Thrombomodulin Concentrations Research Articles

Decreased plasma soluble thrombomodulin levels as a risk factor for pulmonary thromboembolism

To observe the changes of the plasma soluble thrombomodulin (sTM) concentrations in patients with pulmonary thromboembolism (PTE) and assess the association between plasma sTM concentration and the risk of PTE. We measured plasma concentrations of sTM, protein C (PC) and protein S (PS) and examined the association between those plasma markers and the risk of PTE in 72 selected PTE patients and 70 controls. Significant difference was identified in plasma sTM level between overall PTE patients and controls. Female PTE patients had statistically lower sTM concentrations than male patients. A positive linear correlation was found between plasma sTM concentration and age in female patients. Decreased plasma sTM concentration was associated with a continuously and progressively increased risk for PTE in women. The concentrations of plasma PC and PS did not differ between groups and no significant quantitative association was identified between the risk of PTE and the levels of plasma PC or PS. Decreased plasma sTM concentration is associated with an increased risk of PTE in women.

Serum thrombomodulin level relates to the clinical course of disseminated intravascular coagulation, multiorgan dysfunction syndrome, and mortality in patients with sepsis*

To determine serum concentrations of thrombomodulin, the marker of endothelial injury, in patients with sepsis-induced disseminated intravascular coagulation and multiple organ dysfunction syndrome and to investigate the independent association between this marker and the development of disseminated intravascular coagulation, multiple organ dysfunction syndrome, and mortality. A prospective cohort study. A 37-bed intensive care unit of a tertiary care hospital. One hundred consecutive patients with sepsis. Serum thrombomodulin concentrations and the development of disseminated intravascular coagulation and multiple organ dysfunction syndrome were determined in patients on days 1 and 3 of sepsis. These data were used to determine an association between day 1 thrombomodulin concentrations and development of disseminated intravascular coagulation, multiple organ dysfunction syndrome, and mortality during intensive care unit stay. These connections were determined by the Cox proportional hazards model and plotting of receiver operating characteristic curves. Day 1 serum concentrations of thrombomodulin were higher in patients with disseminated intravascular coagulation (11.1 +/- 1.0 vs. 5.3 +/- 0.5 ng/mL; p < .0001) or multiple organ dysfunction syndrome (10.3 +/- 0.7 vs. 4.3 +/- 0.4 ng/mL; p < .0001) than those without, respectively. In patients with resolved disseminated intravascular coagulation (4.9 +/- 0.5 vs. 8.9 +/- 0.9 ng/mL, day 3 vs. day 1, p = .005) or multiple organ dysfunction syndrome (6.3 +/- 1.4 vs. 12.0 +/- 1.6 ng/mL, day 3 vs. day 1, p < .0001) on day 3 of sepsis, day 3 levels of thrombomodulin were down from day 1. Thrombomodulin concentration independently predicted the development of disseminated intravascular coagulation (hazard ratio 1.13, p < .0001), multiple organ dysfunction syndrome (hazard ratio 1.12, p < .0001), and mortality (hazard ratio 1.19, p < .0001) during intensive care unit stay. The area under the receiver operating characteristic curve showed that day 1 serum thrombomodulin levels had good discriminative power in predicting the development of disseminated intravascular coagulation (0.811), multiple organ dysfunction syndrome (0.896), and mortality (0.803) during intensive care unit stay. Endothelial cell injury is critical in the progression from disseminated intravascular coagulation to multiple organ dysfunction syndrome and subsequent mortality in septic patients. Serum concentrations of thrombomodulin may be used in monitoring disseminated intravascular coagulation and multiple organ dysfunction syndrome in these patients.

Thrombomodulin gene polymorphisms in brain infarction and mortality after stroke

Thrombomodulin is expressed at the surface of endothelial cells and controls thrombin generation and thrombin-induced platelets and vascular cell activation. Several thrombomodulin gene polymorphisms have been associated with coronary events and brain infarction. In a previous analysis from the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC) study, we found that soluble thrombomodulin (sTM) concentration modulated the risk of and prognosis for brain infarction. In 474 brain infarction cases and 483 controls from the GENIC study, we investigated the relationship between three thrombomodulin gene polymorphisms (-1748G/C, -1208/-1209delTT, +1418C/T) and sTM levels, brain infarction risk and 5-year mortality after stroke. The three polymorphisms were in linkage disequilibrium and defined three major haplotypes with no influence on sTM concentration (all P values > 0.16). Single locus and haplotype analyses found no significant association with brain infarction, even when the analysis was restricted to individuals without a vascular history. After 5 years of follow-up, we found no relationship with vascular or total mortality (all P values > 0.64). Our results suggest that these three thrombomodulin gene polymorphisms do not contribute to sTM level variations and are not associated with risk of brain infarction and mortality after stroke.

Correlation between circulating endothelial cell counts and plasma thrombomodulin levels as markers for endothelial damage

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

Endothelial cell damage in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia type II is a severe complication of heparin treatment that may result in thrombosis. When thrombosis occurs it carries a 50% mortality rate. The exact pathophysiology is not fully understood but in the majority of cases it is associated with the production of heparin/platelet factor 4 antibodies. The endothelium provides a protective anticoagulant surface over which blood flows. Perturbation of the endothelial cells causes a reversal of the anticoagulant properties of the cells to that of a procoagulant surface. This is often due to release or down-regulation of the anticoagulant membrane proteins such as thrombomodulin and up-regulation of procoagulant factors such as tissue factor. We studied 10 patients in our cardiothoracic institute with clinically and laboratory-confirmed heparin-induced thrombocytopenia type II for evidence of endothelial cell damage. There was a statistically significant rise in the concentrations of von Willebrand factor (P < 0.0001) and soluble thrombomodulin (P = 0.004) when patients with heparin-induced thrombocytopenia type II were compared with healthy laboratory controls and patients having had cardiopulmonary bypass surgery (von Willebrand factor 324 versus 103 versus 108 U/dl and soluble thrombomodulin 9.5 versus 2.3 versus 1.2 ng/ml, respectively). Our findings suggest that endothelial cell damage is a major factor in the pathophysiology of heparin-induced thrombocytopenia.

The Role of Endothelium-Derived Mediators in Exercise-Induced Bronchoconstriction

Background: Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition whose pathogenesis and effect on airways remain unclear. The aim of the study was to evaluate the changes in endothelial-derived mediators following intensive exercise in asthmatic patients. Methods: The study was conducted in a group of 25 asthmatic patients (15 with EIB, 10 without EIB) and 8 healthy volunteers. Changes induced by intensive exercise in the plasma concentrations of endothelin-1 (ET-1), thrombomodulin and soluble E-selectin were determined. Moreover, the possible correlations of these measurements with the results of baseline lung function, bronchial hyperreactivity (BHR), exhaled nitric oxide (F_ENO), baseline eosinophil cationic protein, peripheral blood eosinophilia and total IgE were investigated. Results: In the group of asthmatics with EIB, statistically significantly higher baseline concentrations of ET-1 and soluble E-selectin compared with asthmatics with negative exercise test results were revealed. In this group of patients, ET-1, thrombomodulin and soluble E-selectin concentrations significantly increased after exercise. Baseline concentrations of ET-1 as well as an increase in soluble E-selectin concentrations 60 min after exercise showed a statistically significant correlation with baseline eosinophil cationic protein, F_ENO and BHR. The increase in plasma concentrations of ET-1 15 min after exercise and soluble E-selectin 60 min after exercise showed a statistically significant correlation with the increase in F_ENO and BHR to histamine 24 h after exercise. Conclusions: We suggest that during EIB changes in the function of pulmonary endothelium occur and these changes may influence inflammation of the airway and remodeling in asthmatic patients.

Stimulation of the peroxisome proliferator-activated receptor γ (PPARγ) and the expression of selected blood monocyte cytokine genes in diabetic macroangiopathy

Monocytes and macrophages play a key role in the progression of atheromatous changes. The peroxisome proliferator-activated receptor γ (PPARγ) can limit macroangiopathy through the control of cytokine transcription. The objectives of this study were to examine the influence of PPARγ and its agonist (rosiglitazone) on the TNFα, IL-6, IL-8 and IL-10 gene expression in monocytes of patients with diabetic macroangiopathy and to analyse obtained results in context of selected atherogenic factors ant direct indicators of endothelial lesion. TNFα, IL-6, IL-8, IL-10 and PPARγ gene expression was assessed in peripheral blood monocytes in 45 patients with type 2 diabetes before and following 22 weeks of rosiglitazone therapy (real-time PCR [Applied Biosystems]). As indicators of endothelial lesion, concentration of thrombomodulin (immunoassay [Diagnostica Stago ®]) and amount of circulating blood endothelial cells (immunofluorescence method with MoAb CLB-HEC19) were determined. Following rosiglitazone therapy, a statistically significant downward tendency of TNFα ( p = 0.026) and IL-8 ( p = 0.008) gene expression was noted. Before and following rosiglitazone treatment, PPARγ, IL-6 and IL-10 gene expression was undetectable in studied monocytes in vivo. In conclusion, TNFα and IL-8 play an important role in monocyte atherogenic activity. Rosiglitazone reduces monocyte proinflammatory readiness by influencing the expression of selected atherogenic cytokines (PPARγ-independent pathway).

Plasma Thrombomodulin (TM) Concentration in Cats with Cardiomyopathies

Plasma Thrombomodulin (TM) Concentration in Cats with Cardiomyopathies

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.

Impaired Flow-Mediated Vasodilation, Carotid Artery Intima-Media Thickening, and Elevated Endothelial Plasma Markers in Obese Children: The Impact of Cardiovascular Risk Factors

Childhood obesity contributes to the development of adult obesity and subsequent cardiovascular disease. The present study aimed to assess vascular status (flow-mediated vasodilation [FMD], intima-media thickness [IMT]) and to analyze plasma surrogate endothelial markers (von Willebrand factor [vWf], E-selectin, and thrombomodulin) in obese children as compared with controls. Associations between early morphologic and functional vascular changes, surrogate soluble markers of early atherosclerosis, and the cardiovascular risk profile were determined. We examined 32 obese children versus 20 control subjects. All of the children underwent identical screening, comprehensive risk factor assessment, and measurements of E-selectin, vWf, thrombomodulin, FMD, and IMT. Compared with controls, obese children demonstrated significantly impaired FMD and increased IMT. Concentrations of soluble E-selectin and thrombomodulin were significantly elevated in obese children, whereas vWf showed no significant differences between obese children and controls. FMD, IMT, E-selectin, and thrombomodulin were significantly associated with various risk factors, including the extent of obesity, arterial hypertension, fibrinogen, C-reactive protein, and low physical fitness. The present study documented increased IMT, impaired endothelial function, and elevated plasma markers of endothelial activation and injury in obese children. Morbid obesity, arterial hypertension, subclinical inflammation, and low physical fitness formed a risk profile associated with the risk of early atherosclerosis in these children. Sonographic assessment of vascular status and the estimation of soluble endothelial plasma markers, combined with comprehensive risk factor screening, may form a rationale to identify high-risk children susceptible to early atherosclerotic disease and to monitor vascular changes during follow-up studies and therapeutic measures.

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.

Decreased protein C activation in patients with fulminant hepatic failure

Objective. Abnormalities of the blood coagulation system have an influence on outcome in patients with fulminant hepatic failure (FHF). The protein C (PC) pathway is one of the main modulators of the blood coagulation system. The role of the PC pathway in FHF is not clear. In the present study, we evaluated endothelial cell injury and the grade of activated protein C (APC) generation in FHF patients. Material and methods. The effect of APC on the expression of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 from LI90 stellate cells was also evaluated. This study comprised 5 patients with FHF, 6 with acute hepatitis (AH), 12 with chronic hepatitis (CH) and 20 healthy subjects. Results. The plasma concentrations of thrombin-antithrombin complex and thrombomodulin were significantly increased in FHF patients compared with those in AH patients and healthy subjects. The circulating levels of activated protein C-protein C inhibitor (APC-PCI) complex and the APC-PCI/PC ratio were significantly decreased in patients with FHF compared to healthy controls. APC significantly inhibited in vitro the expression of TNFα and MCP-1 from LI90 stellate cells. Conclusions. This study demonstrated enhanced endothelial cell injury in association with decreased PC activation and hypercoagulability in FHF.

Protecting the Endothelial Integrity of Internal Thoracic Arteries

Previous functional studies on human internal thoracic arteries, comparing the effect of the traditional harvesting method (occlusion with a clip) with a method leaving the artery perfused, revealed considerably impaired endothelial function associated with enhanced contractility after clipping. We have now investigated whether these observations could be correlated (1) with plasma markers of endothelial dysfunction, and (2) with structural changes in the endothelial layer. 32 patients were randomly distributed into groups of clipped and perfused arteries. Arterial blood samples were obtained from both the artery and extracorporeal circulation to determine sP-selectin, sE-selectin, sL-selectin, and thrombomodulin using enzyme-linked immunosorbent assay. Arteries from three patients were examined by scanning electron microscopy. Concentrations of sP-selectin and thrombomodulin were significantly higher in plasma from clipped arteries compared to perfused arteries, whereas sE-selectin and sL-selectin concentrations were similar within the groups. Scanning electron microscopy revealed significant structural changes and loss of endothelial cells in clipped arteries. Biochemical and structural results support our findings that leaving the internal thoracic artery perfused preserves endothelial function in the arterial graft.

Changes in the plasma concentration of soluble thrombomodulin in patients with severe carotid artery stenosis after eversion endarterectomy

The purpose of the study was to investigate the putative role of soluble thrombomodulin (sTM) in severe carotid artery stenosis. We prospectively studied 64 patients who were undergoing carotid endarterectomy (2001-2003). Plasma sTM concentration was determined in each patient before surgery and at 14 months postsurgery. -308 TNF-alpha promoter polymorphism was also determined. Strong negative correlation was found between the preoperative duplex scan values and the plasma sTM concentrations (R = -0.418, p = 0.0006). Patients with 308 A TNF-alpha genotype had significantly lower (p = 0.0415) preoperative sTM values than their counterparts with no such polymorphism. Soluble TM concentrations measured in plasma samples taken at the end of the postsurgical follow-up period of 14 months duration were significantly higher compared to the preoperative values (p < 0.0001). Our present findings indicate that sTM may be adsorbed to the atherosclerotic plaques or inflamed endothelium in carotid arteries. The pathological significance of this adsorption remains to be determined.

Epidural Analgesia Prevents Endotoxin-Induced Gut Mucosal Injury in Rabbits

In the present study, we evaluated the effect of epidural analgesia on the alterations of gut barrier function elicited by endotoxin in rabbits. After the placement of an epidural catheter, 28 male rabbits were randomized into either 0.5% lidocaine (group E) or saline (group C) group. The solutions (0.4 mL/kg) were epidurally injected, followed by continuous infusion (0.1 mL . kg(-1) . h(-1)) throughout the study period. Under a continuous infusion of lipopolysaccharide (15 microg . kg(-1) . h(-1)), mean arterial blood pressure, intramucosal pH, and plasma thrombomodulin concentrations were measured. At 4 h, mean arterial blood pressure was lower (P < 0.05), intramucosal pH was higher (P < 0.01), and the progression of hemodilution more profound (P < 0.05) in group E versus group C, whereas plasma thrombomodulin levels were increased to a similar extent between the groups. With less wet-to-dry weight ratio of ileum, histopathological injury scores of gut mucosa were significantly less in group E versus group C (P < 0.01). In a separate series of experiments (n = 10 each group), mucosal permeability in group E was significantly less compared with group C (P < 0.05). Collectively, these studies showed that despite a significant decrease of perfusion pressure and arterial oxygen content, epidural analgesia minimized endotoxin-induced functional and structural injury of gut mucosa possibly through endothelium-independent mechanisms.

The Affinity of Protein C for the Thrombin·Thrombomodulin Complex Is Determined in a Primary Way by Active Site-dependent Interactions

The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C (PC). Interactions between PC and extended surfaces, likely contributed by TM within the IIa.TM complex, have been proposed to play a key role in PC activation. Initial velocities of PC activation at different concentrations of PC and TM could be accounted for by a model that did not require consideration of direct binding interactions between PC and TM. Reversible inhibitors directed toward the active site of IIa within the IIa.TM complex behaved as classic competitive inhibitors of both peptidyl substrate cleavage as well as PC activation. The ability of these small molecule inhibitors to block PC binding to the enzyme points to a principal role for active site-dependent substrate recognition in determining the affinity of IIa.TM for its protein substrate. Selective abrogation of active site docking by mutation of the P1 Arg in PC to Gln yielded an uncleavable derivative (PC(R15Q)). PC(R15Q) was a poor inhibitor (K(i) >or= 30 microm) of PC activation as well as peptidyl substrate cleavage by IIa.TM. Thus, inhibition by PC(R15Q) most likely results from its ability to weakly interfere with active site function rather than by blocking extended interactions with the enzyme complex. The data suggest a primary role for active site-dependent substrate recognition in driving the affinity of the IIa.TM complex for its protein substrate. Interactions between PC and extended surfaces contributed by IIa and/or TM within the IIa.TM complex likely contribute in a secondary or minor way to protein substrate affinity.

Does activated protein C-resistant factor V contribute to thrombin generation in hemophilic plasma?

In this study we assessed the role of factor V (FV) inactivation in hemophilic plasma with particular reference to the activated protein C (APC)-resistant variants FV-R506Q (FV Leiden) and FV-R306T (FV Cambridge). Purified recombinant full-length FV carrying these single substitutions and FV-R306T/R506Q were used in thrombin generation experiments. Plasma was first immunodepleted of FV, and subsequently of factors VIII, IX, or combinations thereof. Thrombin generation was initiated by low concentrations of recombinant tissue factor. Recombinant soluble thrombomodulin (TM) was used to trigger the APC system. Surprisingly, TM concentrations that reduced thrombin generation in normal plasma by no more than 50% virtually abolished thrombin formation in plasma deficient in the factor VIII/IX complex. This was already apparent at TM levels as low as 0.1 nmol L(-1). By varying the concentrations of purified (activated) protein C to plasma that was additionally depleted of protein C, we confirmed that impaired thrombin generation indeed was the result of the action of APC. In contrast, this did not occur when FV-depleted plasma had been reconstituted with FV-R306T/R506Q. Addition of FV-R306T or FV-R506Q partially reduced prothrombin activation, demonstrating the involvement of both APC cleavage sites. FV inactivation also occurred on the surface of human microvascular endothelial cells. Apparently, these cells express sufficient TM to down-regulate thrombin production via the APC pathway. We further conclude that in hemophilic plasma this pathway can induce a secondary defect because of premature FV inactivation. It therefore seems conceivable that APC-resistant FV has the potential of alleviating hemophilic bleeding.

Active Site-Dependent Substrate Recognition Plays a Primary Role in Determining the Affinity of the Thrombin-Thrombomodulin Complex for Protein C.

The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C. The TM-dependent enhancement of the anticoagulant function of IIa has alternately been considered to primarily arise from an allosteric modulation of the active site of the protease or from the ability of TM to bind protein C and participate in its presentation to the enzyme. We have applied established kinetic strategies to investigate the relative contributions of active site interactions versus interactions with extended surfaces in the action of the IIa-TM complex on human protein C. Initial velocity studies yielded equivalent kinetic constants for the hydrolysis of L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) by IIa alone or IIa saturated with TM. The reversible active site-directed ligands Nα-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581) and 4-amino-phenylpyruvic acid (APPA) were found to act as classical competitive inhibitors of S2366 cleavage by either IIa or IIa-TM. This finding is consistent with the limited interactions expected between S2366 and the active site of either IIa or IIa-TM. Equivalent inhibition constants were obtained for IIa or IIa-TM with either inhibitor that were in good agreement with directly determined equilibrium dissociation constants for I-2581 (IIa: Kd =25 ± 1 nM, IIa-TM: Kd=16 ± 1 nM) or APPA (IIa: Kd = 530 ± 90 nM, IIa-TM: Kd = 370 ± 40 nM). Similar kinetic and equilibrium dissociation constants obtained for IIa and IIa-TM indicate that obvious changes in the active site of IIa induced by TM are not detectable by this series of active-site directed probes. In contrast to peptidyl substrate cleavage, protein C activation by IIa (Km=177 ± 31 μM, Vmax/Et=0.004±0.001 s−1) was greatly enhanced by the addition of saturating concentrations of TM to form the IIa-TM complex (Km=12 ± 1 μM, Vmax/Et=0.28±0.01 s−1). However, both active site-directed reversible inhibitors continued to act as classical competitive inhibitors of protein C activation by IIa-TM (I-2581: Ki = 20 ± 1 nM; APPA: Ki = 470 ± 20 nM). Mutually exclusive binding interactions of protein C and active site-directed inhibitors indicate a principal role for active site-dependent interactions in determining the affinity of IIa-TM for protein C. This conclusion was further investigated by using a recombinant derivative of protein C with the P1 Arg mutated to Gln (PCR169Q) that rendered the zymogen uncleavable by either IIa or the IIa-TM complex. PCR169Q was a poor inhibitor (Ki>>50 μM) of protein C activation or S2236 cleavage implying a greatly reduced affinity for IIa-TM. These data suggest a primary role for active site-dependent substrate recognition in driving the affinity of IIa-TM for protein C. In contrast to the established role for exosites in action of IIa on many of its other substrates, possible interactions between protein C and extended surfaces within the IIa-TM complex likely contribute in a secondary way in determining the affinity of this enzyme complex for its biological substrate.

Correlation of glomerular permeability, endothelial injury, and postoperative multiple organ dysfunction.

To determine whether the degree of microalbuminuria correlates with the extent of endothelial cell injury, the severity of illness, and the magnitude of multiple organ dysfunction in patients who undergo emergency surgery. We measured the urinary albumin : creatinine ratio (ACR) within 24 h after surgery in 31 patients and examined its relationship with various clinical measurements. The ACR increased during the first 24 h postoperatively. The log ACR correlated with the serum thrombomodulin concentration measured on the same day, but not with the level of plasma von Willebrand factor antigen. The increase in the log ACR correlated with the acute physiology and chronic health evaluation score (APACHE III), the simplified acute physiology score, the multiple organ dysfunction score, and the score of sequential organ failure assessment (SOFA) calculated on the same day, and the blood volume lost during the operation. The log ACR did not correlate with the white blood cell count or the serum C-reactive protein measured at the same time. The log ACR correlated with SOFA on postoperative days 3, 7, and 10, and mortality increased in accordance with the increase in log ACR. The urinary ACR correlated with the extent of endothelial cell injury, the severity of illness, and the magnitude of multiple organ dysfunction.

TLR4/Asp299Gly, CD14/C-260T, plasma levels of the soluble receptor CD14 and the risk of coronary heart disease: The PRIME Study.

TLR4 and CD14 are two components of the LPS receptor complex, which are considered to play a key role in the pathogenesis of atherosclerosis. TLR4/Asp299Gly and CD14/C-260T polymorphisms are thought to modulate the activity of this complex. The aim of the study was to examine the association between the TLR4/Asp299Gly and CD14/C-260T polymorphisms, plasma levels of the soluble receptor CD14 (sCD14), and the incidence of coronary heart disease (CHD) in a prospective cohort (the PRIME Study) of 9758 healthy men aged 50-59 years recruited in France and Northern Ireland. A nested case-control design was used, comparing the 249 participants who developed a CHD event during the 5-year follow-up with 492 population- and age-matched control subjects. The two polymorphisms were genotyped and baseline plasma concentrations of sCD14 were measured. None of the two polymorphisms, or sCD14 levels, either considered alone or in combination, were associated with the risk of CHD. The CD14/C-260T allele was associated with increased plasma concentrations of soluble thrombomodulin and vascular cell adhesion molecule-1 and, to a lesser extent, sCD14. No relationship was observed between the TLR4 polymorphism and, any of the inflammatory and endothelial markers measured. The TLR4/Asp299Gly and CD14/C-260T polymorphisms and plasma sCD14 concentrations do not appear as significant predictors of the risk of CHD in healthy individuals.