Comparison of functional assays for protein S: European collaborative study of patients with congenital and acquired deficiency.

Four functional assays for protein S were evaluated by 4 different laboratories, each center using its own method. The aim of this study was to compare these different assays and to establish a relationship with results of immunological assays of total and free protein S antigen and C4bBP. The same plasma samples were distributed to each center and tested in blind. In 47 normal subjects, there was no significant difference between the 4 functional assays, with mean values ranging from 93 to 100%. These values were in good agreement with those of free and total protein S antigen. In 34 patients with a quantitative congenital deficiency of protein S the mean values of protein S activity were decreased with the 4 assays, ranging from 25 to 40%. Free protein S antigen was reduced to a similar extent, whereas total antigen was either normal or decreased. The correlation of protein S activity with free protein S antigen was satisfactory for 3 methods, with coefficients of correlation varying from 0.84 to 0.92 whereas it was only 0.70 in one lab. When total protein S antigen was reduced, protein S activity was decreased in all the patients with the 4 assays. In contrast when total protein S antigen was normal an important overlap of protein S activity between normals and patients was observed in one lab with 12 patients misclassified. In 8 patients with a functional defect, results of protein S activity differed substantially according to the assay used and about half of these patients were misclassified.(ABSTRACT TRUNCATED AT 250 WORDS)

The mutation in fibrinogen Bicêtre II (gamma Asn308-->Lys) does not affect the binding of t-PA and plasminogen to fibrin.

The aim of this study was to investigate the interactions of t-PA and plasminogen with fibrin derived from an abnormal fibrinogen detected in a 40-year-old male patient who had had an episode of thrombophlebitis with pulmonary embolism. An abnormal fibrinogen was diagnosed on the basis of prolonged thrombin and reptilase times also detected in two other family members. Fibrinogen purified from plasma, in the presence of protease inhibitors, by glycine precipitations, gel filtration and affinity chromatography, was devoid of plasminogen, fibronectin, and vWf. SDS-PAGE analysis according to Laemmli under reducing conditions, showed an abnormal gamma chain (approximately 50% of the total) migrating in a more anodic position (M(r) 48 kDa). By PCR amplification and DNA sequencing, the abnormality was identified as an Asn308-->Lys mutation of the gamma chain. Since such a mutation constitutes a new plasmin cleavage site as first reported for fibrinogen Kyoto I, it may modify interactions of plasminogen and t-PA with carboxy-terminal lysine residues. Ligand-binding studies were therefore performed using intact and plasmin-degraded fibrin surfaces obtained from the abnormal fibrinogen. The plasminogen and t-PA binding isotherms obtained with the abnormal fibrinogen were similar to the control. Moreover, the stimulation by fibrin of plasminogen activation by t-PA was not different from the control. These results suggest (i) that the lysine 308 residue may not be exposed to plasmin cleavage in fibrin, and (ii) that the thrombotic accident of the propositus cannot be explained by an abnormality of the plasminogen/t-PA binding to fibrin.

Functional assay of protein S in 70 patients with congenital and acquired disorders.

A functional assay for the selective measurement of the active form of protein S in plasma, based on the prolongation of an APTT, was previously developed. This assay is sensitive, reproducible and specific, not affected by other clotting factors including FVIII. This method was applied to the measurement of protein S activity in congenital and acquired disorders. Results of protein S activity were compared to those of total and free antigen measured by ELISA. In 30 controls, there was an excellent correlation between protein S activity and free antigen. In patients with inflammatory disease, protein S activity and free antigen were normal, despite high levels of both C4b-binding protein and total protein S antigen. In dicoumarol-treated patients, protein S activity was lower than free antigen due to the presence of acarboxylated forms. Surprisingly, in liver cirrhosis, free antigen was only slightly decreased whereas protein S activity was significantly reduced. In 23 patients with congenital deficiency, protein S activity was consistently decreased, from less than 5% to 60% and showed good correlation with the free antigen. This functional assay allows the rapid diagnosis of congenital or acquired deficiency of protein S.

Carrier detection and prenatal diagnosis in 98 families of haemophilia A by linkage analysis and direct detection of mutations.

489 individuals from 98 families with a haemophilia A member were studied with restriction fragment length polymorphisms (RFLPs) for carrier detection and prenatal diagnosis. Five intragenic polymorphisms revealed with the restriction enzymes BclI, XbaI, BglI, HindIII and AlwNI and one extragenic multiallelic polymorphism (St14) at the DXS52 locus were used. The combination of the five intragenic polymorphisms did not add significantly more information than just the BclI and XbaI polymorphisms because of strong linkage disequilibrium. The sequences surrounding the intronic restriction sites of the BclI and XbaI RFLPs are known so they can be rapidly analysed using the polymerase chain reaction (PCR). 68.6% of the women were heterozygous for either the BclI or XbaI RFLP and this heterozygosity rate increased to 98.6% when the St14 extragenic polymorphism was included. Linkage analysis using these RFLPs led to the classification of over 90% of the women as carriers or normal and 98.6% of the carriers were heterozygous. Prenatal diagnosis was successful in the 16 foetuses tested and all could be classified as carrier, normal or haemophiliac. Five TaqI restriction sites in the coding region of the factor VIII gene can detect a C to T transition that results in an in-frame stop codon. These five sites were amplified by PCR in 119 haemophiliacs and tested for an abnormal TaqI restriction pattern. A stop codon was found in three haemophiliacs at exons 18, 22 and 24. The same analysis revealed three deletions, two involving the last exon 26 and one exons 23-26.

Factors affecting the stability of tPA and PAI-1 during storage and handling of human plasma for in vitro studies: implications in the determination of tPA and PAI-1 activities.

Factors that influence the physico-chemical conditions of plasma (e.g. pH, dilution, freezing, storage) and thereby the stability of tPA and PAI-1 activities, have been studied and optimized using a solid-phase fibrin-tPA activity assay. Optimal recovery of tPA activity was at a pH of 6.8 +/- 0.2, while at the pHs usually found in thawed plasma, i.e. pH 7.6-8.2, the activity was lower and showed great variability. Free tPA activity was tested in undiluted plasma, while plasma diluted 1:20 was used to recover maximal tPA activity. The corrected value for the diluted plasma and the value for the euglobulin suspensions were similar. In both cases the pH optimum was 7.4. PAI activity levels were tested in undiluted plasma and showed no variations after venous occlusion. Our results indicate that the in vitro determination of tPA activity is directly related to the pH of thawed plasma and not to the freezing procedure or the temperature of storage. Therefore, thawed plasma should be tested at a pH giving the maximal recovery of tPA activity in a particular assay method.

Familial variant of antithrombin III (AT III Bligny, 47Arg to His) associated with protein C deficiency.

The association of a variant of antithrombin III (AT III Bligny) and protein C deficiency is described in a 36-year-old patient having suffered from severe thrombotic episodes. His mother has protein C deficiency and showed a single episode of thrombophlebitis following surgery. His father, sister and daughter have the variant AT III and are asymptomatic. The abnormal AT III was characterized in plasma by the discrepancy between a normal progressive activity and a reduced heparin cofactor activity. This variant AT III was purified, separated from the normal protein by heparin-Sepharose chromatography and was eluted with increased NaCl concentrations. At pH 7.4, the variant AT III eluted at lower (0.3 to 0.5 M) NaCl concentrations than normal (1 to 1.5 M) AT III, thus demonstrating a decreased affinity for heparin. At pH 6.0, however, the abnormal molecule bound more avidly to heparin-Sepharose and was eluted like normal AT III at pH 7.4. Similarly, the heparin enhancement of intrinsic fluorescence of the variant AT III, markedly reduced at pH 7.4, was normalized at pH 6.0. The abnormal AT III showed a normal antiprotease activity, a normal molecular weight by SDS-PAGE, but displayed only a partial immunological identity with the normal protein. Analysis of amplified genomic DNA from this patient by dot-blot demonstrates a heterozygous substitution of arginine by histidine at position 47.

Prenatal diagnosis in type IIA von Willebrand disease.

A prenatal diagnosis for fetal disease was performed at 20 weeks gestation in a severely affected patient with type IIA von Willebrand disease. In the fetal cord blood sample obtained under ultrasound guidance, the level of von Willebrand ristocetin cofactor activity was similar to that of von Willebrand factor antigen, and all the multimers were present. These results were compared to those obtained in 51 normal fetuses of similar gestational age (19-21 weeks). Normal fetuses showed slightly lower levels of von Willebrand factor than normal adults and in addition to all adult multimers, the presence of unusual large forms. This data compared with the case, allowed the exclusion of the diagnosis of type IIA von Willebrand disease in our patient's fetus. This was confirmed at birth in the cord blood.

Hemophilia: state of the art of hematologic care 1988.

Since 1982, when the World Federation of Hemophilia first published a document on the state of the art of hemophilia diagnosis and care, there have been lights and shadows in this field. Although the widespread infection of hemophiliacs with the human immunodeficiency virus (HIV) contaminating clotting factor concentrates is still a threatening and formidable shadow, the gloomy picture brought about by the AIDS epidemic is partially lightened by spectacular improvements in therapy and diagnosis. Carrier detection and first-trimester prenatal diagnosis can now be performed accurately in most kindreds by analysis of DNA of the factor VIII or IX genes. An important step forward towards the elimination of the risk of blood-borne infections transmitted by plasma products was recently made through the application of virucidal methods to clotting factor concentrates. Since HIV appears more vulnerable to such methods than the hepatitis viruses, currently available concentrates can be considered substantially free from the risk of transmitting HIV infection. Even though transmission of hepatitis is much reduced but not totally abolished, virucidal methods are continuously being improved, so that it can be foreseen that concentrates will become safer and safer. Finally, factor VIII produced by recombinant DNA technology is undergoing the first clinical trials in hemophiliacs. Hopefully, it will free from the risk of transmitting infections and will be available in sufficiently large amounts to meet the need of hemophiliacs worldwide. In 1982, the World Federation of Hemophilia published a message on the status of diagnosis and treatment of hemophilia. Since then, hemophilia care has been complicated by widespread infection of hemophiliacs with human immunodeficiency virus (HIV).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a CpG mutation in the coagulation factor-IX gene by analysis of amplified DNA sequences.

In a family with no previous bleeding history, the sister of a single, severely affected haemophilia B patient requested carrier detection and prenatal diagnosis. In Southern blots, using Taq I digested DNA and a factor-IX cDNA probe, a normal invariant band at 1.6 kb was missing in the haemophiliac suggesting the loss of the Taq I site at the 5' end of exon h. A 162 bp sequence which includes the suspected mutant region was amplified by the polymerase chain reaction in each DNA. Two oligonucleotide probes were synthesized and differed by only one base pair which substituted a T for C in the normal Taq I recognition sequence. The amplified DNA was dot-blotted and hybridized with the labelled probes. The altered sequence hybridized to DNA from the affected individual, his sister and her fetus and not to DNA from the normals. The mutation, involving the haemophiliac, his mother, his sister and her fetus, transforms a CGA codon that encodes for arginine in the catalytic domain of the protein into a UGA stop codon.

Carrier detection in severe (type III) von Willebrand disease using two intragenic restriction fragment length polymorphisms.

DNA from a family with a female member affected with severe (type III) vWD was analysed using three restriction enzymes and a partial vWF cDNA probe. Two restriction fragment length polymorphisms (RFLPs) detected with the enzymes Bgl II and Xba I proved to be informative in this family. A 36.0 Kb allele demonstrated with the enzyme Xba I was rare in the general population but very important in this family for segregation analysis of the alleles and their association with the putative defective chromosome. The propositus was homozygous for the 36.0 Kb Xba I polymorphic band and heterozygous for the Bgl II polymorphism. She was the only member of the family showing this allelic pattern. The linkage of the alleles could be determined because her mother was homozygous for the 9.0 Kb Bgl II polymorphism but heterozygous for the Xba I polymorphism. The segregation of the alleles could be traced to the proband's son and a niece. The genotypic analysis revealed that her niece could be considered as carrying a defective gene for severe vWD.

Haemophilia A in a female: study of a family using intragenic and extragenic restriction site polymorphisms.

Restriction fragment length polymorphisms (RFLPs) were studied in a large Algerian family which includes 6 haemophiliacs and a previously described case of female haemophilia A. The female propositus is 66 years old with a normal karyotype. Her parents are first cousins. Her 3 sons are haemophiliacs and her 3 daughters with affected children are obligate carriers. The proband has an excessive bleeding tendency and markedly reduced levels of F.VIII (VIII C 0.03 U/ml, VIII Ag 0.01 U/ml) with elevated vWF Ag (2.30 U/ml), similar to the levels observed in affected males from the family. Four RFLPs can be identified by Southern blotting after digesting genomic DNA with the restriction enzymes Bcl I, Bgl I, Kpn I/Xba I and Taq I and hybridization with a 647 bp Stu I/Sca I F.VIII genomic probe, a 1.8 Kb EcoRI F.VIII cDNA probe, a 1.0 Kb EcoRI/Sst I fragment of intron 22 and the extragenic probe ST 14, respectively. With these four RFLPs, the propositus was found to be homozygous for the alleles segregating in this family with the abnormal X-chromosome. The carrier status was proven in a granddaughter and excluded in another. In conclusion, this RFLP linkage analysis is another argument to suggest that the propositus, a rare case of female haemophilia, is homozygous for the abnormal gene.

Long-term evaluation of HIV antigen and antibodies to p24 and gp41 in patients with hemophilia. Potential clinical importance.

To investigate the relation between human immunodeficiency virus (HIV) antigenemia and clinical manifestations of HIV infections, we studied 96 patients with hemophilia who were positive for HIV antibody, for a median of 34 months. Every 4 to 10 months a clinical and laboratory examination was performed and serum samples were tested for three HIV markers: HIV antigen, antibody to p24, and antibody to gp41. Twenty-two subjects (23 percent) were found to be positive for HIV antigen: 8 were positive upon entry and remained so (Group 1), and 14 became positive during the study, 4 to 26 months after HIV antibody appeared (seroconversion), 13 of whom remained positive for HIV antigen (Group 2). Most subjects positive for HIV antigen had low or undetectable titers of antibody to p24, whereas the antibody titer to gp41 remained high. In Group 2, patients with low p24 antibody titers had further decreases in their titers before or at the time HIV antigen appeared. Once present, HIV antigen persisted and tended to increase in concentration. In contrast to Group 3 (negative for HIV antigen, low anti-p24 titer) and 4 (negative for HIV antigen, high anti-p24 titer), the groups positive for HIV antigen had significantly higher incidences of acquired immunodeficiency syndrome (P = 0.05), immunodeficiency-related infections (P less than 0.001), and immune thrombocytopenia (P = 0.001), and had more severe disease as measured by the Walter Reed staging system (P less than 0.001). In this study, HIV antigen appeared to be a better predictive marker of HIV-related complications than the absolute T4+ count. These results suggest that HIV antigenemia indicates a poor clinical prognosis.

Purification and further characterization of antithrombin III Milano: lack of reactivity with thrombin.

The functional abnormality of Antithrombin III "Milano", a previously described variant with monomeric and dimeric forms of abnormal AT III, has been further characterized. Affinity chromatography on heparin-Sepharose led to the separation and purification of two distinct fractions: fraction I is identical to normal AT III; fraction II (abnormal AT III) reproduces the abnormalities of the AT III "Milano", i.e. lack of thrombin inhibition, increased mobility by two-dimensional immunoelectrophoresis in the absence of heparin and migration as two bands with molecular weights of 60 K and 120 K by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The interaction of both fractions with purified alpha-thrombin was studied by the formation of complexes as well as by affinity chromatography on thrombin-Sepharose. No thrombin-AT III complexes could be demonstrated with either the monomeric or dimeric forms of purified variant AT III at both concentrations of thrombin used. Similarly, no binding to thrombin-Sepharose was observed, thus indicating that the molecular defect of AT III Milano is related to its absence of reactivity with thrombin.

[Acquired deficiencies in antithrombin III and C protein during treatment with L-asparaginase]. / Déficits acquis en antithrombine III et en protéine C au cours du traitement par la L-asparaginase.

Fourteen children treated by L-Asparaginase for acute lymphoblastic leukemia had sequential coagulation studies performed including cephalin-kaolin time, Quick time, fibrinogen, factors II, VII + X and V, as well as antithrombin III and protein C, the major coagulation inhibitors. A severe antithrombin III and protein C deficiency was observed during therapy, with a coexisting hypocoagulability. This equilibrium partially explains the lack of thrombo-embolic phenomena in these children, despite the risk factors present. Although no substitutive therapy was instituted in these cases, their use in high-risk cases is discussed.

Multicenter comparison of five functional and two immunological assays for protein C.

Five functional assays and two immunoassays for protein C (PC) were evaluated in parallel for the same plasma samples collected from healthy subjects, patients with congenital and acquired PC deficiencies or patients with conditions associated with high PC levels. For 7 patients starting warfarin therapy and for 15 patients during stabilized warfarin therapy, there were significant between-assay differences. For these groups immunoassays gave higher values than most functional assays and the latter also gave varied results, probably depending on their respective capacity for recognizing a carboxylated PC. On the other hand, there were no significant between-assay differences nor discrepancies between PC activity and antigen levels for healthy subjects (n = 39), patients with congenital PC deficiency (n = 10), myocardial infarction (n = 25), chronic liver disease (n = 19), disseminated intravascular coagulation (n = 35), in the post-operative period (n = 20) or in women taking oral contraceptives (n = 20). This comparison of PC assays indicates that PC levels measured by different functional or immunological assays are very close in the majority of clinical conditions, but not for patients on oral anticoagulants.

Synovectomy of the elbow in young hemophilic patients.

Synovectomy of twenty-three elbows was done in eighteen patients, eight to twenty-five years old, who had severe hemophilia and were followed for eighteen to seventy months. Episodes of bleeding recurred in four elbows, and moderate pain persisted in three. A significant improvement in mobility was observed for pronation-supination in nine elbows and for flexion-extension in fourteen. No radiographic evidence of arthritis was seen. Synovectomy of the elbow, performed through a single lateral incision, appears to be a valuable surgical procedure in hemophiliacs in whom non-operative treatment has failed, and resection of the radial head should be done in adults when there is moderate or severe damage to the cartilage of the radial head.

[Antithrombin III]. / Antithrombine III.

The in vivo regulation of coagulation is mainly controlled by plasma inhibitors in which antithrombin III (AT III) plays an importance role. AT III is a glycoprotein which inhibits all serine proteases, except factor, VIIa, generated during the coagulation process. The proteases are inactivated by formation of an equimolecular complex and this reaction is greatly enhanced in the presence of heparin. A similar catalytic process could occur in vivo, involving heparin-like substances present on the surface of the surface of the endothelial cell. The physiological importance of AT III is clearly demonstrated by the high incidence of thromboembolic disease in patients with congenital AT III défficiency.

The role of HIV infectivity and composition of factor VIII concentrates on the immunity of haemophiliacs positive for HIV antibodies.

Forty-six subjects (44 HIV antibody-positive) with some degree of immune deficiency (at least TH/TS ratio below 1) were randomly distributed into 4 treatment groups. Each group was assigned to 1 of 4 products to be used exclusively for a 1-year period: 1 concentrate was of intermediate purity and not heat-treated, and 3 were heat-treated in order to inactivate HIV, 2 of them being of higher purity. At 4-6-month intervals, check-ups, including as markers clinical examination, platelet, lymphocyte and T cell subset counts, IgG levels and delayed hypersensitivity test, were carried out. At entry as well as at the end of the study, groups were not statistically distinguishable. No intra- nor inter-group differences were demonstrable for any of the markers. In contrast, using a scoring system for each marker and the results of check-up at entry as reference, significant differences between groups appeared on subsequent check-ups. Patients receiving intermediate-purity factor VIII, whether heat-treated or not, were mostly steady, while groups receiving heat-treated concentrates of a higher purity significantly worsened. This surprising outcome was no related to differences in anti-HIV titers or specificities. From this study, the potential long-term predictive value of this scoring system could not be established.