Presence Of Thrombomodulin Research Articles

Thrombin generation in patients after acute deep-vein thrombosis

Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels.

Preservation of Beneficial TAFI Functions by an Activated Protein C Variant with Normal Cytoprotective Functions but Minimal Anticoagulant Activity.

Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa), a carboxypeptidase B-like enzyme, both inhibits fibrinolysis and exerts anti-inflammatory effects via removal of C-terminal Arg residues of bradykinin (BK) and the complement anaphylatoxins, C3a and C5a. Proteolytic activation of TAFI is caused by thrombin or thrombin-thrombomodulin; thus, TAFI activation can be down-regulated by the anticoagulant, activated protein C (APC). Recombinant APC reduces mortality in severe sepsis patients (PROWESS trial), but it increases risk of serious bleeding. This risk is presumably due to APC's anticoagulant activity and possibly exacerbated by profibrinolytic effects derived from down-regulation of thrombin-mediated activation of TAFI. In addition, APC therapy might ironically aggravate sepsis by reducing TAFIa generation because TAFIa inactivates BK, C3a and C5a, well known pathological mediators in sepsis. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191–193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, J Exp Med, 2007). This current study compares the effects of this variant to wild type (wt) APC on profibrinolytic and TAFIa's anti-inflammatory activities in plasma-based assays. To determine the effects of APC on TAFIa-mediated inhibition of fibrinolysis, clot lysis was studied in a plasma system of thrombin-induced clot formation and tPA-mediated fibrinolysis. Compared to wt-APC (IC50=0.36 nM APC), almost 10-times more 5A-APC was required for 50% inhibition of clot lysis (IC50=3.1 nM 5A-APC) and 20-times more 5A-APC was needed to inhibit completely TAFI activation under the conditions employed. The effects of wt-APC and 5A-APC on TAFIa's anti-inflammatory activities were determined by following proteolytic inactivation of BK in plasma during tissue factor-induced clotting. In the absence of added thrombomodulin, angiotensin-converting enzyme, carboxypeptidase N, and TAFIa each inactivated approximately 10–15% of BK under the study conditions. But in the presence of thrombomodulin, BK was fully inactivated in plasma and converted to des-Arg9-BK (> 98%), an effect mainly attributable to TAFIa. Analysis of the time course of BK inactivation in the presence of thrombomodulin indicated that TAFIa rapidly converted BK to des-Arg9-BK with only a minor contribution from carboxypeptidase N. Under conditions where TAFIa fully converted BK to des-Arg9-BK, wt-APC, but not 5A-APC, dose-dependently diminished BK inactivation and des-Arg9-BK generation. At the highest tested concentrations of APC (40 nM), wt-APC completely inhibited TAFIa-dependent BK inactivation whereas non-anticoagulant 5A-APC permitted normal TAFI activation and showed no significant inhibition of TAFIa-dependent BK inactivation. Thus, 5A-APC left intact the TAFIa-mediated anti-inflammatory mechanism for inactivation of BK in plasma and, presumably, TAFIa's ability to inactivate the C3a and C5a anaphylatoxins. These studies show that genetic engineering can selectively alter APC's multiple activities to provide an APC variant that retains this enzyme's beneficial anti-inflammatory and cytoprotective effects but that diminishes bleeding risk due to reduction in APC's anticoagulant and profibrinolytic activities. Thus, these data support the hypothesis that preservation of TAFI activation in plasma by normally cytoprotective 5A-APC variant would preserve TAFIa-dependent anti-inflammatory effects that are lost with the use of wt-APC.

Das TAFI-System

The primary focus of the blood coagulation system is the prevention of blood loss. The system is regulated by various inhibitors, and by the fibrinolytic system, which removes the final product of the blood coagulation system, the fibrin clot. The fibrinolytic system is activated in the course of coagulation activation. The thrombin-activated fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis. TAFI is activated by thrombin, and activation is enhanced in the presence of thrombomodulin. TAFIa, the product of TAFI activation, removes lysine residues from fibrin, which are essential for the binding of t-PA, plasminogen, and plasmin to fibrin. The fibrin loses its cofactor activity in t-PA-induced plasminogen activation, resulting in less plasmin, and the remaining plasmin finds less binding sites on fibrin, resulting in an increased resistance of the clot towards plasmin proteolysis. High concentrations of thrombin result in high TAFIa-activity and consequently in highly resistant fibrin clots. Patients with hyperprothrombinaemia consequently display elevated TAFIa-levels, which may contribute to the risk for thrombosis. Treatment with recombinant factor VIIa also leads to high concentrations of thrombin, resulting in fibrin clots with enhanced resistance towards fibrinolysis. At low thrombin concentration, as observed in patients with bleeding disorders or patients treated with anticoagulant drugs, less TAFIa is produced in the course of coagulation activation, and the clots are less resistant towards fibrinolysis. TAFIa-inhibitors are currently being developed for the treatment of throboembolic disorders or hypofibrinolytic DIC. Enhancement of TAFIa-activity may be helpful in patients with bleeding.

The effect of aprotinin on activated protein C‐mediated downregulation of endogenous thrombin generation

Thrombin plays a central role in coagulation and haemostasis. Binding of thrombin to thrombomodulin generates activated protein C (APC), which exerts a negative feedback on thrombin formation. Aprotinin, a natural proteinase inhibitor is used extensively during cardiac surgery because this procedure is often associated with profound activation of coagulation and inflammatory pathways. Some in vitro evidences suggest that aprotinin inhibits APC, but the clinical relevance is unclear. The recombinant human soluble thrombomodulin (rhsTM)-modified thrombin generation (TG) assay was used to investigate the effects of aprotinin on APC in plasma samples obtained from healthy volunteers, aprotinin-treated cardiac surgical patients and in protein C (PC)-depleted plasma. Based on the results of in vitro TG assay, addition of rhsTM (0.75-3.0 microg/ml) to volunteer or patient platelet-poor plasma significantly reduced (70.8 +/- 21.9 and 95.3% +/- 4.6%, respectively) thrombin formation when compared with PC-depleted plasma (8.3% +/- 5.2%). Aprotinin (100-200 KIU) caused a small, statistically insignificant decrease in the peak thrombin formation in normal and PC-deficient plasma (12.0 +/- 6.1%). In cardiac surgical patients, levels of functional PC, factor II, antithrombin and platelet significantly decreased after cardiopulmonary bypass (CPB). Soluble thrombomodulin concentrations were increased after CPB (3.5 +/- 2.2 to 5.0 +/- 2.2 ng/ml), but they were still within the normal range for human plasma. Our results showed that, even though endogenous PC level is decreased after CPB, it retains its activity in the presence of thrombomodulin, and aprotinin has limited inhibitory effect on APC generation.

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.

Evidence of normal thrombin generation in cirrhosis despite abnormal conventional coagulation tests†‡

The role played by coagulation defects in the occurrence of bleeding in cirrhosis is still unclear. This is partly due to the lack of tests that truly reflect the balance of procoagulant and anticoagulant factors in vivo. Conventional coagulation tests such as prothrombin time and activated partial thromboplastin time are inadequate to explore the physiological mechanism regulating thrombin, because they do not allow full activation of the main anticoagulant factor, protein C, whose levels are considerably reduced in cirrhosis. We used a thrombin generation test to investigate the coagulation function in patients with cirrhosis. Thrombin generation measured without thrombomodulin was impaired, which is consistent with the reduced levels of procoagulant factors typically found in cirrhosis. However, when the test was modified by adding thrombomodulin (i.e., the protein C activator operating in vivo), patients generated as much thrombin as controls. Hence, the reduction of procoagulant factors in patients with cirrhosis is compensated by the reduction of anticoagulant factors, thus leaving the coagulation balance unaltered. These findings help clarify the pathophysiology of hemostasis in cirrhosis, suggesting that bleeding is mainly due to the presence of hemodynamic alterations and that conventional coagulation tests are unlikely to reflect the coagulation status of these patients. In conclusion, generation of thrombin is normal in cirrhosis. For a clinical validation of these findings, a prospective clinical trial is warranted where the results of thrombin generation in the presence of thrombomodulin are related to the occurrence of bleeding.

Activated protein C, a natural anticoagulant protein, has antioxidant properties and inhibits lipid peroxidation and advanced glycation end products formation

Introduction Activated protein C (APC) is an important natural anticoagulant that is proteolytically generated from protein C (PC) by the modulation of thrombin activity in the presence of thrombomodulin on an endothelial surface. Recent studies have demonstrated that, beyond its anticoagulant acitivities, APC had anti-inflammatory and cytoprotective properties. The mechanisms underlying APC's anti-inflammatory effects remain unknown. Our goal was to elucidate and confirm these mechanisms. Methods We first examined the effect of APC on reactive oxygen species (ROS) and inflammatory cytokine production in murine macrophage-like RAW264.7 cells. We further examined the effect of APC on chemically induced lipid peroxidation and advanced glycation end-products (AGE) formation. Results and conclusions APC in the range of 10–50 μg/mL could reduce lipopolysaccharide (LPS)-induced ROS generation, nuclear factor κB (NF-κB) activation and resultant proinflammatory cytokine production. Additional cell-free experiments revealed that APC (10–50 μg/mL) had inhibitory effects on lipid peroxidation and AGE formation. These findings suggest that APC, via its intrinsic anti-oxidant properties, may, in settings of oxidant stress, exert important cytoprotective and anti-inflammatory effects that are distinct from its anticoagulant activity as an antioxidant protein. If that is true, APC may contribute to ROS-related chronic disorders including atherosclerosis and diabetes as well as acute shock conditions.

The Anticoagulant Thrombin Mutant W215A/E217A Has a Collapsed Primary Specificity Pocket

The thrombin mutant W215A/E217A features a drastically impaired catalytic activity toward chromogenic and natural substrates but efficiently activates the anticoagulant protein C in the presence of thrombomodulin. As the remarkable anticoagulant properties of this mutant continue to be unraveled in preclinical studies, we solved the x-ray crystal structures of its free form and its complex with the active site inhibitor H-d-Phe-Pro-Arg-CH(2)Cl (PPACK). The PPACK-bound structure of W215A/E217A is identical to the structure of the PPACK-bound slow form of thrombin. On the other hand, the structure of the free form reveals a collapse of the 215-217 strand that crushes the primary specificity pocket. The collapse results from abrogation of the stacking interaction between Phe-227 and Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other notable changes are a rotation of the carboxylate group of Asp-189, breakage of the H-bond between the catalytic residues Ser-195 and His-57, breakage of the ion pair between Asp-222 and Arg-187, and significant disorder in the 186- and 220-loops that define the Na(+) site. These findings explain the impaired catalytic activity of W215A/E217A and demonstrate that the analysis of the molecular basis of substrate recognition by thrombin and other proteases requires crystallization of both the free and bound forms of the enzyme.

Studies on the Different Modes of Action of the Anticoagulant Protease Inhibitors DX-9065a and Argatroban: II. EFFECTS ON FIBRINOLYSIS

The accompanying paper (Nagashima, H. (2002) J. Biol. Chem. 277, 50439-50444) has demonstrated that argatroban can yield a stronger inhibitory effect on thrombin generation than DX-9065a during extrinsic pathway-stimulated human plasma coagulation, while these anticoagulant compounds have comparable abilities to prolong clot time. Since thrombin generation is known to be an important determinant for fibrinolytic resistance of clots formed during coagulation, the two compounds are compared by tissue plasminogen activator-induced clot lysis assays. The results demonstrated that, in the presence of thrombomodulin, argatroban dose dependently accelerated fibrinolysis of the clots, whereas DX-9065a did not. The activation of thrombin activatable fibrinolysis inhibitor (TAFI) determined in separate assays reflected the differential influence on thrombin generation by these compounds. Moreover, TAFI activation correlated closely with the fibrinolytic resistance observed during tissue plasminogen activator-induced clot lysis. This study demonstrates the differential effects of DX-9065a and argatroban on thrombin generation, which in turn results in a differential acceleration of fibrinolysis as well as TAFI activation in the clots formed under the influence of these compounds. The data implicate a possible difference in the antifibrinolytic properties of clots formed during treatment with these compounds.

Effect of Different Types of Thrombin Inhibitors on Thrombin/Thrombomodulin Modulated Activation of Protein C In Vitro

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human α-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl 2. TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant ( K D) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC 50 values±S.D. of 4.4±0.8, 20.0±1.1 and 6.4±0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC 50 value of 290±10 nmol/l, which was enhanced fourfold (IC 50 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.

Rational Design of a Potent Anticoagulant Thrombin

Thrombin acts as a procoagulant when it cleaves fibrinogen and promotes the formation of a fibrin clot and functions as an anticoagulant when it activates protein C with the assistance of the cofactor thrombomodulin. The dual function of thrombin in the blood poses the challenge to turn the enzyme into a potent anticoagulant by selectively abrogating fibrinogen cleavage. Using functional and structural data, we have rationally designed a thrombin mutant, W215A/E217A, that cleaves fibrinogen with a value of k(cat)/K(m) about 20,000-fold slower than wild-type but activates protein C in the presence of thrombomodulin with a specificity comparable with wild-type. This mutant demonstrates for the first time that the relative specificity of thrombin toward fibrinogen and protein C can be completely reversed.

Cellular Cofactors for Amyloid β-Peptide-Induced Cell Stress: Moving from Cell Culture to in Vivo

Molecular mechanisms underlying Alzheimer’s disease (AD) can be divided into inciting pathogenic factors and those more likely to be associated with amplification of cell stress once the disease process is already under way; the latter are termed progression factors. In view of the protracted clinical course of AD, evolving over years, both processes are relevant to the inexorable decline in neuronal function, both mechanistically and therapeutically. Mutations in β-amyloid precursor protein (βAPP) and presenilins 1 and 2, resulting in increased generation of amyloid β-peptide (Aβ) spanning residues 1–42, have been linked to the pathogenesis of familial AD. 1-4 An emerging role for compromised Aβ clearance and degradation, possibly involving the low density lipoprotein receptor-related protein (LRP), apolipoprotein E (apoE), and/or α2-macroglobulin, has been suggested to contribute to sporadic AD. 5-8 Though other mechanisms certainly remain to be discovered, these data contribute to an increasing body of evidence connecting Aβ as a pathogenetic factor central to neuronal dysfunction underlying AD. Although studies of βAPP biology have advanced to sophisticated analyses of presenilin association with intracellular signaling molecules 9 and subcellular compartmentalization of βAPP processing, 10-14 analysis of mechanisms of Aβ-induced cellular stress are at a less advanced stage. Despite our relative lack of knowledge, understanding how Aβ triggers changes in cellular properties is clearly an essential part of any formulation of the amyloid hypothesis. 15 Why, then, has the search for progression factors in the biology of Aβ been so elusive? An analogy with the blood coagulation mechanism is pertinent. For many years, thrombin, the final enzyme in the procoagulant pathway, was known to cleave plasma protein C, forming an important antithrombotic regulator, activated protein C. 16 There was an apparent paradox as, in vitro, the amounts of thrombin required, the concentration of divalent cations, and the rate of activated protein C formation suggested that this reaction, as it occurs in a purified system, was physiologically irrelevant. 16 The solution to this quandary was provided by the identification of a novel endothelial cell cofactor, the integral membrane protein thrombomodulin. In the presence of thrombomodulin, thrombin-mediated activation of protein C occurred rapidly with physiological concentrations of reactants and cations. 16 The contribution of such cellular cofactors to many biological systems is accepted as a given. In this regard, what types of cellular cofactors have been identified based on their interaction with Aβ? Progression factors relevant to AD exacerbate cell stress in an environment created by the pathogenetic factors. There are many candidate progression factors, such as cytokines, 17 complement activation, 18-20 reactive oxygen intermediates (ROIs), 21-24 and other products of activated microglia and/or astrocytes. Such mediators and other mechanisms, including elevated levels of cytosolic calcium, 24 are likely to be placed distally in pathways of cellular dysfunction. Thus, by the time neurons are bathed in proinflammatory cytokines and the environment is characterized by ubiquitous ROIs and elevated intracellular calcium, cellular dysfunction is likely to be quite advanced. However, another class of cell-associated progression factors relevant to AD are those selectively engaged by pathophysiologically relevant forms of Aβ at nanomolar concentrations. These are described below, grouped into several categories.

Fibrinolysis and the plasma carboxypeptidase.

Plasma contains the precursor of a carboxypeptidase B-like enzyme. This zymogen can be activated by high levels of thrombin, such as those that occur after clot formation, or by low levels of thrombin in the presence of thrombomodulin. The activated enzyme suppresses fibrinolysis by removing carboxy terminal lysine residues from partially degraded fibrin, thereby preventing plasmin-mediated positive feedback in the fibrinolytic cascade. The existence of this protein implies a direct molecular link between the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. This connection most likely plays a significant role in maintaining a proper balance between the deposition and removal of fibrin.

The inactivation of single-chain urokinase-type plasminogen activator by thrombin in a plasma milieu: effect of thrombomodulin.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process which may contribute to the maintenance of a fresh blood clot. We have examined the inactivation of scu-PA by thrombin in a plasma milieu to get more insight in the physiological relevance of this phenomenon. Citrated pooled normal plasma was treated with thrombin in the absence and presence of thrombomodulin. After an incubation period of 30 min the concentrations of scu-PA and tcu-PA/T were measured using specific bioimmunoassays. The inactivation of scu-PA in citrated plasma was found to be stimulated fourfold by thrombomodulin. Kinetic experiments showed that the inactivation of scu-PA by thrombin in the absence and presence of thrombomodulin occurred rapidly and declined within 1 min as a result of rapid inhibition by antithrombin III (ATIII) and other possible inhibitors. Calcium had no direct effect on the inactivation of scu-PA by exogenously added thrombin in the absence and presence of thrombomodulin. However, recalcification of plasma induced significant inactivation of scu-PA in plasma as a result of endogenous thrombin generation through the contact activation system. This calcium-induced inactivation of scu-PA was completely abolished in the presence of thrombomodulin, most likely as a result of activation of protein C by the complex formed between thrombomodulin and endogenously generated thrombin. Thrombomodulin thus appeared to play a dual role both by stimulating the inactivation of scu-PA by thrombin, and by inhibiting calcium-induced inactivation of scu-PA in plasma. In the plasma from a patient heterozygous for protein C deficiency, thrombomodulin could not prevent calcium-induced generation of tcu-PA/T, whereas the stimulating effect of thrombomodulin predominated instead. This result implied that disturbance of the protein C pathway may lead to the inactivation of substantial amounts of scu-PA in plasma under (patho)physiological circumstances and may provide an additional explanation for the association found between thromboembolism and deficiencies in the protein C pathway. This study shows that the amount of scu-PA that is inactivated in plasma depends mainly on the generation of thrombin and on thrombomodulin. We conclude that the inhibition of scu-PA-induced fibrinolysis appears to be regulated by activation of the coagulation system, providing a link between coagulation and fibrinolysis.

On the Mechanism of the Antifibrinolytic Activity of Plasma Carboxypeptidase B

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

Effect of Thrombomodulin on the Molecular Recognition and Early Catalytic Events in Thrombin-Protein C Interaction

A viscosity perturbation method allowed to compute the second order rate constant, k+1, for the formation of thrombin-Protein C complex, both in the absence and presence of thrombomodulin (TM) at pH 8.00 and 37 degrees C. In the absence of TM the second order rate constant was found equal to 7.9 +/- 0.6 x 10(3) M-1 sec-1, whereas it was enhanced to 9.9 +/- 0.4 x 10(4) M-1 s-1 by a saturating (100 nM) TM concentration. Addition of 5 mM Ca++ to solution containing 100 nM TM induced a further increase of k+1 value up to 7.3 +/- 0.5 x 10(5) M-1 s-1. Moreover, it was demonstrated that the thrombin-PC complex undergoes the acylation reaction more rapidly than it dissociates to form free thrombin and substrate (stickiness ratio = 2.4 +/- 0.9). This tendency is even favored when thrombin is bound to TM both in the absence and presence of Ca++ (stickiness ratio = 9 +/- 6 in the absence of Ca++ and 16 +/- 10 in the presence of Ca++). Altogether these results demonstrate that TM is able to positively affect both the molecular encounter and the kinetics of the early catalytic events of the thrombin-Protein C interaction.

Characteristic effects of activated human protein C on tissue thromboplastin-induced disseminated intravascular coagulation in rabbits

Protein C (PC) is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating thrombosis and fibrinolysis by inhibiting not only blood coagulation factors Va and Villa but also type-1 plasminogen activator inhibitor (PAI-1). In the present study we examined the effects of human APC on tissue thromboplastin-induced disseminated intravascular coagulation (DIC) in rabbits and compared them with those of heparin. Both APC (300–3000U/kg) and heparin (100–300 IU/kg) inhibited the decreases in platelet count and fibrinogen level equally. APC improved the prolonged bleeding time, but heparin aggravated bleeding with potent prolongation of activated partial thromboplastin time (APTT). Furthermore, in APC-treated animals, fibrin deposition in glomeruli was less than in heparin-treated animals. This result suggests that APC accelerated local fibrinolysis by neutralizing PAI-1. From our findings, we concluded that APC can improve both coagulation and fibrinolysis in a DIC model and should be useful for the clinical remedy of DIC without having an adverse side effect like a bleeding tendency.

Inactivation of receptor-bound pro-urokinase-type plasminogen activator (pro-uPA) by thrombin and thrombin/thrombomodulin complex.

Urokinase-type plasminogen activator (uPA) binds with high affinity to a specific cell surface glycosyl phosphatidyl-inositol (GPI)-anchored receptor, the urokinase receptor (uPAR). Pro-uPA, the enzymatically inactive single-chain form of uPA after having been activated by certain proteases, converts plasminogen into plasmin. This activation of pro-uPA to enzymatically active uPA can be prevented by the action of thrombin on pro-uPA. This inactivation process is accelerated in the presence of thrombomodulin (TM). The present study investigated whether pro-uPA bound to uPAR is still susceptible to inactivation by thrombin in the presence or absence of TM. A truncated soluble form of the uPAR lacking the GPI-anchor was cloned and expressed in CHO-cells (rec-uPAR277). Rec-uPAR277 efficiently inhibited the thrombin-mediated inactivation of pro-uPA up to 90% in a concentration dependent manner. The protective effect of rec-uPAR277 was far less pronounced when thrombin was complexed with TM. Enzyme kinetic experiments with varying concentrations of pro-uPA showed that in the presence of TM the catalytic efficiency (kcat/Km) of thrombin-mediated inactivation raised from 0.010 microM-1 s-1 to 0.50 microM-1 s-1 corresponding to a fifty-fold increase. In the presence of rec-uPAR277, however, the catalytic efficiency dropped by 4.1-fold (0.5 microM-1 s-1 to 0.122 microM-1 s-1). The inactivation kinetics of pro-uPA by thrombin (no TM added) in the presence of an excess of rec-uPAR277 could not be determined since virtually no inactivation occurred. Our data suggest that pro-uPA once bound to uPAR, is significantly protected from inactivation by thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

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 ζ-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to ζ-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-CH 2Cl 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 ζ-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the Dl 99E 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.