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Blood, Vol. 114, Issue 9, 1929-1936, August 27, 2009
High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension
Blood Morris et al.
114: 1929
Supplemental materials for: Morris et al
Files in this Data Supplement:
- Figure S1. Validation of the fibrin B knob release assay (JPG, 24 KB)
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Competition ELISA shows that the synthetic B knob peptide (the standard for the assay) behaves identically to a crude peptide mixture derived from a complete plasmin digest of normal fibrin.
- Figure S2. Mass spectra of the γ R375W fibrinogen variant (JPG, 29.1 KB)
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The Bβ and γ chain mass spectra from patient #1 are compared to those of a typical control. The predominant peaks represent the mono and disialylated isoforms of each chain (mono- and disialylation of the biantennary oligosaccharides linked to Bβ Asn-364 and γ Asn-52). In patient #1, the Bβ and the γ chains have a higher proportion of the disialylated (291 Da heavier peaks) chain compared to all controls. Gene sequencing disclosed a heterozygous C>T mutation in exon 9 of the FGG gene (inset), resulting in a R375W substitution in the mature γ chain. The mass difference affected by this substitution (−30 Da) was not reflected in the γ chain mass spectrum. However, fibrinogen molecules comprised of mutant γ chains are retained in the hepatocytes and never reach the circulation. The codon affected by the mutation is denoted in capital letters. Vertical dashed lines denote positions of the predominant peaks in the typical control mass spectrum, which result from mono- and disialylation of each chain.
- Figure S3. Mass spectrum of the γ Y114H fibrinogen variant (JPG, 26.7 KB)
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The γ chain mass spectrum from patient #2, compared to that of a typical control, disclosed an additional peak pair 28 Da lighter than the typical peak pair observed in both the patient and the control. Gene sequencing disclosed a heterozygous T>C mutation in exon 5 of the FGG gene (inset), resulting in a Y114H substitution in the mature γ chain. The mass change affected by this substitution (−26 Da) is in close agreement with the mass difference between the atypical and typical peak pairs in the patient’s mass spectrum. The codon affected by the mutation is denoted in capital letters. Vertical dashed lines denote positions of the predominant peaks in the typical control mass spectrum, which result from mono- and disialylation of the γ chain.
- Figure S4. Mass spectra of the Bβ P235L fibrinogen variant (JPG, 37.1 KB)
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The Bβ chain mass spectrum from patient #1, compared to that of a typical control, disclosed a predominant peak pair slightly heavier (14 Da) and wider than the peak pair from the control. The Bβ chain mass spectrum from patient #3, who was heterozygous for the common Bβ R448K polymorphism, allowed resolution of an additional peak pair 15 Da heavier than the typical peak pair in the control. Gene sequencing disclosed a heterozygous C>T mutation in exon 5 of the FGB gene from both patients (insets), resulting in a P235L substitution in the mature Bβ chain. The mass change affected by this substitution (+16 Da) is in close agreement with the mass difference between the atypical peak pair in both patients and the typical peak pair in the control. This mutation was also found in patient #2 (mass spectrum not shown). The codon affected by the mutation is denoted in capital letters. Vertical dashed lines denote positions of the predominant peaks in the typical control mass spectrum, which result from mono- and disialylation of the Bβ chain.
- Figure S5. Mass spectrum of the Aα L69H fibrinogen variant (JPG, 35.5 KB)
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The Aα chain mass spectrum from patient #4, compared to that of a typical control, disclosed a peak pattern 22 Da heavier than the typical peak pattern observed in the control. Gene sequencing disclosed a heterozygous T>A mutation in exon 3 of the FGA gene (inset), resulting in a L69H substitution in the mature at Aα chain. The mass change affected by this substitution (+24 Da) is in close agreement with the mass difference between the atypical and typical peak patterns observed in the patient’s spectrum. The codon affected by the mutation is denoted in capital letters. Vertical dashed lines denote positions of the predominant peaks in the typical control mass spectrum, which result from non-, mono-, and di-phosphorylation of the Aα chain.
- Figure S6. Mass spectrum of the Aα R554H fibrinogen variant (JPG, 34.9 KB)
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The Aα chain mass spectrum from patient #5 disclosed an additional (atypical) peak pattern 45 Da lighter than the typical peak pattern observed in both the patient and the control. Gene sequencing disclosed the common heterozygous Aα T312A polymorphism (not shown) and a second heterozygous G>A mutation in exon 5 of the FGA gene (inset), resulting in a R554H substitution in the mature Aα chain. The mass change affected by both substitutions (−49 Da) is in close agreement with the mass difference between the atypical and typical peak patterns observed in the patient’s spectrum. The codon affected by the mutation is denoted in capital letters. Vertical dashed lines denote positions of the predominant peaks in the typical control mass spectrum, which result from non-, mono-, and di-phosphorylation of the Aα chain.
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