Judith S. Greengard

Protein C inhibitor is expressed in tubular cells of human kidney.

Protein C inhibitor (PCI) is a serpin that inhibits a number of proteases. PCI is found in urine and binds to kidney epithelial cells. To determine if kidney is a source of PCI, cDNA was produced from human kidney total RNA. Sequencing and restriction mapping showed identity between kidney and liver PCI cDNA sequences. Similar cDNAs were obtained from rhesus monkey kidney and liver RNAs. Conditioned medium from the rhesus monkey kidney cell line CCL7.1 was analyzed on immunoblots, showing a 57,000-D protein band that comigrated with human plasma PCI. Immunohistochemical staining and in situ hybridization of human kidney tissue sections showed that kidney PCI antigen and RNA were confined to tubular cells. The findings are consistent with the idea that PCI is synthesized and localized in kidney tissue where it may provide protease inhibitory activity and suggest that complexes of PCI with urokinase found in human urine may be produced locally in the kidney.

Two Mutations in the Promoter Region of the Human Protein C Gene Both Cause Type I Protein C Deficiency by Disruption of Two HNF-3 Binding Sites

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by the proteolytic inactivation of factors Va and VIIIa. Individuals affected with protein C deficiency are at risk for thrombosis. Genetic analyses of affected individuals, to determine the cause of the protein C deficiency, revealed a large variety of mutations in the protein C gene, including several in the promoter region of this gene. Comparison of the region around two of these mutations, A G and T A, with transcription factor consensus sequences suggested the presence of two overlapping and inversely oriented HNF-3 binding sites. Direct evidence for the presence of the two HNF-3 binding sites in the protein C promoter was obtained using electrophoretic mobility shift assays and UV cross-linking experiments. These experiments revealed that HNF-3 can bind specifically to both putative HNF-3 sites in the wild-type protein C promoter. Due to the T A mutation, one binding site is completely lost, while the other site still binds HNF-3, but with strongly reduced affinity. As a consequence of the A G mutation, the protein C promoter loses all its HNF-3 binding capacity. Transient transfection experiments demonstrated that the binding of HNF-3 to the protein C promoter is of physiological significance. This followed from experiments in which the introduction of the A G or T A mutation resulted in a 4-5-fold reduced promoter activity in HepG2 cells. Furthermore, transactivation of the wild-type protein C promoter construct with HNF-3 showed a 4-5-fold increased promoter activity in HepG2 cells. In HeLa cells, significant wild-type promoter activity was only observed after transactivation with HNF-3. When a promoter construct containing the T A mutation at position −27 was used, the transactivation potential of HNF-3 was 2-fold reduced in HepG2 cells, whereas in HeLa cells no transactivation was observed. With the promoter construct containing the A G mutation, no transactivation by HNF-3 was found either in HepG2 or in HeLa cells.

Protein S, an Antithrombotic Factor, Is Synthesized and Released by Neural Tumor Cells

Abstract: Protein S, an anticoagulant factor in the protein C antithrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three‐to 11‐fold higher than that of a microvascular endothelial cell line and 36–144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully γ‐carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomain protein beyond its function in control of thrombosis.

Genetic mutations in ten unrelated American patients with symptomatic type 1 protein C deficiency

Symptomatic patients with Type 1 protein C deficiency and venous thrombosis were analysed for defects in this gene using polymerase chain reaction amplification and direct sequencing of all nine exons. Ten different heterozygous point mutations were detected in 19 patients from eleven American families. Seven represent novel mutations. Two of these were found in the TATA box or near the transcription initiation site and presumably lead to loss of transcription, and seven missense mutations were found including G103R, P168L, R169W, I201T, P279L, T298M, and C384Y. These may lead to abnormal folding or thermodynamic instability of the protein C molecule, potentially causing abnormal secretion or rapid clearance from the circulation. Two other protein C mutations, a nonsense mutation at codon Trp-145 and a deletion inducing a frameshift at codon 364 resulting in premature termination at codon 378, likely lead to unstable products. The previously published R169W mutation resulted in a Type 1 deficiency. The data show that diverse molecular defects result in similar phenotypes and emphasize that a wide variety of mutations are responsible for Type 1 protein C deficiency in the American setting of a diverse population.

Exonic polymorphisms in the protein C gene: interethnic comparison between Caucasians and Asians

Plasma protein C deficiency is associated with inherited thrombotic disease. Allelic frequencies of five previously reported DNA polymorphisms and a new polymorhic site (C8480T) were calculated in Asian and American Caucasian individuals by direct genomic sequencing and compared to previous reports.