ABSTRACT
INTRODUCTION: Abnormal fibrinolysis early after injury has been associated with increased mortality in trauma patients, but no studies have addressed patients with burn injury. This prospective cohort study aimed to characterize fibrinolytic phenotypes in burn patients and to see if they were associated with mortality. METHODS: Patients presenting to a regional burn centre within 4 h of thermal injury were included. Blood was collected for sequential viscoelastic measurements using thromboelastography (RapidTEG™) over 12 h. The percentage decrease in clot strength 30 min after the time of maximal clot strength (LY30) was used to categorize patients into hypofibrinolytic/fibrinolytic shutdown (SD), physiological (PHYS) and hyperfibrinolytic (HF) phenotypes. Injury characteristics, demographics and outcomes were compared. RESULTS: Of 115 included patients, just over two thirds were male. Overall median age was 40 (i.q.r. 28-57) years and median total body surface area (TBSA) burn was 13 (i.q.r. 6-30) per cent. Some 42 (36.5 per cent) patients had severe burns affecting over 20 per cent TBSA. Overall mortality was 18.3 per cent. At admission 60.0 per cent were PHYS, 30.4 per cent were SD and 9.6 per cent HF. HF was associated with increased risk of mortality on admission (odds ratio 12.61 (95 per cent c.i. 1.12 to 142.57); P = 0.041) but not later during the admission when its incidence also decreased. Admission SD was not associated with mortality, but incidence increased and by 4 h and beyond, SD was associated with increased mortality, compared with PHYS (odds ratio 8.27 (95 per cent c.i. 1.16 to 58.95); P = 0.034). DISCUSSION: Early abnormal fibrinolytic function is associated with mortality in burn patients.
Subject(s)
Blood Coagulation Disorders/etiology , Blood Coagulation Disorders/mortality , Burns/complications , Fibrinolysis/physiology , Adult , Body Surface Area , Burns/diagnostic imaging , Female , Humans , Incidence , Logistic Models , Male , Middle Aged , Prospective Studies , ThrombelastographyABSTRACT
Essentials Acidosis, an outcome of traumatic injury, has been linked to impaired procoagulant efficiency. In vitro model systems were used to assess coagulation dynamics at pH 7.4 and 7.0. Clot formation dynamics are slightly enhanced at pH 7.0 in blood ex vivo. Acidosis induced decreases in antithrombin efficacy offset impairments in procoagulant activity. SUMMARY: Background Disruption of hydrogen ion homeostasis is a consequence of traumatic injury often associated with clinical coagulopathy. Mechanisms by which acidification of the blood leads to aberrant coagulation require further elucidation. Objective To examine the effects of acidified conditions on coagulation dynamics using in vitro models of increasing complexity. Methods Coagulation dynamics were assessed at pH 7.4 and 7.0 as follows: (i) tissue factor (TF)-initiated coagulation proteome mixtures (±factor [F]XI, ±fibrinogen/FXIII), with reaction progress monitored as thrombin generation or fibrin formation; (ii) enzyme/inhibitor reactions; and (iii) TF-dependent or independent clot dynamics in contact pathway-inhibited blood via viscoelastometry. Results Rate constants for antithrombin inhibition of FXa and thrombin were reduced by ~ 25-30% at pH 7.0. At pH 7.0 (+FXI), TF-initiated thrombin generation showed a 20% increase in maximum thrombin levels and diminished thrombin clearance rates. Viscoelastic analyses showed a 25% increase in clot time and a 25% reduction in maximum clot firmness (MCF). A similar MCF reduction was observed at pH 7.0 when fibrinogen/FXIII were reacted with thrombin. In contrast, in contact pathway-inhibited blood (n = 6) at pH 7.0, MCF values were elevated 6% (95% confidence interval [CI]: 1%-11%) in TF-initiated blood and 15% (95% CI: 1%- 29%) in the absence of TF. Clot times at pH 7.0 decreased 32% (95% CI: 15%-49%) in TF-initiated blood and 51% (95% CI: 35%-68%) in the absence of TF. Conclusions Despite reported decreased procoagulant catalysis at pH 7.0, clot formation dynamics are slightly enhanced in blood ex vivo and suppression of thrombin generation is not observed. A decrease in antithrombin reactivity is one potential mechanism contributing to these outcomes.
Subject(s)
Acidosis/blood , Blood Coagulation Tests/methods , Blood Coagulation/drug effects , Thrombin/pharmacology , Antithrombin III/analysis , Blood Coagulation Disorders , Elasticity , Fibrin/analysis , Fibrinogen/pharmacology , Healthy Volunteers , Humans , Hydrogen-Ion Concentration , Ions , Proteome , Thrombin/antagonists & inhibitors , Thromboplastin/pharmacology , Time Factors , ViscosityABSTRACT
INTRODUCTION: Factor VIII (FVIII) products used in haemophilia A treatment show inter-and intra-product and inter-assay differences in specific activity. The mechanistic basis of these differences remains unclear. AIM: The aim of this study was to mechanistically compare the functional properties of an in-house excipient-free full-length FVIII standard and pharmacologic recombinant products containing full-length (products A and B) or B-domainless (C and D) FVIII. METHODS: Factor VIII protein concentration was quantitated by ELISA. Product potency determinations (APTT, intrinsic tenase assays) and kinetic analyses detailing these products' activations by thrombin and FXa, their spontaneous and activated protein C (APC) catalysed inactivation and their performances in coagulation proteome reconstructions were studied +/- von Willebrand factor (VWF). Computational models were developed to facilitate interpretation of empirical data. RESULTS: Factor VIII protein content per manufacturer activity unit was highest for product C with the other three products similar to the standard. Potency estimates, done five different ways, varied 20-30% in inter- and intra-assay comparisons, with product B consistently showing lower specific activity. Kinetic analyses showed the five FVIII species to differ somewhat in maximum rate of activation, the maximum level of activity achieved, the rate of spontaneous or APC catalysed inactivation and the magnitude of the effect of VWF on these parameters. When evaluated both computationally and empirically in the context of tissue factor initiated thrombin generation, product C appears the most dissimilar. CONCLUSION: Assessments of FVIII activation/inactivation dynamics report larger differences between FVIII products than standard functional assays. However, all FVIII products promote a 'normal' thrombin generation response to TF.
Subject(s)
Factor VIII/metabolism , Catalysis , Coagulants/therapeutic use , Enzyme-Linked Immunosorbent Assay , Factor VIII/genetics , Factor VIII/therapeutic use , Factor Xa/metabolism , Hemophilia A/drug therapy , Humans , Kinetics , Partial Thromboplastin Time , Protein C/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Thrombin/metabolism , von Willebrand Factor/metabolismABSTRACT
SUMMARY BACKGROUND: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca(2+). FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by approximately 10(7). One potential APTT procofactor activator in this setting is FXIa. OBJECTIVE: To test the hypothesis that FXIa can activate FVIII and FV. METHODS: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic analyses of FXIa-catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. CONCLUSIONS: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.
Subject(s)
Factor VIII/metabolism , Factor V/metabolism , Factor XIa/physiology , Binding Sites , Blood Coagulation Tests/methods , Blood Coagulation Tests/standards , Catalysis , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Kinetics , Partial Thromboplastin Time/methods , Partial Thromboplastin Time/standardsABSTRACT
BACKGROUND: Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (F) Xa emerging as a promising approach. OBJECTIVE: To assess anticoagulant strategies targeting FXa. METHODS: A deterministic computational model of tissue factor (Tf)-initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of FXa-directed anticoagulants [an antithrombin (AT)-dependent agent, fondaparinux, and an AT-independent inhibitor, Rivaroxaban] in experimental regimens relevant to long-term (suppression of new Tf-initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. RESULTS: Computational representations of each anticoagulant's efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long-term and acute clinical applications, respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. CONCLUSION: The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an FXa inhibitor. We have reported that FIXa contributes to the long-term capacity of clot-associated catalysts to restart a coagulation process, suggesting that the enhanced anti-FIXa activity of fondaparinux-AT may be critical to its success in acute settings in vivo.
Subject(s)
Anticoagulants/therapeutic use , Blood Coagulation/drug effects , Factor Xa Inhibitors , Computer Simulation , Factor V/chemistry , Factor Xa/chemistry , Fondaparinux , Humans , Morpholines/therapeutic use , Polysaccharides/therapeutic use , Recombinant Proteins/therapeutic use , Rivaroxaban , Software , Thiophenes/therapeutic use , Thrombin/biosynthesis , Time Factors , Treatment OutcomeABSTRACT
We have developed an integrated approach that combines empirical and computational methodologies to define an individual's thrombin phenotype. We have evaluated the process of thrombin generation in healthy individuals and individuals with defined pathologies in order to develop general criteria relevant to assess an individual's propensity for hemorrhage or thrombosis. Three complementary hypotheses have emerged from our work: (i) compensation by the ensemble of other coagulation proteins in individuals with specific factor deficiencies can 'normalize' an individual's thrombin generation process and represents a rationale for their unexpected phenotype; (ii) individuals with clinically unremarkable factor levels may present thrombin generation profiles typical of individuals with hemostatic complications; and (iii) in some hemostatic disorders a specific pattern of expression of a small ensemble of coagulation factors may be sufficient to explain the overall phenotype.
Subject(s)
Empirical Research , Models, Theoretical , Thrombin/physiology , Computational Biology , Hemorrhage , Hemostasis , Humans , Phenotype , Thrombin/biosynthesis , ThrombosisABSTRACT
Our studies involve computational simulations, a reconstructed plasma/platelet proteome, whole blood in vitro and blood exuding from microvascular wounds. All studies indicate that in normal haemostasis, the binding of tissue factor (TF) with plasma factor (F) VIIa (extrinsic FXase complex) results in the initiation phase of the procoagulant response. This phase is negatively regulated by tissue factor pathway inhibitor (TFPI) in combination with antithrombin (AT) and the protein C (PC) pathway. The synergy between these inhibitors provides a threshold-limited reaction in which a stimulus of sufficient magnitude must be provided for continuation of the reaction. With sufficient stimulus, the FXa produced activates some prothrombin. This initial thrombin activates the procofactors and platelets required for presentation of the intrinsic FXase (FVIIIa-FIXa) and prothrombinase (FVa-FXa) complexes which drive the subsequent propagation phase; continuous downregulation of which is provided by AT and the thrombin-thrombomodulin-PC complex. FXa generation during the propagation phase is largely (>90%) provided by the intrinsic FXase complex. TF is required for the initiation phase of the reaction but becomes non-essential once the propagation phase has been achieved. The propagation phase catalysts (FVIIIa-FIXa and FVa-FXa) continue to drive the reaction as blood is resupplied to the wound site by flow. Ultimately, the control of the reaction is governed by the pro- and anticoagulant dynamics and the supply of blood reactants to the site of a perforating injury. Our systems have been utilized to examine the qualities of hypothetical and novel antihaemorrhagic and anticoagulation agents and in epidemiologic studies of venous and arterial thrombosis and haemorrhagic pathology.
Subject(s)
Blood Coagulation/physiology , Hemostasis/physiology , Blood Coagulation Factors/metabolism , Blood Proteins/metabolism , Fibrinogen/metabolism , Humans , Kinetics , Models, Biological , Proteome , Thrombin/metabolismABSTRACT
BACKGROUND: Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). METHODS AND RESULTS: Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age- and sex-matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual's coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. CONCLUSION: Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD.
Subject(s)
Acute Coronary Syndrome/blood , Blood Coagulation Factors/analysis , Coronary Artery Disease/blood , Thrombin/biosynthesis , Acute Coronary Syndrome/diagnosis , Antithrombin III , Coronary Artery Disease/diagnosis , Diagnosis, Differential , Factor VIII , Humans , Models, Statistical , ProthrombinABSTRACT
BACKGROUND: Sodium citrate has been used as an anticoagulant to stabilize blood and blood products for over 100 years, presumably by sequestering Ca(++) ions in vitro. Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor (CTI). OBJECTIVE: To evaluate the influence of citrate anticoagulation on the performance of blood, platelet-rich and platelet-poor plasma assays. METHODS: Blood was anticoagulated in three ways: by collection into citrate, CTI and citrate with CTI. Plasma was prepared using each anticoagulation regimen. Functional analyses included calibrated automated thrombography, thromboelastography, plasma clotting, the synthetic coagulation proteome and platelet aggregation. Coagulation reactions were initiated with tissue factor-phospholipid and Ca(++) (when indicated). RESULTS: In all cases, citrate anticoagulation resulted in reaction dynamics significantly altered relative to blood or plasma stabilized with CTI alone. Subsequent experiments showed that calcium citrate itself impairs coagulation dynamics. CONCLUSION: Coagulation analyses using blood that has been exposed to citrate and recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes.
Subject(s)
Anticoagulants/therapeutic use , Citrates/therapeutic use , Thrombin/chemistry , Blood Coagulation , Blood Coagulation Tests , Calcium/metabolism , Calcium Citrate/pharmacology , Calibration , Humans , Ions , Platelet Activation , Platelet Aggregation , Thrombelastography/methods , Thrombin/metabolism , Thrombin TimeABSTRACT
BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.
Subject(s)
Activated Protein C Resistance/drug therapy , Blood Coagulation/drug effects , Enzyme Inhibitors/chemistry , Hemophilia A/drug therapy , Protein C/antagonists & inhibitors , Activated Protein C Resistance/enzymology , Blood Coagulation Tests/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Factor V/metabolism , Factor Va/metabolism , Factor Xa/metabolism , Hemophilia A/enzymology , Humans , Models, Biological , Protein C/chemistry , Protein C/metabolism , Proteome/chemistry , Proteome/metabolismABSTRACT
We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).
Subject(s)
Factor Va/antagonists & inhibitors , Homocysteine/pharmacology , Protein C/antagonists & inhibitors , Blotting, Western , Humans , Phospholipids/metabolism , Protein C/metabolismABSTRACT
The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.
Subject(s)
Acanthamoeba/drug effects , Antiprotozoal Agents/pharmacology , Distamycins/pharmacology , Genes, Protozoan/drug effects , RNA, Protozoan/biosynthesis , Acanthamoeba/genetics , Acanthamoeba/growth & development , Animals , Cellulose/metabolism , Cytoskeletal Proteins/genetics , DNA-Binding Proteins/genetics , Protein Binding/drug effects , RNA, Messenger/metabolism , TATA-Box Binding Protein , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effectsABSTRACT
The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.
Subject(s)
Acanthamoeba/genetics , RNA Polymerase II , Acanthamoeba/cytology , Acanthamoeba/growth & development , Animals , Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Genes, Protozoan/genetics , Protozoan Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , Transcription, GeneticABSTRACT
alpha-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by alpha-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, alpha-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L alpha-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of alpha-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that alpha-thrombin may regulate repair of the cerebral microvascular system.
Subject(s)
Brain/blood supply , DNA/biosynthesis , Endothelium, Vascular/metabolism , Microcirculation/metabolism , Thrombin/pharmacology , Urokinase-Type Plasminogen Activator/biosynthesis , Blotting, Northern , Cells, Cultured , Endothelium, Vascular/drug effects , Humans , Immunoblotting , Male , Receptors, Thrombin/physiology , Umbilical VeinsABSTRACT
Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.
Subject(s)
Blood Platelets/metabolism , Bone and Bones/metabolism , Collagen/metabolism , Osteonectin/metabolism , Plasminogen/metabolism , Aminocaproic Acid/pharmacology , Binding, Competitive , Collagen/chemistry , Enzyme-Linked Immunosorbent Assay , Humans , Kinetics , Osteonectin/chemistry , Plasminogen/chemistry , Protein Binding , Spectrometry, Fluorescence , Structure-Activity Relationship , Tissue Plasminogen Activator/metabolism , alpha-2-Antiplasmin/pharmacologyABSTRACT
The delivery of a blow to the head represents a transfer of energy, part of which manifests itself as a short-lived pressure change within the skull. An in vitro model was developed to test whether cerebral endothelial cell hemostatic function is altered with exposure to this type of pressure event. Human cerebral microvascular endothelium (HCME) cells were subjected to rapid (2-5 msec) changes in pressure (delta atmosphere = 1.2-10), the sublethal range defined (delta atmosphere < or = 6.5), and the nonthrombogenic status of sublethally percussed HCME cells assessed using the adherence of alpha-thrombin activated platelets as an indicator. The HCME cells had lost their normal capacity to suppress adherence of activated platelets when evaluated 1 hour or 24 hours after percussion. Adherence of activated platelets to percussed HCME cells was blocked by the addition of PGI2, an inhibitor of platelet adherence, when evaluated at 1 hour but not 24 hours after percussion, indicating that percussed HCME cells were undergoing further derangement of their nonthrombogenic mechanisms. Percussed HCME cells cultured for 24 hours in medium containing scavengers of oxygen free radicals recovered their capacity to block platelet adherence. We conclude that sublethal percussion immediately compromises the nonthrombogenic character of HCME cells and initiates the development of a persisting prothrombotic state in HCME cells. This derangement appears linked to increased production of reactive oxygen species by percussed HCME cells.
Subject(s)
Brain Injuries/physiopathology , Brain/blood supply , Endothelium, Vascular/physiopathology , Hemostasis , Platelet Adhesiveness , Endothelium, Vascular/cytology , Epoprostenol/pharmacology , Free Radical Scavengers , Humans , Platelet Adhesiveness/drug effects , Platelet Adhesiveness/physiology , Pressure , Reactive Oxygen Species/metabolismABSTRACT
A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.
Subject(s)
Fluorescent Dyes , Naphthalenesulfonates/chemistry , Serine Endopeptidases/metabolism , Chemical Phenomena , Chemistry, Physical , Enzyme Activation , Factor IX/metabolism , Factor VIIa/metabolism , Naphthalenesulfonates/metabolism , Protein C/metabolism , Structure-Activity Relationship , Substrate Specificity , Thrombin/metabolism , Thromboplastin/metabolismABSTRACT
Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.
Subject(s)
Endothelium, Vascular/metabolism , Fibrinolysis , Oxygen/metabolism , Antigens/biosynthesis , Blotting, Northern , Blotting, Western , Cells, Cultured , Endothelium, Vascular/cytology , Free Radicals , Humans , Hydrogen Peroxide , Plasminogen Inactivators/metabolism , Precipitin Tests , RNA, Messenger/metabolism , Tissue Plasminogen Activator/metabolism , Umbilical Veins , Xanthine , Xanthine Oxidase/metabolism , Xanthines/metabolismABSTRACT
Cardiovascular and peripheral adaptations to an aerobic conditioning program were studied in 30 hypertensive adults taking either placebo, beta 1-selective beta-adrenergic blocker (metoprolol) or beta 1-nonselective beta-adrenergic blocker (propranolol). The placebo group increased aerobic capacity (VO2max) 24% (p less than 0.002), largely explained by an increased peripheral arteriovenous (AV) oxygen difference with minimal changes in cardiac size and function. Resting blood pressure and total systemic resistance also decreased. The group taking a beta 1-selective beta blocker increased VO2max 8% (p less than 0.05), reduced resting blood pressure but had no significant change of AV oxygen difference or cardiac size or function. The group taking the beta 1-nonselective beta blocker propranolol had no increase in VO2max, no decrease in resting blood pressure and no cardiovascular or peripheral adaptations to the exercise program. Thus, beta 1-selective and beta 1-nonselective beta blockers attenuate conditioning in hypertensive patients to differing degrees, in each case by blocking peripheral mechanisms of conditioning.