Relationship of plasmin generation to cardiovascular disease risk factors in elderly men and women. (1/266)

Plasmin-alpha2-antiplasmin complex (PAP) marks plasmin generation and fibrinolytic balance. We recently observed that elevated levels of PAP predict acute myocardial infarction in the elderly, yet little is known about the correlates of PAP. We measured PAP in 800 elderly subjects who were free of clinical cardiovascular disease in 2 cohort studies: the Cardiovascular Health Study and the Honolulu Heart Program. Median PAP levels did not differ between the Cardiovascular Health Study (6.05+/-1.46 nmol/L) and the Honolulu Heart Program (6.11+/-1.44 nmol/L), and correlates of PAP were similar in both cohorts. In CHS, PAP levels increased with age (r=0. 30), procoagulant factors (eg, factor VIIc, r=0.15), thrombin activity (prothrombin fragment F1+2, r=0.29), and inflammation-sensitive proteins (eg, fibrinogen, r=0.44; factor VIIIc, r=0.37). PAP was associated with increased atherosclerosis as measured by the ankle-arm index (AAI) (P for trend, +info)

Alpha2-antiplasmin gene deficiency in mice is associated with enhanced fibrinolytic potential without overt bleeding. (2/266)

alpha2-antiplasmin (alpha2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine alpha2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with the neomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated alpha2-AP allele was observed, and homozygous deficient (alpha2-AP-/-) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of alpha2-AP mRNA in kidney and liver from alpha2-AP-/- mice, in contrast to wild-type (alpha2-AP+/+) mice. Immunologic and functional alpha2-AP levels were undetectable in plasma of alpha2-AP-/- mice, and were about half of wild-type in heterozygous littermates (alpha2-AP+/-). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for alpha2-AP+/+, alpha2-AP+/-, and alpha2-AP-/- mice. After amputation of tail or toe tips, bleeding stopped spontaneously in alpha2-AP+/+, as well as in alpha2-AP+/- and alpha2-AP-/- mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin-labeled plasma clots was significantly higher in alpha2-AP-/- than in alpha2-AP+/+ mice when injecting clots prepared from alpha2-AP+/+ plasma (78% +/- 5% v 46% +/- 9%; mean +/- SEM, n = 6 to 7; P =.02) or from alpha2-AP-/- plasma (81% +/- 5% v 46% +/- 5%; mean +/- SEM, n = 5; P =.008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in alpha2-AP-/- mice, as compared with alpha2-AP+/+ mice (P +info)

Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation. (3/266)

Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion.  (+info)

Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. (4/266)

BACKGROUND: The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or alpha2-antiplasmin (PAI-1(-/-) or alpha2-AP-/-). METHODS AND RESULTS: FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6+/-1.1 mm3 (mean+/-SEM), uPA-/- mice had similar infarcts (7.8+/-1.0 mm3, P=NS), tPA-/- mice smaller (2.6+/-0.80 mm3, P<0.0001), PAI-1(-/-) mice larger (16+/-0.52 mm3, P<0.0001), and Plg-/- mice larger (12+/-1.2 mm3, P=0.037) infarcts. alpha2-AP-/- mice had smaller infarcts (2. 2+/-1.1 mm3, P<0.0001 versus wild-type), which increased to 13+/-2.5 mm3 (P<0.005 versus alpha2-AP-/-) after intravenous injection of human alpha2-AP. Injection into alpha2-AP-/- mice of Fab fragments of affinospecific rabbit IgG against human alpha2-AP, after injection of 200 microg human alpha2-AP, reduced FCI from 11+/-1.5 to 5.1+/-1.1 mm3 (P=0.004). CONCLUSIONS: Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity (tPA gene inactivation) reduces whereas its augmentation (PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity (Plg gene inactivation or alpha2-AP injection) increases whereas its augmentation (alpha2-AP gene inactivation or alpha2-AP neutralization) reduces infarct size. Inhibition of alpha2-AP may constitute a potential avenue to treatment of FCI.  (+info)

Characterization of wild-type and mutant alpha2-antiplasmins: fibrinolysis enhancement by reactive site mutant. (5/266)

During human blood clotting, alpha2-antiplasmin (alpha2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha2AP and a specific epsilon-lysyl group in each of the alpha-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified alpha2AP, which lacked plasmin-inhibitory activity, competed effectively with native alpha2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant alpha2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant alpha2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant alpha2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native alpha2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of alpha2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native alpha2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant alpha2AP displayed full plasmin inhibitory activity, while mutant alpha2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type alpha2AP. When our mutant alpha2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant alpha2AP augmented lysis by competing with native alpha2AP for FXIIIa-catalyzed incorporation into fibrin.  (+info)

Hepatitis C virus NS3 serine protease interacts with the serpin C1 inhibitor. (6/266)

Both NS3 protein (1007-1657) and its protease moiety (NS3p, 1027-1207) were able to interact in vitro with C1 Inhibitor (C1Inh) to give a 95-kDa Mr C1Inh cleavage product similar to that obtained upon proteolysis by complement protease C1s. High-Mr reaction products were also detected after incubation of C1Inh with NS3 but not with NS3p; they correspond to ester-bonded complexes from their hydroxylamine lability. Similar reactivity of NS3 was observed upon incubation with alpha2-antiplasmin. Serpin cleavage was prevented by treatment of NS3 with synthetic serine protease inhibitors. This interaction between viral NS3 and host serpins suggests that NS3 is likely to be controlled by infected cell protease inhibitors.  (+info)

Solvent/detergent-treated plasma has decreased antitrypsin activity and absent antiplasmin activity. (7/266)

Solvent/detergent (S/D)-treated plasma is currently marketed by the American Red Cross as a virally inactivated alternative to fresh-frozen plasma (FFP). The serpin-type serine proteinase inhibitors have a flexible reactive site loop (RSL) that can convert from the active conformation to the inactive latent or polymerized conformations when exposed to heat and/or detergents. We have compared the conformational stability and inhibitory activity of 3 plasma serpins-antithrombin, antitrypsin, and antiplasmin-in S/D plasma and FFP. In S/D plasma, virtually 100% of the antiplasmin and approximately 50% of the antitrypsin are in either the latent or polymerized conformation and lack inhibitory activity, while in FFP only the active conformation is present. Interestingly, antithrombin is not affected by S/D treatment and remains fully active. These data demonstrate that S/D plasma is not simply a virally inactivated equivalent of FFP. The lack of antiplasmin activity and decreased antitrypsin activity in S/D plasma suggest that it may not be as effective as FFP for the treatment of bleeding in patients with systemic activation of proteolytic cascades, such as disseminated intravascular coagulation and sepsis, acquired fibrinolytic states, and large-volume transfusion. Although there has been extensive use of S/D plasma in several European countries with no reports of adverse effects, clinical studies directly comparing the efficacy of these 2 plasma products are needed to directly evaluate the relative therapeutic efficacy of FFP and S/D plasma for the treatment of these diseases.  (+info)

Comparative effects of tissue plasminogen activator, streptokinase, and staphylokinase on cerebral ischemic infarction and pulmonary clot lysis in hamster models. (8/266)

BACKGROUND: The effects of alteplase (rtPA), streptokinase, and staphylokinase (rSak) on focal cerebral ischemia (FCI) and on pulmonary clot lysis (PCL) were studied in hamsters. METHODS AND RESULTS: ++FCI was produced by ligation of the left middle cerebral artery (MCA) and common carotid artery (CCA) and a 10-minute occlusion of the right CCA. FCI was measured after 24 hours by 2,3, 5-triphenyltetrazolium chloride staining. (125)I-fibrin-labeled plasma clots were injected via the jugular vein, and clot lysis was determined from residual radioactivity at 90 minutes. Study drugs were given intravenously over 60 minutes. FCI increased from 1.2 (0. 27 to 2.3) mm(3) (median and 17th to 83rd percentile range, n=24) in controls to 19 to 27 mm(3) with thrombolytic agent, with maximal rates at 0.13+/-0.05 mg/kg rtPA, 0.23+/-0.09 mg/kg streptokinase, and 0.037+/-0.025 mg/kg rSak. PCL increased from 18+/-2% (mean+/-SEM, n=27) in controls to approximately 85% with thrombolytics, with maximal rates at 0.12+/-0.03 mg/kg rtPA, 0.17+/-0.05 mg/kg streptokinase, and 0.018+/-0.002 mg/kg rSak. All agents caused maximal FCI and PCL rates at similar doses without alpha(2)-antiplasmin and fibrinogen depletion. Injection of 6 mg/kg human plasminogen combined with streptokinase caused a "systemic fibrinolytic state" with fibrinogen depletion. Maximal rates of FCI were obtained with 0.097+/-0.077 mg/kg streptokinase (P=0.26 versus streptokinase alone) and of PCL with 0.010+/-0.002 mg/kg (P=0.006 versus streptokinase alone). CONCLUSIONS: Thrombolytic agents cause similar dose-related extension of FCI after MCA ligation and PCL, irrespective of the agent or systemic plasmin generation.  (+info)

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