The Fab Fragment of a Novel Anti-GPVI Monoclonal Antibody, OM4, Reduces In Vivo Thrombosis Without Bleeding Risk in Rats

SummaryGram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin- induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees.Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 μg/kg (n = 2) or 100 μfig/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1+2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels. Endotoxin administration resulted in activation of the fibrinolytic system as reflected by a rapid increase in plasma plasmin- α2-antiplasmin complexes. Administration of 12D10 was without effect on the endotoxin-induced fibrinolytic activation.In conclusion, this study confirmed the importance of the TF: VII complex in the initial, endotoxin-induced activation of coagulation which was completely blocked by neutralizing all free and tissue factor-complexed factor VII/VIIa by a specific monoclonal Fab fragment. Activation of fibrinolysis was not influenced. Therefore, neutralization of factor VII/VIIa might be a promising therapeutic option in preventing endotoxin-induced microthrombosis during Gram-negative sepsis.

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Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.745360 ◽

2021 ◽

Vol 8 ◽

Author(s):

Gian Pietro Pietri ◽

Marta Tontini ◽

Barbara Brogioni ◽

Davide Oldrini ◽

Stefania Robakiewicz ◽

...

Keyword(s):

Monoclonal Antibody ◽

Capsular Polysaccharide ◽

Hydrophobic Interactions ◽

Rabbit Serum ◽

Antigenic Determinants ◽

Enzyme Linked Immunosorbent Assay ◽

Salt Bridges ◽

Fab Fragment ◽

Preclinical Trials

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

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Quantification of TCR/CD3 complex expression on human T-lymphocytes after in vivo tretment with murine IgA monoclonal antibody to human CD3

Immunology Letters ◽

10.1016/s0165-2478(97)88346-0 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 373

Author(s):

R Meijer

Keyword(s):

Monoclonal Antibody ◽

T Lymphocytes ◽

Human T Lymphocytes ◽

Complex Expression

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

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.

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Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

Author(s):

A W J Stuttle ◽

M J Powling ◽

J M Ritter ◽

R M Hardisty

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Aggregation ◽

Calcium Ions ◽

Human Platelet ◽

Human Platelets ◽

In Vivo Detection ◽

Fibrinogen Binding ◽

Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648952 ◽

1994 ◽

Vol 72 (05) ◽

pp. 745-749 ◽

Cited By ~ 7

Author(s):

Elza Chignier ◽

Maud Parise ◽

Lilian McGregor ◽

Caroline Delabre ◽

Sylvie Faucompret ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Peripheral Blood ◽

Vascular Trauma ◽

Activated Platelets ◽

Group I ◽

Group Ii ◽

Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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Anti-thrombotic Effect of a PAI-1 Inhibitor in Rats Given Endotoxin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650231 ◽

1996 ◽

Vol 75 (01) ◽

pp. 118-126 ◽

Cited By ~ 27

Author(s):

T Abrahamsson ◽

V Nerme ◽

M Strömqvist ◽

B Åkerblom ◽

A Legnehed ◽

...

Keyword(s):

Plasminogen Activator Inhibitor ◽

Rat Plasma ◽

Clot Lysis ◽

Fibrin Deposition ◽

Plasminogen Activator Inhibitor 1 ◽

Fab Fragment ◽

Dose Dependent Decrease ◽

Pai 1

SummaryThe aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i. v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus + infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

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Determination and Treatment of Disorders of Primary Haemostasis

Hämostaseologie ◽

10.1055/s-0038-1660415 ◽

1999 ◽

Vol 19 (04) ◽

pp. 168-175 ◽

Cited By ~ 6

Author(s):

M. Weippert-Kretschmer ◽

V. Kretschmer

Keyword(s):

Platelet Function ◽

Bleeding Risk ◽

Bleeding Complications ◽

Bleeding Tendency ◽

Platelet Counts ◽

Platelet Function Disorders ◽

Perioperative Bleeding ◽

Before And After ◽

Low Sensitivity

SummaryPerioperative bleeding complications due to disorders of primary haemostasis are often underestimated. Routine determination of primary haemostasis is still problematic. The in vivo bleeding time (BT) shows low sensitivity and high variability. In this contribution the results and experiences with the IVBT having been obtained in various studies and during 10 years of routine use are reported. Patients and Methods: Blood donors before and after ASA ingestion, patients with thrombocytopenia as well as congenital and acquired platelet function disorders. Monitoring of desmopressin efficacy. IVBT with Thrombostat 4000 (tests with CaCl2 = TST-CaCl2 and ADP = TST-ADP) and PFA-100 (test cartridges with epinephrine = PFA-EPI and ADP = PFA-ADP). Results and Conclusions: IVBT becomes abnormal with platelet counts <100,000/μl. With platelet counts <50,000/μl the results are mostly outside the methodical range. IVBT proved clearly superior to BT in von Willebrand syndrome (vWS). All 16 patients with vWS were detected by PFA-EPI, whereas with BT 7 of 10 patients with moderate and 1 of 6 patients with mild forms of vWS were spotted. The majority of acquired and congenital platelet function disorders with relevant bleeding tendency were detectable by IVBT. Sometimes diagnostic problems arose in case of storage pool defect. Four to 12 h after ingestion of a single dose of 100 mg ASA the TST-CaCl2 became abnormal in all cases, the PFA-EPI only in 80%. However, the ASA sensitivity of TST-CaCl2 proved even too high when looking for perioperative bleeding complications in an urological study. Therefore, the lower ASS sensitivity of the PFA-100 seems to be rather advantageous for the estimation of a real bleeding risk. The good efficacy of desmopressin in the majority of cases with mild thrombocytopenia, congenital and acquired platelet function disorders and even ASS-induced platelet dysfunction could be proven by means of the IVBT. Thus IVBT may help to increase the reliability of the therapy. However, the IVBT with the PFA-100 is not yet fully developed. Nevertheless, routine use can be recommended when special methodical guidelines are followed.

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Faculty Opinions recommendation of Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergen-induced asthma.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717961811.793464520 ◽

2012 ◽

Author(s):

Mathur Kannan

Keyword(s):

Monoclonal Antibody ◽

Fab Fragment

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