Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder

Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.     

Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.