Stimulation of monocyte tissue factor expression in an in vitro model of bacterial endocarditis

The coagulation system plays a major role in the formation of the infected endocardial vegetation in bacterial endocarditis. Since monocytes can express tissue factor (TF) on their surfaces, they are thought to be responsible for the extrinsic activation of the coagulation cascade during this disease. The present study used an in vitro model in which fibrin plates, isolated adherent monocytes, and Streptococcus sanguis were used as an analog for endocardial vegetations. Adherence to fibrin by itself was found to stimulate TF expression on the monocytes, but stimulation by S. sanguis significantly increased TF expression, which was found to be maximal at a bacterium-to-monocyte ratio of 9 or more.     

A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis

We have examined the prothrombin gene as a candidate gene for venous thrombosis in selected patients with a documented familial history of venous thrombophilia. All the exons and the 5'- and 3'-UT region of the prothrombin gene were analyzed by polymerase chain reaction and direct sequencing in 28 probands. Except for known polymorphic sites, no deviations were found in the coding regions and the 5'-UT region. Only one nucleotide change (a G to A transition) at position 20210 was identified in the sequence of the 3'-UT region. Eighteen percent of the patients had the 20210 AG genotype, as compared with 1% of a group of healthy controls (100 subjects). In a population-based case-control study, the 20210 A allele was identified as a common allele (allele frequency, 1.2%; 95% confidence interval, 0.5% to 1.8%), which increased the risk of venous thrombosis almost threefold {odds ratio, 2.8; 95% confidence interval, 1.4 to 5.6}. The risk of thrombosis increased for all ages and both sexes. An association was found between the presence of the 20210 A allele and elevated prothrombin levels. Most individuals (87%) with the 20210 A allele are in the highest quartile of plasma prothrombin levels (> 1.15 U/mL). Elevated prothrombin itself also was found to be a risk factor for venous thrombosis.     

Preparation of lyophilized partial thromboplastin time reagent composed of synthetic phospholipids: usefulness for monitoring heparin therapy

To contribute to the development of a reference reagent for monitoring heparin therapy, a lyophilized partial thromboplastin time (PTT) reagent was prepared from synthetic dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and dioleoylphosphatidylethanolamine, with colloidal silica as activator. The reagent, coded 91/558, was contained in sealed glass ampoules; it deteriorated in a heat degradation experiment, but its activity remained constant for at least 4 years when stored at -70 degrees C. Within- and between-run precision with this reagent complied with the requirements proposed by the International Committee for Standardization in Haematology (ICSH) Panel on PTT. The response of this reagent and of two other reagents to heparin added to pooled normal plasma was nonlinear. Citrated samples from 58 patients receiving intravenous heparin and from 24 apparently healthy volunteers were tested with reagent 91/558, with Automated APTT (Organon Teknika), with Manchester APTT reagent, with an antifactor Xa assay, and with an anti-factor IIa assay. The correlation of APTT with anti-Xa and anti-IIa activity was poor. The best correlation was observed between reagent 91/558 and the Organon Teknika reagent. Correlations were improved when individual patients' samples were replaced by pooled plasmas from heparinized patients, in whom the effect of oral anticoagulation was minimal. These results suggest that preparation of a lyophilized synthetic phospholipid reagent is feasible for use in monitoring heparin therapy.     

Involvement of amino acid residues 423-429 of human protein S in binding to C4b-binding protein

Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP.     

Compound heterozygosity for two novel missense mutations in the prothrombin gene in a patient with a severe bleeding tendency

The abnormal prothrombin gene of an Italian patient with a severe bleeding tendency and hypoprothrombinemia was selected for study and compared with the prothrombin genes of healthy controls. All the coding and their flanking regions and the 5'- and 3'-UT regions of the prothrombin gene were screened by analyzing the nucleotide sequence of the corresponding PCR products. The patient was found to be heterozygous for two novel point mutations: one at nucleotide 4251 in exon 6, which changes the codon for cysteine-138 (TGC) in the kringle 1 domain to that for tyrosine (TAC), and one at nucleotide 8812 in exon 10, which results in the replacement of tryptophan-357 (TGG) by cysteine (TGT) in the catalytic domain. Her mother was heterozygous for the Cys-138 Tyr mutation and her father heterozygous for the Trp-357 Cys mutation. Several other sequence variations were identified in the prothrombin genes from control individuals. Only the variations at nucleotide 4203 and 10253 could be established as polymorphisms.     

Factor V Leiden and other coagulation factor mutations affecting thrombotic risk

Five genetic defects have been established as risk factors for venous thrombosis. Three are protein C, protein S, and antithrombin deficiencies, defects in the anticoagulant pathways of blood coagulation. Together they can be found in approximately 15% of families with inherited thrombophilia. Their laboratory diagnosis is hampered by the large genetic heterogeneity of these defects. The other two genetic risk factors, resistance to activated protein C associated with the factor V Leiden mutation and increased prothrombin associated with the prothrombin 20210 A allele, are much more prevalent and together can be found in 63% of the thrombophilia families. Because both defects are caused by a single mutation, DNA analysis is the basis of their laboratory diagnosis.     

Two novel mutations in the prothrombin gene cause severe bleeding in a compound heterozygous patient

Hypoprothrombinemia is a rare hereditary coagulation defect characterized by low levels of biologically active prothrombin. In this paper we report the laboratory and genetic analysis of a patient with a severe hypoprothrombinemia and some of her relatives. Laboratory analysis showed very low levels of prothrombin antigen. Molecular analysis of the prothrombin genes of the patient resulted in the identification of two novel sequence variations in heterozygous state, a 20079 G to A transition, which predicts a Trp 569-->Stop mutation, and a 1261C-->G change within intron B near the acceptor splice site. A cosegregation of prothrombin deficiency in family members with the two genetic defects was observed.