Tissue type plasminogen activator antigen and activity in sickle cell disease.

Abstract Background: Sickle cell nephropathy (SCN) is one of the most common complications of SCD, leading in most cases to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Despite the high prevalence of CKD in sickle cell disease (SCD) patients, there remains a poor understanding of the pathophysiological mechanism of SCN and a lack of biomarkers for early detection of SCD-associated CKD. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker of CKD. suPAR is a member of the fibrinolytic system, which is dysregulated in SCD patients. Objective: To evaluate suPAR as a biomarker of SCD-associated nephropathy and identify plasma proteases responsible for its increase in SCD. Methods: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior to the sample collection. Whole blood and urine samples were collected from 77 SCD patients and 10 healthy individuals, and plasma was isolated. Levels of creatinine and cystatin C in plasma and albumin and creatinine in urine were measured by ELISA. eGFR was calculated using CKD-EPI creatinine-cystatin equation, and CKD stages were assigned. Plasma suPAR was measured by ELISA and was correlated with CKD stages. The activities of candidates uPAR proteases: Neutrophile elastase (NE), urokinase-type plasminogen activator (uPA) and plasmin in plasma samples from SCD patients were measured and compared to healthy participants. Results: The average age of SCD patients was 42.5 years (range 18-67 years). Most patients had HbSS genotype (67.5%),19.5% of patients were HbSC (hemoglobin C sickle cell compound heterozygous), and 13% had HbS β-thalassemia. More than half (53.2 %) were females. We observed an increased level of plasma suPAR (>3ng/ml) in more than 60% of SCA patients without renal disease, representing a risk factor for CKD progression. Plasma suPAR levels further increased in the patients with CKD and positively correlated with stages of CKD (r=0.419, R2=0.1696). Analysis of plasma proteases that cleaved uPAR producing soluble peptides (suPAR) demonstrated increased urokinase-type plasminogen activator (uPA) activity without significant changes in neutrophile elastase. Conclusion: This study validated plasma suPAR as a potential marker of CKD in SCD patients and identified plasma uPA as a uPAR protease that may increase circulating suPAR in SCD. Future longitudinal analysis of suPAR levels in patients with SCA is needed. Acknowledgments: We thank Drs. Namita Kumari and Xiaomei Niu for their help in samples identification. This work was supported by NIH Research Grants 1R01HL125005-06A1, 5U54MD007597, 1P30AI117970-06,1UM1AI26617, and 1SC1HL150685. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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DEFECTIVE FIBRINOLYSIS IN SICKLE CELL DISEASE

10.1055/s-0038-1644838 ◽

1987 ◽

Author(s):

P Glas-Greenwalt ◽

J Palascak ◽

R Gruppo ◽

D Stroop ◽

V Pollak

Keyword(s):

Sickle Cell Disease ◽

Plasminogen Activator ◽

Sickle Cell ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Cell Disease ◽

Normal Range ◽

Clearing System ◽

Functionally Impaired ◽

Predominant Factor

Vasocclusive crises (VOC) cause significant morbidity and mortality in sickle cell disease (SCD). Although sickling is thought to be the predominant factor in VOC, investigators have examined the possible role of the hemostatic mechanism in the process. The data are, however, inconsistent. We studied, functionally with the fibrin plate method, the fibrinolytic system in 36 adults in the steady state and in 8 children, 7 of whom suffered from painful crises. Values in 240 normal blood donors were: tissue-type plasminogen activator activity (t-PA); 3-25 activator units/ml, corresponding to 0.04 to 0.4 ng/ml; plasminogen activator inhibitor (PA-I): 649-885 inhibitor units/ml; and α2-antiplasmin (α2-AP) : 718-970 inhibitor units/ml. In patients with sickle cell disease t-PA levels were below the normal range in 24/44. PA-I and α2-AP levels were elevated in 35/44 and 23/44 respectively. The prevalence was similar in patients in crises and quiescent. Functionally impaired t-PA was associated with either low, normal or high t-PA antigen levels measured immunologically with an ELISA technique (normal range: 3-6 ng/ml). This indicates various degrees of endothelial dysfunction, ranging from impaired synthesis to functionally defective protein to complex formation with PA-I. Fibrin has been implicated in the intravascular sludging of sickle cells. It is suggested that a defective fibrin-clearing system contributes to the syndrome of SCD.

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Acute Ischemic Stroke in Sickle Cell Disease Challenges for Thrombolysis

Dubai Medical Journal ◽

10.1159/000510873 ◽

2020 ◽

Vol 3 (4) ◽

pp. 173-177

Author(s):

Amal Al Hashmi ◽

Sanjith Aaron

Keyword(s):

Ischemic Stroke ◽

Sickle Cell Disease ◽

Acute Ischemic Stroke ◽

Sickle Cell ◽

Heart Association ◽

Tissue Type ◽

Cell Disease ◽

Excellent Outcome ◽

Type Plasminogen Activator ◽

Recommended Therapy

Sickle cell disease (SCD) is a hemoglobinopathy disorder that was recognized in 1949. Stroke is one of the most devastating complications of this disorder. The risk of stroke in SCD patients varies with the genotype; the highest rate has been reported with hemoglobin S. Tissue type plasminogen activator (t PA) is class I recommended therapy for acute ischemic stroke (AIS) since 1996. Although it is unclear if SCD patients were included in the t PA trail, SCD has never been identified as contraindication. The debate of offering t PA for adult SCD patient presenting with AIS continued for at least 2 decades. It is not up and until 2 years ago when the American Heart Association and American stroke association (AHA/ASA) announced that intravenous (IV) t PA can be offered to adult SCD patients presenting with AIS. Here, we report a case where successful IV thrombolytic therapy was offered to an adult SCD patient presenting with AIS with excellent outcome.

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P1705 Increase in venous tissue-type plasminogen activator (tPA) by exercise predicts local endothelial tPA release in vivo in healthy men

European Heart Journal ◽

10.1016/s0195-668x(03)94843-3 ◽

2003 ◽

Vol 24 (5) ◽

pp. 322

Author(s):

T GUDNASON

Keyword(s):

Plasminogen Activator ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Healthy Men ◽

Type Plasminogen Activator ◽

Venous Tissue

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Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

Nuklearmedizin ◽

10.1055/s-0038-1628893 ◽

1987 ◽

Vol 26 (05) ◽

pp. 224-228 ◽

Cited By ~ 2

Author(s):

Y. Isaka ◽

H. Etani ◽

K. Kimura ◽

S. Yoneda ◽

T. Kamada ◽

...

Keyword(s):

Plasminogen Activator ◽

Abdominal Aorta ◽

Half Life ◽

Balloon Catheter ◽

Biochemical Properties ◽

Tissue Type ◽

Fibrin Deposition ◽

Tissue Type Plasminogen Activator ◽

High Affinity ◽

Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

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The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647041 ◽

1988 ◽

Vol 60 (02) ◽

pp. 255-261 ◽

Cited By ~ 81

Author(s):

A Hotchkiss ◽

C J Refino ◽

C K Leonard ◽

J V O'Connor ◽

C Crowley ◽

...

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Sodium Periodate ◽

Glycosylation Site ◽

Tissue Type ◽

Carbohydrate Structure ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

High Mannose ◽

Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

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Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646342 ◽

1992 ◽

Vol 68 (06) ◽

pp. 672-677 ◽

Cited By ~ 4

Author(s):

Hitoshi Yahara ◽

Keiji Matsumoto ◽

Hiroyuki Maruyama ◽

Tetsuya Nagaoka ◽

Yasuhiro Ikenaka ◽

...

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Half Life ◽

Tissue Type ◽

Clot Lysis ◽

Amino Acid Substitutions ◽

Wild Type ◽

Plasma Clot ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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Enhanced Effective Fibrinolysis following the Neutralization of Heparin in Open Heart Surgery Increases the Risk of Post-Surgical Bleeding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645202 ◽

1990 ◽

Vol 63 (02) ◽

pp. 241-245 ◽

Cited By ~ 49

Author(s):

Jørgen Gram ◽

Thomas Janetzko ◽

Jørgen Jespersen ◽

Hans Dietrich Bruhn

Keyword(s):

Blood Loss ◽

Plasminogen Activator ◽

Heart Surgery ◽

Degradation Products ◽

Open Heart Surgery ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Chest Tube Drain ◽

Median Blood Loss

SummaryThe tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery. The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours. Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracor-poreal circulation (p <0.01), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors. Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p <0.01) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p <0.01). Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p <0.05).We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery

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Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646886 ◽

1989 ◽

Vol 62 (02) ◽

pp. 699-703 ◽

Cited By ~ 8

Author(s):

Rob J Aerts ◽

Karin Gillis ◽

Hans Pannekoek

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Binding Sites ◽

Umbilical Vein ◽

Tissue Type ◽

Single Chain ◽

Human Endothelial Cells ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

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