Cofactor For Activated Protein Research Articles

A missense mutation in lectin domain of thrombomodulin causing functional deficiency

Thrombomodulin (TM) functions in coagulation, fibrinolysis and inflammation by its cofactor activity for protein C, thrombin-activatable fibrinolysis inhibitor (TAFI) activation and high mobility group box 1 (HMGB1) degradation induced by thrombin. It has been widely reported that mutations in TM are related to thromboembolic diseases but hardly in lectin domain. Here we report our findings about the functional deficiencies in TM caused by substitution of aspartate with tyrosine at residue 126. Three patients suffering from recurrent thromboembolic diseases were identified with this mutation and their plasma soluble TM levels were decreased. Transfected cells expressing wild-type TM or the variant and corresponding proteins were used to examine TM functions in vitro. The cofactor activity of the mutant for protein C, TAFI activation was reduced to approximately 50% and 60% respectively. Loss in anti-inflammation due to weakened HMGB1 degradation was also observed. And the study with thrombosis models of mice suggested the decreased inhibition of thrombus development of the mutant. Together the results showed deleterious changes on TM function caused by this mutation, which may explain the thrombophilia tendency of the patients. This work provided supportive evidence that mutation in lectin domain of TM might be related to thrombotic diseases and may help us better understand the physiological roles of TM.

Modulation of Substrate Specificity of the Thrombin-Thrombomodulin Complex for Protein C Versus TAFI Activation by Platelet Factor 4 and Non-Anticoagulant Heparin-Derivatives.

Abstract 3181 Poster Board III-108 The endothelial cell receptor thrombomodulin (TM) is an essential cofactor for thrombin-mediated activation of protein C and the generation of activated protein C (APC), an enzyme with anticoagulant and multiple cytoprotective activities. TM also stimulates thrombin-mediated activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI), resulting in anti-fibrinolytic and anti-inflammatory effects. Based on these properties of TM, several potential strategies for therapeutic intervention have been proposed. In hemophilia patients with inhibitory antibodies, stimulation of TM-dependent TAFI activation might diminish bleeding complications by protection of clots against premature lysis. Alternatively, stimulation of endogenous APC generation in patients with severe sepsis might prove to augment the beneficial effects of APC, as pharmacologic applications of APC in these patients increased survival. Platelet Factor 4 (PF4), a platelet alpha-granule protein, acts as a soluble cofactor for protein C activation by the thrombin-TM complex and stimulated endogenous generation of APC in vivo. Here we show that in contrast to stimulation of protein C activation, PF4 dose-dependently inhibited activation of TAFI by thrombin-TM in a purified system, in normal pooled plasma and on TM-expressing cells. Functional consequences of inhibition of TAFI activation by PF4 were determined in hemophilia A plasma by analysis of clot resistance to tPA-induced fibrinolysis. In hemophilia A plasma, activation of TAFI was dependent on TM due to the ineffective amplification of thrombin generation via the tenase complex. PF4 diminished TM-dependent prolongation of clot lysis times but did not affect clot lysis in the absence of TM. Inhibition of TM-dependent prolongation of clot lysis time by PF4 was dependent on both the TM and tPA concentration used, consistent with the proposed threshold mechanism for inhibition of fibrinolysis by TAFIa. To determine the effect of PF4 on TM-dependent TAFI activation in normal plasma, the inactivation of bradykinin (BK) in plasma by TAFIa during tissue factor induced coagulation was analyzed. Addition of TM to plasma accelerated BK cleavage, i.e., generation of des-Arg9-BK, consistent with TM-mediated stimulation of TAFI activation and subsequent BK cleavage by TAFIa. PF4 neutralized the TM-enhanced BK inactivation and reversed BK and des-Arg9-BK levels to those seen in the absence of TM. Thus, PF4 alters the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, such that PF4 prevents generation of TAFIa's anti-fibrinolytic and anti-inflammatory activities. To find an antidote for PF4's inhibitory effects on TAFI activation, heparin-derivatives were screened for high affinity PF4 binding but greatly reduced anticoagulant activity. N-acetylated heparin (NAc-Hep), but not N-desulfated without N-acetylation or N-acetylated and O-desulfated heparins, retained high affinity binding to PF4 and effectively blocked PF4 binding to immobilized TM. Consistent with high affinity binding of NAc-Hep to PF4, sulfylamine group oxygens were relatively minor determinants of the interactions between heparin and PF4 based on the available structural model. NAc-Hep did not show detectable anticoagulant activity in APTT clotting assays but restored BK conversion to des-Arg9-BK by TAFIa in the presence of PF4, similar to that seen in the absence of PF4. In addition, NAc-Hep reversed PF4-mediated inhibition of TAFI activation and TAFIa's antifibrinolytic functions in clot lysis assays on TM-expressing cells using hemophilia A plasma. Thus, NAc-Hep reversed PF4-mediated inhibition of TAFI activation. In summary, PF4 modulates the substrate-specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation. NAc-Hep, a non-anticoagulant heparin derivative with high affinity for PF4, effectively reversed PF4-mediated inhibition of TM-dependent TAFI activation and restored TAFIa's antifibrinolytic and anti-inflammatory functions. Accordingly, NAc-Hep or similar non-anticoagulant heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis. Disclosures No relevant conflicts of interest to declare.

The Role of Thrombomodulin in Atherosclerosis: From Bench to Bedside

Atherosclerosis remains a leading cause of morbidity and mortality worldwide. In addition to the deposition of cholesterol in the arterial wall, inflammation, cell proliferation and migration play important roles in the pathogenesis of atherosclerosis. Thrombomodulin (TM) is a cell surface-expressed glycoprotein which is predominantly synthesized by vascular endothelial cells and a critical cofactor for thrombin-mediated activation of protein C. Activated protein C is best known for its natural anticoagulant and anti-inflammatory properties. Recent evidence has revealed that TM also has protein C- and thrombin-independent physiological function. This review summarizes recent investigations of TM, giving an overview on the TM unique effects on cellular proliferation, adhesion and inflammation, all of which are important steps in atherosclerosis. The current evidence of TM in the pathogenesis of atherosclerosis will be reviewed, and the associations of TM gene polymorphisms with atherosclerosis are presented. Newly emerging data of the TM in mouse atherosclerosis model demonstrates that TM potentially may have therapeutic role in atherosclerosis.

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin

Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in approximately 60% of the animals and a clinical score of approximately 2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neitherAb(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED(50), nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10- to 100-fold enhancement of the anticoagulant potency, relative to the ED(50) in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.

Antithrombotic Effects of Recombinant Human Soluble Thrombomodulin in a Rat Model of Vascular Shunt Thrombosis

Thrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTM alpha, 0.15 mg/kg rsTM beta, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTM alpha or 0.4 mg/kg rsTM beta resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM.(ABSTRACT TRUNCATED AT 250 WORDS)

The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.

Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.

Protein S and C4b-binding protein levels in patients with stroke: implications for protein S regulation.

Protein S circulates either free or bound to C4b-binding protein (C4b-BP). Only free protein S possesses cofactor activity for protein C, a physiologic anticoagulant. Deficiencies of either protein C or protein S are associated with increased thrombotic risk. Over a 23-month period, 40 patients with low free protein S were identified. Eight of these patients were found to have suffered a stroke. This study examined the relationship between total S, free S, and C4b-BP in 15 healthy adult volunteers, in 20 patients with normal protein S levels, in 40 patients with decreased free protein S levels, and in 8 patients with combined low free S levels and stroke. Total and free protein S and C4b-BP levels were determined using the method of Laurell. In healthy adults, free protein S increased with increasing total protein S (r = 0.60). In patients with normal free S, the total S level increased as C4b-BP increased (r = 0.74), and the free S level remained constant. In patients with low free S, total S did not increase with increasing C4b-BP. In stroke patients, the correlation between free S and total S was actually negative (r = -0.449). Evaluation of dissociation constants for the protein S-C4b-BP complex revealed enhanced binding in patients with low levels of free protein S. A non-C4b-BP protein S binding protein, a previously undescribed regulatory factor which modulates S binding to C4b-BP, or shifts in the amount of non-protein S binding C4b-BP are possible explanations of these results.

Determination of total, free and complexed protein s in plasma by ELISA, and comparison with a standard electroimmunoassay

An enzyme-linked immunosorbent assay (ELISA) for measuring total, free and complexed protein S in plasma was developed. To assay free protein S, C4b-binding protein-bound protein S (C4b-BP-PS) was extracted by addition of polyethyleneglycol (PEG) 6000 (5%, final concentration) to plasma samples. Microtiter plates were coated with rabbit anti-human protein S, and bound protein S was detected with labelled anti-protein S antibody. Diluted plasma samples were incubated in the plates overnight at 22 °C to permit C4b-BP-PS complexes to dissociate. Mean variation coefficients of 2.1 and 3.2% (intra-assay) and 4.3 and 7.9% (inter-assay) were found for total and free protein S assays, respectively. The ELISA measures free and complexed protein S with equal efficiency as is demonstrated by the fact that the sum of free protein S and C4b-BP-PS complex levels in normal individuals, women in their third trimester of gestation and patients with acute deep vein thrombosis (DVT), equaled the level of total protein S present in the corresponding plasma. Total protein S values obtained with the ELISA, in all groups studied, correlated well with those obtained with a standard electroimmunoassay (EIA) (r=0.93; n=40). However, total protein S levels measured by EIA were lower than those assayed by ELISA in pregnant women and in DVT patients. Furthermore, addition of several amounts of purified C4b-BP to NHP, which reduced the recovery of free protein S, did not influence the total protein S values measured by ELISA but slightly decreased the recovery of total protein S measured by EIA. These results indicate the necessity of using assays which accurately and reliably measure the total amount of protein S antigen. After addition of C4b-BP to NHP, the residual functional protein S level was lower than the residual level of free protein S antigen; this lends support to the idea that C4b-BP-PS complex inhibits the activated protein C cofactor activity of protein S.

Binding site for vitamin K-dependent protein S on complement C4b-binding protein.

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Activated protein C (APC) acts as a potent anticoagulant enzyme by inactivating Factor V and Factor VIII. In this study, protein S was shown to increase the inactivation of purified Factor VIII by APC ninefold. The reaction rate was saturated with respect to the concentration of protein S when protein S was present in a 10-fold molar excess over APC. The heavy chain of Factor VIII was cleaved by APC and protein S did not alter the degradation pattern. Factor VIII circulates in a complex with the adhesive protein von Willebrand factor. When purified Factor VIII was recombined with von Willebrand factor, the inactivation of Factor VIII by APC proceeded at a 10-20-fold slower rate as compared with Factor VIII in the absence of von Willebrand factor. Protein S had no effect on the inactivation of the Factor VIII-von Willebrand factor complex by APC. After treatment of this complex with thrombin, however, the actions of APC and protein S towards Factor VIII were completely restored. In hemophilia A plasma, purified Factor VIII associated with endogenous von Willebrand factor, resulting in a complete protection against APC (4 nM). By mixing hemophilic plasma with plasma from a patient with severe von Willebrand's disease, we could vary the amount of von Willebrand factor. 1 U of von Willebrand factor was needed to provide protection of 1 U Factor VIII. Also in plasma from patients with the IIA-type variant of von Willebrand's disease, Factor VIII was protected. In von Willebrand's disease plasma, which was depleted of protein S, APC did not inactivate Factor VIII. These results indicate that protein S serves as a cofactor in the inactivation of Factor VIII and Factor VIIIa by APC and that von Willebrand factor can regulate the action of these two anticoagulant proteins.

Plasma protein s activity measured using protac, a snake venom derived activator of protein C

Functional activity of protein S, a cofactor of activated protein C-dependent inhibition of blood coagulation, in human plasma was measured by using Protac, a snake venom derived activator of protein C. This assay appeared to be specific for protein S, because 1) the activated partial thromboplastin time of protein S-depleted plasma depended on the purified protein S added in the presence of Protac; and 2) the level of protein C in plasma sample (0 to 10 μg/ml) had no influence on the clotting time. The cofactor activity of protein S in the plasma of normal men (n=16) and women (n=14) was 99.4 ± 23.8% and 98.6 ± 24.5% respectively. The protein S activity in the plasma of pregnant women at pre- and post-partum (n=14), and that in the plasma of patients under warfarin therapy (n=20) were 46.2 ± 18.9%, 45.8 ± 19.6% and 24.0 ± 15.7%, respectively. In these plasmas, the levels of protein S activity were lower than those of total protein S antigen, but were similar to those of free protein S antigen. In 16 patients out of two families with congenital protein S deficiency, the protein S activity, the free antigen and the total antigen were 9.4 ± 6.9%, 13.3 ± 4.6% and 57.4 ± 20.7%, respectively. There was no significant relationship between the level of protein S activity and that of a complemental C4b-binding protein antigen in any of these patients.