Platelet-type von Willebrand disease (PTvWD) is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of von Willebrand factor (vWF) by patient platelets. Although the platelet glycoprotein (GP) Ib/IX complex is known to constitute the platelet's ristocetia-dependent receptor for vWF, a unique structural abnormality within this complex has not previously been identified in PT-vWD. Using the polymerase chain reaction to amplify genomic DNA coding for the a chain of GP lb (GP Ibta) and then sequencing the amplified DNA following cloning into M13mpl8 and M13mpl9 phage vectors, we have found a single point mutation in the GP Iba coding region of PT-vWD DNA resulting in the substitution of valine for glycine at residue 233. This substitution within the vWF-binding region of GP Iba is likely to exert a significant influence on the conformation of the resulting protein. Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Iba mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-tpe alleles in the affected PT-vWD patients. Absence in the normal population, tight linkage with phenotypic expression of disease, and absence of any additional abnormality of GP Iba in these patients identify the glycine-to-valine substitution as a point mutation underlying functional abnormality of the vWF receptor in PT-vWD.Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patients characteristically show prolonged bleeding times, borderline thrombocytopenia, and decreased von Willebrand factor (vWF) high molecular weight multimers and functional activity (1-5). PT-vWD appears to result from an abnormality of the platelet receptor for vWF, whereby patient platelets show an abnormally increased binding of circulating vWF. In the laboratory, this platelet hyperresponsiveness may be demonstrated with the use of low concentrations of ristocetin. Whereas normal platelets show little or no aggregation at ristocetin concentrations as low as 0.5 mg/ml, patient platelets typically show significant binding of vWF, together with strong aggregation, following stimulation by 0.5 mg/ml, or even lower, concentrations of ristocetin (1-3). The unique ability of desialylated vWF (asialo-vWF) to agglutinate patient platelets in the presence of the divalent-cation chelator EDTA has additionally been demonstrated (6). Platelets from patients with PT-vWD also show a characteristically increased binding of the monoclonal antibody C-34, which is directed against an epitope within the platelet glycoprotein (GP) lb/IX complex (7). Although this complex is known to constitute the platelet's ristocetin-dependent receptor for vWF (8), identification of a u...

Summary. The majority of patients with idiopathic thrombocytopenic purpura (ITP) have antiplatelet autoantibodies that are most frequently directed against platelet glycoproteins IIb/IIIa or Ib/IX/V. However, there is some debate whether the immune response is oligoclonal or polyclonal in nature. We investigated the subclass distribution of anti-IIb/IIIa IgG autoantibodies in 59 prospectively studied patients with ITP. We also tested patients with a variety of thrombocytopenic disorders (n ¼ 31) and healthy controls (n ¼ 30). Platelet lysates were tested for IgG anti-IIb/IIIa autoantibodies, and the specific IgG subclass distribution was measured using antigen capture assays. All testing was done blinded to diagnosis and other assay results. After unblinding, we found that 43 of the 59 ITP patients had anti-IIb/IIIa autoantibodies (sensitivity ¼ 73%). Anti-IIb/IIIa autoantibodies were also detected in five of the 31 non-ITP patients, but in none of the 30 healthy controls (specificity ¼ 91%). The IgG subclass assay was positive in 39 of the 43 ITP patients with anti-IIb/IIIa antibodies (sensitivity ¼ 92%) and in 12 samples that had no detectable anti-IIb/IIIa antibodies including two ITP patients (specificity ¼ 83%). The most common subclass in the ITP patient samples was IgG1 (77%), either alone (n ¼ 14) or with other IgG subclass antibodies (n ¼ 19). However, there were also patients with only IgG2 (n ¼ 2), IgG3 (n ¼ 3) or IgG4 (n ¼ 3) antibodies. Our results are consistent with the hypothesis that ITP is a heterogeneous disorder and that some patients have evidence of oligoclonality, whereas other patients have polyclonal autoantibodies.

Decreased von Willebrand factor (VWF)-cleaving protease activity (<5%) has been implicated in patients with congenital thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (Upshaw-Schulman syndrome) and associated with mutations within the ADAMTS13 gene. In this report, we describe longitudinal studies in a patient with congenital TTP who ultimately developed end-stage renal failure and required plasma therapy from infancy. The patient was deficient in plasma high molecular weight (HMW)-VWF multimers during acute disease but had increased amounts of the HMW-VWF multimers during periods of remission. DNA analysis of this patient detected homozygosity for the R692C mutation on exon 17 of the ADAMTS13 gene, previously linked to congenital TTP. The level of VWF-cleaving protease activity in the patient was remarkably low (<5%) throughout her disease, even after she entered complete remission. However, despite no improvement in the level of VWF-cleaving protease activity, this patient had complete resolution of disease following splenectomy and commencing hemodialysis, without need for ongoing plasma therapy. The patient has remained in remission for over 4 years. These observations suggest that there are other factors in conjunction with severe deficiency of VWF protease activity that participate in the platelet-mediated thrombotic complications and other disease manifestations of congenital TTP. In addition, it is possible that splenectomy could be an effective treatment option for some patients with severe, congenital TTP. Am.

The underlying molecular basis for Bernard-Soulier Disease (BSD) is currently unknown. Platelets from patients with this autosomal recessive bleeding disorder have multiple abnormalities, including a markedly reduced von Willebrand factor-dependent adhesiveness due to a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex. In the present studies, we have used an intragenic restriction fragment length polymorphism (RFLP) for Taq I in the GPIb alpha gene to study linkage between this gene and the inheritance of BSD in a family with two affected siblings. Whereas the proband was heterozygous, showing both the 0.7 and 4.0 kb bands of this polymorphism (A/B), her affected brother was homozygous for the 0.7 kb band (A/A). Accordingly, these siblings did not inherit the same pair of GPIb alpha alleles from their parents. Additionally, one child of the proband was A/A, while the second studied child was A/B, with neither showing any evidence of BSD. No construct of heterozygosity or homozygosity for GPIb alpha alleles in this family is consistent with a model in which one or more defective GPIb alpha alleles could produce BSD. RFLP analysis with BamHI or HindIII showed entirely normal patterns in the patients, indicating the absence of any gross deletion of the GPIb alpha gene. GPIb alpha mRNA from patient platelets was reverse transcribed and subsequently amplified by the polymerase chain reaction, demonstrating the presence of GPIb alpha transcript. Furthermore, trace amounts of GPIb could be shown on the surface of patient platelets. Based on these results, a defect in the GPIb alpha gene is unlikely to be the cause of BSD in this family.