Studies on the interaction between vitamin K-dependent protein S and complement regulator C4b-binding protein: localization of binding sites and identification of a possible function of the complex

Complement is a cascade-like system that is part of the innate immune defence. It is an explosive system, potentially harmful also for the host cells, and needs to be strictly regulated. An important down-regulator of complement is C4bbinding protein (C4BP). C4BP contains two different types of subunits, seven identical &#102 -chains and one unique &#103 -chain. The &#102 -chains bind to C4b, C4BP's target in the complement system. The &#103 -chain binds to vitamin K-dependent protein S. Approximately 70% of all protein S in plasma circulates in a high affinity complex with C4BP. Free protein S, the remaining 30%, functions as an important cofactor in the anticoagulant system. The reason for the complex formation between C4BP and protein S has remained an intriguing enigma. Protein S has a very high affinity to negatively charged phospholipids for protein S. One area where such phospholipids are present is the surface of the apoptotic cell, where the exposure of phosphatidylserine is an early event. Physiological apoptosis is characterized by a lack of inflammatory response in surrounding tissues, indicating that cells are rapidly cleared before leaking cytoplasmic components into the extracellular space. A number of studies demonstrate that early complement proteins are important for the removal of apoptotic cells, but that subsequent assembly of later complement components and anaphylatoxin release must be prohibited in order not to provoke an inflammatory response. We demonstrate that protein S localizes C4BP to the surface of apoptotic cells via binding to the exposed phosphatidylserine. The C4BP attached to the apoptotic cell through protein S was still able to bind C4b, suggesting that C4BP retains its physiological function also when localized to the apoptotic cell surface. In addition, we have also pinpointed a hydrophobic binding site for protein S on C4BP. The binding studies between C4BP and protein S were performed on recombinant proteins where mutations had been introduced. Mutations were chosen based on a 3D-homology model of the C4BP &#103 -chain.     

Potential of macrolide antibiotics to inhibit protein synthesis of Pseudomonas aeruginosa: suppression of virulence factors and stress response

Recently we have reported that sub-minimum inhibitory concentrations (MICs) of macrolide antibiotics, such as erythromycin, clarithromycin, and azithromycin, induce loss of viability of Pseudomonas aeruginosa with longer incubation periods. In the present study we examined the effects of sub-MICs of macrolide antibiotics on protein synthesis and the expression of heat shock proteins (Gro-EL) in P. aeruginosa and the association of these factors with the viability of P. aeruginosa. In seven strains of P. aeruginosa clinical isolates, inhibition of protein synthesis was generally observed in bacteria grown on agar with sub-MIC azithromycin (8 μg/ml) at 24 h, and this was followed by loss of viability after an additional 24-h incubation. The inhibition of protein synthesis was shown in bacteria treated with sub-MICs of erythromycin and clarithromycin, but not with sub-MICs of other antibiotics examined (josamycin, tobramycin, ofloxacin, clindamycin, and ceftazidime) even at relatively high sub-MICs. In the heat shock condition (45°C), strong expression of the heat shock protein Gro-EL was induced in bacteria grown on antibiotic-free medium, whereas there was a delay of such a response in bacteria exposed to 4 μg/ml of azithromycin. Reflecting these results, an abrupt reduction of viability in azithromycin-treated bacteria was observed within 3 h in the heat shock condition. Western blot analysis, using specific antibody for Gro-EL, demonstrated that erythromycin, clarithromycin, and azithromycin, at concentrations of 0.5–2 μg/ml, inhibited the expression of lower-molecular weight Gro-EL bands in the constitutive state. These results indicated that macrolides, at concentrations far below the MICs, suppressed protein synthesis in P. aeruginosa, an effect which may be associated with the inhibition of P. aeruginosa virulence and its loss of viability with longer incubation. Moreover, it is likely that the macrolides may sensitize bacteria to stresses, as these antibiotics induced alterations in a major stress protein, Gro-EL, in constitutive and inducible states. Received: September 13, 1999 / Accepted: December 7, 1999     

Hemostatic parameters and platelet activation by flow-cytometry in normal pregnancy: a longitudinal study

Summary Nineteen pregnant women with uncomplicated pregnancies were studied during the first, second, and third trimesters. We measured the following hemostatic parameters: prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, protein C, protein S, platelet number and volume. Platelet function was examined by a cytofluorimetric method, using an anti-GPM-140 antibody which is directed against a platelet α granule membrane protein. Activated platelets were expressed as a percentage of the GMP-140-positive platelets over total platelets. Fibrinogen levels showed a steady increase during pregnancy; conversely prothrombin time, activated partial thromboplastin time, protein C, and antithrombin III showed no significant modifications and remained within the reference range. There was a decrease of protein S activity throughout pregnancy, although protein S antigen did not follow this trend. The decrease occurred early in pregnancy and persisted during the second and third trimesters, reaching a stable plateau. We observed no platelet volume change or activation: the percentage of activated platelets was within the normal reference range, even in late pregnancy.     

Screening for inherited thrombotic disorders

Summary In order to determine a scheme for the screening of inherited thrombotic disorders, abnormalities considered as predisposing to thrombosis have been reviewed. Owing to the low prevalence of biological alterations, a selection of patients is required: documented venous thromboses, possibly at unusual sites (mesenteric vein, portal, cerebral veins), occurring before the age of 40 in patients with a positive family history of thromboses are relatively frequently associated with coagulation abnormalities. In addition, patients with skin necrosis at the initiation of oral anticoagulants, or with repeated superficial vein thrombosis or unexplained arterial occlusions at a young age might be included for screening. Tests have also to be selected. Some abnormalities, such as congenital deficiencies in antithrombin III, protein C and protein S, are recognized risk factors and have to be searched. Some others cannot be at present considered as definite risk factors (e.g., dysfibrinogenemias or deficiencies in factor XII), but their detection is easy by routine tests: prothrombin time, fibrinogen assay. Other abnormalities are recognized risk factors (or not) and need specific uncommon tests (e.g., study of fibrinolysis). Each time a biological abnormality is found, it is important to verify it is isolated since combined deficiencies have been observed and we should be able to answer the question whether the abnormality is the cause or the consequence of thrombosis, or a coincidence. Finally, in our experience, even in well selected patients, a coagulation disorder is detected in less than 30% of patients, so that new tests are needed to improve our knowledge in this field. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Treatment with stanazolol of type I protein C deficiency in an Italian family

Summary Functional and immunological assays specific for protein C were employed in the study of a family with congenital protein C deficiency associated with venous thromboembolism. By both assays, four members of the family belonging to two generations had half-normal PC levels. These findings, as well as the normal mobility of the protein in crossed immunoelectrophoresis, suggest that PC deficiency in this family is due to the decreased synthesis of a functionally normal protein. In one member of the family oral administration of the anabolic steroid stanazolol increased PC levels until normal values, suggesting that the defect can be overcome by pharmacological stimulation of protein synthesis. This work was supported in part by grants from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy, ‘Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Genetiche.     

Protein S and protein C gene mutations in Japanese deep vein thrombosis patients

OBJECTIVES: Coagulation factor V Leiden has not been detected in Japanese patients suffering from thrombosis. Hitherto, the constitutional background of Japanese thrombotic patients has never been systematically examined. We have performed a systematic investigation to determine pathogenesis for deep vein thrombosis in a Japanese population. DESIGN AND METHODS: Routine coagulation and fibrinolysis tests were performed to determine the activities of protein S, protein C, antithrombin, plasminogen and fibrinogen. Gene analysis was performed in thrombotic patients having low activities of these factors. RESULTS: Our study indicates that the frequency (19/85 = 0.22) of mutations of protein S gene in the Japanese patients was 5-10 times higher than that of mutations of protein S gene in Caucasian patients, and the frequency (8/85 = 0.09) of mutations of protein C gene was almost three times higher than that of Caucasian patients. The frequency of antithrombin gene mutation was similar in both populations. CONCLUSION: Our study reinforces that the genetic anomaly in the protein S/protein C anticoagulation system is an important risk factor for thrombophilia in the Japanese population.     

Familial protein S deficiency is associated with recurrent thrombosis.

Recent studies have demonstrated that protein C deficiency is associated with recurrent familial thrombosis. In plasma, activated protein C functions as an anticoagulant. This anticoagulant response requires a vitamin K-dependent plasma protein cofactor, referred to as protein S. Since the anticoagulant activity of activated protein C is dependent on protein S, we hypothesized that patients lacking functional protein S might have associated thrombotic disease. Two related individuals with otherwise normal coagulation tests are described whose plasma is not effectively anticoagulated with activated protein C. Addition of purified human protein S to their plasma restores a normal anticoagulant response to activated protein C. We have developed a rapid one-stage clotting assay for protein S to quantitate the level of protein S in their plasma. Plasma is depleted of protein S by immunoadsorption with immobilized antiprotein S antibodies. The resultant plasma responds poorly to activated protein C, but is effectively anticoagulated in a dose-dependent fashion upon addition of purified protein S or small quantities of plasma. The affected individuals possess less than 5% protein S activity. Using Laurell rockets, protein S antigen was detected in the plasma but was at reduced levels of 13 and 18% in the two individuals. When the barium eluate of the patient plasma was chromatographed on quaternary aminoethyl Sephadex, a single peak of protein S antigen devoid of protein S anticoagulant cofactor activity was detected early in the chromatogram. In contrast, the barium eluate from normal donors separated into two peaks, one emerging early and also devoid of anticoagulant cofactor, and the second peak with anticoagulant activity emerging later. The first peak of protein S antigen, from both the normal donor and the patient, chromatographed in the region of the complement component C4-binding protein-protein S complex. These studies suggest that protein S deficiency may result in recurrent thrombotic disease.     

抗磷脂综合征合并静脉血栓和蛋白C活性异常1例研究报告

目的：探讨抗磷脂综合征（antiphospholipid syndrome，APS）患者静脉血栓形成的原因。方法：对1例APS患者用发色底物法测定蛋白C活性（PC：A）、蛋白S活性（PS：A）和抗凝血酶活性；用ELISA方法测定PC、血浆纤溶酶原、血浆组织型纤溶酶原激活物、血浆纤溶酶原激活抑制物-1、α2-抗纤溶酶抗原和抗心磷脂抗体（ACA）。活化蛋白C抵抗（APC—R）检测结果以受检者血浆加入APC后的APTT与未加APC血浆的APTT的比值表示，比值〈2．0时为APC-R阳性。狼疮抗凝物质（LA）检测使用以dRVVT为基础的商品化试剂盒。采用PCR扩增和直接测序，检测PC的基因及FVLeiden和凝血酶原G20210A的突变。结果：本例患者LA和APC—R阳性，PC：A降低，PC抗原量增加，其他结果正常．PC基因所有外显子测字结果正常，FVLeiden突变和凝血酶原G20210A突变未检出。结论：LA可能通过抑制PC途径导致患者发生血栓，联合检测ACA、APC-R、抗凝蛋白抗原及活性有利于血栓性疾病的病因学诊断。     

两个终止密码子导致的遗传性蛋白C和蛋白S缺陷症

目的:研究1个蛋白C(PC)和1个蛋白S(PS)缺陷症家系的表型诊断和基因特征。方法:PC活性(PC:A)和PS活性(PS:A)用发色底物法测定;PC抗原(PC:Ag)和PS抗原(PS:Ag)用ELISA方法测定。用PCR扩增PC和PS基因各个外显子及其侧翼序列,用直接测序法检测突变点。利用逆转录PCR(RT-PCR)分析PCmRNA水平变化。同时利用蛋白印迹分析血浆中PC含量的变化。结果:先证者1的PC:A为49%,PC:Ag为1.34mg/L,基因检测发现PC基因9号外显子的8831有G→A杂合无义突变,导致Trp372Stop;先证者2的PS:A为29%,PC:Ag为8.3mg/L,14号外显子Gln522(CAG)→Stop(TAG)。结论:G8831A杂合突变引起Trp372Stop,其可导致遗传性PC缺陷症;Gln522Stop可导致遗传性PS缺陷症。     

Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals.

BACKGROUND: Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. OBJECTIVES: Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. METHODS: An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. RESULTS: Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). CONCLUSIONS: Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen.     

Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture

The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied. Caffeine (0.2–2 mM) inhibited both DNA and protein synthesis of the cells. Addition of EDTA in the growth medium inhibited both DNA and protein synthesis. Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10μM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA). Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc. Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis. The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.     

Laboratory diagnosis of von Willebrand disease

Von Willebrand disease is the most-common inherited bleeding disorder, including both quantitative (types 1 and 3) and qualitative (type 2) defects of von Willebrand factor. Among patients with suspected von Willebrand disease, the laboratory diagnosis requires three levels of testing: screening tests, specific assays for von Willebrand factor to establish the diagnosis, and discriminating tests to allow accurate characterization of the numerous types and subtypes of the disease. Because of their poor sensitivity, normal screening tests do not exclude the diagnosis. In most cases, specific measurements of von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity, and factor VIII levels in plasma allow differentiation of quantitative (proportionately decreased levels) and qualitative (discrepant levels) deficiencies of von Willebrand factor. Among the latter, a decreased von Willebrand factor ristocetin cofactor activity/von Willebrand factor antigen ratio is in favor of the three subtypes (2A, 2M, and 2B) defined by an abnormal interaction between von Willebrand factor and platelet glycoprotein Ib, whereas a decreased factor VIII/von Willebrand factor antigen ratio suggests subtype 2N, defined by a defective binding of von Willebrand factor to factor VIII. Several discriminating tests are available to definitively characterize each subtype. Moreover, for all variants, the link between phenotype and genotype is established using DNA analysis. In all cases, the precise characterization of type and subtype of von Willebrand disease remains essential for the choice of optimal therapeutic monitoring of each patient. Presented at the Joint Meeting of the World Health Organization and the International Society for Thrombosis and Hemostasis “Impact, Prevention and Control of von Willebrand’s disease” London, October 12–14, 1998     

Dynamic equilibrium between protein S and C4b binding protein is important for accurate determination of free protein S antigen.

Protein S in circulation is in a dynamic equilibrium with C4b binding protein (C4bBP), thus affecting the measurement of free protein S antigen. We addressed the issue of overestimation of the free protein S concentration with current immunoassays due to the dynamic equilibrium and propose a new method for its accurate determination. Our assay system was tested at different reaction temperatures using purified free protein S, protein S-C4bBP complexes, plasma samples, and a commercially available free protein S assay kit. At a reaction temperature of 37 degrees C, the free protein S fraction increased from 0.5 ng/ml (at 4 degrees C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4 degrees C) to 56 ng/ml when the concentration of the assayed protein S-C4bBP complexes was 20 ng/ml and 200 ng/ml, respectively. In plasma samples, free protein S levels were approximately 0.8 microg/ml and 6 pg/ml higher at 25 degrees C and 37 degrees C, respectively compared to measurements at 4 degrees C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 microg/ml higher at 25 degrees C than measurements performed at 4 degrees C. Dynamic equilibrium between protein S and C4bBP affects the measurement of free protein S antigen. Measurement of free protein S antigen should be performed under conditions where protein S is not dissociated from protein S-C4bBP complexes, as exemplified by assay at low temperature (4 degrees C).     

Evaluation of diagnostic methods for Candida albicans translocation in a mouse model: seminested polymerase chain reaction, blood culture, and serological assays

For the rapid diagnosis of systemic Candida infection, we compared the performance of an established seminested polymerase chain reaction (snPCR), serological tests for (1 → 3)-β-D-glucan assay and Candida mannan antigen assay, and blood culture in our murine model for Candida albicans translocation. In this mouse model, C. albicans disseminated to the liver from the intestine after day 6.5; the snPCR and blood culture results became positive from days 8 to 8.5 in about 60% of infected mice with culture-proven translocation, and in 100% on day 9. Both (1 → 3)-β-D-glucan and Candida mannan antigen were elevated in the serum as early as day 6.5 of infection, though they did not identify Candida species. Because the established snPCR can differentiate four clinically important Candida species and conventional microbiological methods require at least 48 h to identify Candida species in blood samples, the snPCR assay is advantageous for rapidly identifying Candida species in the blood. Therefore, the combination of the serological assays and the snPCR seems to be valuable for the early diagnosis of systemic C. albicans infection.     

Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma

Summary. Background: Protein S and tissue factor pathway inhibitor (TFPI) act together in down‐regulating coagulation. Objective: To investigate the TFPI/protein S system in hereditary and acquired protein S deficiency. Methods: Plasma antigen levels of protein S and full‐length TFPI were determined in heterozygous type I protein S‐deficient individuals (n = 35), patients on oral anticoagulant treatment (OAT) (n = 29), oral contraceptive (OC) users (n = 10) and matched controls. Thrombin generation was determined using calibrated automated thrombography. Results: Full‐length TFPI levels were lower in type I protein S‐deficient individuals (76.8 ± 33.8%) than in age‐ and sex‐matched controls (128.0 ± 59.4%, P < 0.001). Among protein S‐deficient individuals with thrombosis, those on OAT had not only lower total protein S levels (25.7 ± 8.2% vs. 54.7 ± 8.2%, P < 0.001), but also lower full‐length TFPI levels (52.6 ± 15.0% vs. 75.4 ± 22.9%, P = 0.009) than those not on OAT. Similarly, OC users had lower protein S (73.8 ± 11.5% vs. 87.9 ± 10.8%, P = 0.005) and full‐length TFPI levels (73.7 ± 27.7% vs. 106.4 ± 29.2%, P = 0.007) than non‐users. When triggered with tissue factor, plasma from protein S‐deficient individuals generated 3–5‐fold more thrombin than control plasma. The difference was only partially corrected by normalization of the protein S level, full correction requiring additional normalization of the TFPI level. Protein S‐immunodepletion experiments indicated that free protein S and full‐length TFPI form a complex in plasma, and the protein S/TFPI interaction was confirmed by surface plasmon resonance analysis. Conclusions: Full‐length TFPI binds to protein S in plasma and is reduced in genetic and acquired protein S deficiency. The concomitant TFPI deficiency substantially contributes to the hypercoagulable state associated with protein S deficiency.     

Protein C and S deficiency in severe infectious purpura of children: A collaborative study of 40 cases

We studied, in 40 children (mean age: 52 months) with severe infectious purpura, the relationships between protein C (PC) and protein S (PS) levels, and shock, disseminated intravascular coagulation (DIC) and outcome. We determined, on admission, PC antigen (ELISA) and activity (chromogenic test), and total PS (ELISA). Results were expressed as % of normal adult values. Statistical analysis was performed with SAS. Thirty children were in shock, 20 had DIC. All children with DIC, and 10 without DIC were in shock. Of 20 children who were in shock and had DIC, 7 died and 3 had an amputation. PC antigen was significantly decreased in shock children (p<0.05), in children with DIC (p<0.0005). and in non-survivors (p<0.05). PC activity was significantly decreased in shock children (p<0.05), in children with DIC (p<0.0005), and in non-survivors (p<0.005). Total PS was not decreased in shock children, but was significantly decreased in children with DIC (p<0.005), and in non-survivors (p<0.005). We conclude that PC and PS levels were decreased in our children, and that PC levels were significantly decreased in the presence of shock, DIC, and fatal outcome. PC and antithrombin III (AT III) supplementation, should be evaluated in children with severe infectious purpura with shock and DIC.Presented in part at the 5th European Congress on Intensive Care Medicine, Amsterdam, The Netherlands, June 5–8, 1990 (published in abstract form (Intensive Care Med [Suppl] 1, 1990; 16:p73)     

Aging affects cellular zinc and protein synthesis in the femoral diaphysis of rats

To clarify the effect of aging on bone metabolism, alteration of the cellular zinc content and protein synthesis was examined in the femoral diaphysis of 3- and 30-week-old male rats. The cellular zinc content in bone tissue markedly decreased in 30-week-old compared to 3-week-old rats. When the bone tissue from older rats were cultured with [³H]leucine, incorporation of [³H]leucine into the acid-insoluble residues was less than for weanling rats. This decrease was partly restored by the oral administration of zinc sulfate (0.5, 1.0, and 2.0 mg Zn/100 g body weight) to elderly rats for 3 days. An increase of in vitro [³H]leucine incorporation by bone tissues obtained from the rats that had received zinc (2.0 mg/100 g) was blocked by cycloheximide (10⁻⁶ M) or dipicolinate (10⁻³ M), a chelator of zinc. These results suggest that bone protein synthesis declines with age, and that this decline may be based partly on the decrease in bone cellular zinc.     

Bactericidal permeability increasing protein gene variants in children with sepsis

Objective To evaluate the role of genetic polymorphisms of the bactericidal permeability increasing protein (BPI) in pediatric patients with sepsis. Design Prospective, single-center, case-control study at the pediatric intensive care unit (PICU) of a university hospital. Patients 345 consecutive pediatric patients admitted to the PICU with fever, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, or multiple organ distress syndrome (MODS). Interventions DNA was isolated and two BPI gene polymorphisms BPI (G545 > C) Taq and BPI (A645 > G) 216 were studied in patients and compared with healthy controls. Measurements and results Genetic analysis of the BPI Taq gene revealed significant differences between healthy controls and the subgroup of febrile patients (p = 0.0243), the subgroup of SIRS and sepsis (p = 0.0101), and the subgroup of severe sepsis, septic shock, and MODS (p = 0.0027), respectively. No statistically significant differences for the BPI 216 gene polymorphism were found between patient and healthy control groups. A statistically significant predisposition to Gram-negative sepsis in patients carrying the BPI Taq GG variant together with the BPI 216 AG or GG variant was revealed (p = 0.0081), and these haplotypes were also associated with death due to sepsis-related complications. Conclusion BPI Taq gene polymorphism is the accurate predictor of the severity of sepsis in children admitted to the PICU.     

Meningococcemia and purpura fulminans in adults: acute deficiencies of proteins C and S and early treatment with antithrombin III concentrates

It has been recently suggested that an acquired deficiency of proteins C and S could contribute to the pathogenesis of meningococcemic purpura fulminans (PF) in children. Our study was designed to measure the levels of antithrombin III (AT III), protein C, and protein S during adult PF and to determine the effects of an early infusion of high doses of AT III concentrates on clinical and biological alterations of PF. We studied five consecutive adult patients with meningococcemia (type B) and PF. The levels of AT III, protein C (antigen and activity), and protein S (total and free) were measured at admission and 24 h and 1 month later. The treatment included in each case: amoxycillin, dobutamine and high doses of AT III concentrates. All patients survived and were discharged without any sequelae. At admission, biological data were consistent with severely depressed protein C and protein S levels and moderately decreased AT III levels, without any discrepancy between protein C antigen and activity. After 24 h, AT III and protein S levels were within normal ranges, whereas protein C levels were still depressed. These data are consistent with the theory of a particular imbalance in the anticoagulant systems during meningococcemic PF, contrasting with the usual findings observed during septic disseminated intravascular coagulation. The possibility must be considered that high doses of one anticoagulant (AT III concentrates) could compensate for the acute decrease in the other (protein C system).     

Detection of histamine-induced capillary protein leakage and hypovolaemia by determination of indocyanine green and glucose dilution method in dogs

Objective: The plasma volume of histamine-induced protein capillary leakage may be overestimated when this is determined using the indocyanine green (ICG) dilution method (Vd-ICG), since this dye binds to plasma proteins. The initial distribution volume of glucose (IDVG) has been shown to indicate the central extracellular fluid volume including plasma. Accordingly, the overestimation would be detected by a higher Vd-ICG/IDVG ratio. Our study was intended to examine whether the simultaneous measurement of these two variables can evaluate histamine-induced protein leakage and associated hypovolaemia. Design: Prospective animal study. Setting: Institutional animal research laboratory. Subjects: Twenty-four anaesthetized and ventilated mongrel dogs. Interventions: Anaesthetized animals were mechanically ventilated and received infusions of normal saline (n = 8), histamine 50 μg/kg per h (n = 8), or histamine 100 μg/kg per h. The Vd-ICG and IDVG were calculated using a one-compartment model by simultaneous administration of ICG 0.5 mg/kg, and glucose 100 mg/kg followed by serial arterial blood sampling. Measurements and results: In both histamine groups, a significant elevation of haematocrit and a decrease of plasma albumin concentration were found (p < 0.05). Although the IDVG decreased following histamine administration (p < 0.05), the Vd-ICG remained unchanged. The Vd-ICG/IDVG ratio increased in a dose-dependent manner after histamine administration (p < 0.05), but remained unchanged following normal saline administration. Conclusion: The results suggest that the Vd-ICG/IDVG ratio and the IDVG are useful in evaluating the magnitude of the leakage and hypovolaemia. Received: 1 July 1998 Accepted: 29 December 1998 Final revision received: 8 December 1998