A Case of Successful Thromboelastographic Guided Resuscitation after Postpartum Hemorrhage and Cardiac Arrest.

Amniotic fluid embolism (AFE) is an unusual cause of life threatening peri partum hemorrhage (PPH). AFE resuscitation is often associated with renal and respiratory insufficiency, and a coagulopathy similar to disseminated intravascular coagulation (DIC). Resuscitation requires immediate recognition and limited use of crystalloid. We present a case of PPH caused by AFE with resultant cardiac arrest, renal and respiratory failure, and DIC-like coagulopathy, whose successful resuscitation was guided by perfusionist-directed serial thromboelastography (TEG). Viscoelastic tests (VET)s, including the TEG and rotational thromboelastometry (ROTEM), may provide more individualized blood component therapy (BCT) in the treatment of severe PPH associated with AFE as has been previously noted with trauma resuscitation in the literature. However, VET's efficacy is often limited by a lack of standardization, quality assurance norms, and consistent operator proficiency. We suggest that there may be a role for perfusionsts adept at utilizing TEG in the optimization of BCT and adjunctive hemostatic agents in severely hemorrhagic patients. This patient's successful resuscitation demonstrates the importance of resuscitation guided by the perfusionist or other medical professionals with expertise in TEG guided resuscitation and how the administration of specific blood products and hemostatic agents guided by the TEG can help optimize patient outcomes in comparison to traditional 1:1:1 packed red blood cells (PRBC) /fresh frozen plasma (FFP) /platelets ratios given to severely hemorrhaging patients.

Severe deficiency of coagulation Factor VII results in spontaneous cardiac fibrosis in mice.

Mice genetically modified to produce low levels (approximately 1% of wild-type) of coagulation FVII presented with echocardiographic evidence of heart abnormalities. Decreases in ventricular size and reductions in systolic and diastolic functions were found, suggestive of a restrictive cardiomyopathy and consistent with an infiltrative myopathic process. Microscopic analysis of mouse hearts showed severe patchy fibrosis in the low-FVII mice. Haemosiderin deposition was discovered in hearts of these mice, along with increases in inflammatory cell number, ultimately resulting in widespread collagen deposition. Significant increases in mRNA levels of TGFbeta, TNFalpha and several matrix metalloproteinases in low-FVII mice, beginning at early ages, supported a state of cardiac remodelling associated with the fibrotic pathology. Mechanistic time-course studies suggested that cardiac fibrosis in low-FVII mice originated from bleeding in heart tissue, resulting in the recruitment of leukocytes, which released inflammatory mediators and induced collagen synthesis and secretion. These events led to necrosis of cardiomyocytes and collagen deposition, characteristics of cardiac fibrosis. The results of this study demonstrated that haemorrhagic and inflammatory responses to a severe FVII deficiency resulted in the development of cardiac fibrosis, observed echocardiographically as a restrictive cardiomyopathy, with compromised ventricular diastolic and systolic functions.

Regulation of plasminogen activation on cell surfaces and fibrin.

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma hemostasis cofactors. Vascular endothelial cells (VECs) retain tissue-type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI-1), regulates fibrinolytic potential via inhibition of the VEC surface-bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C-terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin-activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI-1, TAFI, TM, and the fibrin cross-linking enzyme, factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed.

The plasminogen receptor, Plg-RKT, is essential for mammary lobuloalveolar development and lactation.

Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. SUMMARY: Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

The influence of the nature of the asparagine 289-linked oligosaccharide on the activation by urokinase and lysine binding properties of natural and recombinant human plasminogens.

Several strategies have been used to obtain recombinant (r) human plasminogens (HPg) containing different oligosaccharide side chains on its sole N-linked glycosylation site, present at Asn289. The approaches included expression of the cDNA for HPg in insect cell lines under various conditions, addition of glycosidase inhibitors during expression, and purification of specific glycoforms of HPg using affinity chromatography on an insolubilized lectin column. The activation kinetics for urokinase (UK) of each of the purified HPgs, as well as their relative abilities to bind to the ligand, epsilon-aminocaproic acid (EACA), were then determined. Removal of both N- and O-linked oligosaccharide from HPg resulted in a slight increase in the Kcat/Km for its activation, while a glycoform containing tetrasialyl-tetra-antennary complex oligosaccharide on Asn289 was a slightly poorer substrate for UK than plasma HPg, which contains bisialyl-biantennary complex carbohydrate on Asn289. The most dramatic differences were observed for HPgs with high mannose-type glycans on Asn289. (Man9GlcNAc2)-HPg possessed only approximately 6% of the kcat/Km of plasma HPg, whereas (Glc3Man9GlcNAc2)-HPg did not undergo activation at a significant rate by UK. Differences were also found in the relative abilities of the HPg glycoforms to interact with EACA. The most effective interactions were observed with HPgs containing complex-type glycans, and the least effective binding was found for HPgs with high mannose-type oligosaccharides. The full range of the binding effects is represented by a fourfold difference between HPg containing tetrasialyl-tetra-antennary glycan and HPg with (Glc3Man9GlcNAc2) assembled on Asn289. These results clearly demonstrate that the nature of the N-linked glycan assembled on HPg dramatically influences its ability to be activated by UK and to bind to omega-amino acid effector molecules.

Combined factor VII/protein C deficiency results in intrauterine coagulopathy in mice.

To determine whether an additional loss of the coagulation factor VII (FVII) gene influenced the coagulopathy observed in protein C gene-deficient (PC(-/-)) embryos and neonates, we crossed mice doubly heterozygous for the factor VII (FVII(+/-)) and protein C (PC(+/-)) genes to produce offspring possessing the 9 predicted genotypic combinations. FVII(-/-)/PC(-/-) embryos, although present at their expected Mendelian frequency, displayed a phenotype that had not been observed in either the FVII or PC singly deficient embryos. At E12.5 days postcoitum (dpc), FVII(-/-)/PC(-/-) embryos demonstrated an intra- and extravascular coagulopathy that progressed with substantial concomitant hemorrhage and peripheral edema by E17.5dpc, resulting in mortality immediately after birth. FVII(+/-)/PC(-/-) embryos showed a less severe phenotype, suggesting a gene dosage effect. The lack of rescue of PC(-/-) embryos and neonates and augmented coagulopathy resulting from an additional heterozygous or homozygous FVII deficiency are probably due to increased factor Xa and thrombin generation, resulting from loss of FVIIa-dependent tissue factor pathway inhibitor function and the absence of control at the levels of factors Va and VIIIa. The presence of fibrin in embryos in the absence of fetal FVII suggests that significant clot-generating potential exists outside of the embryonic factor VII-dependent pathway.

Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice.

Matings of mice heterozygous for a protein C (PC) deficient allele, produced by targeted PC gene inactivation, yielded the expected Mendelian distribution of PC genotypes. Pups with a total deficiency of PC (PC-/-), obtained at embryonic day (E) 17.5 and at birth, appeared to develop normally macroscopically, but possessed obvious signs of bleeding and thrombosis and did not survive beyond 24 h after delivery. Microscopic examination of tissues and blood vessels of E17.5 PC-/- mice revealed their normal development, but scattered microvascular thrombosis in the brain combined with focal necrosis in the liver was observed. In addition, bleeding was noted in the brain near sites of fibrin deposition. The severity of these pathologies was exaggerated in PC-/- neonates. Plasma clottable fibrinogen was not detectable in coagulation assays in PC-/- neonatal mice, suggestive of fibrinogen depletion and secondary consumptive coagulopathy. Thus, while total PC deficiency did not affect the anatomic development of the embryo, severe perinatal consumptive coagulopathy occurred in the brain and liver of PC-/- mice, suggesting that a total PC deficiency is inconsistent with short-term survival.

Factor VII deficiency rescues the intrauterine lethality in mice associated with a tissue factor pathway inhibitor deficit.

Mice doubly heterozygous for a modified tissue factor pathway inhibitor (TFPI) allele (tfpi delta) lacking its Kunitz-type domain-1 (TFPI+/delta) and for a deficiency of the factor VII gene (FVII+/-) were mated to generate 309 postnatal and 205 embryonic day 17.5 (E17. 5) offspring having all the predicted genotypic combinations. Progeny singly homozygous for the tfpidelta modification but with the wild-type fVII allele (FVII+/+/TFPIdelta/delta), and mice singly homozygous for the fVII deficiency and possessing the wild-type tfpi allele (FVII-/-/TFPI+/+), displayed previously detailed phenotypes (i.e., a high percentage of early embryonic lethality at E9.5 or normal development with severe perinatal bleeding, respectively). Surprisingly, mice of the combined FVII-/-/TFPIdelta/delta genotype were born at the expected mendelian frequency but suffered the fatal perinatal bleeding associated with the FVII-/- genotype. Mice carrying the FVII+/-/TFPIdelta/delta genotype were also rescued from the lethality associated with the FVII+/+/TFPIdelta/delta genotype but succumbed to perinatal consumptive coagulopathy. Thus, the rescue of TFPIdelta/delta embryos, either by an accompanying homozygous or heterozygous FVII deficiency, suggests that diminishment of FVII activity precludes the need for TFPI-mediated inhibition of the FVIIa/tissue factor coagulation pathway during embryogenesis. Furthermore, the phenotypes of these combined deficiency states suggest that embryonic FVII is produced in mice as early as E9.5 and that any level of maternal FVII in early-stage embryos is insufficient to cause a coagulopathy in TFPIdelta/delta mice.

Tumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1.

In vivo tumor progression in mice with targeted deficiencies in urokinase-type plasminogen activator (UPA-/-) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1-/-), was studied using a fibrosarcoma tumor model. Murine T241 fibrosarcoma cells were s.c. implanted into three groups of mice with the following genotypes, wild-type (WT), UPA-/-, and PAI-1-/-. A significantly diminished primary tumor growth in UPA-/- and PAI-1-/- mice occurred, relative to WT mice. Tumors in UPA-/- and PAI-1-/- mice displayed lower proliferative and higher apoptotic indices and displayed a different neovascular morphology, as compared with WT mice. These results are consistent with the decreased growth rates of this tumor in these gene-deleted mice. Immunohistochemical analyses of the tumors revealed a decrease in vascularity and vascular endothelial growth factor expression only in tumors in PAI-1-/- mice. Analyses of the relative extents of corneal angiogenesis in these same animals, induced by basic fibroblast growth factor, corroborated the resistance of PAI-1-/- mice to neovascularization. The results obtained suggest that the host fibrinolytic system plays an important role in tumor growth in this model. Alterations in host expression of components of this system may alter tumor growth and dissemination by affecting the balance between tumor cell death and proliferation, as well as extracellular matrix changes needed for invasiveness and angiogenesis.

The role of the lysine binding sites of human plasmin in the hydrolysis of human fibrinogen.

The importance of the lysine binding sites of human plasmin for its ability to digest human fibrinogen has been assessed by analyzing the nature and rate of the products formed in the presence of epsilon-aminocaproic acid. No major differences in this regard were found when comparing Lys77-plasmin and Val442-plasmin, in the absence of epsilon-aminocaproic acid, the latter plasmin being devoid of the lysine binding sites present on residues Glu1-Val441 (K 1-4). The presence of epsilon-aminocaproic acid, at concentrations ranging from 0.5-5.0 mM, results in progressively stronger inhibition of the digestion of fibrinogen and in appearance of fibrinogen degradation products Y, D and E, for both Lys77-plasmin, and Val442-plasmin, showing the importance of lysine binding regions in this property. However, since both plasmin forms were inhibited equally well at all levels of epsilon-aminocaproic acid, these studies show that lysine binding sites other than those present on region K 1-4 of Lys77-plasmin are of primary importance to fibrinogenolysis by plasmin.

Interaction of lysophosphatidylcholine with phosphatidylcholine bilayers. A photo-physical and NMR study.

Several photo-physical methods together with 31P-NMR have been used to investigate the effect of lysophosphatidylcholine on phosphatidylcholine bilayers. 31P-NMR shows that the permeability of the vesicle to Eu3+ increases sharply above approx. 40% lysophosphatidylcholine: fluorescence-quenching studies also show this type of behavior. Similar sharp changes in vesicle properties are observed via the photo-physical technique at this lysophosphatidylcholine/phosphatidylcholine composition. Fluorescence spectra of pyrene and pyrene carboxaldehyde show that increasing lysophosphatidylcholine composition increases the polarity of the environments of these probes up to 40% lysocompound. Above this composition the photo-physical properties of the probes slowly revert to those characteristic of the micellar lyso-compound. The pyrene fluorescence lifetime, the fine structure of the fluorescence, and the case of formation of pyrene excimer in these bilayer mixtures suggest that pyrene complexes weakly with the charged nitrogen of the choline group of the phosphatidylcholine and that the physical state of the system has a striking effect on this complexation process. Similar experiments with simple quaternary compounds lend strong support to this suggestion. The studies monitor in several ways the effect of bilayer composition on movement of molecules in these systems. The degree or site of solubilization of carcinogens is also uniquely affected by composition.

The effects of ligand binding on the backbone dynamics of the kringle 1 domain of human plasminogen.

The internal motions of the backbone nitrogen atoms of the kringle 1 domain of human plasminogen (K1(Pg)) were examined in the absence and presence of the ligand, epsilon-aminocaproic acid. These dynamic properties were determined from (15)N NMR relaxation data in terms of the extended model-free parameters. The model of isotropic reorientation was found sufficient to account for overall molecular tumbling for both apo and EACA-bound K1(Pg). The global rotational correlation time (tau(m)) for apo-K1(Pg) was 5.87(+/-0.01) ns, while the tau(m) for ligand-bound K1(Pg) was 5.20(+/-0.01) ns, suggesting that perhaps some small degree of aggregation occurred in the apo form of the kringle module. Complexation of K1(Pg) with ligand mainly reduced those internal motions that occurred on a 100 ps to 5 ns time-scale. The magnitude of the chemical exchange was also attenuated upon ligand binding. These data are consistent with studies employing other approaches that suggest that the binding pocket is preformed in K1(Pg).

Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein.

The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2Pg[C4G/E56D/L72Y] (mK2Pg), containing an upregulated lysine-binding site, bound to a functional 30 residue internal peptide (VEK-30) from an M-type protein of a group A Streptococcus surface protein, has been determined by molecular replacement methods using K4Pg as a model, and refined at 2.7 A resolution to a R-factor of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a nearly end-to-end alpha-helix in the complex with mK2Pg. The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for Asp54 and Asp56 of the consensus lysine-binding site of mK2Pg, while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2Pg. The hydrophobic ligand-binding pocket in mK2Pg, consisting primarily of Trp60 and Trp70, situated between the positive and negative centers of the lysine-binding site, is utilized in a novel manner in stabilizing the interaction with VEK-30 by forming a cation-pi-electron-mediated association with the positive side-chain of Arg17 of this peptide. Additional lysine-binding sites, as well as exosite electrostatic and hydrogen bonding interactions involving Glu9 and Lys14 of VEK-30, were observed in the structural model. The importance of these interactions were tested in solution by investigating the binding constants of synthetic variants of VEK-30 to mK2Pg, and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most critical amino acid binding determinants. With regard to the solution studies, circular dichroism analysis of the titration of VEK-30 with mK2Pg demonstrated that the peptidic alpha-helical structure increased substantially when bound to the kringle module, in agreement with the X-ray results. This investigation is the first to delineate structurally the mode of interaction of the lysine-binding site of a kringle with an internal pseudo-lysine residue of a peptide or protein that functionally interacts with a kringle module, and serves as a paradigm for this important class of interactions.

Human protein C and activated protein C Components of the human anticoagulation system.

Protein C, the zymogen form of the anticoagulant protein, activated protein C, is a member of the vitamin K-dependent class of proteins that function in generation and control of formation of blood clots. This plasma protein consists of a series of domain regions that are qualitatively similar to those present in human coagulation factors VII, IX, and X, and that appear to be related to specific properties of these proteins. With the recent advent of rigorous application of genetic engineering strategies to this system, and the continuing discoveries and characterization of genotypes and phenotypes of protein C in patients, great progress has been made in understanding structure-function relationships of protein C and activated protein C. This review is a summary and synthesis of recent pertinent studies with an emphasis on these topics.

NMDA-receptor antagonist requirements in conantokin-G.

A series of variants of the neuroactive 17-residue gamma-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-D-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.

Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R.

Conantokin-R (con-R) is a gamma-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [(3)H]MK-801, to rat brain membranes, and yielded an IC(50) of 93 nM. This value represents a 2-5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low alpha-helical content. There is an increase in alpha-helicity upon the addition of divalent cations, such as Ca(2+), Mg(2+), or Zn(2+). Isothermal titration calorimetry experiments showed one detectable Mg(2+) binding site with a K(d) of 6.5 microM, and two binding sites for Zn(2+), with K(d) values of 150 nM and 170 microM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional (1)H-NMR. Analyses of the alpha-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an alpha-helical conformation for residues 1-19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The alpha-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.

The NMR solution structure of the NMDA receptor antagonist, conantokin-T, in the absence of divalent metal ions.

The solution conformation of conantokin-T, a Gla-containing 21-residue peptide, (G1 EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-amide), in the absence of divalent metal ions, was studied by use of two-dimensional proton NMR spectroscopy. The peptide is helical from the N-terminus to the C-terminus, as defined by upfield-shifted alpha-proton resonances and by characteristic NOE connectivities. Extensive interactions among the amino acid side-chains were identified from the NOESY spectra of this peptide in a buffered aqueous solution. Four hydrophobic residues Tyr5, Met8, Leu9, and Leu12 contact one another in a stable cluster, even in the presence of 6 M urea. The solution structure of conantokin-T is a well-defined alpha-helix, having RMSD values for the backbone and all heavy atoms of 0.40 A and 0.77 A, respectively. Potential repulsion between the negatively-charged side chains of Gla10 and Gla14 is minimized by a Gln6-Gla10 hydrogen bond and by an Arg13-Gla14 ion-pair interaction. The C-terminal amide and the Asn20 side-chain amide both interact with the backbone and minimize fraying at the C-terminal end of the alpha-helix. This study provides a basis to evaluate the changes in peptide conformation concomitant upon the binding of divalent metal ions. In addition, this investigation demonstrates that apo-conantokin-T has almost all of the favorable interactions that are known to contribute to helical stabilization in proteins and monomeric helices.

Endogenous plasmin converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface.

Recently, we showed that localization of Glu-plasminogen on cell surfaces enhances its conversion to Lys-plasminogen by exogenous plasmin. This leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human plasminogen mutant, [D(646)E]Pg, which can be cleaved by plasminogen activators, but cannot catalyze the generation of Lys-plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, alpha2-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that this enzyme is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Activated protein C attenuates coagulation-associated over-expression of fibrinolytic activity by suppressing the thrombin-dependent inactivation of PAI-1.

Several activated coagulation factors have been reported to enhance fibrinolysis by neutralizing plasminogen activator inhibitor type 1 (PAI-1) activity. We evaluated the physiological relevance of this mechanism using the euglobulin clot lysis time (ECLT) assay in the presence and absence of Ca2+, which is controlled by PAI-1 and mimics physiological thrombolysis. We found that the ECLT (18.5 +/- 0.6 h) was shortened by Ca2+ (5 mm) (6.6 +/- 0.1 h). A significant difference was observed in thrombin generation by the presence of Ca2+ in the euglobulin fraction. Prothrombin was almost fully converted to thrombin within 15 min in the presence of Ca2+, whereas essentially no conversion was observed without Ca2+. The presence of activated protein C (aPC) suppressed thrombin generation, and attenuated the shortening of ECLT in a dose-dependent manner, an effect enhanced by phospholipid and protein S. In the absence of Ca2+, aPC did not prolong the ECLT. After addition of biotin-labeled recombinant PAI-1 to the euglobulin fraction, PAI-1 was cleaved to lower molecular weight forms only in the presence of Ca2+. This cleavage did not occur in the presence of aPC, suggesting that thrombin was the catalyst for PAI-1 cleavage. The cleavage and inactivation of PAI-1 by generated thrombin is proposed to be responsible for the shortening of ECLT by Ca2+ and for coagulation-associated over-expression of fibrinolysis. Under such conditions, aPC appeared to suppress thrombin generation and to normalize highly activated fibrinolysis.

A central role for plasminogen in the inflammatory response to biomaterials.

The inflammatory response to implanted biomaterials severely limits their deployment in patients. Plasminogen has been shown to play a central role in cell migration, and therefore could regulate this inflammatory response. We sought to determine if plasminogen influences recruitment of inflammatory cells to a biomaterial implanted into plasminogen-deficient (Plg(-/-)) mice. Small disks of polyethylene terephthalate, a material used in vascular grafts, were surgically implanted into the peritoneum of wild-type and Plg(-/-) mice. Recruitment of neutrophils and monocytes/macrophages into the peritoneum and onto the disks was measured, primarily at 18 h. Monocyte/macrophage recruitment was markedly blunted in Plg(-/-) mice compared with wild-type mice. Unexpectedly, neutrophil recruitment was also markedly decreased in the Plg(-/-) mice. While recruitment of leukocytes into the peritoneum was plasminogen-dependent, the adhesion of the emigrating cells to the implants was not. In contrast, adhesion but not recruitment was reduced in fibrinogen-deficient mice. Reconstitution of Plg(-/-) mice with intravenous or intraperitoneal plasminogen differentially restored monocyte/macrophage and neutrophil recruitment. Tranexamic acid, an inhibitor of the lysine binding sites of plasminogen, suppressed leukocyte recruitment in wild-type mice, but aprotinin, a plasmin inhibitor, did not. Plasminogen exerts a marked influence on both neutrophil and monocyte/macrophage recruitment to implanted biomaterials. This role is distinct from that of fibrinogen, and the two inflammatory cell types use plasminogen in different ways. Plasminogen represents a therapeutic target for controlling the inflammatory response to implanted materials.