HEPARIN DIRECTS THE INACTIVATION OF ANTITHROMBIN BY ELASTASE

1987 ◽  
Author(s):  
R E Jordan ◽  
J Kilpatrick ◽  
J Nelson ◽  
J O New gren ◽  
M A Fournel
Keyword(s):  
Alternative Explanation ◽  
Anticoagulant Activity ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Apparent Contradiction ◽  
Platelet Factor ◽  
Complete Inactivation

In apparent contradiction to its anticoagulant activity, we have observed a previously undetected, and potentially opposing function for heparin: a distinct heparin-dependency for the in vitro inactivation of highly-purified human antithrombin by neutrophil elastase. Similar to its ability to accelerate antithrombin-mediated inhibition of coagulation enzymes, anticoagulantly-active heparin was also found to stimulate the rate of inactivation of antithrombin by the neutrophil enzyme.In the absence of heparin, or in the presence of the heparin antagonists platelet factor 4 or polybrene, little or no inactivation of antithrombin occurred. Catalytic amounts of heparin and elastase caused the complete inactivation of antithrombin (approximate molar ratio of 1:1:400 respectively) in 5-10 minutes. The loss of heparin binding affinity by the elastase-cleaved form of antithrombin permitted its separation from active antithrombin by heparin-agarose chromatography.The purified elastase-inactivated antithrombin was injected into rabbits for determination of its comparative clearance behavior. In contrast to intact, functional antithrombin (t 1/2 >30 hours) and the thrombin-antithrombin (T-AT) complex (t 1/2 previously shown to be minutes), elastase-inactivated antithrombin circulated for approximately 13 hours. This prolonged clearance relative to the T-AT complex may suggest an alternative explanation for the circulating, non-functional antithrombin observed in certain coagulopathic states. In summary, these results point to a potential and unexpected role for heparin in directing the inactivation of antithrombin and suggest a possible in vivo mechanism for neutralizing the usually non-thrombogenic nature of the vascular lining.

scholarly journals Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)

2004 ◽  
Vol 383 (2) ◽  
pp. 361-370 ◽  
Author(s):  
Elena S. DREMINA ◽  
Victor S. SHAROV ◽  
Keshava KUMAR ◽  
Asma ZAIDI ◽  
Elias K. MICHAELIS ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Conformational Transition ◽  
Direct Interaction ◽  
Molar Ratio ◽  
Endoplasmic Reticulum Calcium ◽  
Oxidative Inactivation ◽  
Complete Inactivation

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2Δ21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2Δ21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2Δ21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2Δ21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2Δ21 to SERCA. In contrast, Bcl-2Δ21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2Δ21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2Δ21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Abstract 451: Platelet Activation and Thrombosis by Antibodies against VEGF and CD40 Ligand: Extending the mechanism of HIT

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ali Amirkhosravi ◽  
Todd Meyer ◽  
Florian Langer ◽  
Theresa Robson ◽  
Liza Robles ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Cd40 Ligand ◽  
Serotonin Release ◽  
Heparin Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
Cardiovascular Comorbidity ◽  
Platelet Aggregometry

Introduction: The vascular endothelial growth factor (VEGF) monoclonal antibody, bevaci-zumab (Avastin), has been associated with arterial thromboembolic events in some cancer patients. Another therapeutic antibody, hu5c8, which targets CD40L, also produced unexpected thrombosis in lupus clinical trials. Because platelets play a crucial role in arterial thrombosis, we hypothesized that antibodies against VEGF and CD40L may activate platelets via a mechanism similar to that responsible for thrombosis in Heparin-Induced Thrombocytopenia (HIT). Methods: Immune complexes (ICs) were prepared by combining these monoclonal IgG antibodies with their antigens: M90 (or hu5c8) with CD40L (“M90+CD40L”), or bevacizumab with VEGF and heparin (“BVH”). VEGF binds heparin (as does platelet factor 4, the HIT antigen). We measured platelet activation by serotonin release assay, platelet aggregometry, and flow cytometry. We also evaluated IC-induced thrombosis in hFc mice, transgenic for the human IgG receptor, CD32. Results: Similar to HIT antibodies, these ICs potently induced platelet activation dependent on CD32, IC concentration (>10nM) and optimal stoichiometry. Intravenous injection of M90+CD40L or BVH into hFc, but not wild-type mice rapidly produced signs of thrombotic shock, thrombocytopenia and pulmonary thrombosis. However, wild-type control mice (lacking platelet CD32) were unaffected by IC injection. Similarly as with HIT antibodies, bevacizumab IC activity was reduced in the absence or excess of heparin both in vitro and in vivo, whilst M90+CD40L-induced platelet activation was abolished in vitro by blockade of the platelet CD40L receptor, CD40, demonstrating a requirement for Fab-dependent anchoring. Furthermore, VEGF 121 (which lacks the heparin-binding domain of VEGF 165 ) and bevacizumab with or without heparin failed to activate platelets or cause thrombosis in hFc mice. Conclusions: Together, these findings demonstrate that Fab-dependent anchoring of anti-CD40L and anti-VEGF ICs is required for potent platelet activation and thrombosis, as is the case in HIT, suggesting common mechanistic elements. Clinical implications may apply in patients with cardiovascular comorbidity receiving immunotherapy.

Pressure-Induced Metabolic Activation of Platelets Demonstrated In Vitro by Determination of Malondialdehyde Formation

1984 ◽  
Vol 52 (01) ◽  
pp. 004-006 ◽  
Author(s):  
Alessandro Torsellini ◽  
Luca Doni ◽  
Wulf Palinski ◽  
Giovanni Guidi ◽  
Valeriano Lombardi
Keyword(s):  
Arachidonic Acid ◽  
Atmospheric Pressure ◽  
Platelet Rich Plasma ◽  
Metabolic Activation ◽  
Platelet Factor ◽  
Malondialdehyde Formation ◽  
Shape Changes

SummaryPlatelet rich plasma (PRP) exposed in vitro to 200 mm Hg above atmospheric pressure showed a significant increase in malondialdehyde (MDA) formation compared to PRP at atmospheric pressure. This difference is also evident when platelets are incubated with arachidonic acid.The increase of MDA demonstrates that the increased beta- thromboglobulin and platelet factor 4 in plasma and the shape changes of platelets after pressure stimulation in vitro that were described in a previous paper result from the release reaction.Pressure-induced effects in vivo are discussed

Binding of Platelet Factor 4 to Cultured Human Endothelial Cells

1979 ◽  
Author(s):  
C. Busch ◽  
J. Dawes ◽  
D.S. Pepper ◽  
Å Wasteson
Keyword(s):  
Endothelial Cells ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Human Endothelial Cells ◽  
Platelet Factor ◽  
Platelet Release

Platelet factor 4(PF-4) is a product of the platelet release reaction. A well known property of PF-4 is to interact with sulphated glycosaminoglycans (CAGs), including heparin; binding to heparin leads to neutralization of the anticoagulant activity.This study was undertaken to examine the possible binding of PF-4 to monolayers of cultured human endothelial cells(EC). The EC surface has been shown to expose GAG in particular heparan sulphate. Cultures incubated at 37°C with various amounts of 125I-PF-4 bound 125I-radioactivity in a dose-dependent manner. Maximum binding was about 500 ng per 0-7 cm2 dish (0.4x106 cells). About 50% of this was associated with the cells. In the presence of 200 ug/ml of unlabelled PF-4 or 6 ug/ml of heparin the binding was less than 5%. Chondroitin-4-sulphate (30 ug/ml) had little influence on the binding.The observed EC-PF-4-binding in vitro may reflect a similar interaction in vivo. Such a phenomenon may influence the non-thrombogenic properties of the vascular lining. Furthermore it could affect the reliability of the PF-4 measurements as indicators of intravascular platelet release.

Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

1994 ◽  
Vol 71 (05) ◽  
pp. 609-614 ◽  
Author(s):  
Laxminarayana N Korutla ◽  
Gwendolyn J Stewart ◽  
Elizabeth C Lasz ◽  
Theodore E Maione ◽  
Stefan Niewiarowski
Keyword(s):  
Half Life ◽  
Anticoagulant Activity ◽  
Protamine Sulfate ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Liver And Kidney ◽  
Kinetics Of ◽  
Heparin Neutralization

SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodina- ted and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivitized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Sign in / Sign up

Export Citation Format

Share Document