Predictors of Adherence Among Vulnerable Populations of Adults Assigned to Smoke Very Low Nicotine Content Cigarettes.

INTRODUCTION: Regulators are considering reducing the nicotine content in cigarettes to a minimally addictive level. This could particularly benefit smokers from populations vulnerable to heavy smoking and difficulties quitting. We assessed predictors of adherence among adults from vulnerable populations assigned to use very low nicotine content cigarettes (VLNCs) in randomized clinical trials, to identify characteristics of those who require additional assistance if a nicotine reduction policy were implemented. AIMS AND METHODS: Data came from three populations of vulnerable adult smokers assigned to use VLNC cigarettes (0.4 mg/g nicotine) during 12-week randomized controlled trials (n = 286): Socioeconomically disadvantaged women of reproductive age, opioid-maintained adults, and adults with affective disorders. Logistic and linear regressions modeled predictors of adherence based on changes in cotinine at week-6 and week-12 assessments relative to baseline, and as a 90% reduction in cotinine relative to baseline (full adherence: yes/no). Predictors included satisfaction with study cigarettes, craving, nicotine dependence severity, withdrawal, population membership, baseline affective-disorder symptoms, and sociodemographic characteristics. RESULTS: Dependence severity was negatively associated with both adherence measures at week 6 (p < .01), whereas increased satisfaction with study cigarettes and age were positively associated with both measures at weeks 6 and 12 (p < .01). Opioid-maintained adults exhibited reduced adherence and were less likely to reach full adherence at week 12 compared to disadvantaged women (p = .02). CONCLUSIONS: Factors associated with VLNC adherence in vulnerable populations are similar to those in the general population of smokers. Furthermore, studies are indicated investigating nicotine supplements (e.g., e-cigarettes, NRT) to support highly dependent adults faced with using VLNCs. IMPLICATIONS: This study identified factors predicting difficulty maintaining adherence to a regimen of very low nicotine content cigarettes (VLNC) among adults from vulnerable populations. Findings suggested that factors predicting difficulty maintaining adherence (greater nicotine dependence and low satisfaction with study-provided VLNC) were common across vulnerable smokers and the general population of adults who smoke. Furthermore, research should investigate alternatives to support highly dependent adults, such as pairing VLNC with supplemental, noncombusted nicotine. Some vulnerable populations (e.g., opioid-maintained adults) may be especially in need of supplemental, noncombusted nicotine.

Physiologic variations in blood plasminogen levels affect outcomes after acute cerebral thromboembolism in mice: a pathophysiologic role for microvascular thrombosis.

UNLABELLED: Essentials Physiologic variations in blood plasminogen (Pg) levels may affect ischemic stroke outcomes. We tested Pg effects in a model with translational relevance to human thromboembolic stroke. A dose-response exists between Pg levels and brain injury, fibrinolysis, barrier breakdown. Higher Pg levels reduce microvascular thrombosis and improve outcomes in ischemic stroke. SUMMARY: Background and Objectives Plasminogen appears to affect brain inflammation, cell movement, fibrinolysis, neuronal excitotoxicity, and cell death. However, brain tissue and circulating blood plasminogen may have different roles, and there is wide individual variation in blood plasminogen levels. The aim of this study was to determine the integrated effect of blood plasminogen levels on ischemic brain injury. Methods We examined thromboembolic stroke in mice with varying, experimentally determined, blood plasminogen levels. Ischemic brain injury, blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis were determined. Results Within the range of normal variation, plasminogen levels were strongly associated with ischemic brain injury; higher blood plasminogen levels had dose-related, protective effects. Higher plasminogen levels were associated with increased dissolution of the middle cerebral artery thrombus. Higher plasminogen levels decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis in the ischemic brain. In plasminogen-deficient mice, selective restoration of blood plasminogen levels reversed the harmful effects of plasminogen deficiency on ischemic brain injury. Specific inhibition of thrombin also reversed the effect of plasminogen deficiency on ischemic injury by decreasing microvascular thrombosis, blood-brain barrier breakdown, and matrix metalloproteinase-9 expression. Conclusions Variation in blood plasminogen levels, within the range seen in normal individuals, had marked effects on experimental ischemic brain injury. Higher plasminogen levels protected against ischemic brain injury, and decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression, and microvascular thrombosis. The protective effects of blood plasminogen appear to be mediated largely through a decrease in microvascular thrombosis in the ischemic territory.

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

BACKGROUND: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. OBJECTIVE: We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. METHODS AND RESULTS: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. CONCLUSION: These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Fibrinolysis is amplified by converting alpha-antiplasmin from a plasmin inhibitor to a substrate.

alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.

The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase.

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.

Catalytic life of activated factor XIII in thrombi. Implications for fibrinolytic resistance and thrombus aging.

BACKGROUND: Because the increased fibrinolytic resistance of older thrombi may be caused by the continuous cross-linking action of fibrin-bound activated factor XIII (FXIIIa), we examined the persistence of FXIIIa catalytic activity in clots of various ages. METHODS AND RESULTS: The time-related changes in FXIIIa activity in clots was measured with (1) alpha(2)-antiplasmin (alpha(2)AP), a physiological glutamine substrate; (2) alpha(2)AP(13-24), a peptide; and (3) pentylamine, a nonspecific lysine substrate. The cross-linking of alpha(2)AP, alpha(2)AP(13-24), and pentylamine into fibrin by clot-bound FXIIIa declined rapidly with half-lives of 19, 21, and 26 minutes, respectively. Mutational studies showed that glutamine 14 (but not glutamine 3 or 16) and valine 17 of alpha(2)AP(13-24) were required for efficient cross-linking to fibrin. The loss of activity was not due primarily to FXIIIa proteolysis and was partially restored by reducing agents, suggesting that oxidation contributes to the loss of the enzyme's activity in clots. In vivo, the ability of thrombus-bound FXIIIa to cross-link an infused alpha(2)AP(13-24) peptide into existing pulmonary emboli also declined significantly over time. CONCLUSIONS: FXIIIa cross-links alpha(2)AP and an alpha(2)AP peptide, in a sequence-specific manner, into formed clots with a catalytic half-life of approximately 20 minutes. This indicates that FXIIIa activity is a hallmark of new thrombi and that the antifibrinolytic cross-linking effects of FXIIIa are achieved more rapidly in thrombi than previously believed.

Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation.

The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.

Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets.

We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.

Zymogen activation in the streptokinase-plasminogen complex. Ile1 is required for the formation of a functional active site.

Plasminogen (Plgn) is usually activated by proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK.Plgn) which subsequently rearranges to an active complex (SK.Plgn*) although the Arg561-Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (muPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK.Plgn* by 100-fold (Ile1Val) to >/= 104-fold (Ile1-->Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK.Plgn but is necessary for SK.Plgn*. The substitution of Lys698 of muPlgn decreases the activity of SK.Plgn* by only 10-60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15-50-fold. These observations suggest that Lys698 is involved in formation of the initial SK.Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms.

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.

Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages.

Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-alpha did not induce SR-A transcript levels, indicating that a TNF-alpha autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.

Extreme resistance to Potato leafroll virus in potato cv. Russet Burbank mediated by the viral replicase gene.

High levels of field resistance to Potato leafroll virus (PLRV; Genus: Polerovirus; Family: Luteoviridae) were achieved by expression of the unmodified, full-length PLRV replicase gene in potato plants cv. Russet Burbank. A high degree of resistance was also achieved, but less frequently, by expression of a truncated construct of the replicase gene. In limited testing, neither miss-frame nor antisense constructs of the replicase gene conferred resistance. The degree of resistance expressed among different transformant lines ranged from near immunity to full susceptibility. Resistance to the Colorado potato beetle (Leptinotarsa decemlineata Say) was combined with resistance to PLRV by expression of the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis in combination with the unmodified, full-length, viral replicase gene. Resistance was expressed as a reduced incidence of infection detectable by foliage symptoms or serological tests. Reduced incidence of infection was not associated with a decrease in virus antigen concentration in the few plants of resistant lines that became infected. Virus was not detected in the foliage of symptomless plants but was detected in progeny plants produced from the tubers of inoculated but symptomless test plants of some resistant lines. The resistance was effective under natural exposure and against plant-to-plant spread of PLRV by the aphid vector, Myzus persicae Sulzer. Three of the resistant lines selected in these studies were released and are now in commercial production.

Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution.

In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.

A critical role for N-ethylmaleimide-sensitive fusion protein (NSF) in platelet granule secretion.

The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both alpha-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant alpha-SNAP-stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant alpha-SNAP-stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion.

A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator.

Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH(2)-terminal "catalytic switch" that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSKDelta59) fusion protein lacking the 59 NH(2)-terminal residues was no longer capable of fibrin-independent Pg activation (k(cat)/K(m) decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH(2)-terminal peptide rSK1-59. Deletion of the NH(2) terminus made rSKDelta59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSKDelta59 activator complex apparently resulted from selective catalytic processing of fibrin-bound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSKDelta59) of the SK NH(2)-terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSKDelta59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH(2) terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH(2) terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.

Plasminogen is not required for neointima formation in a mouse model of vein graft stenosis.

Recent studies of mice that lack plasminogen have identified a critical role for this zymogen in arterial remodeling. To permit the use of these (and other) genetically modified mice in the analysis of venous injury, we developed a model in which a patch cut from the external jugular vein of a mouse is grafted to repair a surgically created defect in its carotid artery. In wild-type mice, the venous graft showed initial endothelial denudation and formation of a neointima that progressively and reproducibly expanded in a manner analogous to human vein graft disease, albeit at an accelerated pace. This neointima occupied 37+/-4.6% of the vessel lumen at day 7 and 66+/-5.7% at day 20. The proliferative index of neointimal cells assessed by proliferating cell nuclear antigen staining was 50.6+/-3. 6% at day 7 and 15.2+/-2.0% at day 20. CD45-positive leukocytes and alpha-actin-positive smooth muscle cells accounted for 9.5+/-1.0% and 9.9+/-1.1% of intimal area at day 7, respectively, with the latter increasing to 40.9+/-2.6% at day 20. Collagen accounted for 6.8+/-0.7% of intimal area at day 7 and 20.7+/-1.8% at day 20. Surprisingly, even though arterial neointima formation due to electrostatic and immune-mediated injury is impaired in plasminogen -/- mice, in our study vein graft neointima formation in these mice was not significantly different from that in controls (70.9+/-6.4 versus 65.6+/-4.4% luminal occlusion, P=NS). Thus, plasmin proteolysis, although critical in extracellular matrix degradation and cellular migration after arterial injury, does not appear to be so important in vein graft neointima formation, perhaps because of the relative lack of structural barriers to cellular migration in the normal vein wall. This novel model of vein graft injury should be useful for further studies of differences in the response to injury of arterial and venous tissues.

Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.

Plasminogen (Plgn) is usually activated by proteolytic cleavage of Arg561-Val562. The new N-terminal amino group of Val562 forms a salt bridge with Asp740, creating the active protease plasmin (Pm). However, streptokinase (SK) binds to Plgn, generating an active protease in a poorly understood, nonproteolytic process. We hypothesized that the N-terminus of SK, Ile1, substitutes for the N-terminal Val562 of Pm, forming an analogous salt bridge with Asp740. SK initially forms an inactive complex with Plgn, which subsequently rearranges to create an active complex; this rearrangement is rate limiting at 4 degrees C. SK.Plgn efficiently hydrolyzes amide substrates at 4 degrees C, although DeltaIle1-SK. Plgn has no amidolytic activity. DeltaIle1-SK prevents formation of wild-type SK.Plgn. These results indicate that DeltaIle1-SK forms the initial inactive complex with plasminogen, but cannot form the active complex. However, when the experiment is performed at 37 degrees C, amidolytic activity is observed when DeltaIle1-SK is added to plasminogen. SDS-PAGE analysis demonstrates that the amidolytic activity results from the formation of DeltaIle1-SK.Pm. To further demonstrate that the activity of DeltaIle1-SK requires the conversion of Plgn to Pm, we characterized the reaction of SK with a mutant microplasminogen, Arg561Ala-microPlgn, that cannot be converted to microplasmin. Amidolytic activity is observed when Arg561Ala-microPlgn is incubated with wild-type SK at 37 degrees C; however, no amidolytic activity is observed in the presence of DeltaIle1-SK. These observations demonstrate that the amidolytic activity of DeltaIle1-SK at 37 degrees C requires the conversion of Plgn to Pm. Our findings indicate that Ile1 of SK is required for the nonproteolytic activation of Plgn by SK and are consistent with the hypothesis that Ile1 of SK substitutes for Val562 of Pm.

Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion.

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.

The contribution of activated factor XIII to fibrinolytic resistance in experimental pulmonary embolism.

BACKGROUND: The resistance of thrombi to fibrinolysis induced by plasminogen activators remains a major impediment to the successful treatment of thrombotic diseases. This study examines the contribution of activated factor XIII (factor XIIIa) to fibrinolytic resistance in experimental pulmonary embolism. METHODS AND RESULTS: The fibrinolytic effects of specific inhibitors of factor XIIIa-mediated fibrin-fibrin cross-linking and alpha2-antiplasmin-fibrin cross-linking were measured in anesthetized ferrets with pulmonary emboli. Five experimental groups were treated with heparin (100 U/kg) and/or tissue plasminogen activator (TPA, 1 mg/kg) and the percent (mean+/-SD) lysis of emboli was determined: (1) control, normal factor XIIIa activity (14.1+/-4. 8% lysis); (2) inhibited factor XIIIa activity (42.7+/-7.4%); (3) normal factor XIIIa activity+TPA (32.3+/-7.7%); (4) inhibited factor XIIIa activity+TPA (76.0+/-11.9%); and (5) inhibited alpha2-antiplasmin-fibrin cross-linking+TPA (54.7+/-3.9%). Inhibition of factor XIIIa activity increased endogenous lysis markedly (group 1 versus 2; P<0.0001), to a level comparable to that achieved with TPA (group 2 versus 3; P<0.05). Among groups receiving TPA, selective inhibition of factor XIII-mediated alpha2-antiplasmin-fibrin cross-linking enhanced lysis (group 3 versus 5; P<0.0005). Complete inhibition of factor XIIIa also amplified lysis (group 3 versus 4; P<0.0001) and had greater effects than inhibition of alpha2-antiplasmin cross-linking alone (group 4 versus 5; P<0.0005). No significant fibrinogen degradation occurred in any group. CONCLUSIONS: Factor XIIIa-mediated fibrin-fibrin and alpha2-antiplasmin-fibrin cross-linking both caused experimental pulmonary emboli to resist endogenous and TPA-induced fibrinolysis. This suggests that factor XIIIa may play a critical role in regulating fibrinolysis in human thrombosis.