Pulmonary function and health-related quality of life after COVID-19 pneumonia.

BACKGROUND: The COVID-19 pandemic has led to many cases of pneumonia with extensive lung abnormalities on CT-scans. The consequences of COVID-19 pneumonia on survivors' pulmonary function and quality of life are unknown. The purpose of this study is to examine the impact of COVID-19 pneumonia on pulmonary function, health-related quality of life (HRQoL) and perceived dyspnoea. METHODS: A prospective longitudinal cohort study regarding patients discharged from our hospital after PCR-proven, non-critical COVID-19 pneumonia was conducted. Cases were classified as moderate or severe pneumonia according to WHO definitions. Six weeks post-discharge subjects underwent interviews and pulmonary function tests, and completed questionnaires to assess their HRQoL, perceived dyspnoea (Borgscale and mMRC), and symptoms of depression and anxiety (HADS). RESULTS: 101 patients were included. Twenty-eight (27.7%) pneumonias were classified as moderate cases of COVID-19 pneumonia and 73 (72.3%) were classified as severe cases. Diffusion limitation (DLCOc < 80% of predicted value) was found in 66 (71.7%) of 92 cases, obstruction in 26 (25.7%) of 101, and restriction in 21 (21.2%) of 99. Diffusion capacity was significantly lower in cases after severe pneumonia. In the entire group, HADS scores ≥8 for depression were found in 16.6% and in 12.5% for anxiety. Across all SF-36 domains, except for bodily pain, significant impairment was found. FEV1 and DLCOc showed significant positive correlations with mMRC scores and multiple SF-36 domains, especially physical functioning. CONCLUSION: COVID-19 non-critical pneumonia survivors have significant impairment in diffusion capacity and HRQOL six weeks after being discharged from hospital.

Treatment selection of early stage non-small cell lung cancer: the role of the patient in clinical decision making.

BACKGROUND: The objective of this study is to investigate the role and experience of early stage non-small cell lung cancer (NSCLC) patient in decision making process concerning treatment selection in the current clinical practice. METHODS: Stage I-II NSCLC patients (surgery 55 patients, SBRT 29 patients, median age 68) were included in this prospective study and completed a questionnaire that explored: (1) perceived patient knowledge of the advantages and disadvantages of the treatment options, (2) experience with current clinical decision making, and (3) the information that the patient reported to have received from their treating physician. This was assessed by multiple-choice, 1-5 Likert Scale, and open questions. The Decisional Conflict Scale was used to assess the decisional conflict. Health related quality of life (HRQoL) was measured with SF-36 questionnaire. RESULTS: In 19% of patients, there was self-reported perceived lack of knowledge about the advantages and disadvantages of the treatment options. Seventy-four percent of patients felt that they were sufficiently involved in decision-making by their physician, and 81% found it important to be involved in decision making. Forty percent experienced decisional conflict, and one-in-five patients to such an extent that it made them feel unsure about the decision. Subscores with regard to feeling uninformed and on uncertainty, contributed the most to decisional conflict, as 36% felt uninformed and 17% of patients were not satisfied with their decision. HRQoL was not influenced by patient experience with decision-making or patient preferences for shared decision making. CONCLUSIONS: Dutch early-stage NSCLC patients find it important to be involved in treatment decision making. Yet a substantial proportion experiences decisional conflict and feels uninformed. Better patient information and/or involvement in treatment-decision-making is needed in order to improve patient knowledge and hopefully reduce decisional conflict.

Reliability and validity of the Cancer Therapy Satisfaction Questionnaire in lung cancer.

PURPOSE: To test the reliability and validity of the Cancer Treatment Satisfaction Questionnaire (CTSQ), to assess its relation with quality of life (QoL), and to assess the interpretability of the domain scores in lung cancer patients receiving intravenous chemotherapy. METHODS: Patients with stage IIIB and IV non-squamous non-small cell lung carcinoma treated with pemetrexed were enrolled in our study. They completed the 16-item CTSQ and two other (health-related) QoL questionnaires. Information about sociodemographic characteristics, cancer stage, and the experience of adverse events was collected. Internal consistency, construct validity, and clinical interpretability were calculated. RESULTS: Fifty-five patients completed the CTSQ. Correlations of the CTSQ items with its domain were all above 0.40. A high correlation between item 8 and the expectations of therapy and satisfaction with therapy domain was observed (0.50 and 0.48, respectively). The CTSQ domains demonstrated good internal consistency and low to moderate correlations of the CTSQ with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 and World Health Organization Quality of Life-BREF. No significant differences in mean domain scores were observed in relation to the number and severity of different adverse events and chemotherapy-related adverse events. CONCLUSIONS: The Dutch version of the CTSQ was found to be a reliable and valid instrument to assess satisfaction and expectations of treatment in lung cancer patients receiving intravenous chemotherapy. Furthermore, the CTSQ proved to be of additional informative value as not all of its domains correlated with the various domains of the existing HRQoL instruments.

Targeted deletion of the cytosolic domain of tissue factor in mice does not affect development.

The role of the cytosolic domain of tissue factor (TF) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neo(R) selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neo(R) cassette by in vivo Cre-mediated loxP recombination yielded TF(+/deltaCT) and TF(deltaCT/deltaCT) mice. In contrast to TF(-/-) mice, TF(+/deltaCT) and TF(deltaCT/deltaCT) mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF(+/+) and TF(deltaCT/deltaCT) fibroblasts. These data, based on expression of a TF(deltaCT) molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.

Depletion of circulating alpha(2)-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization.

In the absence of arterial recanalization, thrombolytic agents induce a dose-related extension of focal cerebral ischemic injury (FII) in experimental animals. However, FII is smaller in mice lacking alpha(2)-antiplasmin (alpha(2)-AP), the physiologic inhibitor of plasmin, suggesting its depletion might reduce FII in the absence of reperfusion. Therefore, the effect of human plasmin (Pli), human miniplasmin (mPli), and an Fab fragment neutralizing murine alpha(2)-AP (Fab-4H9) on FII after middle cerebral artery (MCA) ligation was studied in mice and in hamsters. In BALB/c mice, the median FII after 24 hours was 28 microL (range, 20-34) (n = 10) with saline and 23 microL (range, 17-26) (n = 9) with a single bolus of 0.07 mg Pli, given after MCA ligation (P =.010), which reduced alpha(2)-AP to 44% and fibrinogen from 0.75 to 0.44 g/L. FII was 20 microL (range, 13-26) (n = 6, P =.025) with 0.2 mg mPli and was 24 microL (range, 20-27) (n = 6, P =.020) with 1.7 mg Fab-4H9. Neuronal atrophy and reduction of laminin immunoreactivity were comparably observed in the infarct area after saline and Pli. In hamsters, a single bolus injection of 1 mg Pli, after MCA ligation, depleted alpha(2)-AP and fibrinogen and reduced FII at 24 hours from 20 microL (range, 9.9-38) (n = 6) to 7.0 microL (range, 0.44-31) (n = 7, P =.032). Thus, reduction of circulating alpha(2)-AP, with a single bolus of plasmin or of a neutralizing antibody fragment, significantly reduced FII after MCA ligation in mouse and hamster models, suggesting that, provided these observations can be extrapolated to human beings, transient depletion of circulating alpha(2)-AP might reduce ischemic stroke in the absence of reperfusion.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice.

BACKGROUND: The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or alpha2-antiplasmin (PAI-1(-/-) or alpha2-AP-/-). METHODS AND RESULTS: FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6+/-1.1 mm3 (mean+/-SEM), uPA-/- mice had similar infarcts (7.8+/-1.0 mm3, P=NS), tPA-/- mice smaller (2.6+/-0.80 mm3, P<0.0001), PAI-1(-/-) mice larger (16+/-0.52 mm3, P<0.0001), and Plg-/- mice larger (12+/-1.2 mm3, P=0.037) infarcts. alpha2-AP-/- mice had smaller infarcts (2. 2+/-1.1 mm3, P<0.0001 versus wild-type), which increased to 13+/-2.5 mm3 (P<0.005 versus alpha2-AP-/-) after intravenous injection of human alpha2-AP. Injection into alpha2-AP-/- mice of Fab fragments of affinospecific rabbit IgG against human alpha2-AP, after injection of 200 microg human alpha2-AP, reduced FCI from 11+/-1.5 to 5.1+/-1.1 mm3 (P=0.004). CONCLUSIONS: Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity (tPA gene inactivation) reduces whereas its augmentation (PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity (Plg gene inactivation or alpha2-AP injection) increases whereas its augmentation (alpha2-AP gene inactivation or alpha2-AP neutralization) reduces infarct size. Inhibition of alpha2-AP may constitute a potential avenue to treatment of FCI.

Vascular wound healing and neointima formation induced by perivascular electric injury in mice.

Vascular interventions for atherothrombotic disease frequently induce neointima formation, which can contribute to restenosis of blood vessels. As the molecular mechanisms of this process remain largely unknown, quantitative models of arterial injury in transgenic animals may be useful to study this process at the genetic level. Here, an injury model is proposed in which surgically exposed femoral arteries in mice were injured perivascularly via a single delivery of an electric current. Transmission electron microscopy, light microscopy, and immunohistochemistry revealed that electric injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and transiently induced platelet-rich mural thrombosis. A vascular wound-healing response resulted that was characterized by degradation of the mural thrombus, transient infiltration of the vessel wall by inflammatory cells, and progressive removal of the necrotic debris. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the uninjured borders and progressing into the necrotic center. Within 3 weeks after injury, a neointima of 0.026 +/- 0.003 mm2 (n = 7 arteries) was formed that contained a maximum of 12 +/- 1 layers of smooth muscle alpha-actin-immunoreactive cells. Evans blue staining in five electrically injured arteries revealed a denuded distance of 2.8 +/- 0.2 mm immediately after injury, which became progressively re-endothelialized from the uninjured borders to 2.2 +/- 0.08 mm (P = 0.013 vs freshly injured by analysis of variance), 0.8 +/- 0.22 mm (P < 0.001), and 0.005 +/- 0.003 mm (P < 0.001) within 2, 7, and 14 days after injury, respectively. Analysis of 5'-bromo-2'-deoxyuridine incorporation revealed that a maximum of 35 +/- 10% endothelial cells proliferated within 2 days after injury and that in the media and neointima, a maximum of, respectively, 12 +/- 2% and 18 +/- 3% smooth muscle cells proliferated within 2 weeks after injury. Thus, electric injury of arteries provides a model of vascular wound healing with arterial neointima formation and re-endothelialization that may be useful for the genetic analysis of its molecular mechanisms in transgenic mice.

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.

Reactivated recombinant plasminogen activator inhibitor-1 (rPAI-1) effectively prevents thrombolysis in vivo.

The effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (approximately 2 units of t-PA neutralizing activity per micrograms protein) and reactivated rPAI-1 (approximately 150 units/micrograms). Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity approximately 0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.

Studies of recombinant plasminogen activator inhibitor-1 in rabbits. Pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo.

The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.

Recombinant plasminogen activator inhibitor-1 reverses the bleeding tendency associated with the combined administration of tissue-type plasminogen activator and aspirin in rabbits.

The major side effect of thrombolytic therapy is bleeding; however, the pathogenesis of this potential complication is not well understood. Accordingly, we examined the effects of aspirin and recombinant human tissue-type plasminogen activator (rt-PA) on serial template bleeding times and on hemostasis parameters in rabbits. The administration of intravenous aspirin (15 mg/kg) produced a slight prolongation in bleeding times, from 2.1 +/- 0.5 to 2.6 +/- 0.5 min (mean +/- SD, n = 26, P less than 0.01), whereas rt-PA (1 mg/kg per h for 2 h) lengthened the bleeding time from 2.4 +/- 0.3 to 3.2 +/- 0.6 min (n = 5, P = NS). Combination of aspirin with 0.5 mg/kg per h of rt-PA for 2 h prolonged the bleeding time from 2.5 +/- 0.4 to 6.2 +/- 0.9 min (n = 10, P less than 0.01), with an associated fibrinogen decrease of approximately 15%. The combination of aspirin with 1 mg/kg per h of rt-PA for 2 h prolonged the bleeding time from 3.0 +/- 0.3 to 8.3 +/- 1.4 min (n = 8, P less than 0.01) and simultaneously induced a decrease of plasma fibrinogen by approximately 40%. Virtually all animals treated with rt-PA and aspirin manifested a bleeding tendency, as evidenced by spontaneous rebleeding at sites of previously performed template bleeding times or oozing at the femoral venous catheterization site. Intravenous bolus injection of 1 mg/kg of guanidine hydrochloride-reactivated recombinant human plasminogen activator inhibitor-1 (rPAI-1) at the end of the rt-PA infusion resulted in complete reversal, within 5 min, of the prolongation of the bleeding time, and in a disappearance of the bleeding tendency. Nonreactivated rPAI-1 and tranexamic acid were significantly less potent in reversing the bleeding time prolongation. These findings indicate that aspirin and rt-PA given separately do not markedly affect the template bleeding time, but in combination induce a marked prolongation associated with a significant bleeding tendency. This bleeding time prolongation can be rapidly normalized by the administration of reactivated rPAI-1.

Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin).

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1).

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.

Fibrinolytic response and fibrin fragment D-dimer levels in patients with deep vein thrombosis.

An enzyme-linked immunosorbent assay for fragment D-dimer was developed with the use of two monoclonal antibodies directed against specific non-overlapping antigenic determinants, present in fragment D-dimer of crosslinked fibrin but not in fragment D of non crosslinked fibrin or of fibrinogen. The lower limit of sensitivity of the assay when applied to human plasma, is 25 ng/ml. Concentration of fragment D-dimer in plasma from healthy individuals was 177 +/- 83 ng/ml (mean +/- SD). In plasma of 11 out of 12 patients with phlebographically confirmed acute deep vein thrombosis, fragment D-dimer levels were significantly increased. Fragment D-dimer was not increased in 9 out of 10 patients with recurrent idiopathic deep vein thrombosis during clinically silent episodes. Total t-PA antigen and free t-PA antigen concentrations were measured using previously developed ELISAs. Nine of the 12 patients with acute deep vein thrombosis showed a significant increase of total t-PA antigen (from 8.6 +/- 6.9 ng/ml to 21 +/- 16 ng/ml) after venous occlusion but in 3 of these free t-PA remained undetectable. Five of the 10 patients with recurrent deep vein thrombosis responded to venous occlusion with a significant increase of total t-PA antigen (from 6.7 +/- 3.2 ng/ml to 14 +/- 7.9 ng/ml) but, in all patients, free t-PA antigen remained undetectable. It is concluded that the combined assays of total and free t-PA antigen and of fragment D-dimer may be useful for the evaluation of the dynamics of the fibrinolytic system in physiological and pathological conditions.

Covalent complexes between low molecular weight heparin fragments and antithrombin III - inhibition kinetics and turnover parameters.

Two high affinity heparin fragments (Mr 4,300 and Mr 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401--3408) with an apparent 1:1 stoichiometry and a 30--35% yield. The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin. The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t1/2) of both low Mr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 congruent to 7.7 hr), approaching that of free antithrombin III (t1/2 congruent to 11 +/- 0.4 hr) and resulting in a 30 fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 congruent to 0.25 +/- 0.04 hr).