High-molecular-weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, 3 copies exist in the rat (1 encoding K-kininogen and 2 encoding T-kininogen). Here, we confirm that the mouse genome contains 2 homologous kininogen genes, mKng1 and mKng2, and demonstrate that these genes are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1 Ϫ/Ϫ mice were viable, but lacked plasma HK and low-molecular-weight kininogen (LK), as well as ⌬mHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Moreover, despite normal tail vein bleeding times, mKng1 Ϫ/Ϫ mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser-induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma HK contributes to induced arterial thrombosis in mice. (Blood. 2008;111:1274-1281)

Summary Plasminogen activator inhibitor‐1 (PAI‐1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) activity. Although the principal function of PAI‐1 is the inhibition of fibrinolysis, PAI‐1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI‐1 functions in vivo and in vitro. The use of Serpine1‐deficient mice has particularly enhanced our understanding of the functions of PAI‐1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI‐1 and its role in clinical conditions are discussed.

A genetically-transmissible factor (F) XII-inactivated allele has been produced in mice by targeted replacement of exons 3-8 of the FXII gene with the neomycin resistance gene. Interbreeding of these mice provided offspring homozygous for two inactivated FXII alleles (FXII(-/-)). Male and female FXII-deficient mice bred normally in all genotypic combinations of the heterozygous and homozygous states, and the offspring survived to adulthood, suggesting that a total FXII deficiency does not affect embryonic development and survival. Neither FXII transcripts nor FXII antigen was found in various tissues of adult FXII(-/-) mice. No obvious unchallenged coagulopathies were present in FXII(-/-) adult mice, despite greatly prolonged activated partial thromboplastin times in this mouse cohort. FXII(-/-) mice were then used to assess the in vivo importance of the plasma FXII/prekallikrein/kininogen pathway in provision of resting plasma bradykinin (BK) levels and in generation of plasma BK stimulated by contact with an artificial surface, using a new and greatly improved plasma BK assay developed during these studies. It was found that approximately 50% of resting BK, and all of the contact-stimulated plasma BK, was provided by this FXII-dependent pathway, without a requirement for FXI. These results provide clear evidence that surface-stimulated BK production, in mice, is dependent on the activation of FXII.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Active metabolism of clopidogrel is mainly mediated by CYP2C19. There are genetic differences in the activity of CYP2C19. Therefore, active metabolism of clopidogrel is affected by CYP2C19 genotypes.• The main metabolizing enzyme of proton pump inhibitors (PPIs) is CYP2C19. Therefore, the anti-platelet function of clopidogrel is attenuated by concomitant use of PPIs.• There are differences in the metabolic disposition among different PPIs. Affinity to CYP2C19 differs among different PPIs. WHAT THIS STUDY ADDS• Whether a PPI attenuates the efficacy of clopidogrel depends on CYP2C19. Individuals who are decreased metabolizers, i.e. carriers the allele of CYP2C19 *2 and/or *3, are more likely to convert from 'responder' to 'non-responder' to clopidogrel when placed on a concomitant PPI.• We found that rabeprazole, whose affinity to CYP2C19 has been considered lower, attenuated the efficacy of clopidogrel.• We tested whether the separate dosing of a PPI and clopidogrel decreased the risk of attenuation of clopidogrel efficacy. We unfortunately found that separate dosing did not avoid the problematic interaction between clopidogrel and a PPI in subject's with CYP2C19 *2 and/or CYP2C19 *3. AIMSThe efficacy of clopidogrel is influenced by CYP2C19 genotypes and substrates of CYP2C19, such as proton pump inhibitors (PPIs). We assessed the influence of three different PPIs on the anti-platelet function of clopidogrel in relation to CYP2C19 genotype status. METHODSThirty-nine healthy volunteers with different CYP2C19 genotypes took clopidogrel 75 mg with or without omeprazole 20 mg, lansoprazole 30 mg or rabeprazole 20 mg in the morning for 7 days. The influence of the three PPIs on the anti-platelet function of clopidogrel was determined. A less than 30% inhibition of platelet aggregation (IPA) during clopidogrel dosing was defined as a 'low responder' . We also examined whether evening dosing of omeprazole could prevent the interaction with clopidogrel dosed in the morning. RESULTSIn rapid metabolizers (RMs, *1/*1, n = 15) of CYP2C19, omeprazole and rabeprazole significantly attenuated the anti-platelet function of clopidogrel. In decreased metabolizers (DMs, carriers of *2 and/or *3, n = 24), there was a large variation in IPA and there was a trend but no significant decrease in IPA when placed on a concomitant PPI. Some DMs became 'low-responders' when placed on a concomitant PPI. Evening omeprazole dose in RMs did not seem to cause a significant decrease in IPA in contrast to morning dosing, but did so in DMs. CONCLUSIONSThe three PPIs affected the efficacy of clopidogrel to different degrees. Both omeprazole and rabeprazole significantly decreased IPA in RMs but not DMs, although there was a trend towards lower IPA in DMs.Morning and evening dosing of omeprazole were both associated with lower IPA in DMs.

Fibrinogen (Fg) is a 340-kd symmetrical heterodimeric protein consisting of three pairs of nonidentical polypeptide chains, and has an overall structure of (A␣/B␤/␥) 2 . This protein is the precursor of fibrin, formed during the hemostatic response. Three separate genes, FGA, FGB, and FGG, located on a 50-kb region of chromosome 4q28-q31, are translated in a coordinated fashion to code for the A␣-, B␤-, and ␥-chains, respectively, of Fg. 1 For its conversion into fibrin, thrombin first catalyzes release of fibrinopeptides A and B from the A␣ and B␤ chains, thus leading to exposure of polymerization sites that organize the initially formed fibrin monomers into a polymeric network of fibers that form the major protein component of the blood clot. This clot is ultimately covalently stabilized by crosslinking of specific ⑀-NH 2 groups of Lys and ␥-COOH groups of Glu residues of fibrin monomer, a process catalyzed by a transglutaminase, namely, activated Factor XIII (FXIIIa). 2

A model of gram-negative lethal endotoxin shock, involving continuous peritoneal infusion of lipopolysaccharide (LPS), has been applied to wild-type (WT) mice and mice with a severe deficiency of endothelial protein C receptor (EPCR ␦/␦ ). The survival of EPCR ␦/␦ mice was significantly diminished as compared to WT mice after administration of LPS via this route. Heart rates and central blood pressures also were significantly more depressed in EPCR ␦/␦ mice, indicating that the receptor-based protein C (PC) pathway functions in regulation of hemodynamic properties in the mouse. Further, heart muscle damage was more severe in EPCR ␦/␦ mice as compared to WT mice after endotoxin administration, as revealed by the more elevated plasma myoglobin levels in EPCR ␦/␦ mice and by microscopic examination of stained heart sections. Neutrophil infiltration was more pronounced in heart tissue of EPCR ␦/␦ mice, perhaps in response to the greatly increased expression level of the chemokine, MIP-2, which also significantly more up-regulated in the LPS-treated EPCR ␦/␦ mouse cohort. In conclusion, a severe deficiency of EPCR adversely affects survival of mice subjected to continuous infusion of endotoxin, via contributions of more responsive hemodynamic and cardiac alterations, thus suggesting that, among its other functions, the PC-based receptor system has a cardioprotective role after acute inflammatory challenge.

The fibrinolytic system consists of a balance between rates of plasminogen activation and fibrin degradation, both of which are finely regulated by spatio-temporal mechanisms. Three distinct inhibitors of the fibrinolytic system that differently regulate these two steps are plasminogen activator inhibitor type-1 (PAI-1), α2-antiplasmin, and thrombin activatable fibrinolysis inhibitor (TAFI). In this review, we focus on the mechanisms by which PAI-1 governs total fibrinolytic activity to provide its essential role in many hemostatic disorders, including fibrinolytic shutdown after trauma. PAI-1 is a member of the serine protease inhibitor (SERPIN) superfamily and inhibits the protease activities of plasminogen activators (PAs) by forming complexes with PAs, thereby regulating fibrinolysis. The major PA in the vasculature is tissue-type PA (tPA) which is secreted from vascular endothelial cells (VECs) as an active enzyme and is retained on the surface of VECs. PAI-1, existing in molar excess to tPA in plasma, regulates the amount of free active tPA in plasma and on the surface of VECs by forming a tPA-PAI-1 complex. Thus, high plasma levels of PAI-1 are directly related to attenuated fibrinolysis and increased risk for thrombosis. Since plasma PAI-1 levels are highly elevated under a variety of pathological conditions, including infection and inflammation, the fibrinolytic potential in plasma and on VECs is readily suppressed to induce fibrinolytic shutdown. A congenital deficiency of PAI-1 in humans, in turn, leads to life-threatening bleeding. These considerations support the contention that PAI-1 is the primary regulator of the initial step of fibrinolysis and governs total fibrinolytic activity.

Summary. Background: Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological regulator of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) activity. A number of studies have shown that elevated levels of PAI-1 are related to pathological states such as an increased risk of arterial thrombotic events and a poor prognosis for cancer patients; however, there are few reports about PAI-1 deficiency in humans because the disorder is very rare. Objective: To understand the in vivo impact of a complete PAI-1 deficiency, Serpine1 )/) mice were generated; a number of in vivo studies have been conducted to elucidate the function of PAI-1 using Serpine1 )/) mice. The phenotypes demonstrated in Serpine1 )/) mice, however, were quite different from those in humans. Therefore, it is necessary to find out and analyze SERPINE1 deficiency in humans. Patient and methods: The patient is a 47-year-old woman who has had multiple episodes of major bleeding. Although most of the patientÕs blood coagulation factors were functionally normal, her PAI-1 antigen levels were undetectable. Therefore, DNA sequencing of the SERPINE1 gene were analyzed. Results: The proband had a homozygous 1-bp duplication (C) at exon 3 (c.356dupC; p.Ile120AspfsX42). Both wild-type PAI-1 (42.7 kDa) and mutated (Mut) PAI-1 (14.7 kDa) were expressed in COS-1 cells, although the level of Mut PAI-1 expressed in the cell lysates was much lower. Wild-type PAI-1 was observed in the culture supernatant, whereas no Mut PAI-1 was detected in the supernatant. Conclusions: Considering the results of the present study, the translation of mouse studies to humans must be performed with great care.