Thrombin-thrombomodulin Complex Research Articles

Amino Acid Residues in the P6-P′3 Region of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Do Not Determine the Thrombomodulin Dependence of TAFI Activation

Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P'3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen Bbeta-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90-95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P'3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.

Recombinant human activated protein C in severe sepsis.

The role of activated protein C (APC) in coagulation, inflammation, and fibrinolysis and the pharmacology, pharmacokinetics, and trials of recombinant human activated protein C (rhAPC), or drotrecogin alfa (activated), in sepsis are described. Protein C, a naturally occurring vitamin K-dependent serine protease in the blood, remains inactive until exposed to the thrombin-thrombomodulin complex. This change between the inactive and active forms occurs constantly in humans and serves to balance the coagulation cascade. APC functions in concert with protein S as an anticoagulant, a fibrinolytic agent, and an antiinflammatory agent. In response to serious infection, a procoagulant process is activated leading to thrombin and fibrin deposition in small vessels that results in decreased blood flow, decreased oxygen delivery, and organ failure. The body's natural defense during severe sepsis is to activate protein C through the thrombin-thrombomodulin complex in an attempt to restore the imbalance of the hemostatic systems. However, APC has a short half-life, and the pool of circulating protein C is rapidly depleted in severe sepsis. Low protein C levels have been correlated with poor outcome in patients with severe sepsis and in animal models. These observations led to a Phase III safety and efficacy trial of drotrecogin alfa (activated) that demonstrated a significant improvement in mortality compared with placebo (24.7% versus 30.8%). This 6.1% absolute difference in mortality translates to a 19.4% reduction in relative risk of death in the treated patients. The proper use of drotrecogin alfa (activated) will require careful consideration of appropriate patients to treat and further studies in patient populations that were excluded from the Phase III trial, as well as possible modification of dosing schemes on the basis of patient response.

Measurement of carboxypeptidase R by colorimetric assay

Carboxypeptidases (CP), carboxypeptidase N (CPN) and carboxypeptidase R (CPR), have been reported as a protease, which can cleave carboxy-terminal arginine or lysine residues from biologically active peptides, such as C3a and C5a, and regulate their activity. CPN is present in the active form in plasma, but CPR is generated from its zymogen during coagulation. CPR (identical to carboxypeptidase U [CPU], plasma carboxypeptidase B [plasma CPB]) has also been described as an inhibitor of fibrinolysis, and termed TAFI (thrombin activatable fibrinolysis inhibitor). ProCPR is activated by thrombin, thrombin-thrombomodulin complex (T-TM), plasmin, and trypsin. Today, the T-TM complex pathway has been taken notice because of effectiveness of Protein C for sepsis. Protein C has been recognized as a mediator between inflammation and coagulation. About CPR, some recent clinical studies have been shown that CPR is an acute phase protein and proCPR have been reduced in DIC. Similar to Protein C, CPR may be a mediator between inflammation and fibrinolysis. Colorimetric assay is one of the methods for measuring CP activity. Although it is convenient for determining CP activity, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction. If one will pay attention to this point, colorimetric assay will be a good method for measuring CP activity and give us further findings.

Two Naturally Occurring Variants of TAFI (Thr-325 and Ile-325) Differ Substantially with Respect to Thermal Stability and Antifibrinolytic Activity of the Enzyme

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated to TAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37 degrees C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30-50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant.

A myriad of responses

Severe sepsis is normally caused by bacterial infection, and can result in organ failure. Increased concentrations of inflammatory cytokines contribute to vascular and organ damage. Leucocytes are activated and adhere to the vessel wall. Leucocyte clumping contributes to impaired blood flow and oxidant injury to blood vessels. Blood pressure drops. Vascular permeability is increased, leading to oedema. In severe cases, there is disseminated intravascular coagulation (figure). Most patients who die from sepsis have low concentrations of anticoagulants protein C and antithrombin, indicating an active coagulation process even in the abscence of disseminated intravascular coagulation. Therefore, replacement therapy with these proteins might be effective. Many challenge this strategy, however, since patients who die, generally do not have extensive thrombosis at necropsy. However, an understanding of the protein C pathway, through animal experiments, provides a basis for this strategy. Protein C is activated by the thrombin-thrombomodulin complex on the endothelium. The thrombomodulin concentration, and hence the rate of protein C activation, is higher in the microvasculature than in the macrovasculature because of a high endothelial cell-to-blood volume ratio. Activated protein C (APC) inactivates factors Va and VIIIa inhibiting thrombin generation. Hinshaw and Taylor showed that dogs placed on an extracorporeal pump developed a circulating anticoagulant, APC, probably caused by thrombin generated by the pump. When these animals were challenged with a lethal dose of endotoxin, they survived. Seemingly paradoxically, we found that thrombin infusion before infusion of a lethal dose of endotoxin could also protect animals from severe sepsis. APC was then shown to protect baboons from sepsis. APC not only blocked the coagulant response, but also reduced the concentrations of various inflammatory markers, minimised the drop in blood pressure, and was protective when given hours after infusion of bacteria. Thrombin activation of protein C therefore probably plays a part in defense against severe sepsis. Studies from Hancock’s laboratory provided evidence for a cell surface receptor for APC on monocytes. Engagement of this receptor blocks endotoxin mediated calcium fluxes in the cell and prevents CD14 down-regulation. Thus, APC attenuates over-expression of some of the cytokines that contribute to organ dysfunction. Joyce and colleagues have shown that APC decreases expression of adhesion molecules responsible for leucocyte interaction with the endothelium. APC also altered gene expression in a pattern that would prevent apoptosis. The prevention of leucocyte adhesion to the endothelium might decrease endothelial cell injury in severe sepsis. Prevention of apoptosis would also decrease the endothelial cell dysfunction seen in the disease. The endothelial cell protein C receptor might be involved in cellular signalling. The receptor translocates from the plasma membrane into the nucleus and can carry APC in the process. Nuclear translocation seems to modulate gene expression, but the relative importance of the contributions of nuclear translocation and cell surface signalling to the protective functions of APC on the endothelium are unknown. Investigation of the protein C pathway has identified possible new treatments for severe sepsis, and combination therapies for sepsis might be possible. These include the addition of anticytokines, which by themselves have little or no protective effects, or inhibitors of platelet activating factor, which have shown promise in clinical studies. Since these agents work at different stages of the disease, combination therapies might improve survival without increased risk. Severe sepsis induces release of cytokines, oxidants, and proteases from leucocytes, injuring the vessel, triggering clotting, and inhibiting the protein C pathway (A). APC limits many of these events, protecting the vessels and organs from injury (B).

Activity and Antigen Levels of Thrombin-Activatable Fibrinolysis Inhibitor in Plasma of Patients With Disseminated Intravascular Coagulation

We measured the plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) activity and antigen in patients with disseminated intravascular coagulation (DIC) to examine the relationship between hypofibrinolysis and the pathogenesis of DIC. TAFI activity and antigen levels in the plasma were both significantly low in patients with DIC. TAFI activity in plasma was correlated with TAFI antigen, indicating that activity and antigen correspond well. The decrease of TAFI activity in DIC may be due to enhanced consumption. Since the plasma thrombin–antithrombin III complex (TAT) level was found to be elevated in DIC, increase of thrombomodulin–thrombin complex generation is suggested in this state. TAFI activity and antigen levels were negatively correlated with TAT and D-dimer, suggesting that the plasma levels of TAFI are reduced by thrombin generation. Since TAFI was not correlated with fibrinogen, plasma–α 2plasmin inhibitor complex (PPIC) and tissue type plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complex, TAFI might be a secondary modulator of fibrinolysis. The TAFI activity in plasma was significantly low in patients with infection and in those with organ failure, suggesting that TAFI may play an important role in the mechanism of organ failure in DIC-associated sepsis. In brief, TAFI may play an important role in the pathogenesis of DIC and organ failure.

Endothelial cell protein C receptor plays an important role in protein C activation in vivo

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

Identification of the Protein C/Activated Protein C Binding Sites on the Endothelial Cell Protein C Receptor: IMPLICATIONS FOR A NOVEL MODE OF LIGAND RECOGNITION BY A MAJOR HISTOCOMPATIBILITY COMPLEX CLASS 1-TYPE RECEPTOR

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.

Prevalence of a 23bp Insertion in Exon 3 of the Endothelial Cell Protein C Receptor Gene in Venous Thrombophilia

Summary Background: The endothelial cell protein C receptor (EPCR) enhances protein C activation by the thrombin-thrombomodulin complex. As evidence is accumulating that EPCR is an important component of the protein C anticoagulant pathway, polymorphisms in the EPCR gene might be candidate risk factors predisposing to venous thromboembolism (VTE). Recently, a 23bp insertion in exon 3 of the EPCR gene has been identified, which duplicates the preceding 23 bases and results in a STOP codon downstream from the insertion point. However, the clinical significance of this mutation in VTE remains to be clarified. Methods and Results: In this study we evaluated the EPCR 23bp insertion in 889 patients with documented VTE and in 500 healthy controls. The prevalence of the EPCR insertion among patients was 0.1%, which was not significantly different compared to controls (0.6%, p = 0.1). Conclusions: Our findings showed that the EPCR 23bp insertion is very rare in both patients with VTE and the general population and failed to support an association between the EPCR 23bp insertion and an increased risk of VTE.

Protein C Inhibitor Regulates the Thrombin-Thrombomodulin Complex in the Up- and Down Regulation of TAFI Activation

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a carboxy-peptidase B-like proenzyme that after activation by thrombin down regulates fibrinolysis. Thrombomodulin (TM) stimulates the activation of both TAFI and protein C whereas activated protein C (APC) inhibits the activation of TAFI by down regulating thrombin generation. Recently, protein C inhibitor (PCI) was identified as a potent inhibitor of thrombin bound to TM and it can thereby regulate the balance between TAFI activation, and inhibition of TAFI activation by APC. Both in a purified system and in plasma, activation of TAFI and protein C by [Ia-TM could be inhibited by PCI. Previously we found in plasma that at low concentrations (approximately 1 nM), TM predominantly stimulated the activation of TAFI whereas at higher concentrations of TM (approximately 10 nM) the activation of protein C resulted in inhibition of the activation of TAFI. In agreement with this. PCI inhibited the activation of TAFI at 1 nM TM whereas at 10 nM TM PCI inhibited the activation of protein C resulting in an increase in the activation of TAFI. This suggests that PCI can up regulate TAFI activation by inhibiting the protein C activation. PCI may therefore be an important regulator in the balance between coagulation and fibrinolysis by differentially inhibiting the activation of TAFI and of protein C. The local TM concentration plays an important role in the outcome of this process.

Relationship between soluble thrombomodulin in plasma and coagulation or fibrinolysis in type 2 diabetes

Serum concentration of soluble thrombomodulin (TM) is thought to be a marker for endothelial damage. Although several studies have reported that serum TM concentrations are increased in patients with diabetes mellitus, there is little information on the physiological function of soluble TM in human plasma. To evaluate the relationship of soluble TM in plasma between coagulation and/or fibrinolysis system in patients with diabetes, we measured plasma soluble TM, protein C activity (a natural anticoagulant induced by thrombin–TM complex), prothrombin F1+2 (a direct marker of thrombin generation), and plasmin–α2-antiplasmin complex (PAP) and D dimer (measures of fibrinolytic activity) in 55 patients with type 2 diabetes mellitus. The plasma concentrations of soluble TM ( P<0.01), protein C activity ( P<0.01), prothrombin F1+2 ( P<0.05), PAP ( P<0.001) and D dimer ( P<0.001) were significantly higher in the diabetic patients than the 48 age-matched control subjects. The plasma concentrations of TM and PAP were obviously increased in patients with diabetic nephropathy. In the diabetic patients, the plasma concentrations of soluble TM were inversely correlated with the protein C activity ( r=−0.43, P<0.005), and were positively correlated with the plasma concentrations of prothrombin F1+2 ( r=0.63, P<0.0001) and the plasma PAP concentrations ( r=0.30, P<0.05). The present study demonstrated that both coagulation and fibrinolysis are enhanced concomitantly in patients with type 2 diabetes mellitus, and that an increase in plasma concentration of soluble TM is associated not only with hypercoagulability but also with enhanced fibrinolysis in diabetic patients.

Molecular cloning and partial characterization of rat procarboxypeptidase R and carboxypeptidase N.

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy-terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin-thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte-specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.

Cardiac operation in a patient with combined homozygous protein C and protein S deficiency

PROTEIN C IS a vitamin K-dependent plasma protein that is an important natural antithrombotic component. After activation by a thrombin-thrombomodulin complex on the endothelial cells, activated protein C inhibits coagulation by inactivating factors V and VIII and leads to inhibition of thrombin formation. Protein S serves as a cofactor for protein C in its anticoagulant properties. 1 Protein C and S deficiencies are rare diseases, characterized by recurrent venous thrombosis. Their deficiency may be inherited, either homozygous (purpura fulminans in neonates) or heterozygous or acquired. Inherited protein C and S deficiencies are transmitted in an autosomal codominant fashion and present during infancy. If not treated, homozygous individuals die of thrombosis in early infancy. 2 An infant is described with a highly unusual case of combined congenital homozygous protein C and S deficiency who also had cardiac defects (transposition of the great arteries and subpulmonic stenosis) and inferior vena cava thrombosis.

Structural basis for the anticoagulant activity of the thrombin–thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor-alpha (TNF-alpha) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF-alpha failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo. (Blood. 2000;95:1687-1693)

Mechanisms by Which Soluble Endothelial Cell Protein C Receptor Modulates Protein C and Activated Protein C Function

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Bovine Protein C Inhibitor Has a Unique Reactive Site and Can Transiently Inhibit Plasmin

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg-Ser) of other species' PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system.

Organization and chromosomal localization of the human endothelial protein C receptor gene.

Endothelial protein C receptor (EPCR), present on endothelial cells of relatively large veins and arteries, plays a role in the enhancement of protein C activation by the thrombin-thrombomodulin complex. In the present study, we determined the organization and the complete nucleotide sequence of the human EPCR gene using polymerase chain reaction-direct sequencing method. The transcription initiation site of the EPCR gene was also determined by the cap site hunting method, using a cap site cDNA prepared from human placenta. The human EPCR gene spanned approx. 6 kb and was composed of four exons and three introns. All exon-intron boundaries agreed with the GT-AG rule. The 5'-flanking region (300 bp) of the EPCR gene contained a putative AP1-binding site, two Sp1-binding sites and two AP2-binding sites, but not definite TATAA or CCAAT sequences. Fluorescence in situ hybridization analysis showed that the EPCR gene is located in chromosome 20q11.2.

Thrombin Inhibitors Suppress the Thrombin-Thrombomodulin– Mediated Generation of Activated Protein C

In the treatment of unstable coronary artery disease, direct thrombin inhibitors have shown no or only limited benefit as compared with heparin, despite theoretical advantages. One explanation may be that the direct thrombin inhibitors to a greater extent than heparin have an inhibiting effect on the generation and activity of activated protein C. In the present study, this hypothesis was tested in an in vitro, “purified” system, where human protein C underwent activation to activated protein C by the thrombin-thrombomodulin complex. Direct thrombin inhibitors, inogatran and hirudin, unfractionated heparin+antithrombin, or dalteparin+antithrombin, were added to the system before activation to evaluate their inhibitory effect on the generation of activated protein C. At inhibitor concentrations well below the achieved plasma levels in major clinical trials, the thrombin-thrombomodulin–mediated activation of protein C was inhibited by all the studied inhibitors in a dose-dependent manner, but, contrary to our hypothesis, to a greater extent by unfractionated heparin+antithrombin and dalteparin+antithrombin than by the direct thrombin inhibitors, hirudin and inogatran. Despite difficulties to draw conclusions for the in vivo situation, the in vitro inhibition, by all studied inhibitors, of the generation of activated protein C, found in this study may be a possible explanation for ongoing cardiovascular events despite adequate treatment with thrombin inhibitors, in patients with unstable coronary artery disease. This inhibition of the generation of activated protein C may also contribute to the rebound in cardiovascular events after withdrawal of effective antithrombotic treatment.

Association of Plasma Levels of Activated Protein C with Recanalization of the Infarct-Related Coronary Artery after Thrombolytic Therapy in Acute Myocardial Infarction

Protein C is one of the most important antithrombotic components. After activation by the thrombin-thrombomodulin complex on endothelial cells, activated protein C (APC) inactivates factors Va and VIIIa, which leads to the inhibition of thrombin formation. We examined the association of plasma levels of APC with the responsiveness to coronary thrombolytic therapy of the infarct-related coronary artery in patients with acute myocardial infarction (AMI). Plasma levels of APC, thrombin-antithrombin III complex (TAT), and plasminogen activator inhibitor (PAI) activity were measured in 32 consecutive AMI patients who underwent coronary angiography followed by thrombolytic therapy, and compared to the measurements in 23 control subjects. On admission, APC levels (ng/mL) were significantly elevated in patients with AMI, as compared with controls (2.5±0.4 vs. 1.2±0.2, 1.3±0.2, respectively, p<0.01). At discharge, plasma levels in AMI patients decline to values not significantly different from those in controls. (1.2±0.2, 1.3±0.2, respectively). TAT levels (ng/mL) were different among the groups in a fashion similar to that of APC (14.1±3.1 on admission vs. 3.3±0.4 at discharge, 1.8±0.1 in the control subjects, respectively, p<0.01). PAI activity levels (IU/mL) were higher on admission than at discharge and higher than the control subjects (19.7±1.8 vs. 10.5±1.0, 5.4±0.7, respectively, p<0.01). Thirty-two patients with AMI were classified into two groups according to the results of thrombolysis: the success group (24 patients) and the failure group (eight patients). APC levels were higher in the failure group than in the success group (5.1±0.7 vs. 1.6±0.2, p<0.01). TAT levels were also higher in the failure group than in the success group (30.8±9.6 vs. 8.6±1.7, p<0.01). PAI activity levels (IU/mL) were lower in the failure group than in the success group (13.5±3.1 vs. 21.7±2.1, p<0.05). There were correlations between APC and TAT levels both on admission r=0.75, p<0.0001 and at discharge r=0.71, p<0.0001 . Elevated APC was thought to correlate with increased thrombin generation in patients with AMI. This study demonstrated that there was a significant relation between plasma APC level and the responsiveness to thrombolytic therapy of the infarct artery. This study may also indicate that increased thrombin generation is a cause of the resistance to thrombolytic therapy.