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Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (betaER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22-24 months) WT and estrogen receptor beta knockout (betaERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17beta-estradiol were comparable between WT and betaERKO mice. Number of spontaneous deaths was greater in the betaERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc ( 1 ) complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from betaERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in betaERKO compared to WT mice. These results suggest that deficiencies in betaER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females.

Objective: To test the hypothesis that statins will acutely inhibit platelet thrombus formation, intravenous lovastatin was assessed in our well-characterized porcine carotid injury model., Methods and Results: The first carotid artery was crush-injured and harvested after 30 minutes. Pigs then received intravenous lovastatin (100 microg/kg bolus+100 microg/kg/h infusion, n=6) or saline (n=11) before injury of the second carotid artery. Thrombus size was quantified by scintillation detection of autologous (111)In-platelets. Sequential carotid injury produced a thrombus more than 50% greater in volume in the second (3149+/-2053 x 10(6)/cm(2)) relative to the first injured artery (2081+/-1552 x 10(6)/cm(2); P=0.04) in control pigs. This augmentation was inhibited by intravenous lovastatin which acutely reduced platelet deposition (944+/-246 x 10(6)/cm(2)) relative to saline control (P=0.02). Flow chamber closure times increased on average by 2.45-fold in response to whole blood lovastatin incubation. Lovastatin (P<0.05) and simvastatin (P<0.05) reduced platelet dense granule secretion in vitro., Conclusions: Sequential arterial injury augments the thrombotic response suggesting that the propensity for arterial thrombosis is at least partially acquired. This thrombotic augmentation can be acutely attenuated by intravenous lovastatin which may result from a pleiotropic impact on platelet function. These results appear to be a class effect of 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors.

While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC) was scored using 64-slice computed tomography scanners. The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score < 10%, range 1-3%), and high-sensitivity C-reactive protein did not differ significantly among women with low (0 < 35; range, 0.3-32 Agatston units) or high (>50; range, 93-315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis.

Background: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD) and for monitoring therapy. However, current manual or automated VWF:RCo assay methods have relatively poor operating characteristics. Our goal was to develop and validate a simple, accurate, specific and sensitive platelet-based VWF:RCo assay., Methods: Using green or red fluorochrome-labeled, fixed normal platelets and normal or patient plasma, ristocetin-dependent and VWF-mediated platelet aggregation was detected by flow cytometry. VWF:RCo activity was assayed as the number of double-positive events (green and red) among all green or red events, relative to the calibrator plasma signal (6-150% or IU dL(-1)), and reported as percent or IU dL(-1). We tested plasma samples from normal donors (n = 51) and known VWD patients (type 1, n = 16; type 2, n = 17) based on clinical history, levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregometry/agglutination assay), factor (F) VIII activity and VWF multimer analysis., Results: For normal donors and type 1 VWD patients, VWF:RCo activity by flow cytometry vs. manual platelet aggregation correlated closely (R2 = 0.74), and VWF:RCo/VWF:Ag ratios did not differ significantly. In contrast, VWF:RCo/VWF:Ag ratios for type 2 VWD subtypes were significantly lower using VWF:RCo by flow cytometry vs. manual platelet aggregation assay (P < 0.01), especially for type 2A VWD patients., Conclusions: This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17beta-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17beta-estradiol and CEE treatments; eosinophils increased only with 17beta-estradiol treatment. Content of TF protein increased in platelets from 17beta-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17beta-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17beta-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate TF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans.

Changes in platelet functions could contribute to thrombotic risk associated with estrogen treatments. This study was designed to test the hypothesis that three clinically relevant estrogenic treatments affect platelet function comparably. Adult female pigs were ovariectomized and randomized to either no treatment or treatment with oral 17 beta-estradiol (2 mg/day), conjugated equine estrogen (0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Platelet turnover, aggregation, and secretion were assessed before and after treatment. Platelet turnover and mRNA increased significantly only in pigs treated with 17 beta-estradiol. Expression of estrogen receptors increased with ovariectomy and decreased with all treatments. Platelet aggregation and secretion of ATP, platelet-derived growth factor, and matrix metalloproteinase-2 increased with ovariectomy. All treatments reduced both aggregation and secretion. Expression of mRNA for constitutive endothelial nitric oxide synthase (eNOS), but not eNOS protein, increased with ovariectomy. Only eNOS mRNA decreased with all treatments, but only treatment with 17 beta-estradiol increased secretion of nitric oxide from intact platelets. Platelets from 17 beta-estradiol-treated animals caused relaxation of coronary arteries, which was sensitive to inhibition of nitric oxide. Although three different estrogenic treatments reversed increases in platelet aggregation caused by ovariectomy, only 17 beta-estradiol increased platelet RNA and release of platelet-derived nitric oxide. These differences reflect transcriptional and posttranscriptional regulation of protein synthesis in bone marrow megakaryocytes and circulating platelets.

Dysfunction of the protein C anticoagulant system is associated with venous thromboembolism (VTE) and thrombomodulin (TM) is a critical cofactor within the protein C system. The aim of this study was to test the hypotheses that polymorphisms or haplotypes within the TM gene are common risk factors for VTE. We screened the TM putative promoter, exon and 3'-untranslated region for sequence variations in a random sample (n = 266) of consecutive idiopathic, objectively confirmed non-Olmsted County VTE patients referred to the Mayo Clinic. We then genotyped a sample of Olmsted County, MN residents with a first lifetime, objectively confirmed VTE in the 25-year period, 1966-90 (n = 223), and a sample of Olmsted County residents without VTE (n = 237) for polymorphisms either discovered in the screening population or previously published, and tested for an association of VTE with TM genotype or haplotypes using unconditional logistic regression and generalized linear models, respectively. We also genotyped these Olmsted County cases and controls at 20 'null' genetic maker loci and tested for population admixture. Nine novel and three previously described mutations were identified in the screening population. Mutations within the TM promoter, EGF(1-5), serine/threonine-rich, transmembrane, and cytoplasm regions were absent or uncommon. TM845G-->A (Ala25Thr; lectin region), TM2136T-->C (Ala455Val; EGF(6) region), TM2470C deletion (3'-untranslated region), and 4363A-->G (3'-flanking region) were more common, but were not associated with VTE by genotype or haplotype. Null genetic marker allele frequencies did not differ significantly among cases and controls. We conclude that polymorphisms or haplotypes within the TM gene are not common risk factors for incident VTE.

Background/objective: The efficacy of a direct factor (F)Xa inhibitor, ZK-807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet-rich thrombi in a well-characterized porcine carotid injury model., Methods: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK-807834-Dose 1 (40 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; n=6); or saline (n=6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In-platelets., Results: The prothrombin time ratio was 2.2 +/- 0.1; 1.4 +/- 0.3; 1.2 +/- 0.9 and 1.1 +/- 0.2, respectively. ZK-807834-Dose 1 significantly inhibited carotid platelet deposition (525 +/- 226 x 10(6) cm(-2); P = 0.008), whereas ZK-807834-Dose 2 (2325 +/- 768) and enoxaparin (1236 +/- 383) were not different from saline (2776 +/- 642). Thrombus deaggregation was greatest for animals receiving ZK-807834-Dose 1 (473 +/- 185). Neither ZK-807834-Dose 2 (1588 +/- 480) nor enoxaparin (1618 +/- 686) was different from saline control (2222 +/- 598)., Conclusions: Direct FXa inhibition with ZK-807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.

Background/objective: Thromboembolism secondary to atrial fibrillation accounts for approximately one-fourth of all strokes. Although considerable resources have been targeted to pharmacologic prophylaxis, neither the cellular nor the biochemical composition of atrial thrombi is known. Quantitative immunohistochemistry was undertaken to define the composition of atrial thrombi and to explore morphological differences between atrial appendage thrombi and those that embolize., Patients/methods: Serial sections of thrombi obtained during valve replacement surgery or embolectomy from 22 patients with atrial fibrillation were stained with antibodies against fibrin, integrin beta3, or tissue factor and analyzed with NIH-image., Results: Thrombi showed distinct regions staining for either fibrin or platelets and on average, the fibrin-rich regions predominated (P < 0.0001). The platelet content of embolized thrombi was nearly twice that of atrial thrombi (P = 0.02). Non-staining amorphous material comprised nearly half of atrial thrombi in situ, but was rare in embolized thrombi (P < 0.001). Tissue factor colocalized to areas rich in platelets and granulocytes., Conclusions: The abundance of fibrin relative to platelets underscores the enhanced efficacy of warfarin prophylaxis in clinical trials. The finding of tissue factor localized to platelet-leukocyte clusters suggests its blood-borne origin. Compositional differences between in situ and embolized thrombi suggest directions for investigating propensity for embolization.

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.

Objective: Occurrence of arterial thrombosis secondary to vascular disease in an individual is not easily predicted. After establishing that this poor predictability arises at least in part from an intrinsic thrombosis propensity of the individual, we sought to determine whether the propensity for arterial thrombosis is governed by blood or arterial wall factors., Methods and Results: To evaluate the variability arising from the blood, autologous 111In-labeled platelet deposition was measured after high-shear perfusion of compressed aortic strips, prepared from a single pig, with heparinized blood from 25 pigs. To evaluate the variability arising from the vessel wall, aortic strips from 8 pigs were superfused with blood from a single animal. Blood samples from 25 animals superfused over aortic substrate from a single source yielded a 24-fold range of platelet deposition. In contrast, when aortic substrates from 8 different animals were superfused with blood from a single animal, platelet deposition spanned a 3-fold range. Platelet deposition was significantly correlated with whole-blood lymphocyte counts and with platelet counts., Conclusions: Individual propensity for arterial thrombosis in pigs is more greatly influenced by blood components than by elements within the arterial wall.

Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGF(AB) and PDGF(BB)), transforming growth factors (TGF-beta(1) and TGF-beta(2)), and vascular endothelial growth factor (VEGF(165)) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGF(BB), but not PDGF(AB), TGF-beta(1), and TGF-beta(2), increased with ovariectomy. VEGF(165) was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGF(BB) in platelets. Higher levels of PDGF(BB) in platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy.

Atherosclerosis manifests as a systemic disease with near global involvement of the named segments of the arterial tree. Acute thrombotic arterial occlusion, however, is not equally distributed. To evaluate intra-individual regional differences in arterial thrombogenicity, we compared (111)In-platelet deposition in porcine carotid and femoral arteries after a standardized crush injury. Within the unidirectional flow conditions of elastic carotid arteries, platelet deposition was more than 3-fold higher compared with predominantly muscular femoral arteries with triphasic arterial flow. To determine the influence of rheology on platelet deposition after crush injury, carotid arteries were transplanted into the femoral position and compared with the paired native carotid and femoral arteries. Similarly, femoral arteries transposed to the carotid position were compared with the paired native carotid artery. In each of these experiments, arterial transposition to a new anatomic location imparts a predilection for platelet deposition indigenous to the new location. In the controlled environment of two high-shear thrombin-independent and -dependent flow chambers, porcine carotid and femoral arterial substrates were indistinguishable from one another with respect to platelet deposition. Regional differences in arterial hemodynamics may account for substantial differences in thrombosis arising from deep arterial injury.

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

Arterial thrombophilia independent of vascular pathology has not been previously defined either experimentally or epidemiologically. To address the existence of an individual propensity to arterial thrombosis, we exploited a previously developed procedure entailing traumatic (crush) injury of paired porcine carotid arteries for generating platelet-rich thrombi. Porcine carotid arteries were injured bilaterally by serial hemostat crushes. Thrombus generation was monitored by local accumulation of autologous 111In-labeled platelets and Doppler blood flow. Within this cohort of animals of similar age and size, the lowest to the highest responders in thrombus mass spanned a 7-fold range, showing no correlation with shear, platelet or leukocyte count, or plasma concentrations of fibrinogen or von Willebrand factor. However, there was strong intra-individual correlation (r2=0.80; P<0.001) of thrombus deposition between carotid artery pairs. The wide variation in thrombotic response to a standardized stimulus, not accounted for by shear stress or typical hematological variables, appears to be an intrinsic propensity of the individual. The experimental system for thrombus generation is sufficiently quantitative for assessment of variables determining this propensity.

We have analyzed the properties of cAMP response element-binding protein (CREB) in solution with emphasis on dimerization and effects of phosphorylation. Using a purified CREB fusion protein, a novel dye-label technique, and sedimentation equilibrium analysis, we directly and conclusively demonstrate that, unlike Jun and Fos, CREB dimerization is DNA-dependent. CREB exists primarily as a monomer in solution and cooperatively assembles on DNA to form dimers. Sedimentation equilibrium analysis also indicates that dimerization is unaffected by cAMP-dependent protein kinase-phosphorylation or by the symmetry of the cAMP-responsive element binding site. Filter binding assays reveal that CREB binding is unaffected by phosphorylation regardless of the symmetry of the cAMP-responsive element binding site. Our results suggest that structurally similar members of the same bZIP superfamily may differ significantly in their regulation at the level of dimerization.

1. We examined signal, noise and response properties of salamander rod photoreceptors by measuring: (a) the circulating current of rods which were adapted to darkness and to a wide range of backgrounds; (b) contrasts of natural environments; (c) the effect of adaptation on the linear response range of rods; and (d) the behaviour of rods responding to dynamically modulated stimuli having a range of contrasts found in nature. 2. In the dark, the circulating current contained two noise components analogous to those described in toad. A discrete noise component consisted of events occurring at a rate of 1 event per 32 s (21 degrees C) and had a variance of 0.036 pA2. A continuous noise component contributed 0.022 pA2 to the dark current, roughly equal to the discrete noise variance. 3. Exposure to a wide range of steady backgrounds (suppressing up to 80% of the circulating current), elicited a sustained fluctuating photocurrent having a power spectrum which resembled those of single photon responses and was consistent with the linear summation of single photon events; this indicates that the primary source of noise in the current is caused by the light. 4. Eighty-nine per cent of the contrasts (C) measured in natural environments had magnitude of C < 50%, where C = magnitude of I - Imean/magnitude of Imean. The linear response range elicited by brief flashes expanded with brighter backgrounds, well-encompassing flash contrasts of 100%. 5. Dynamically modulated stimuli and incremental flashes having contrasts similar to those in natural scenes elicited small currents which deviated by a few picoamps about the mean and the transfer functions computed from each type of stimulus-response pair closely corresponded to one another. These results indicate that in natural environments, rods behave as linear small-signal transducers of light.

Immunohistochemical analysis of surgically obtained porcine tissue samples reveals ubiquitous staining for prothrombin in organs rich in smooth muscle content and universal staining of smooth muscle in tissue vasculature. The native character of tissue prothrombin is verified first by chromogenic substrate hydrolysis and hirudin inhibition after incubation of tissue extracts with taipan snake venom and phospholipid. Western analysis of tissue extracts confirms the native zymogen molecular weight. In addition, prothrombin purified in good yield from porcine uterus is activated by Echis carinatus venom which, like taipan venom, is 4-carboxyglutamic acid-sensitive. After correction for blood (gross heme) and interstitial fluid (albumin), excess functional prothrombin is observed in extracts of tissues having abundant smooth muscle. In contrast with factor X, the yield of prothrombin purified from porcine uterus greatly exceeds that attributable to contamination by whole blood. Northern blot analysis from selected bovine tissues extracted for polyadenylated messenger RNA is equivocal for prothrombin mRNA with the exception of liver, which is positive. It is concluded that functionally intact prothrombin is widely distributed among tissues owing to smooth muscle content, although the mechanism of emplacement and physiologic significance of prothrombin in these tissues remains unclear.

Myosin accelerates plasminogen activation by tissue-type plasminogen activator (tPA), and is degraded extensively by plasmin. Myosin binds both tPA and plasminogen, and enhances activation of des1-77-plasminogen by tPA but not by urokinase-type plasminogen activator (uPA). Myosin decreases K(M) and increases k(cat) for des1-77-plasminogen activation by tPA, to yield catalytic efficiencies in excess of 8000 M-1 s-1. The effect of myosin is attributed to its C-terminal portion, the myosin rod. With a K(M) of 3 microM, myosin is a high-affinity substrate for plasmin. The findings indicate that myosin is a cofactor for plasminogen activation and a substrate for plasmin.

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.

Addition of exogenous plasminogen activator inhibitor-1 (PAI-1) to fibrin clots inhibits fibrinolysis in vivo. However, it is unknown whether the localized concentrations of active PAI-1 necessary to produce this antifibrinolytic effect can be recruited to acute arterial thrombi by endogenous mechanisms. We measured PAI-1 activity and antigen in porcine coronary artery thrombi that formed in response to acute vascular injury. Mean PAI-1 activity in thrombi (n = 5) was 36 +/- 5.1 micrograms/mL, which is > 2000 times its concentration in normal porcine plasma. The presence of markedly elevated concentrations of active PAI-1 in thrombi was confirmed by an immunoactivity assay and by demonstrating formation of sodium dodecyl sulfate-stable complexes after addition of 125I-urokinase to thrombus extracts. Comparative analysis of PAI-1 antigen by Western blotting and urokinase inhibition assay suggested that approximately one third of thrombus-associated PAI-1 was active. Histological examination of coronary thrombi revealed that they consisted predominantly of dense aggregates of platelets with interspersed islands of fibrin, which closely resemble the histological appearance of thrombi in patients with myocardial infarction and unstable angina pectoris. Washed porcine platelets prepared from peripheral blood contained sufficient PAI-1 antigen and activity to account for the concentrations observed in coronary artery thrombi. However, the specific activity of human platelet PAI-1 was lower than that of porcine platelet PAI-1 (2% versus 50% active, respectively), and human platelets inhibited in vitro fibrinolysis to a lesser extent than did porcine platelets. These results indicate that active PAI-1 accumulates in porcine coronary artery thrombi in concentrations markedly higher than those present in plasma and that PAI-1 may be an important determinant of the known resistance of platelet-rich thrombi to lysis by tissue-type plasminogen activator. These studies also underscore the importance of considering possible species differences in protein function when comparing animal models of thrombosis to acute coronary thrombosis in humans.

Background: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin., Methods and Results: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline)., Conclusions: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.

We employed the fluorescent calcium indicator Fura-2, loaded into intact retinas of the bullfrog Rana catesbeiana, to measure free calcium concentrations in the rod outer-segment cytosol. We determined that traditional methods of calculation yielded erroneous values of calcium. This error results from the presence of at least two distinct pools of Fura-2 in rod outer segments. Application of manganese quenches each pool, but quenching occurs at different rates. Using this fact, we show that the pools can be isolated by brief exposure to manganese and examined separately. One of these pools has the same fluorescent properties as the free salt of Fura-2 we use in our in vitro calibrations. The other source of fluorescence has more unusual properties. Although insensitive to calcium concentrations in the physiological range, it contributes significant anomalous fluorescence when cytosolic free calcium concentrations are elevated by application of IBMX. Nevertheless, the experimentally isolated, classic pool of Fura-2 is well behaved and allows us to calculate calcium concentrations relative to the Kd of Fura-2 by the usual ratio method. We show that when rods are exposed to saturating light, the free calcium concentration in their outer segments falls to a level not significantly different from zero within 20-30 s.

The lectin from Eranthis hyemalis has been previously characterized as consisting of two polypeptide chains covalently linked by disulfide bond(s) (Cammue, B. P., Peeters, B., and Peumans, W. J. (1985) Biochem. J. 227, 949-955). We have further characterized the biochemical properties of the lectin and demonstrated that it possesses the property of inhibition of protein synthesis using in vitro eukaryotic translation systems. The protein also possesses antiviral activity against the plant virus, alfalfa mosaic virus, and larvicidal activity against the southern corn rootworm, Diabrotica undecimpunctata howardii, a major insect pest of the maize plant. Both isoelectric focusing on gels and chromatofocusing indicated heterogeneity of the protein, with three species having isoelectric points in the range 4-5. The disulfide bond(s) can be rapidly reduced with beta-mercaptoethanol under native conditions. The reduced alkylated polypeptide chains remain associated under native conditions to form a species, EHL', that elutes at the same position as the native protein and has the same molecular weight by sedimentation equilibrium experiments. However, circular dichroism and fluorescence measurements indicated conformational differences between the species.

The amount of thrombin active in vivo in the intravascular space (blood and endothelial surface), both basally and in experimental intravascular coagulation, is measured by way of the accessibility of thrombin to intravascular hirudin. Blood samples from pigs given intravenous 125I-labeled hirudin contain 125I-labeled hirudin-thrombin complex in concentrations indicative of a basal thrombin concentration in vivo of 0.5 nmol/liter. Intravenous infusion of Salmonella endotoxin elicits an increase in the circulating concentration of hirudin-thrombin complex that begins within 15 min and is 20-30 times basal after 4 hr. Induction of mild intravascular coagulation is evidenced by a modest reduction in plasma fibrinogen concentrations. It is concluded that there is a basal pool of hirudin-accessible thrombin in the intravascular space that, were it free in the plasma phase, would be sufficient in principle to sustain intravascular coagulation.

1. We studied the effects of 2-amino-4-phosphonobutyric acid (APB) on the response properties of rods, horizontal cells and bipolar cells in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. A concentration of 100 microM was found to be sufficient to elicit maximal effects. 2. Rods hyperpolarized slightly upon exposure to 100 microM-APB and their response amplitudes were slightly reduced. The amplitude of the cone-generated component of the rod's response to 700 nm light was not significantly affected by APB. 3. Horizontal cells hyperpolarized by 2-5 mV upon exposure to 100 microM-APB. The rod-driven component of the horizontal cell response increased in amplitude while the cone-driven component decreased in amplitude. APB thus causes an increase in voltage gain between rods and horizontal cells and a decrease in cone/horizontal cell gain. These findings can be explained in terms of an APB-induced reduction in transmitter release from the cones. 4. APB at a concentration of 100 microM caused an increase in the length constant of the horizontal cell syncytium. Our analysis shows this to be due primarily to a 50% reduction in the coupling impedance between the cells of the syncytium. 5. The effects of APB on off-centre bipolar cells were qualitatively similar to those on horizontal cells. APB increased the amplitudes of rod-driven responses and reduced those of cone-driven responses. The length constants, both of the receptive field centre and of the surround, were increased and the strength of the surround relative to the centre was reduced by about 20%. 6. APB abolished the depolarizing light responses of the receptive field centres of on-centre bipolar cells. A hyperpolarizing response remained whose spatial properties were similar to those of the receptive field surround. We believe this response to reflect a direct (feedforward) input to on-centre bipolar cells from horizontal cells.

Recent experiments indicate that the dark-adapted vertebrate visual system can count photons with a reliability limited by dark noise in the rod photoreceptors themselves. This suggests that subsequent layers of the retina, responsible for signal processing, add little if any excess noise and extract all the available information. Given the signal and noise characteristics of the photoreceptors, what is the structure of such an optimal processor? We show that optimal estimates of time-varying light intensity can be accomplished by a two-stage filter, and we suggest that the first stage should be identified with the filtering which occurs at the first anatomical stage in retinal signal processing, signal transfer from the rod photoreceptor to the bipolar cell. This leads to parameter-free predictions of the bipolar cell response, which are in excellent agreement with experiments comparing rod and bipolar cell dynamics in the same retina. As far as we know this is the first case in which the computationally significant dynamics of a neuron could be predicted rather than modeled.

1. The spatial properties of rods, horizontal cells and bipolar cells were studied by intracellular recording in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. Low stimulus intensities were used in order to keep cell responses close to, or within, their linear intensity/response range. 2. Spatial properties of bipolar cell receptive fields, measured while perfusing with normal Ringer solution, were compared with those measured during exposure to agents that eliminated the bipolar cells' receptive field surround (RFS). In this way, the spatial properties of the receptive field centre (RFC) and those of the RFS could be characterized independently. 3. To a good approximation, the contribution to the horizontal cell's response of unit area of its receptive field declined exponentially with distance from the centre of the receptive field. The (apparent) length constant describing this decay was 200 microns. The one-dimensional length constant of the horizontal cell syncytium was thus 248 microns. The variation of response amplitude with the radius of a centred circular stimulus was consistent with this finding. 4. This was true also of the RFCs of bipolar cells. The one-dimensional length constant of the RFC of off-centre bipolar cells averaged 124 microns. That of the RFC of on-centre cells averaged 62 microns though values were more variable, the RFCs of some on-centre cells being comparable to those of off-centre cells. These values were independent of the class of photoreceptor driving the bipolar cell. 5. The large size of the RFCs of off-centre cells and many on-centre cells cannot by explained by light scatter within the retina or by voltage spread within the rod syncytium. We proposed that off-centre cells are tightly coupled in a syncytium. On-centre cells, on average, are less tightly coupled. 6. The spatial properties of the bipolar cell's RFS were consistent with the notion that the RFS represents a convolution of the horizontal cell's receptive field and the bipolar cell's RFC. 7. The spatial properties of bipolar cell receptive fields were reconstructed from the measured properties of their RFCs and the measured properties of horizontal cell receptive fields. Under the conditions of our experiments, the bipolar cell's response could be described by a linear difference between a component generated by the RFC and a component generated by the RFS. 8. The spatial filtering characteristics of the bipolar cells were calculated from our data.(ABSTRACT TRUNCATED AT 400 WORDS)

A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the reversible, high affinity, high capacity binding of thrombin to the vascular endothelium.

The role of the microvascular endothelium in the integration of inhibitory and catabolic pathways of hemostasis is discussed in light of recent findings of direct biochemical links between endothelium and regulatory plasma proteins. These findings include the following: (1) On the vascular endothelium, a cofactor for antithrombin III (with an activity comparable to stationary phase heparin) catalyzes thrombin inhibition in vivo. (2) A second cofactor on endothelium binds thrombin in a manner that enhances by several orders of magnitude the ability of thrombin to activate protein C. (3) Activated protein C has both anticoagulant and catabolic activities; anticoagulant activity results from the susceptibility of factors Va and VIIIa to inactivation by activated protein C, whereas catabolic activity arises from the stimulation by activated protein C of the release from endothelium of fibrin-dependent plasminogen activator. (4) Because it requires fibrin as a cofactor, the plasminogen activator lyses clots without provoking fibrinogenolysis. Location of these activities on endothelium separates coagulation in time and space from catabolic pathways, and provides for their expression after the initiation of hemostasis.

1. Intracellular recordings of the voltage responses of rods and both functional classes of bipolar cell were made in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. 2. Brief, dim flashes of 519 nm light delivered to the receptive-field centres were used to measure the flash sensitivities of twenty-one on-centre bipolar cells and thirty-six off-centre cells. In each experiment the flash sensitivity of a rod was also measured using diffuse illumination of the same duration and wave-length. 3. The mean flash sensitivity of the rods (fifty-nine cells) was 4.47 mV photon-1 micron 2 flash. The mean flash sensitivity of the off-centre bipolar cells was 35.4 mV photon-1 micron 2 flash (thirty-six cells). The mean flash sensitivity of the on-centre bipolar cells was 12.5 mV photon-1 micron 2 flash. 4. The ratio of the flash sensitivity of the bipolar cell to that of a rod recorded in the same retina defined the gain of voltage transfer from rod to bipolar cell. For signal transfer to on-centre bipolar cells the mean value of the voltage gain was 5.05 +/- 1.34 (S.E. of mean). For signal transfer to the off-centre bipolar cells, the mean value of the gain was 10.4 +/- 1.29. 5. The on-centre cell gain in the salamander was smaller by a factor of 27 than that of the on-centre cells in the dogfish retina (Ashmore & Falk, 1980 a), while the off-centre cell gain was comparable in the two species. Possible reasons for the large difference between the voltage gains of on-centre cells in the dogfish and salamander are considered.

Two in vitro systems were used to identify an antithrombin III cofactor activity on vascular endothelium. Langendorff rat heart preparations or columns packed with endothelium cultured on microcarrier beads were perfused with mixtures of purified thrombin and antithrombin III. With each preparation, accelerated inhibition of thrombin by antithrombin III occurred during passage over endothelium. Platelet factor 4, protamine sulfate and diisopropylphosphoryl thrombin, all antagonists of the antithrombin III cofactor activity of heparin, significantly reduced the capacity of the preparation to inhibit thrombin. It is concluded that a substance with the functional properties of a stationary phase cofactor for antithrombin III is present on the microvascular endothelium and there catalyzes the inactivation of circulating free thrombin.

The dysthrombin, Thrombin Quick, is chromatographically separable into two components designated Thrombin Quick I and Thrombin Quick II. Thrombin Quick II lacks observable catalytic activity toward thrombin substrates. The steady-state kinetics of hydrolysis of benzoylarginine ethyl ester and Tos-Gly-Pro-Arg-p-nitroanilide by Thrombin Quick I are equivalent to those of thrombin. These results, in addition to binding studies with the active site titrant N2-(5-dimethylaminonaphthalene-1-sulfonyl)arginine N-(3-ethyl-1,5-pentanediyl)amide, indicate that binding interactions at the catalytic site of Thrombin Quick I are unaltered. Thrombin Quick I is inhibited by anti-thrombin III at the same rate as thrombin. Steady-state kinetic parameters for the release of fibrinopeptide A indicate defects in both kcat and Km for Thrombin Quick I with kcat/Km equal to 0.012 of the value for thrombin, corresponding to the relative fibrinogen clotting activity of 0.013. The results are interpreted as indicating a defect in Thrombin Quick I at a binding site, external to the catalytic site, which is essential for determining specificity toward fibrinogen. The defect in kcat may result secondarily from small perturbations in the steric relationship of the catalytic triad residues. The rate of hydrolysis by Thrombin Quick I of the protein substrates bovine prothrombin and bovine protein C (in the absence of cofactors) is about one-third of that observed for thrombin, indicating that hydrolysis of these substrates by thrombin involves different specificity determinants than does the hydrolysis of fibrinogen.

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.

The electrical spread of excitation in the network of rod photoreceptors was studied by intracellular recording in the isolated, perfused retina of the toad, Bufo marinus. Experiments with dim, bar-shaped flashes of light revealed that the rod network behaves as a high-pass filter to laterally propagating small signals. Such a behavior had been found earlier in the turtle (Detwiler et al., 1980). Three electrical equivalent circuit models that can explain this behavior were considered and analytical solutions to the network equations were obtained. By fitting these analytical expressions to linear responses elicited by weak light flashes and to voltage excursions elicited by extrinsic current injections, values for the circuit parameters were determined. Values obtained by independent methods were consistent. The effects of changing each of these parameters in turn upon the high-pass filtering of small signals were then predicted. These predictions provided a framework for an analysis of the ionic basis of the underlying mechanism, which is described in the following paper.

The effect of thrombin and its derivative, diisopropylphosphoryl-thrombin on [3H]arachidonic acid metabolism is studied in cultured umbilical vein endothelial cell monolayers. Thrombin causes a dose-dependent release of radioactivity from endothelial cells fed [3H]arachidonate. Thin layer radiochromatography of acidified supernatants reveals that most of the radio-activity is [3H]arachidonate and its metabolites, 6-ketoprostaglandin F1 alpha and prostaglandin E2. Diisopropylphosphoryl-thrombin, which is enzymatically inactive, does not cause release of arachidonic acid or metabolites. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98% inhibition of binding of 125I-thrombin to its high affinity binding sites, does not inhibit thrombin-induced release. We conclude that the high affinity, active site-independent thrombin binding sites are not involved in thrombin-induced mobilization of esterified arachidonic acid.

All of the vitamin K-dependent plasma proteins with domains that are homologous to the epidermal growth factor (EGF) precursor have 1 hydroxylated aspartic acid residue in the NH2-terminal EGF-homology region. In addition, protein S has 1 hydroxylated asparagine residue in each of the three COOH-terminal EGF-homology regions. All of these proteins have been found to have the amino acid sequence, CX(D or N)XXXX(F or Y)XCXC (corresponding to residues 20 to 33 in EGF), where the Asp or Asn residue is hydroxylated. This sequence also appears in two of the three EGF-homology regions of the human low density lipoprotein receptor and in two of the six EGF-homology regions of bovine thrombomodulin so far identified, suggesting that they may have the modified amino acid. We have now identified beta-hydroxyaspartic acid in acid hydrolysates of both these proteins.