Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3346-3346
Author(s):  
Jaa Yien New ◽  
Jose Perdomo ◽  
Xing-Mai Jiang ◽  
Beng Chong
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Heparin Therapy ◽  
Human Platelets ◽  
Binding Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Heparin Induced Thrombocytopenia ◽  
E Coli

Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity

Biologicals ◽  
2018 ◽  
Vol 56 ◽  
pp. 54-62 ◽  
Author(s):  
Surachet Benjathummarak ◽  
Chonlatip Pipattanaboon ◽  
Khwanchit Boonha ◽  
Waranya Wongwit ◽  
Pongrama Ramasoota ◽  
...  
Keyword(s):  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli

HEPARIN-INDUCED THROMBOCYTOPENIA: IN VITRO STUDIES WITH LOW MOLECULAR WEIGHT HEPARINOID, ORG 10172

1987 ◽  
Author(s):  
B H Chong ◽  
F Ismail ◽  
J Cade ◽  
A S Gallus ◽  
S Gordon ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Heparin Therapy ◽  
Cross Reactivity ◽  
Low Molecular Weight ◽  
Heparin Induced Thrombocytopenia ◽  
Normal Platelet

Heparin-induced thrombocytopenia (HIT), an adverse effect of heparin therapy, may be associated with serious thrombosis resulting in severe disability or death. An IgG heparin-dependent antibody may be demonstrated in HIT by platelet aggregation studies with patient serum/plasma and standard (s) heparin. A recent study showed high cross-reactivity of the antibody with low molecular weight (LMW) heparins as most of the 22 patient sera tested gave a positive reaction with various LMW heparins including CY222, CY216, PK10169 and Kabi 2165 (Messmore HL et al, Blood 64(5), 1984 suppl). However, cross-reactivity rate with Org 10172, a LMW heparinoid which has strong anti-Xa but negligible antithrombin activity, is unknown. We tested the plasma of 17 patients with HIT for cross-reactivity with Org 10172. Although all 17 patient plasmas reacted positively with s.heparin (0.2 1.0 IU/ml), only 3 patient plasmas gave a positive but weaker reaction with Org 10172 (0.2-1.0 IU/ml).Further studies were performed to investigate the effect of adding Org 10172 (0.2 to 2.0 anti-Xa U/ml) to a reaction mixture of normal platelet-rich plasma, patient plasma and s.heparin (0.2 IU/ml). With 7 patient plasmas which showed no cross-reactivity with Org 10172, the antibody-induced platelet aggregation was inhibited when the concentration of Org 10172 exceeded 0.5 to 1.0 anti-Xa U/ml. When the study was repeated with other s.heparin concentrations (0.05, 0.1, 0.4 IU/ml), this inhibitory effect was again present provided the ratio of Org 10172 concentration (anti-Xa U/ml) to heparin concentration (IU/ml) exceeds 2.5 to 5. However, this inhibitory effect was not observed with the 3 patient plasmas which showed cross-reactivity with the heparinoid whqp. the same concentrations of Org 10172 were added. This inhibitory effect appeared to be specific for platelet aggregation induced by the heparin-dependent antibody as Org 10172 (<10 anti-Xa U/ml) did not affect platelet aggregation induced by collagen (2 ug/ml) and ADP (2.5 uM). These findings together with our experience of 3 cases of HIT successfully treated with Org 10172 suggest that this LMW heparinoid may be a useful drug for the treatment of HIT.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
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

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

The Clinical Usefulness of the Platelet Aggregation Test for the Diagnosis of Heparin-Induced Thrombocytopenia

1993 ◽  
Vol 69 (04) ◽  
pp. 344-350 ◽  
Author(s):  
B H Chong ◽  
J Burgess ◽  
F Ismail
Keyword(s):  
Platelet Aggregation ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Point System ◽  
Control Groups ◽  
Heparin Induced Thrombocytopenia ◽  
Heparin Concentration ◽  
Release Test ◽  
The Mean ◽  
Normal Controls

SummaryThe platelet aggregation test is widely used for the diagnosis of heparin-induced thrombocytopenia (HIT), a potentially serious complication of heparin therapy. We have evaluated its sensitivity and specificity in comparison with those of the 14C-serotonin release test. The sensitivity of the platelet aggregation test was found to vary with the heparin concentration and the donor of the platelets used in the test. The optimal heparin concentrations were between 0.1 and 1.0 U/ml. Using these heparin concentrations, the mean sensitivity varied from 39% (with the least reactive platelets) to 81% (with the most reactive platelets). In comparison, the sensitivity of the release test ranged from 65% to 94%. The specificities of the platelet aggregation test were 82%, 90% and 100% for the following control groups: (1) non-thrombocytopenic patients given heparin, (2) patients with thrombocytopenia due to other causes, and (3) normal controls not given heparin, respectively. The corresponding specificities for the release test was 94%, 90% and 100%. The specificities can be further increased to 100% for all controls with the adoption of a two-point system which defines a positive result as one in which platelet aggregation occurs with a low heparin concentration (0.5 U/ml) but not with 100 U heparin/ml. For optimal results, a two-point platelet aggregation test should be performed with heparin concentrations of 0.5 and 100 U/ml and using platelets of more reactive donors.

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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