT by 2.1 times, with no significant change in bleeding time (BT); in contrast, increasing the dose of ximelagatran to 10 mg/kg, po prolonged PT by 3.9 times and significantly (P<0.025) increased BT.Our data suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Increased Factor VIII Levels Have a Thrombogenic Effect In Vitro and In Vivo.

The procoagulant co-factor, factor VIII (FVIII), plays a crucial role in the intrinsic blood coagulation cascade. Epidemiologic studies have established a causal association between elevated FVIII levels and venous thrombosis incidence, whereas no such association has been confirmed with arterial thrombosis. Importantly, no objective in vitro or in vivo examinations of this association have previously been performed to establish a mechanistic role for elevated FVIII levels and thrombogenicity. To establish the in vitro thrombogenic effect of elevated FVIII activity, platelet-poor C57Bl/6 hemophilia A mouse plasma was spiked with recombinant human FVIII (r-huFVIII) to FVIII levels 100% and 250% that of normal plasma. Physiological concentrations of tissue factor and calcium chloride were added to the plasma to stimulate the formation of thrombin-antithrombin (TAT) complexes. Over 25 minutes, at 5-minute intervals, the plasma was sub-sampled, and the TAT complex concentration measured using an ELISA. The results showed that as FVIII concentration was increased, the rate of formation of TAT complexes was increased and the maximum concentration of complex was increased. As a second in vitro test of thrombogenicity, FVIII deficient human plasma was spiked with r-huFVIII to FVIII levels similar to those in the TAT experiments. Thromboelastography (TEG) was performed on the samples following the addition of physiological concentrations of tissue factor and calcium chloride. As FVIII levels were increased, the time to initial fibrin formation decreased significantly, in a negative logarithmic manner. As well, the speed with which the clot formed increased significantly, in a logarithmic manner. Although the clot strength (MA) of the FVIII spiked samples differed significantly to the 0% FVIII samples, there was no significant change in MA as FVIII levels were elevated to levels greater than 100%, likely as a result of the platelet poor nature of the samples. To examine the effect of increasing FVIII levels in vivo, fluorescence intravital microscopy was used to visualize the arterioles of the cremaster muscle. Circulating platelets were labeled in vivo with rhodamine 6G (200ng/mL). C57Bl/6 normal mice (n=5), C57Bl/6 hemophillia A mice (n=5), and C57Bl/6 mice hemophilia A mice whose FVIII levels were elevated to 200% (n=3) through an intravenous infusion of r-huFVIII were examined. Arterioles were injured for 3 minutes with 10% ferric chloride soaked filter paper and observed for 40 minutes. None of the hemophilic mice arterioles occluded in the observation period, whereas the normal mice occluded at 25 minutes and the mice with 200% FVIII activity occluded at 20 minutes. Fluorescence analysis revealed that the normal mice and those with 200% FVIII activity had stable platelet accumulation, as there was little variation in the fluorescence intensity over time, but a gradual, and persistent, increase in overall intensity. In contrast, there was strong variation in the accumulation of platelets within the injured arterioles of the hemophilic mice and no persistence in the intensity of fluorescence throughout the observation period. Together, these in vitro and in vivo data indicate that elevated FVIII levels produce a thrombogenic effect that increases with FVIII elevations. However, it is necessary to further examine this relationship to determine whether the thrombogenicity of FVIII is proportional to the FVIII increase and whether the thrombogenicity is affected by the duration of FVIII elevation.

Neuropeptide Y (NPY) cleaving enzymes: Structural and functional homologues of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.

Particulate Matter Induces Tissue Factor and Attenuates Thrombomodulin in Rat Lungs.

Exposure to particulate matter (PM) is an environmental determinant of chronic lung- and cardiovascular disease (CVD). Gene array expression analysis in rats exposed to PM showed a marked increase in tissue factor in the lung (Kooter et al, Inhal Toxicol. 2005; 17(1):53–65). We hypothesised that pro-inflammatory effects of PM would affect the cardiovascular system, in part through a prothrombotic action. To functionally characterize such effects we determined tissue factor (TF) activity and effects on thrombin generation in tissues from animals challenged with PM. Spontaneous hypertensive rats (SHR) rats were exposed via intratracheal instillation to different concentrations of PM collected from a highway tunnel (road tunnel dust: 0.3–1–3–10 mg PM/kg bodyweight (BW)) for 4 and 48 hours. For comparison we also tested organ- homogenates of mice that received 2 mg/kg endotoxin i.p. for 6 hours, as a well characterized inflammation driven prothrombotic animal model. Tissue factor activities show an increase with increasing concentrations of road tunnel dust. For 10mg/kg BW there is a significant difference both after 4 and 48 hours of exposure compared to controls (1044.5 ± 471.6, 1020.8 ± 61.6 vs. 397 ± 231.1 pM, p<0.02 ). For comparison, tissue factor activity in lungs of endotoxemic mice (25.8 ± 5.28 pM) was significantly higher than in lungs of control mice (12.7 ± 1.73 pM, p<0.05), whereas the activity in other organs (brain, heart, spleen, liver, kidney) was comparable between both groups. TF in the lung is known to increase under inflammatory conditions. We adapted the Calibrated Automated Thrombogram (CAT) that measures the thrombin generating potential in human platelet poor plasma (PPP) so that we were able to add tissue homogenates and measure the effect on thrombin generation. There is an 8-fold reduction in the endogenous thrombin potential (ETP) upon addition of lung homogenate from untreated rats and mice compared to the ETP of pooled PPP from 80 healthy volunteers (174.5 ± 61 vs. 1436.6 ± 112 nM min). We established that this inhibition is primarily due to the presence of thrombomodulin in the lungs, becausethe inhibitory effect was absent in protein C deficient human plasma andlungs from TM pro/pro mice that have a 100-fold reduction in thrombomodulin cofactor activity show a complete recovery of the ETP (1685.5 ± 208.8 nM min).With increasing concentrations of road tunnel dust the ETP increases with significant differences at 10mg/kg BW after 4 hours (389.5 ± 147.7 vs. 174.5 ± 61 for saline, p<0.05). After 48 hours the ETP augments further for the highest concentrations (1440.7 ± 288.8 nM min vs. saline: 163.9 ± 64 nM min, p<0.0001). This indicates a concentration- and time dependent reduction in thrombomodulin activity after PM exposure. Endotoxin challenge caused an increase in ETP (940.8 ± 523.1 vs. 194.3 ± 158.7nM min) for lung homogenates, whereas no changes were observed for brain (1794.8 ± 49.6 vs. 1801.8 ± 42.8 nM min) and heart (784.6±90 vs. 873.8 ± 74.3 nM min) compared to control tissues. The combination of these measurements indicates that the procoagulant state in the lungs after exposure to PM is related to inflammation and can be (partially) explained by the ratio of tissue factor and thrombomodulin activities in these organs.

Studies of surface activated coagulation: antisera binding onto methyl gradients on silicon incubated in human plasma in vitro

Human plasma proteins factor XII, high molecular weight kininogen, prekallikrein, factor XI and fibrinogen, participate in surface-initiated coagulation. Antisera binding to methyl gradients made on hydrophilic silicon was studied after immersion in normal and deficient human blood plasma. Scanning ellipsometry was used to quantify the adsorbed organic material. The hydrophilic part of the gradient deposited anti-factor XII and anti-high molecular weight kininogen, but low amounts of anti-fibrinogen. Increased amounts of anti-fibrinogen bound onto the hydrophobic part, and the intermediate gradient region with mixed polar-nonpolar surface characteristics bound low amounts of anti-factor XII, anti-high molecular weight kininogen and anti-fibrinogen. Tentatively, in this gradient region, simultaneous polar and non-polar surface characteristics result in a low-level of surface-activated coagulation. Surfaces immersed in heparinized-and EDTA-plasma indicate different antisera depositions.

Influence of recombinant hirudin and unfractionated heparin on thrombin and factor Xa generation in extrinsic and intrinsic activated systems

Using biochemically defined conditions on a fast kinetic centrifugal analyzer the effect of recombinant hirudin (rH) and unfractionated heparin (UH) on thrombin and factor Xa generation was investigated. Diluted fibrinogen deficient human plasma was incubated with increasing concentrations of the anticoagulants and protease generation was initiated either by extrinsic (EA;thromboplastin/calcium chloride) or intrinsic (IA;ellagic acid/cephaloplastin/calcium chloride) activation of the coagulation process. Generation of thrombin or factor Xa was measured continuously by amidolytic assay using the specific chromogenic substrates Spectrozyme™ and Spectrozyme™ FXa. By means of calibration curves for thrombin and factor Xa the IC 50 values for the inhibition of the proteases were calculated. It was found that rH and UH were nearly equally effective in inhibiting both the thrombin and factor Xa formation after IA, whereas in EA system rH produced a stronger inhibition on thrombin generation than UH, which in general showed a more pronounced effect after intrinsic than after extrinsic activation. The results suggest that, with regards to thrombin and factor Xa generation, rH does not exhibit a much higher activity than UH. This may be an expression that thrombin-mediated positive feedback-reactions are not influenced by rH as strongly as expected when using a highly specific and selective thrombin inhibitor. Furthermore, it can be concluded that protease generation assays may be useful in the characterization of anticoagulants/antithrombotics.

The Production of a Factor X Activator by a Methylcholanthrene-Induced Rat Fibrosarcoma

The association between neoplasia and thrombosis has been well documented. We have studied the production of a procoagulant which is a factor X activator in a rat fibrosarcoma model. Extracts of excised tumor were assayed in a one stage clotting assay using normal and factor deficient human plasmas. The activity was not due to tissue factor, as acceleration of clotting was observed in FVII deficient plasma. No activity was noted in FX deficient plasma. The activator was capable of cleaving 125I-FX in the absence or presence of calcium. A radioimmunoassay (RIA) demonstrated a 5,100-fold increase in the levels of FX activation peptide after exposure to sarcoma extract. The FX activation occurs in the absence of calcium although the effect is greatly accelerated in the presence of 5 mM CaCl2. Using a two-stage amidolytic assay, functional activation of FX by the sarcoma was demonstrated. Inhibitor studies suggest that the sarcoma-derived procoagulant is a serine protease. The methylcholanthrene-induced rat sarcoma may serve as a useful model for investigating the regulation and effects of cancer procoagulants.

Enhanced procoagulatory activity (PCA) of human monocytes/macrophages after in vitro stimulation with chemically modified LDL

In cultured human monocytes/macrophages, surface expression of procoagulatory activity (PCA) was induced by chemically modified LDL (acetyl-LDL and MDA-LDL) in a dose- and time-dependent manner. Maximum PCA (30-fold increase) was detected after 24 h of culture with modified LDL at doses of 25–750 μ protein/ml. Using factor VII deficient human plasma and phospholipase C this PCA was identified as tissue thromboplastin activity (factor III). These results suggest a further atherogenic potential for modified LDL through stimulation of the conversion of fibrinogen to fibrin in the atheromatous lesion.

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

An alternative extrinsic pathway of human blood coagulation

AbstractTo study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.