Reversible binding of actin to gelsolin and profilin in human platelet extracts.

This paper documents the reversible appearance of high-affinity complexes of profilin and gelsolin with actin in extracts of platelets undergoing activation and actin assembly. Sepharose beads coupled to either monoclonal anti-gelsolin antibodies or to polyproline were used to extract gelsolin and profilin, respectively, from EGTA-containing platelet extracts and determine the proportion of these molecules bound to actin with sufficient affinity to withstand dilution (high-affinity complexes). Resting platelets (incubated for 30 min at 37 degrees C after gel filtration) contained nearly no high-affinity actin/gelsolin or actin/profilin complexes. Thrombin, within seconds, caused quantitative conversion of platelet profilin and gelsolin to high-affinity complexes with actin, but these complexes were not present 5 min after stimulation. The calcium-dependent actin filament-severing activity of platelet extracts, a function of free gelsolin, fell in concert with the formation of EGTA-stable actin/gelsolin complexes, and rose when the adsorption experiments indicated that free gelsolin was restored. The dissociation of high-affinity complexes was temporally correlated with the accumulation of actin in the Triton-insoluble cytoskeleton.

Human platelets contain gelsolin. A regulator of actin filament length.

Morphologic and biochemical studies suggest that actin in human platelets polymerizes in response to various stimuli and that shortening of actin filaments can be regulated by calcium. We report that human platelets contain gelsolin, a protein of Mr 91,000 that binds reversibly to actin in the presence of calcium. Platelet gelsolin exhibits immunologic crossreactivity with rabbit macrophage gelsolin and shortens actin filaments as demonstrated by viscosity measurements and gel point determinations. Gelsolin is active in micromolar calcium concentrations and its effects upon actin filaments are reversible. Gelsolin may be a dynamic regulator of actin filament length in the human platelet.

Role of plasma gelsolin and the vitamin D-binding protein in clearing actin from the circulation.

We determined the plasma kinetics of both actin and complexes of actin with the two high affinity actin-binding proteins of plasma, gelsolin, and vitamin D-binding protein (DBP). Actin is cleared rapidly from the plasma by the liver (half-disappearance time, 0.5 h). Using radiolabeled actin-binding proteins, we found that actin accelerated the clearance of both plasma gelsolin and the vitamin D-binding protein. In separate experiments we found that DBP-actin complexes were cleared more quickly than gelsolin-actin complexes, at a rate comparable to the clearance of actin from the blood. A low affinity interaction (dissociation constant, 2.9 X 10(-4) M) between actin and fibronectin was found, suggesting that little actin will bind to fibronectin in plasma. We conclude that while plasma gelsolin and DBP may both clear actin from the circulation, DBP appears to play a more important role. By so doing, DBP may conserve the filament-severing activity of plasma gelsolin.

Relapsing hypocupraemic myelopathy requiring high-dose oral copper replacement.

Adult-onset copper deficiency with neurological manifestations is a newly recognised syndrome. Long-term oral copper replacement therapy has been the mainstay of treatment in the literature. A case of relapsing hypocupraemic myelopathy responsive to increased doses of copper replacement is reported. Standard doses of copper may not be sufficient for all patients.

Adenosine-mediated killing of cultured epithelial cancer cells.

Because micromolar concentrations of adenosine (Ado) have been documented recently in the interstitial fluid of carcinomas growing in animals, we examined the effects of low concentrations of Ado on the growth of cultured human carcinoma cells. Ado alone had little effect upon cell growth. In the presence of one of a number of Ado deaminase (ADA) inhibitors, Ado led to significant growth inhibition of all cell lines tested. Similar effects were found when ATP, ADP, or AMP was substituted for Ado. Surprisingly, the ADA inhibitor coformycin (CF) had a much greater potentiating effect than did 2'-deoxycoformycin (DCF), although DCF is a more potent ADA inhibitor. The growth inhibition of the Ado/CF combination was not abrogated by pyrimidines or caffeine, a nonspecific Ado receptor blocker. Toxicity was prevented by the addition of the Ado transport inhibitor dipyridamole or the Ado kinase inhibitor 5'-amino 5'-deoxyadenosine. S-Adenosylhomocysteine hydrolase is not involved because neither homocysteine thiolactone nor an S-adenosylhomocysteine hydrolase inhibitor (adenosine dialdehyde) potentiated toxicity of the Ado/CF combination. Unexpectedly, substitution of 2'-deoxyadenosine (the toxic moiety in congenital ADA deficiency) for Ado, did not lead to equivalent toxicity. The Ado/CF combination inhibited DNA synthesis and brought about morphological changes consistent with apoptosis. Together, these findings indicate that the Ado-mediated killing proceeds via an intracellular route that requires the action of Ado kinase. The enhanced cofactor activity of CF may be attributable to its being a more potent inhibitor of AMP deaminase than is DCF.

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation.

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.

Diagnosing liver metastases: a Bayesian analysis.

Clinicians frequently perform tests to determine whether patients have liver metastases. Optimal use of a laboratory test requires that the clinician know the test's operating characteristics (its sensitivity and specificity) and have an estimate of the pretest probability that disease is present. We have surveyed studies that examined the value of four biochemical and three imaging tests in establishing a diagnosis of hepatic metastases in patients who underwent an invasive procedure to establish the presence or absence of disease. We have pooled the data from these studies to arrive at values for the sensitivity and specificity of each of these tests, and calculated the predictive values for these tests over a wide range of pretest probabilities of disease. Several examples illustrate how this information may be used clinically. We provide a framework for the optimal interpretation of these commonly ordered tests and indicate the data needed for their complete analysis.

Telling the diagnosis of cancer.

Although a concensus has emerged in this country that patients should be told when cancer is discovered, no data is available to indicate how and where patients are currently told that they have cancer. Fifty-five patients undergoing anticancer therapy were therefore interviewed to learn how this process occurs. The majority of patients were told by surgeons (74%) and only a minority by primary care physicians (11%). Most were told in a traditional medical setting (42% in the doctor's office, 17% in a hospital room), but 23% were told over the telephone and 19% in the recovery room. Two indicators of patient satisfaction with the telling process suggested that different sites of telling were not equivalent. Patients told over the telephone or in the recovery room were more likely to describe the telling in negative terms and less likely to describe their doctors as being helpful in understanding their illness than those told in a doctor's office or in their hospital bed. This pilot study indicates considerable variation in this aspect of patient care and suggests directions for future research. To determine whether interviews that explore these issues with cancer patients are unpleasant or stressful, patients' reactions to being subjects in this study were sought. Patients asked directly at the completion of the interview or surveyed 2 to 4 months later said the interview had been helpful and/or a positive experience. None expressed negative feelings about participating. Concerns about the psychological harm resulting from such study of this patient group do not appear to be warranted and should not impede future research.

Prolonged bleeding time.

The bleeding time is the most frequently used test of platelet function. This review of the literature relating to the bleeding time outlines the causes and management of prolonged bleeding time. The bleeding time appears to have its greatest utility in evaluation of a patient with active bleeding or one with a well-documented bleeding history. It should not be used as a substitute for a clinical history, since there is insufficient information available to calculate its sensitivity, specificity, or predictive value with regard to peri- or postoperative hemorrhage.

Use of a modified activated partial thromboplastin time to detect lupus anticoagulants.

Laboratory evidence for the presence of lupus anticoagulants (LAs) is sought when patients experience thrombotic events or when coagulation assays are abnormal. Although a number of tests for LAs have been proposed, none detect all LAs, and laboratories may be confronted with the need to perform more than one test to confirm a suspected LA. Recently, a modification of the aPTT, performed by varying the initial time of incubation of the aPTT reagent with the patient's plasma, was reported to detect LAs. The difference in clotting times when plasma is subjected to a 1- or 10- minute incubation (called here the "Delta one minus ten" or DOT) using a particular micronized silica-based aPTT reagent was shown to provide good discrimination between normal and LA plasmas. Because of the low cost of this test and its relative ease of performance, we attempted to replicate the results of this test using previously characterized LA plasmas. The DOT of 23 normal plasmas was 5.1 +/- 2.1 seconds, with a range of 0.5 - 9.3 seconds. The DOT of 20 of 34 LA samples tested (59%) was > 11 seconds. The DOT was abnormal in 8 of 22 (36%) samples diagnosed with a dilute Russell's viper venom time. It was abnormal in 12 of 12 patients diagnosed by other criteria, prior to the use of the dilute Russell's viper venom time. The DOT performed with a kaolin or ellagic acid-based aPTT reagent failed to discriminate normal from LA plasma. We conclude that the DOT performed with a specific silica-based reagent is an apparently simple and moderately sensitive test for detecting the lupus anticoagulant that deserves further evaluation.

Clinically significant differences in the International Normalized Ratio measured with reagents of different sensitivities. SPAF Investigators. Stroke Prevention in Atrial Fibrillation.

The International Normalized Ratio (INR) system was introduced a decade ago as a way of standardizing the results of prothrombin time testing for patients taking oral anticoagulants. A strong emphasis has been placed upon using thromboplastin reagents that are very sensitive to the effects of oral anticoagulants upon the prothrombin time [i.e. reagents with low International Sensitivity Index (ISI)]. In order to assess how well the INR system functions as currently used in clinical laboratories, we compared the INRs determined using thromboplastins of differing ISIs in samples collected during a large clinical trial of oral anticoagulation for atrial fibrillation (Stroke Prevention in Atrial Fibrillation III trial). Frozen plasma was subjected to prothrombin time testing using thromboplastins with ISIs ranging from 0.97 to 2.49. INRs were calculated using machine-specific ISIs and Westgard's rules were followed to maintain quality control. An unanticipated coagulometer failure allowed a determination of the effect of machine recalibration upon the INR of control plasmas. The correlation between each pair of INRs obtained from 1181 plasmas was high (> 0.9), but the differences between reagents were statistically different from zero (P<0.001 for pairwise comparisons). Plasmas had INRs within the therapeutic range (2.0-3.0) with one reagent but not with another in an average of 20% of instances. Among the 20% discordant pairings, 43% (8.5% of the total tested) showed a difference in INR of more than 0.2 INR units above or below the target range. Low ISI thromboplastins did not perform better in this pairwise comparison than other reagents or the locally determined INR. Recalibration of a coagulometer resulted in a significant change in the INRs obtained from control plasmas (P<0.0001), which confirms and extends the observations of other authors concerning the sensitivity of the INR to coagulometer-related variables. There was a clinically significant difference in the INRs obtained with different thromboplastins, and low ISI reagents did not perform better than others. Since the risk of thrombosis rises sharply below the lower limit of the currently recommended target ranges, consideration should be given to narrowing the recommended range, or advising clinicians to aim for its mid-point. These findings illustrate the difficulties in imposing standardization upon coagulation testing after a test is in widespread use.

Plasma levels of factors II, VII and X and their relationship to the international normalized ratio during chronic warfarin therapy.

Monitoring of oral anticoagulant therapy is usually undertaken with the prothrombin time (PT), which is influenced by factors II, X, and VII. A number of studies have suggested that the prothrombin (factor II) level may be the most important determinant of the therapeutic efficacy of these drugs. Although some studies suggest that oral anticoagulants induce a similar residual level of plasma vitamin K-dependent proteins, others have called this into question. We therefore measured plasma levels of factors II, X, and VII in 50 patients undergoing chronic Warfarin therapy. The plasma levels of factors II, X, and VII were significantly different. Although the factor X levels of all plasmas were < 30%, levels of factors II and VII were > 30% in 14% and 50% of the samples, respectively. Multivariable analysis showed factor II levels to be the least significant of the three factors measured in determining the international normalized ratio of plasma or whole blood. Thus, plasma levels of the vitamin K-dependent coagulation factors are not equal in patients on chronic Warfarin therapy. If factor II (prothrombin) levels are indeed the major determinants of the therapeutic efficacy of Warfarin, alternative means of monitoring that more accurately reflects prothrombin levels should be evaluated.

Assessment of coagulation system activation using spot urine measurements.

Coagulation system activation is most commonly assessed by measuring levels of one or more proteins in peripheral blood. Because faulty blood-drawing can cause activation of the coagulation system, artifactual elevations of such markers have been reported. We have therefore investigated the possibility of using randomly collected ('spot') urine samples as a non-invasive means of assessing the state of coagulation system activation. Using a commercially available enzyme-linked immunosorbent assay kit designed to measure plasma levels of fragment 1 + 2, we found immunoreactive fragment 2 in healthy control subjects, and significantly increased levels in diabetic and non-diabetic pregnant subjects, and patients with venous thromboembolism, prostate cancer, and diabetes. Measurements of excretion of immunoreactive fragment 2 are worth further study as an adjunct or alternative to plasma-based assays designed to detect or quantify coagulation system activation.

The insidious development of symptomatic secondary hormone syndromes in patients with malignant endocrine tumors.

Endocrine tumors may produce secondary or "ectopic" hormones that cause paraneoplastic syndromes. Such syndromes may be confused with more common complications related to a patient's tumor, and thus escape detection and appropriate treatment. The secondary hormone secretion responsible for these syndromes often occurs late in the course of such diseases and presents in an insidious manner. Two patients are presented that illustrate these points. The first, a woman with medullary carcinoma of the thyroid (MCT), developed a syndrome secondary to ACTH secretion that was confused initially with the changes caused by the massive diarrhea that accompanies MCT. The second, a man with malignant glucagonoma, is the first with this disease to have developed symptomatic hyperinsulinemia as a late complication. We stress the clinical courses of these patients and note that treatment of these syndromes may improve the quality of patients' lives.

Actin is a noncompetitive plasmin inhibitor.

Actin, one of the most abundant cellular proteins, circulates at micromolar concentrations in peripheral blood. Because actin released from dying cells may be trapped in fibrin clots that form at sites of tissue injury, we examined the effects of actin upon lysis of fibrin clots in vitro. Incorporation of native rabbit skeletal muscle actin into fibrin clots slowed their rates of lysis for periods of up to 24 h, an effect not seen when comparable concentrations of human IgG or bovine serum albumin were added instead. Actins isolated from a variety of sources inhibited plasmin's hydrolysis of the synthetic substrate S-2251 in a noncompetitive manner, with a Ki of a 0.6-3.1 microM. Inhibition was rapid, but covalent actin-plasmin complexes were not formed. Both epsilon-aminocaproic acid and tranexamic acid prevented actin's inhibition of plasmin, suggesting that accessible lysine residues of actin interact with the kringle (lysine-binding) regions of plasmin. Neither of the high-affinity actin-binding proteins of plasma (plasma gelsolin and vitamin D-binding protein) prevented actin from inhibiting plasmin. These findings suggest that actin released into the extracellular space following cell death may modulate plasmin action, and hence a number of plasmin-dependent biological responses, at sites of inflammation and tissue injury.

Actin accelerates plasmin generation by tissue plasminogen activator.

Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.

Capacity of human serum to depolymerize actin filaments.

Human blood depolymerizes filamentous (F-)actin. The interaction of actin filaments and monomers with human serum was studied by following the kinetics and extent of the depolymerization of pyrene-labeled F-actin and by analysis of serum proteins adhering to immobilized actin monomers. In physiologic Ca2+ concentrations, the depolymerization of F-actin proceeds in two stages: a rapid phase, attributed to direct severing of filaments by plasma gelsolin, and a slow phase attributed to the binding of actin monomers to vitamin D-binding protein (DBP). Without Ca2+, only the slow phase is observed. Human serum can completely depolymerize 10 to 18 mumol/L of actin, of which approximately 5 mumol/L occurs rapidly. Depolymerization can be accounted for by the normal serum concentrations of gelsolin and DBP. Fibrin(ogen) and fibronectin, which bind actin in vitro, do not contribute to the kinetics or extent of its depolymerization. Affinity chromatography and functional assays for the presence of gelsolin-actin complexes show that addition of G-actin to serum results in preferential formation of actin-DBP complexes, but that addition of F-actin to serum produces both gelsolin-actin complexes and DBP-actin complexes. The distinctive binding of actin monomers and polymers to these two serum proteins suggests a means by which their coordinated actions are maximized in vivo, from the standpoint of depolymerizing filaments and clearing monomers from the circulation.