Are integrins involved in the aggregatory and phagocytic behaviour of fish haemostatic cells?

1998 ◽  
Vol 201 (4) ◽  
pp. 599-608 ◽  
Author(s):  
D J Hill ◽  
A F Rowley
Keyword(s):  
Monoclonal Antibodies ◽  
Human Platelet ◽  
The Other ◽  
Active Process ◽  
Thrombocyte Aggregation ◽  
Fibrinogen Receptor ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Fibrinogen Binding ◽  
Test Particles ◽  
Inhibitor Peptide

The involvement of a putative integrin-like fibrinogen receptor in the aggregatory and phagocytic behaviour of thrombocytes (platelet equivalents of fish) from the rainbow trout Oncorhynchus mykiss was studied. Aggregation of trout thrombocytes was induced by the thromboxane mimetic U-46619 in the presence of trout fibrinogen. Thrombocyte aggregation was inhibited by the tetrapeptide RGDS, but not by RGES or fibrinogen binding inhibitor peptide (HHLGGAKQAGDV). A range of monoclonal antibodies against the human platelet integrin alphaIIbbeta3 (anti-CD41a, anti-beta3 and LK7r) showed no reactivity with trout thrombocytes. Subsequently, a panel of monoclonal antibodies was raised against thrombocyte membrane preparations in an attempt to obtain an antibody against the putative integrin fibrinogen receptor. Of these monoclonal antibodies, four were found to inhibit thrombocyte aggregation, namely 12G2, 30D8, 32F8 and 32H10. The antibody 32H10 was shown significantly to inhibit the attachment of thrombocytes to immobilised trout fibrinogen, suggesting that it and the other antibodies recognise the putative fibrinogen receptor on trout thrombocytes. FITC-labelled Bacillus cereus were employed as test particles to prove that thrombocytes internalise bacteria via an active process and not simply by passive sequestration into the open canalicular system. Preincubation of bacteria with trout fibrinogen resulted in a significant increase in the number of thrombocytes exhibiting phagocytosis. This enhancement of phagocytosis by preincubation of B. cereus with trout fibrinogen could be inhibited by the tetrapeptide RGDS, but not by RGES, hence implicating the putative fibrinogen receptor in the internalisation of microorganisms. The relevance of these findings to the possible existence of an integrin-like receptor on trout thrombocytes is discussed.

STIMULUS-DEPENDENT CHANGES IN THE SURFACE EXPRESSION OF GPIIb/IIIa AND FIBRINOGEN RECEPTORS. RELATIONSHIP TO PLATELET AGGREGATION

1987 ◽  
Author(s):  
K Niiya ◽  
E Hodson ◽  
R Bader ◽  
V Byers-Ward ◽  
E F Plow ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Divalent Cation ◽  
Surface Expression ◽  
The Other ◽  
Fab Fragment ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dissociation Constant Kd

Platelet stimulation altered the binding of three monoclonal antibodies (monovalent Fab’ fragment) directed against the glycoprotein (GP)IIb/IIIa complex. We found that 47,600-60,300 molecules of antibody bound per platelet before stimulation, as compared to 89,200-146,500 molecules per platelet after thrombin stimulation. These changes were observed in parallel with a small but significant increase in the dissociation constant (Kd) of two antibodies. In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 3040% in the presence of EDTA at 22-25°C, suggesting the occurrence of divalent-cation mediated, activation-dependent changes in the corresponding GPIIb/IIIa epitope. Platelets stimulated by thrombin bound more fibrinogen than those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with different platelet agonists. When the pool of GPIIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, thrombin stimulation still induced substantial binding and aggregation. This effect of thrombin required exposure of platelets to the active agonist and was not mediated by molecules released by thrombin into the medium. Therefore, platelets activated with “strong” agonists exhibit increased number of surface-oriented epitopes associated with GPIIb/IIIa. The GPIIb/IIIa molecules bearing these newly exposed epitopes are functional in that they bind fibrinogen and mediate platelet aggregation.

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

scholarly journals Further studies on the topography of human platelet glycoprotein IIb. Localization of monoclonal antibody epitopes and the putative glycoprotein IIIa- and fibrinogen-binding regions

1991 ◽  
Vol 273 (3) ◽  
pp. 767-775 ◽  
Author(s):  
J J Calvete ◽  
J Arias ◽  
M V Alvarez ◽  
M M Lopez ◽  
A Henschen ◽  
...  
Keyword(s):  
Human Platelet ◽  
Early Stage ◽  
Platelet Glycoprotein ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Platelet Receptor ◽  
Glycoprotein Iiia ◽  
Membrane Bound ◽  
Antibody Epitopes ◽  
Putative Binding Site

Glycoprotein IIb (GPIIb) is a major glycoprotein of the human platelet plasma membrane, which together with glycoprotein IIIa (GPIIIa) forms a Ca2(+)-dependent heterodimer, GPIIb/IIIa, which serves as the major fibrinogen receptor in activated platelets. The precise localization of the epitopes for six anti-GPIIb monoclonal antibodies (M1-M6) has been determined by a combination of enzymic and chemical cleavage procedures, peptide isolation, N-terminal sequence analysis, peptide synthesis and enzyme immunoassay. The following localizations were found: M1, beta 1-16-36, beta 2-4-24; M2, alpha 747-755; M alpha 2, alpha 837-843; M3, alpha 849-857; M4, alpha 143-151; M5, alpha 550-558; M6, alpha 657-665. Besides considerations of the degree of exposure of these epitopes, several remarkable features are readily apparent. The earliest and main chymotryptic cleavage site of GPIIb in whole platelets is between alpha cysteine-545 and alpha phenylalanine-551. The epitope for M3 was located within the same sequence (alpha 842-857) as is the epitope for PMI-1 [Loftus, Plow, Frelinger, D'Souza, Dixon, Lacy, Sorge & Ginsberg (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7114-7118] in spite of the fact that the exposure of the latter in whole platelets is EDTA-dependent whereas that in the former is not. The epitope for M5 shares full homology with the 540-548 peptide stretch of the alpha-subunit of the vitronectin receptor, and this antibody cross-reacts with endothelial cells. The M6 epitope is located in the 25 kDa membrane-bound fragment of GPIIb, which is most epitope is destroyed at an early stage of chymotrypic digestion. This suggests that this region of GPIIb, somewhere between the epitope for M5 (alpha 550-558) and the epitope for M2 (alpha 747-755), may carry the surface of interaction of GPIIb with GPIIIa in the GPIIb/IIIa heterodimer. Finally, the sequence where the epitope for M6 has been located (alpha 657-667) was the only one found to be hydropathically complementary to the gamma 402-411 peptide of fibrinogen within the amino acid sequence of both GPIIb and GPIIIa. This complementariness, the EDTA- or thrombin-dependence of the exposure of the alpha 657-665 stretch in whole platelets to M6 and the ability of this antibody to inhibit platelet aggregation led us to postulate that this peptide stretch is a putative binding site for fibrinogen in the platelet receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571 ◽  
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

Abstract The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

Functional heterogeneity of alloantibodies against the human platelet antigen (HPA)-1a

2005 ◽  
Vol 94 (12) ◽  
pp. 1224-1229 ◽  
Author(s):  
Hartmut Kroll ◽  
Gabriele Penke ◽  
Sentot Santoso
Keyword(s):  
Cell Adhesion ◽  
Clinical Presentation ◽  
Human Platelet ◽  
Severe Bleeding ◽  
Cell Binding ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Platelet Antigen ◽  
Human Platelet Antigen ◽  
Alloimmune Thrombocytopenia

SummaryThe integrin αIIbβ3 is the major fibrinogen receptor on the platelet membrane and plays a crucial role for platelet aggregation. The β3-subunit carries the human platelet alloantigen (HPA)-1a, which is the main target for alloantibodies (alloabs) responsible for foetal and neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP).Whereas PTP is almost invariably associated with severe bleeding, the clinical presentation of FNAIT ranges from mild thrombocytopenia to severe haemorrhagic diathesis. However, this clinical heterogeneity is not fully understood as it is not explained solely by the variability of the platelet count. Here, we examined the ability of HPA-1a alloabs from mothers with FNAIT (n = 43) and PTP patients (n = 8) to inhibit cell adhesion to fibrinogen and asked if this inhibition was correlated with the heterogeneity of the clinical picture. Stably transfected cells expressing HPA-1a (β3-Leu33) and –1b (β3-Pro33) isoforms were incubated with sera containing HPA-1a alloabs and were allowed to adhere to immobilised fibrinogen. The inhibitory activity was measured as percentage of cell adhesion in the presence of patient sera versus normalAB serum. Only two FNAIT sera specifically inhibited the adhesion of HPA-1a, but not HPA-1b cells. Two other FNAIT sera blocked the adhesion of HPA-1a as well as HPA-1b cells. Interestingly, all four neonates with inhibitory HPA-1a alloabs (9% of all sera) suffered severe bleeding. In comparison, the majority of PTP sera (75%) inhibited cell binding to fibrinogen, four PTP sera selectively inhibited the adhesion of HPA-1a cells whilst 2 sera impaired the binding of both allotypes. Our observations indicate that 1) HPA-1a alloabs are heterogeneous in their ability to interfere with fibrinogen binding, and 2) inhibition of the αIIbβ3 fibrinogen receptor by HPA-1a alloabs may contribute to pronounced bleeding in patients with alloimmune thrombocytopenia.

Potentiation by Adrenaline of Ca2+ Influx and Mobilization in Stimulated Human Platelets: Dissociation from Thromboxane Generation and Aggregation

1988 ◽  
Vol 59 (02) ◽  
pp. 212-215 ◽  
Author(s):  
M J Powling ◽  
R M Hardisty
Keyword(s):  
Human Platelets ◽  
The Other ◽  
Fibrinogen Binding ◽  
Thromboxane Production

SummaryIn a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 pM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA ([Ca2+o] >100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists

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

Human Platelet Antigen 3 (HPA-3): Localization of the Determinant of the Alloantibody Leka (HPA-3a) to the C-Terminus of Platelet Glycoprotein IIb Heavy Chain and Contribution of O-Linked Carbohydrates

1993 ◽  
Vol 69 (05) ◽  
pp. 485-489 ◽  
Author(s):  
Isabelle Djaffar ◽  
Didier Vilette ◽  
Dominique Pidard ◽  
Jean-Luc Wautier ◽  
Jean-Philippe Rosa
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Fibrinogen Receptor ◽  
C Terminus ◽  
Platelet Antigen ◽  
Human Platelet Antigen ◽  
Determinant Structure ◽  
Polymerase Chain

SummaryThe human platelet antigen (HPA) 3 system is expressed on GPIIb, one subunit of GPIIb-IIIa, the platelet fibrinogen receptor. It was recently shown that HPA-3 was associated with an Ile843/Ser polymorphism. To investigate further HPA-3 determinant structure, we localized an HPA-3a determinant, recognized by the alloantiserum Leka, within the last 29 amino acids of GPIIbα. This region encompasses the polymorphic Ile843, which, as expected, is substituted into Ser in Leka-negative individuals, as shown by DNA sequence after polymerase chain reaction on platelet RNA. In addition, contribution of glycosylation to the determinant structure was demonstrated since the Leka antigenicity was strongly decreased after specifically removing nonterminal O-linked sugars, but not terminal sialic acids. We have thus refined the localization of an HPA-3a determinant within the last 29 amino acids, including Ile843, of GPIIb heavy chain, and shown that the Leka HPA-3a determinant is dependent, in part, upon the serine-linked carbohydrates adjacent to Ile/Ser843.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

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