Specific 5-HT2A receptor blockade with sarpogrelate immediately after MCT inhibited PAH and prolongs survival in rats. These effects were accompanied by anti-inflammatory and anti-proliferative effects in the lung tissue and marked improvement of pulmonary vascular endothelial dysfunction and activation.

To examine the role of the fibrinogen ␥ chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated ␥ chains, terminating between residues ␥379 and the C-terminus, ␥411. The variant fibrinogens were synthesized from altered ␥-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with ␥ chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the ␥ chain, from 1.4 g/mL for normal fibrinogen to 0.39 g/mL for ␥ 387 fibrinogen. Immunoassays of cell lysates showed that all variant ␥ chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the ␥-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the ␥ chain are essential for fibrinogen assembly, and more specifically, that ␥387 is critical. We propose that the loss of residue ␥387 destabilized the structure of ␥ chain, preventing assembly of ␣␥ and ␤␥ dimers, essential intermediates in the assembly of normal fibrinogen. IntroductionFibrinogen is a 340-kd plasma glycoprotein consisting of 2 copies of 3 polypeptide chains, A␣, B␤, and ␥, linked by an extensive network of 29 intrachain and interchain disulfide bonds. 1,2 A separate gene encodes each polypeptide chain. The 3 chains are synthesized, assembled into the 6-chain molecule, and secreted from hepatocytes into the plasma. Studies of fibrinogen expressed from the endogenous genes in human hepatocytes or from transfected complementary DNAs (cDNAs) in baby hamster kidney (BHK) cells have shown that assembly occurs through specific intermediates. 3,4 These intermediates, ␣␥ dimers, ␤␥ dimers, and ␣␤␥ half-molecules, all include the ␥ chain.Normal fibrinogen levels are 2 to 4 mg/mL in plasma. Hypofibrinogenemia or afibrinogenemia, defined as reduced or immeasurable levels of fibrinogen in plasma, can be hereditary. Although the first case of afibrinogenemia was reported in 1920, 5 the genetic basis of these abnormalities was first demonstrated in 1999. 6 Since that time, 26 different mutations have been identified in cases of afibrinogenemia or hypofibrinogenemia. [7][8][9][10][11][12][13][14][15][16][17][18][19] These mutations were found in all 3 genes, and include missense, nonsense, and frameshift mutations; splice-site abnormalities; and large deletions. Thus, fibrinogen deficiencies are associated with a variety of genetic chan...

SummaryFibrinogen Matsumoto I is a novel hereditary dysfibrinogen identified in a 1-year-old boy with Down’s syndrome. Though he showed no apparent bleeding or thrombotic tendency, he had a congenital heart disease. Preoperative coagulation tests of his plasma revealed a prolonged thrombin time and the fibrinogen level determined by the thrombin time method was markedly decreased. Molecular weight of fibrinogen chains showed apparently normal Aα, Bβ-, and γ-chains. The rate of fibrinopeptide release was normal, whereas fibrin polymerization was delayed. Fibrinogen γ-chain gene fragments from the propositus were amplified by polymerase chain reaction then sequenced. The triplet GAT, coding for the amino acid residue γ364, was replaced by CAT, resulting in the substitution of Asp→His. This residue is adjacent to the Tyr-363 that is demonstrated to be the primary site for fibrin polymerization. Our results indicate that the residue γ364 Asp is essential for normal polymerization of fibrin monomer.

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of γ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.

Introduction: We examined a 6-month-old girl with inherited fibrinogen abnormality

Summary. Background: Fibrin polymerization is mediated by interactions between knobs ÔAÕ and ÔBÕ exposed by thrombin cleavage, and holes ÔaÕ and ÔbÕ always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. Objectives: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ÔaÕ: c364Ala, c364His or c364Val. Methods: We examined thrombin-and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Results: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of c364Val and c364His were more delayed than c364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ÔAÕ. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes ÔaÕ and ÔbÕ, respectively. FXIIIa-catalyzed crosslinking between c-chains was markedly delayed for all the variants. Conclusion: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ÔaÕ. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

We investigated whether nicorandil might prevent and reverse monocrotaline (MCT)-induced pulmonary arterial hypertension. Rats were injected with 50 mg/kg of MCT subcutaneously and randomized to either 7.5 mg/kg/d of nicorandil in drinking water or placebo for 3 weeks. Animals that were treated with MCT and survived for 3 weeks were assigned to either nicorandil or placebo. Nicorandil markedly attenuated pulmonary arterial hypertension with severe structural remodeling of the pulmonary vessels. The survival rate at 3 weeks after treatment was significantly increased in the nicorandil group compared with the placebo group (73% versus 39%, P<0.05). In the placebo group, endothelial nitric oxide synthase (eNOS) protein was significantly decreased, the numbers of the CD45-positive cells and those of the proliferating cell nuclear antigen-positive cells were increased in the lung tissue, and P-selectin was intensely expressed on the endothelium of the pulmonary arteries. These features were prevented by nicorandil. Late treatment with nicorandil did not palliate established pulmonary arterial hypertension nor improved survival. Thus, nicorandil inhibited development of MCT-induced pulmonary arterial hypertension but failed to reverse it. These effects were associated with marked up-regulation of diminished lung eNOS protein along with improvement of pulmonary vascular endothelial activation and anti-inflammatory and anti-proliferative effects in the lung tissue.

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