Emily S. Barrow

Use of a simple visual assay of Willebrand factor for diagnosis and carrier identification.

Summary. A visual assay of factor VIII‐related Willebrand factor (VIIIR:WF) is described which utilizes formaldehyde‐fixed platelets, end points being read in microflocculation tiles. Four dilutions of a sample can be assessed simultaneously, and the correlation with aggregometric assays is high (r=0.91). Measurement error is 8.0% for a single assay in triplicate and less than 5% if an assay is repeated three times. The method has been used for 2 years by the coagulation genetics group at Chapel Hill for diagnosing subjects with von Willebrands disease and assigning genotypes to members of families transmitting this disorder. Its utility in classifying known carriers of haemophilia A has also been examined, both in conjunction with assays of VIII:C and in a three‐way test with assays of VIII:C and VIIIR:Ag. As predicted by the Lyon hypothesis, the rate of false negative diagnosis was higher than false positive diagnosis, but the overall rate of misclassification on single plasma samples was 7/51=13.7%. The error rate was the same whether discrimination was based upon assays of VIII:C vs. VIIIR:Ag, VIII:C vs. VIIIR:WF, or VIII:C vs. VIIIR:Ag vs. VIIIR:WF, the same individuals being misclassified by each method. The observed rate of misclassification was well within the rates reported by others and very similar to our previous experience. We have concluded that this method of assaying VIIIR:WF is highly useful for diagnosing vWd, detecting inhibitors to VIIIR:WF, and examining large numbers of column fractions. It is a useful supplement, although it cannot yet substitute for, assays of VIIIR:Ag in detecting carriers of haemophilia A.

Genetic counselling in haemophilia by discriminant analysis 1975-1980

Between January 1975 and January 1980 we counselled 214 possible or obligatory carriers of haemophilia A in an attempt to provide the counsel they needed and to determine the utility of a probabilistic method of assigning the heterozygous genotype (carrier detection). We found the method of assignment to be quick and easy to use, and the single output (the final probability favouring carriership P(C)) to be understood by most counsellees. The final probabilities obtained were either very high or very low in 80% of the women, which allowed us to give clear counsel in four instances in five. The final probabilities could also be used to relate Mendelian expectation to observation within each of three subsets of women (18 mothers of sporadic haemophiliacs, 78 sisters, and 62 more distant relatives), while the aberrant likelihood ratios of 6/36 (17%) of the obligatory carriers provided an estimate of the false negative diagnostic rate owing to lyonisation. There was no significant age effect on VIII:C or VIIIR:Ag levels of obligatory carriers, and the VIII:C levels of the obligatory carriers who had received the gene from their fathers did not differ from those of the obligatory carriers who had received the gene from their mothers. The ratios of high:low probabilities among the sisters and distant relatives of haemophiliacs conformed to genetic expectation, while the ratio among mothers of sporadic haemophiliacs suggested that their expectations of carriership were greater than 80%. Twenty of the 214 counsellees (9%) were pregnant on the first visit, and 13 of those with low P(C)s (0·0-0·33) went to term and delivered 11 non-haemophilic children. Four with P(C)s between 0·50 and 1·00 requested amniocenteses, and one male was aborted. Three who were obligatory carriers also requested amniocentesis which led to the abortion of a second male. Seven women who were assessed before pregnancy and found to have high P(C)s returned after becoming pregnant. All requested amniocentesis (one twice) and fetoscopy was requested for six of the seven males discovered, one male having been aborted before fetoscopy became available. Of the six males who were fetoscoped, three normal males reached term, one normal male was born prematurely, and one haemophilic male and one normal male did not survive fetoscopy.

The genetics of blood coagulation

Hemostasis is the term applied to the process that regulates the loss of blood from the circulatory system following injury. It involves three interrelated physiological mechanisms: constriction of blood vessels, aggregation of blood platelets to damaged subendothelial surfaces, and the formation of fibrin clots. Together these produce the vascular plug that prevents further bleeding. Abnormal function of one or more of the separate mechanisms may result in excessive bleeding or hemorrhage.

The Separation of Willebrand Factor from Factor VIII‐Related Antigen

Summary. The three activities associated with factor VIII—coagulant (VIII:C), antigenic (VIIIR:Ag), and platelet agglutinating or Willebrand factor (VIIIR:WF)— have been separated by sequential antibody affinity chromatography, utilizing a rabbit antibody to factor VIII and a spontaneous human antibody to VIII:C. Normal plasma differentially lost its factor VIII‐related antigen following passage over the rabbit antibody column. Subsequent passage of the VIIIR:Ag‐depleted plasma over the human antibody column resulted in the loss of VIII:C activity, with retention of the Willebrand factor activity, antigen being partially recovered from the heterologous antibody column. These experiments demonstrate that it is possible to separate two of the factor VIII activities, VIIIR:Ag and VIIIR:WF, which are usually regarded as properties of a single molecule.

Polyglutamic and Polyaspartic Acids: Synthetic Polypeptides with Predominantly Factor VIII-like Coagulant Activity

Summary and discussion We have shown that polyglutamic and polyaspartic acids are quite active in specific assays for F. VIII, much less so in specific assays for F. IX, and inactive in four other systems (Table I). The F. VIII and IX activities are demonstrable only when the deficient substrate plasmas have been activated previously with kaolin and when phospholipid is also present (Tables II and III). Furthermore, polyglutamic acid “potentiates” F. VIII activity, two- to fivefold, when it is added to normal human plasma or F. VII-rich fractions of normal human plasma (Table IV). “Potentiation” of F. VIII by PGA is apparently different from activation or enhancement of F. VIII activity by thrombin. Very low concentrations of thrombin are known to activate F. VIII in a time-dependent reaction in which maximum activation does not occur until several minutes after mixing (8). This increase in F. VIII activity is unstable, decreasing to the original value upon further incubation. In contrast, the “potentiating” effect upon plasma F. VIII of polyglutamic acids (as well as kidney AHF and AHF3 prepared from albumin) is observed immediately upon mixing and remains stable for hours at room temperature (unpublished observations). Our experiments suggest that polyglutamic acid does not decrease clotting times by activating factors XII, XI, or IX, but that, once these factors have been activated by kaolin, polyglutamic acid is able to “substitute” for F. VIII. The fact that the PGA shows a slight corrective effect in previously activated F. IX deficient plasma suggests a possibly even wider role, i.e., that polyglutamic acid may mimic the “intrinsic F. X activator.” Kaolin-activation of the F. VIII deficient substrate is an absolute requirement for expression of the activity of the polypeptides.

Identifying Carriers of Mild Haemophilia

Summary. The problems of carrier identification in mild haemophilia were examined by studying a large kindred transmitting VIII:C levels which averaged 17 u/dl in affected males. Fifteen obligatory carriers and 13 normal women from this kindred were used as reference groups to produce a set of linear discriminants. The utility of these discriminants in identifying carriers of mild haemophilia was compared with that of a similar set of discriminants which had been prepared for use with carriers of severe haemophilia, each set of discriminants being tested against both data bases. It was found that both sets of discriminants had large error rates when applied to the mild data base, and that both had much smaller error rates when applied to the severe data base. This outcome resulted from the greater overlap between the factor VIII‐related activities of mild carriers and normal women than the overlap of these activities between severe carriers and normal women.

Elevation of total progressive antithrombin in Von Willebrand's disease

Abstract Plasmas from 30 normals, 46 patients with von Willebrands disease (vWd) and 17 with hemophilia A were examined for levels of Total Progressive Antithrombin (TPA) and for three Factor VIII-related activities: coagulant (VIII:C), antigenic (VIIIR:Ag) and Willebrand factor (VIIIR:WF). The mean of TPA for the normals was 102.1%, and for the hemophiliacs 102.6% while that for the vWd group was 133.4%. The difference was statistically highly significant. The 1 3 elevation of TPA in vWd was accompanied by reduction to 44% of the average of all the F. VIII activities. The normal TPA in hemophilia A plasmas suggests that the inverse relationship between TPA and F. VIII in vWd is not the result of the reduced coagulant activity.