Calcium-dependent proteolysis of actin during storage of platelet concentrates

In an ongoing study of the changes that occur in platelet concentrates during storage, we investigated two 28-26-Kd proteins designated SP-1 and SP-2, respectively, which increase markedly during blood-bank storage of platelet concentrates at room temperature. Formation of SP-1 and SP-2 was inhibited by storage at 4 degrees C as well as by treatment of the concentrates with leupeptin, N-ethylmaleimide, and EDTA; DFP and PPACK had no effect. The calcium ionophore A23187 markedly stimulated production of SP-1 and SP-2. After partial purification, the two proteins were found to be associated with platelet cytoskeletal protein. Two-dimensional peptide mapping and amino acid sequencing identified SP-1 and SP-2 as fragments of actin formed by cleavage on the N-terminal side of residues Thr-106 and Ala- 114, respectively. Generation of SP-1 and SP-2 during storage of platelet concentrate is likely attributable to calcium-dependent neutral protease degradation of actin and may have implications for development of the platelet storage lesion.     

The Apple 1 and Apple 4 domains of factor XI act synergistically to promote the surface-mediated activation of factor XI by factor XIIa

Binding sites for high molecular weight kininogen (HK) and for factor XIIa are present in the Apple 1 (A1) and the A4 domains of factor XI, respectively. To define the roles of these two sites in surface- mediated factor-XI activation we prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) and determined their effects on the activation of factor XI by factor XIIa in the presence of HK and either kaolin or dextran sulfate. Surface-mediated factor-XI activation by factor XIIa was inhibited by a conformationally constrained A4 peptide (Ala317-Gly350), by an A1 peptide (Phe56-Ser86), and by rA1 (Glu1-Ser90). When used in combination at equimolar concentrations, rA1 and A4 peptide were 10-fold more effective than either one alone in inhibiting surface-mediated activation of factor XI by factor XIIa. The A4 peptide was a competitive inhibitor of factor XIIa amidolytic activity and a noncompetitive inhibitor of factor-XI activation by factor XIIa, whereas rA1 and the A1 peptide did not inhibit factor XIIa. The rA1 domain inhibited factor XI binding to HK, whereas the A4 peptide did not. We conclude that specific sequences exposed on the surfaces of the A1 (Val59-Lys83) and A4 (Ala317-Gly350) domains of factor XI act synergistically to promote surface-mediated factor-XI activation by factor XIIa in the presence of HK by binding factor XI to surface-bound HK (A1 domain) and by binding factor XIIa near the cleavage site (Arg369-Ile370) of factor XI (A4 domain).     

The deletion in both common types of hereditary persistence of fetal hemoglobin is approximately 105 kilobases

The most common forms of hereditary persistence of fetal hemoglobin (HPFH) involve large deletions that remove the adult delta and beta genes but leave the paired fetal genes (G gamma and A gamma) intact. The size of these deletions has previously eluded exact definition. Using pulsed-field gel electrophoresis and the enzyme SfiI, which cuts only rarely in genomic DNA, we have constructed a large-scale restriction map of the beta-globin cluster in normal and HPFH DNA. The deletions in HPFH-1, which occurs in American blacks, and in HPFH-2, which occurs in Ghanaian blacks, are found to be approximately 105 kilobases (kb) in length, though the endpoints are staggered by approximately 5 kb. The fact that two previously reported gamma delta beta-thalassemia deletions to the 5' side of the beta-globin cluster are also about 100 kb suggests a common mechanism, possibly involving the loss of a complete chromatin loop.     

Parathyroid hormone 1–34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

Objective

Parathyroid hormone 1–34 (PTH[1–34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone–related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1–34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA).

Methods

We studied the effect of PTH(1–34) on human articular chondrocytes with azacytidine (azaC)–induced terminal differentiation in vitro and on papain‐induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl‐2, and Bax by real‐time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees.

Results

AzaC induced terminal differentiation of human chondrocytes, including down‐regulation of aggrecan, type II collagen, and GAG and up‐regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3–10 days of treatment with 10 nM PTH(1–34). SOX9 expression was not changed by either azaC or PTH(1–34) treatment. Bcl‐2 and Bax were up‐regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1–34) treatment did not reverse this effect. Furthermore, PTH(1–34) treatment reversed papain‐induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats.

Conclusion

Our findings indicate that PTH(1–34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA.