Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties

Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with kcat = 0.55 s-1, K(m) = 0.54 microM, and Kd = 6.0 nM for rTAFI and kcat = 0.61 s-1, K(m) = 0.55 microM, and Kd = 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37 degrees C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to approximately 62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (delta Ho++ approximately equal to 45 kcal/mol and delta So++ approximately equal to 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and epsilon-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.     

Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.     

Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.     

Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.     

A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.     

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.     

Importance of the Arg-Gly-Asp triplet in human thrombin for maintenance of structure and function

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.     

Application of NMR spectroscopy in biochemical studies of tumor cells sensitive and resistant to anticancer drugs

The anticoagulant activities of human urinary soluble thrombomodulin (UTM) in blood taken from various species using several anticoagulant assay systems were compared; it was examined which coagulant assay system is appropriate for evaluation of the antithrombotic effects of UTM and how the species specificity of UTM is involved in the mechanisms of action of UTM. When anticoagulant activities were compared using activated partial thromboplastin time (APTT), thromboelastography (TEG), and thrombin generation test (TGT), the effect of UTM was found to be the strongest in humans among various species tested. Among the anticoagulant assays tested, TGT reflecting protein C (PC) activation by UTM, appeared to be more sensitive than APTT and TEG in detection of thrombomodulin activity. In the study of the mechanisms of action of UTM, UTM exhibited nearly the same antithrombin activity against human and rat thrombin; the rate of activation of human PC by thrombin/UTM complex was much higher than that of rat PC. Therefore, the species specificity of the anticoagulant activity of UTM may be attributable to thrombin/UTM-PC interaction, but not to UTM-thrombin interaction. From these results, we concluded that TGT reflecting PC activation by UTM will be a more useful assay than APTT and TEG for estimating the antithrombotic effects of UTM in humans. Furthermore, our findings suggest that UTM will exhibit more potent antithrombotic effects in humans than those in rats by strongly enhancing thrombin-catalyzed PC activation.     

Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats

Adult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dansyl Glu Gly Arg chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase. Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.     

Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C

Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.     

Rapid activation of protein C by factor Xa and thrombin in the presence of polyanionic compounds

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (approximately 50 to 100 micromol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations ( approximately 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.     

Evidence that the age at diagnosis of IDDM is genetically determined

Recent studies have suggested that clot-bound thrombin plays an important role in thrombus growth. In this study, we examined the effects of recombinant human soluble thrombomodulin (rhsTM) on clot-induced coagulation. rhsTM enhanced the activation of protein C by clots, and attenuated clot-induced thrombin generation and fibrinopeptide A (FPA) production in a dose-dependent manner. The inhibitory effect of rhsTM was abolished by anti-protein C antibody. The inhibitory effect of rhsTM on clot-induced thrombin generation continued for over 60 min after the addition of the clot, while an active site-directed thrombin inhibitor, argatroban, produced a more transient inhibition. rhsTM also inhibited the regrowth of the clot in (125)I-fibrinogen-supplemented plasma. We also examined the effect of rhsTM by thromboelastography, rhsTM reduced the growth of the clot but had little effect on the time to begin clotting, while heparin and Fragmin (low molecular weight heparin) had effects opposite to those of rhsTM. These findings suggest that rhs-TM attenuates the growth of the clot by activating protein C and inhibiting further thrombin generation in the clot.     

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.     

Coagulation and thrombomodulin in response to exercise of different type and duration

PURPOSE: The present study was conducted to evaluate the role of the duration of exercise and the impact of the exercise type for exercise-induced activation of coagulation. METHODS: Eleven male triathletes were subjected to stepwise maximal (17 min) and 1-h maximal exercise in swimming, cycling, and running. Changes of hemostatic variable sand of plasma thrombomodulin, a marker of endothelial cell activation, were monitored. RESULTS: Irrespective of the type of exercise, alterations in markers of thrombin (prothrombin fragment 1 + 2, thrombin-antithrombin III complexes) and fibrin formation (fibrinopeptide A) were more pronounced after 1-h exercise than after stepwise maximal exercise. Hemostatic parameters rose to the highest levels after running resulting in substantial fibrin formation as indicated by fibrinopeptide A increasing from 1.33 ng.mL-1 to 2.25 ng.mL (P < 0.05) after 1-h exercise testing. Significant changes of plasma thrombomodulin were detected exclusively after running with increases from 38.2 ng.mL-1 to 44.2 ng.mL-1 (1 h, P < 0.01). CONCLUSIONS: The data demonstrated that prolonged exercise is necessary for exercise-induced activation of coagulation resulting in thrombin and fibrin formation and suggested that endothelial cell activation possibly due to mechanical factors associated with running might play a role.     

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Characterization of the mechanical behaviour parameters of the costo-vertebral joint

Humic acid in the drinking water of blackfoot disease endemic areas in Taiwan has been implicated as one of the aetiological factors of the disease. For this report we examined the effects of humic acid on the expression of thrombomodulin (TM) cofactor activity by cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with humic acid (HA) isolated from the drinking water, as a synthetic humic acid polymer (SHA) or with commercial HA, resulted in a dose-dependent reduction of cell surface thrombomodulin activity. Characterization of the mechanism by which humic acid reduced the protein C activation indicated that inhibition was not caused by production or release of a protein C inhibitor. Kinetic analysis showed that binding affinities of TM to thrombin and of TM-thrombin complex to protein C was unchanged upon humic acid treatment. However, the cell surface TM activity was reduced by humic acid, which functions as an irreversible noncompetitive inhibitor of thrombin binding. Down-regulation of TM was inhibited by non-selective protein kinase C inhibitors and a selective inhibitor. These results suggest that protein kinase C is intricately involved in HA-induced TM down-regulation. Down-regulation of TM was also inhibited by free radical scavengers. All these changes occurred in the absence of significant cytotoxic effect. In conclusion, our results suggest that HA induces down-regulation of TM by directly increasing permeability of the cell membrane, thus causing elevation in [Ca2+]i. This species functions as a second messenger to activate protein kinase C, and/or Ca-dependent enzymes eventually inducing down-regulation of TM. Attenuation of vascular endothelial cell TM cofactor activity by humic acid may play a role in the humic acid-induced thrombotic vascular disorders of blackfoot disease.     

Antibodies to protease-activated receptor 3 inhibit activation of mouse platelets by thrombin

Recent studies of mice deficient in the thrombin receptor, protease-activated receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin receptor in mouse platelets. We recently identified a new thrombin receptor designated protease-activated receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.     

Endothelium, thrombosis and fibrinolysis

Vascular endothelium is involved in the control of thrombosis by influencing among other things, platelet functions, coagulation and fibrinolysis. Fibrinolysis is due to plasminogen conversion into plasmin by activators, which are efficiently and rapidly controlled by a specific inhibitor. Endothelium plays a pivotal role in these associations. It is a constitutive and inducible source of activators and inhibitor. It provides a favorable microenvironment for plasmin generation at the vessel surface. Furthermore, the fibrinolytic system participating in angiogenesis is implicated in plaque survival. Subjected to a pathological environment, endothelial activation leads to change in endothelial properties especially by increasing the production of inhibitor of plasminogen activators. These modifications could combine to create locally or distantly, a state predisposing to thrombosis.     

Fibrinolysis, antithrombin III, and protein C in neonates during cardiac operations

Fibrinolysis and coagulation were studied in 10 neonates undergoing cardiac operations for congenital heart defects. Coagulation was activated during cardiopulmonary bypass as evidenced by highly increased prothrombin fragment 1 + 2 levels compared with preoperative values. Prothrombin fragment 1 + 2 levels remained elevated until postoperative day 3. Unlike coagulation, fibrinolysis was not activated during cardiopulmonary bypass but did show late activation on postoperative day 3, as evidenced by elevated levels of the fibrin degradation product D-dimer. Lack of fibrinolytic activation during bypass and its appearance on postoperative day 3 were partly explained by changes observed in tissue plasminogen activator and its inhibitor. During bypass, levels of tissue plasminogen activator and its inhibitor increased by 3.4-fold and 3.2-fold, respectively. In the postoperative period, levels of plasminogen activator inhibitor normalized rapidly whereas tissue plasminogen activator remained elevated, resulting in late fibrinolytic activation on postoperative day 3. In accordance with elevated prothrombin fragment 1 + 2, platelet count, antithrombin III, protein C, prothrombin, and factor VII were decreased on postoperative day 2, indicating ongoing consumptive coagulopathy. Nine patients had antithrombin III and six had protein C levels below age-specific normal ranges, consistent with an acquired deficiency state. Three had central venous thrombosis by postoperative day 4 or 5. In all three, thrombosis was preceded by antithrombin III deficiency, protein C deficiency, and highly elevated plasminogen activator inhibitor (3.7 to 37 times the mean of the other patients) on postoperative days 1 to 3. In conclusion, cardiopulmonary bypass in neonates caused rapid and profound alterations in the coagulation and fibrinolytic systems and initiated consumptive coagulopathy lasting until at least postoperative day 3. Thrombophilic abnormalities in antithrombin III, protein C, and fibrinolysis were frequently found and were associated with serious thrombotic complications.