Platelet membrane glycoproteins implicated in ristocetin-induced aggregation. Studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand's disease.

The antibiotic ristocetin only aggregates platelets in the presence of plasma von Willebrand factor. Platelets from patients with Bernard-Soulier syndrome do not aggregate upon addition of ristocetin although, in contrast to von Willebrand's disease, plasma levels of factor VIII complex (factor VIII clotting activity, von Willebrand factor activity, and von Willebrand antigen) are normal. The membrane surface of normal platelets was modified and compared to the surface of platelets from a patient with Bernard-Soulier syndrome in an attempt to identify the receptor involved in von Willebrand factor-ristocetin-induced aggregation. After the incubation of washed normal platelets with a preparation of ristocetin previously shown to contain a proteolytic contaminant, the aggregation response is significantly decreased on addition or normal plasma. Analaysis by gel electrophoresis of such platelets when stained for carbohydrate revealed a decrease in the relative amounts of membrane glycopro-eins. Chymotrypsin-treated normal platelets had less membrane glycoproteins in addition to giving a reduced aggregation response in ristocetin-induced aggregation. Staining of gels for protein and carbohydrate indicated that there was an extensive change in the surface of Bernard-Soulier platelets, whereas those from patients with von Willebrand's disease appeared the same as normal. Platelets from patients were labeled by the lactoperoxidase iodination technique. Not only was the relative intensity of staining of platelet-specific proteins and glycoproteins changed in Bernard-Soulier platelets, but the iodination of the glycoproteins on the membrane surface relative to other membrane constituents was lower. In contrast, platelets from patients with von Willebrand's disease showed a normal exposure of membrane components. These data suggest therefore that membrane glycoproteins may play a functional role in ristocetin-induced aggregation.

Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding.

It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.

Homozygous variant of antithrombin III: AT III Fontainebleau.

A qualitative defect of antithrombin III (AT III) was demonstrated in four members of a large Tunisian family by the discrepancy between a normal amount of antigen and decreased or absent heparin cofactor activity. The propositus, a 3-year-old girl, died from massive intracardiac thrombosis despite oral anticoagulant therapy. Heparin cofactor activity measured in the presence of thrombin or F. Xa was undetectable in her plasma. Anti-F. Xa activity was also absent when using low molecular weight heparin or a synthetic pentasaccharide, representing the binding site to AT III. The lack of affinity of the propositus AT III for heparin was demonstrated by two-dimensional immunoelectrophoresis and chromatography on heparin-Sepharose. The parents, first cousins, and the sister of the propositus also demonstrated a qualitative abnormality of AT III, with levels of heparin cofactor activity close to 50% of the normal range. Our data support the view that the abnormal protein was present at the heterozygous state in the parents and sister and at the homozygous state in the propositus. None of the affected family members had thrombotic episodes, except for the propositus. The name of AT III Fontainebleau is proposed for this variant.

Catabolism of human fibrinogen fragment D in normal subjects and patients with liver cirrhosis.

The catabolism of human fragment D, (FgD), obtained by plasmin digestion of fibrinogen has been investigated in normal subjects and patients with liver cirrhosis and the results compared with those obtained for fibrinogen (Fg). Fg was labelled with I-125 and Fg D with I-131 using the chloramine T method. The plasma disappearance curves of both labelled proteins fitted a two exponential curve. In controls the plasma clearance rate of Fg D was greater than that of Fg as shown by the marked difference between the half-lives of these two tracers: 8,9 and 83,5 hours for Fg D and Fg respectively. The fractional catabolic rate of Fg D was 3.38 times the plasma pool per day. In nine patients with liver cirrhosis, catabolism of Fg was not modified. In contrast, catabolism of Fg D was significantly reduced with a half life of 13.0 hours and a low fractional catabolic rate. These results suggest the role of the liver in the catabolism of Fg D in man.

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.

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.

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.

The half-life of plasmic degradation products of human fibrinogen in rabbits.

Human fibrinogen and purified plasmic degradation fragments X (stages 1 and 2), D and E were labelled with 125-I using the lactoperoxidase method. The chromatographic, electrophoretic and immunologic properties of the labelled proteins were found to be similar to those of non-labelled fragments. All the degradation products diffused rapidly (T 1/2 0.27-0.75 hours) from the intravascular space of rabbits, as compared with fibrinogen (4.26 hours). In addition, the metabolic half-life was found to be 49.3 hours for fibrinogen, as compared with only 5.6, 6.1, 2.3 and 1.4 for fragments X (stage 1), X (stage 2), D and E, respectively. The metabolic half-life roughly reflects the molecular size of the degradation products.

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.

An enzyme immunoassay (ELISA) for the quantitation of human factor VII.

A new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab')2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21% to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VII C was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

Clinical application of a chromogenic substrate method for determination of factor VIII activity.

A chromogenic substrate kit for the determination of factor VIII activity (COATEST Factor VIII) has been evaluated in five different laboratories, one of them using a semi-automated procedure. This chromogenic method was compared to one-stage clotting assays for factor VIII determination in plasmas from healthy subjects, carriers of hemophilia A, severe, mild and moderate hemophilia A as well as von Willebrand's patients. In all these cases, a high correlation between these two methods was obtained (r = 0.96-0.99, n = 385) with a good agreement of the assigned potencies at all levels of factor VIII. A good correlation (r = 0.94) was also obtained for the levels of factor VIII after infusion of concentrates in six severe hemophiliacs or after administration of DDAVP to von Willebrand's patients. The chromogenic method is insensitive to preactivation of factor VIII by thrombin, thus yielding valid potency assignments also in these situations. The precision was higher with the chromogenic method than with the one-stage clotting assays (C.V. = 2-5% vs 4-15%). Altogether, the new chromogenic substrate method has proven itself suitable for determination of factor VIII in plasma and concentrates.

Von Willebrand factor and platelet adhesiveness.

A modification of Salzman's method has been used in an attempt to provide an assay in vitro for the von Willebrand factor. Platelet adhesiveness was increased in von Willebrand's disease by previously coating the beads with normal or haemophilic plasma or cryoprecipitate, whereas von Willebrand plasma had no corrective effect. Antihaemophilic factor (AHF) concentrates were studied in the same way and results compared with experiments in vivo.

Cross-reacting material in genetic variants of haemophilia B.

Cross-reacting factor IX material (CRM) was immunologically detected in the plasma of 38 normal individuals and 21 out of 22 haemophilia B patients using a rabbit antibody to factor IX. The same reacting material was detected in only nine of these patients using a human antibody. These results indicate that the plasma of the majority of haemophilia B patients contains a protein-lacking biological activity but having antigenic determinants in common with normal factor IX.

Biological findings in Von Willebrand's pedigrees: implications for inheritance.

Thirty-one subjects from three families affected by Von Willebrand's disease have been investigated with the following tests: Ivy's bleeding time; platelet adhesiveness according to Salzman; two-stage factor VIII assay. Twelve patients have a complete form of the disease, i.e., a prolonged bleeding time with low platelet adhesiveness, and a reduced factor VIII level. Eight subjects have an isolated low platelet adhesiveness associated in three cases with a prolonged bleeding time. The low platelet adhesiveness in these subjects was corrected, as in Von Willebrand's disease, by infusion of haemophilia A plasma. The dominant autosomal mode of inheritance appears to be due to a pleiotropic gene, expressed in a variety of ways

Problems in the detection of carriers of haemophilia A.

Factor VIII activity and factor VIII related--or Willebrand--antigen were studied in 49 known carriers of haemophilia A and 31 normal women, and the data were analysed by four statistical approaches. Sixteen per cent of normals and 18% of carriers were misclassified, overlapping with the other group. Although the percentage of carriers detected is higher when taking into account the results of both biological and immunological factor VIII, it is lower than others recently reported, and the discrepancies between the results obtained are discussed.

A new method for the estimation of protein C by ELISA.

A new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human protein C antigen. Anti-protein C F(ab')2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing protein C, was detected by incubation with peroxidase-labeled anti-protein. C-IgG followed by the addition of hydrogen peroxyde and 0-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.02%) and accurate (variation coefficient: 3 to 10%). When results are compared to those obtained by the Laurell technique (electroimmunodiffusion, EID), the correlation coefficient is 0.95 in all tested plasmas. Protein C antigen was measured by ELISA and EID in plasma from 40 controls, 14 patients with congenital protein C deficiency, 15 patients with liver cirrhosis and 40 dicoumarol-treated cases. In normal plasma, protein C ranged from 70 to 126%. In congenital deficiency, protein C was between 35 and 58% in 13 cases and 9% in one of them. In patients with liver cirrhosis and dicoumarol-treated cases, levels of protein C antigen were compared to those of other vitamin K dependent factors, i.e. Factors II and IX measured by EID and Factor X assayed by EID and ELISA. In liver cirrhosis, the amount of protein C was significantly lower than that of Factors II, IX and X. In short-term and long-term dicoumarol-treated patients, the highest correlation (r = 0.72) was observed between protein C and Factor X levels. In the plasma of patients undergoing oral anticoagulant therapy, protein C decreased more rapidly than Factors X or II and migrated in presence of calcium as a double peak, one with a normal mobility and one more anodal corresponding to the non carboxylated form of protein C.

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.

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.