OBJECTIVE: To explore the predictive value of four items of new thrombus markers combined with conventional coagulation tests for thrombosis in antiphospholipid syndrome. METHODS: A total of 121 antiphospholipid syndrome (APS) patients who hospitalized at Peking University People's Hospital from March 2022 to January 2023 were selected and divided into thrombus group (50 cases) and nonthrombus group (71 cases) according to whether thrombosis occurred. The differences of laboratory characteristics including antiphospholipid antibodies were compared between the thrombotic and non-thrombotic groups. Chemiluminescent immunoassay was used to detect thrombomodulin (TM), thrombin-antithrombin complex (TAT), Plasmin-α2 plasmin inhibitor complex (PIC), and tissue plasminogen activator inhibitor complex (t-PAIC) in plasma from venous. The independent risk factors of thrombosis in patients with APS were determined using binary Logistic regression. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the efficacy of each index on the prediction of thrombosis. RESULTS: Compared with the patients without thrombosis, the patients with thrombosis were older [49 (32, 64) years vs. 36 (32, 39) years, P < 0.05]. The percentages of male, smoking, hypertension, and global antiphospholipid syndrome score (GAPSS)≥10 in the patients with thrombosis were significantly higher than those in the patients without thrombosis (P < 0.05). The positive rates of anticardiolipin antibody (aCL) and lupus anticoagulant (LA) in the thrombotic group were significantly higher than those in the non-thrombotic group (P < 0.05), and the levels of prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin degradation product in the thrombotic group were significantly higher than those in the non-thrombotic group (P < 0.05).Among the thrombosis group, venous thrombosis accounted for 19 (38.00%), including deep vein thrombosis (16, 84.21%) and pulmonary embolism accounted (5, 26.32%); Arterial thrombosis accounted for 35 (70.00%), including myocardial infarction (6, 17.14%) cerebral infarction (30, 85.71%). The patients in the thrombotic group had significantly greater TM levels than those in the non-thrombotic group (P < 0.05).There were no significant dif-ferences between the two groups in TAT (Z=-1.420, P=0.156), PIC (Z=-0.064, P=0.949), and t-PAIC (Z=-1.487, P=0.137). Univariate and binary Logistic regression analysis of relevant variables showed that advanced age [OR=1.126, P=0.002], elevated TM [OR=1.325, P=0.048], prolonged prothrombin time (PT) [OR=4.127, P=0.008] were independent risk factors for thrombosis in the patients with APS. ROC curve analysis of the above three independent risk factors showed that the combined detection of age, PT and TM had the highest Yoden index (0.727) and sensitivity (83.0%), with a specificity of 89.7%. CONCLUSION TAT, PIC, TM, and t-PAIC may reflect thrombus formation from the coagulation system, fibrinolysis system, and endothelial system. The combined of age TM and PT is superior to the application of a single marker, which has diagnostic value for the early identification of APS thrombosis.
Humans ; Male ; Antiphospholipid Syndrome/diagnosis* ; Tissue Plasminogen Activator ; Thrombosis/etiology* ; Antibodies, Antiphospholipid/analysis* ; Blood Coagulation Tests/adverse effects*

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.
Amiloride/*pharmacology ; Analysis of Variance ; Animals ; Arginine/toxicity ; Cell Death/drug effects ; Cells, Cultured ; Cyclic GMP/pharmacology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Retina/cytology/*drug effects ; Tissue Plasminogen Activator/*toxicity

OBJECTIVE: Leukoaraiosis (LA) has been suggested to be related to the poor outcome or the occurrence of symptomatic intracerebral hemorrhage (sICH) after acute ischemic stroke. We retrospectively investigated the influences of LA on long-term outcome and the occurrence of sICH after thrombolysis in acute ischemic stroke (AIS). METHODS: In this study, we recruited 164 patients with AIS and magnetic resonance image (MRI)-detected thrombolysis. The presence and extent of LA were assessed using the Fazekas grading system. The National Institutes of Health Stroke Scale score was used to assess the baseline measure of neurologic severity, and the modified Rankin Scale score assessment was used up to 1 year after thrombolysis. RESULTS: Of 164 subjects, 56 (34.2%) showed LA on MRI. Compared to the 108 patients without LA, the patients with LA were of much older age (p<0.01), had a higher prevalence of hypertension (p<0.01), and had a much poorer outcome at 90 days (p=0.05) and 1 yr (p=0.01) after thrombolysis. There were no significant differences in sICH between patients with and without LA on MRI. In univariate analysis for the occurrence of poor outcome at 90 days after thrombolysis, the size of ischemic lesion on diffusion weighted images (DWI), [odds ratio (OR), 1.03; 95% confidence interval (95% CI), 1.01-1.04; p<0.01], recanalization (OR, 0.03; 95% CI, 0.01-0.10; p<0.01), sICH (OR, 12.2; 95% CI, 1.54-95.8), neurologic severity (OR, 1.17; 95% CI, 1.09-1.25; p<0.01), blood glucose level (OR, 1.01; 95% CI, 1.00-1.02; p=0.03), and the presence of LA on MRI (OR, 2.01; 95% CI, 1.04-3.01; p=0.04) were statistically significant. In multivariate analysis, neurologic severity (OR, 1.14; 95% CI, 1.04-1.24; p<0.01), recanalization (OR, 0.03; 95% CI, 0.01-0.11; p<0.01), lesion size on DWI (OR, 1.02; 95% CI, 1.01-1.03; p=0.02), serum glucose level (OR, 1.01; 95% CI; 1.01-1.02; p=0.03), and the presence of LA on MRI (OR, 3.2; 95% CI, 1.22-8.48; p<0.01) showed statistically significant differences. These trends persisted up to 1 yr after thrombolysis. CONCLUSION: In this study, we demonstrated that the presence of LA on MRI might be related to poor outcome after use of intravenous tissue plasminogen activator in AIS.
Blood Glucose ; Cerebral Hemorrhage ; Diffusion ; Glucose ; Humans ; Hypertension ; Leukoaraiosis ; Magnetic Resonance Imaging ; Magnetic Resonance Spectroscopy ; Magnetics ; Magnets ; Multivariate Analysis ; National Institutes of Health (U.S.) ; Prevalence ; Retrospective Studies ; Stroke ; Tissue Plasminogen Activator

To investigate the effects of reactive oxygen species (ROS) on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) plasma levels, and their possible implications on clinical outcome, we measured tPA and PAI-1 levels in 101 patients with acute paraquat (PQ) intoxication. The control group consisted of patients who ingested non-PQ pesticides during the same period. tPA and PAI-1 levels were higher in the PQ group than in the controls. PQ levels were significantly correlated with ingested amount, timelag to hospital, tPA level, and hospitalization duration. tPA levels were correlated with PAI-1, fibrin degradation product (FDP), and D-dimer. D-dimer levels were lower in the PQ group than in the controls. Univariate analysis indicated the following significant determinants of death: age, ingested amount, PQ level, timelag to hospital, serum creatinine, lipase, pH, pCO2, HCO3-, WBC, FDP, PAI-1, and tPA. However, multivariate analysis indicated that only PQ level was significant independent factor predicting death. In conclusion, tPA and PAI-1 levels were higher, while D-dimer levels were lower in the PQ group than in the controls, implying that ROS stimulate tPA and PAI-1, but PAI-1 activity overrides tPA activity in this setting. Decreased fibrinolytic activity appears to be one of the clinical characteristics of acute PQ intoxication.
Acute Disease ; Adult ; Aged ; Female ; Fibrin Fibrinogen Degradation Products/analysis ; Herbicides/blood/*poisoning ; Humans ; Male ; Middle Aged ; Paraquat/blood/*poisoning ; Plasminogen Activator Inhibitor 1/*blood ; Reactive Oxygen Species/metabolism ; Risk Factors ; Tissue Plasminogen Activator/*blood ; Tomography, X-Ray Computed

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange ; methods ; Disulfides ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Protein Refolding ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Tissue Plasminogen Activator ; analysis ; chemistry

BACKGROUNDCigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract (CSE) on fibrinolytic activity of human umbilical vein endothelial cells (HUVECs). Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well.

METHODSThe fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAI-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAI-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAI-1 mRNA and protein.

RESULTSAfter 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly ((0.0365 +/- 0.0083) ng/ml, (0.0255 +/- 0.0087) ng/ml) when compared with that of control group ((0.0660 +/- 0.0120) ng/ml) (P < 0.05). In contrast, the levels of PAI-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably ((13.3225 +/- 0.5680) ng/ml, (14.2675 +/- 1.5380) ng/ml, (14.4292 +/- 1.6230) ng/ml) when compared with that of control group ((8.5193 +/- 0.7537) ng/ml) (P < 0.05). After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAI-1 protein increased over time and reached the peak at 24 hours ((14.6400 +/- 1.0651) ng/ml), which was significantly higher than that of control group ((12.0656 +/- 0.6148) ng/ml) (P < 0.05). Additionally, CSE could up-regulate PAI-1 expression at both the mRNA and the protein levels. The levels of PAI-1 mRNA and protein increased significantly in 5% CSE-treated group ((8.8030 +/- 0.4745) ng/ml, (1.8155 +/- 0.0412) ng/ml) compared with those of control groups ((5.0588 +/- 0.2315) ng/ml, (1.3030 +/- 0.0647) ng/ml) (P < 0.01), and decreased after 2-hour simvastatin pre-treatment ((5.4875 +/- 0.3166) ng/ml, (1.3975 +/- 0.0297) ng/ml) (P < 0.01). No significant difference was found at the levels of t-PA protein and mRNA (P > 0.05).

CONCLUSIONSCSE inhibits the fibrinolytic activity of HUVECs in vitro. Simvastatin plays a protective role in CSE-induced fibrinolytic malfunction.

Cells, Cultured ; Endothelial Cells ; metabolism ; Fibrinolysis ; drug effects ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Plasminogen Activator Inhibitor 1 ; analysis ; biosynthesis ; genetics ; Simvastatin ; pharmacology ; Smoke ; adverse effects ; Tissue Plasminogen Activator ; analysis ; biosynthesis ; genetics ; Tobacco ; adverse effects ; Umbilical Veins ; cytology

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.
Amino Acid Sequence ; Chromatography, Liquid ; methods ; Chymotrypsin ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Molecular Sequence Data ; Molecular Weight ; Peptide Fragments ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods ; Tandem Mass Spectrometry ; methods ; Tissue Plasminogen Activator ; analysis ; chemistry ; Trypsin ; chemistry

OBJECTIVEThe mechanism of neonatal hypoxic-ischemic brain damage (HIBD) remains unclear and effective treatment approach is limited for this disorder. Many studies have shown that tissue-type plasminogen activator (tPA) plays an important role in nervous system. This study investigated the effect of tPA in HIBD in neonatal rats.

METHODSSeven-day-old Wistar rat pups were used for the Rice-Vannucci model of neonatal hypoxia-ischemia (HI). Brain samples were collected 1, 4, and 24 hrs after HI. FITC-Dextran was injected into the left ventricle of pups after HI to observe reperfusion defects of the neonatal brain. RT-PCR and tPA zymogram were used to detect the expression and activity of tPA. Double immunostaining, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DAPI staining were used to detect the expression of integrin GPIIb and fibrin and neuronal apoptosis.

RESULTSFITC-Dextran perfusion analysis indicated there was obvious infarct area in the neonatal brain and the expression of integrin GPIIb and fibrin increased significantly 1 hr after HI compared with the contralateral side. The infarct area decreased and the expression of integrin GPIIb and fibrin were reduced 4 hrs after HI. The expression and activity of tPA increased significantly in neonatal rats after HI, and peaked at 4 hrs after HI. The number of apoptotic neural cells increased progressively with the prolonged reperfusion time following HI.

CONCLUSIONSThe increase of tPA in the acute phase after HIBD may be helpful to clot dissolving, but it induces neuronal apoptosis and aggravates brain injury.

Animals ; Animals, Newborn ; Apoptosis ; Fibrin ; analysis ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; Immunohistochemistry ; In Situ Nick-End Labeling ; Platelet Membrane Glycoprotein IIb ; analysis ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Plasminogen Activator ; analysis ; physiology

BACKGROUND: The intravenous thrombolysis is a well established treatment of acute ischemic stroke. However, baseline prognostic factors were poorly identified by previous studies. METHODS: From January 2001 to May 2006, prospective data of 121 patients treated with intravenous tissue plasminogen activator (tPA) were collected. The clinical, radiologic, transcranial Dopper (TCD) and laboratory finding were evaluated. Clinical assessment was done by National Institutes of Health Stroke Scale (NIHSS) for one week, and by modified Rankin Scale (mRS) at baseline for three months. Early improvement was defined as the complete resolution of the neurological deficit or an improvement of > or =4 points by NIHSS within 24 hours, and good outcome as mRS score of < or =2 at three months. We assessed the possible relationship of the factors with early improvement and good outcome, and also analyzed the correlation of TCD grade with NIHSS score. RESULTS: On univariate analysis, younger age, absence of abnormal CT findings (hyperdense middle cerebral artery sign [HMCAS], focal hypodensity >33% of total MCA territory) were significantly associated with early improvement. Good outcome was associated with younger age, lower levels of baseline NIHSS score, mean blood pressure, fasting glucose, lipoprotein (a), and normal CT finding. Multivariate analysis revealed age <66 years and no HMCAS as independent predictors of early improvement. Thrombolysis in brain ischemia grade by TCD monitoring significantly correlated with NIHSS score for 24 hours. CONCLUSIONS: These results suggest that younger age and normal CT findings are important prognostic factors of acute thrombolytic therapy.
Blood Pressure ; Brain Ischemia ; Fasting ; Glucose ; Humans ; Lipoprotein(a) ; Middle Cerebral Artery ; Multivariate Analysis ; National Institutes of Health (U.S.) ; Prognosis ; Prospective Studies ; Stroke* ; Thrombolytic Therapy* ; Tissue Plasminogen Activator

OBJECTIVETo study the clinical implications of changes of coagulation function and fibrinolytic system in patients with gestational diabetes mellitus (GDM).

METHODSTwenty non-pregnant women, 20 with normal pregnancy and 46 with GDM were enrolled in this study for examinations of platelet alpha-granule membrane protein (GMP-140), Von Willebrand factor (vWF), antithrombin III activity (AT-III), plasminogen activity (PLG), activity of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI).

RESULTSvWF, GMP-140, PLG, D-dimer, PAI were obviously elevated while t-PA was lower in GDM patients as compared with the measurement in non-pregnant women and women with normal pregnancy (P<0.01). AT-III and ProC measurement showed no significant differences between GDM patients and women of the other two groups.

CONCLUSIONGDM patients may have elevated platelet activation and fibrinolyic activity as well as vascular endothelial injuries, and antenatal assessment of the coagulation function can be of value for prevention and treatment of GDM.

Adult ; Blood Coagulation ; physiology ; Diabetes, Gestational ; blood ; physiopathology ; Endothelium, Vascular ; physiopathology ; Female ; Fibrin Fibrinogen Degradation Products ; analysis ; Fibrinolysis ; physiology ; Humans ; P-Selectin ; blood ; Plasminogen Activator Inhibitor 1 ; blood ; Platelet Activation ; physiology ; Pregnancy ; Pregnancy Trimester, Third ; blood ; Tissue Plasminogen Activator ; blood ; von Willebrand Factor ; analysis