British Journal of Haernatology, 1990, 76, 387-394

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation '

THEODORE E. WARKENTIN,' M. J. POWLING'.* A N D R . M. HARDISTY''Department of Pathology, McMaster University Medical Centre, Hamilton, Ontario. Canada, and lKatharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital School of Medicine, London Received 20 February 1990; acceptedfor publication 7 6 July 7 Y Y O

Summary. Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (F1TC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibnnogen binding. Half-maximal fibrinogen binding occurred a t about 0.4 VM ADP. and ADP-induced fibrinogen binding

continued progressively during 2 0 min incubation with 1 0 VM ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb-TIIa expression in members of a family with Glanxmann's thrombasthenia, and, in double labelling experiments, with the binding of PACI, a monoclonal antibody that binds to GP Ilb-llla only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by rnetins of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet-bound fibrinogen. despite the presence of a n approximately 100-fold excess of the former.

Amongst the earliest changes resulting from the binding of agonists to their receptors on the platelet membrane is the expression of fibrinogen binding sites o n the glycoprotein (GP) IIb-IIIa complex-a necessary prerequisite for platelet aggregation (Peerschke, 1985). The characteristics and kinetics of the fibrinogen binding reaction were originally established using ' >'I-fibrinogen and washed platelet suspensions (Bennett & Vilaire, 1979; Marguerie r t d, 1979). but more recently flow cytometry has been used to study the binding of fluorescein isothiocyanate (F1TC)-conjugated fibrinogen (Kasahara ut al, 1987),and the binding of autologous fibrinogen in platelet-rich plasma (PKP) by means of a FITCconjugated antifibrinogen antibody (Jackson & Jennings. 1989). These flow cytometric methods have the advantage over isotopic methods in that they detect the binding of

fibrinogen to individual cells. and thus provide an indication of heterogeneity of response to activation. The centrifugation of blood to obtain PKP or washed platelets, however. may itself cause some damage to the platelet membrane and consequent fibrinogen binding. While this is of minor importance when the response to agonists in vitro is to be studied, it seriously impairs the value of t.he method for the detection of in-vivo platelet activation. For the latter purpose, Abrams et al (1 990) have measured fibrinogen binding in whole blood by means of a monoclonal antibody which reacts with platelet-bound, but not with free plasma fibrinogen. We describe here a simple and rapid method for the measurement of fibrinogen binding in 5 p1 volumes of whole blood, using a commercially available FITC-conjugated polyclonal antifibrinogen antibody which appears to bind indiscriminately to platelet-bound and free plasma fibrinogen. By means of twocolour immunofluorescence, the binding of fibrinogen measured in this way is shown closely to follow the induction ofits receptor on GP Ilb-IIIa, as determined by the binding of PAC1 , a monoclonal antibody which recognizes a n epitope expressed a t or near the fibrinogen receptor on platelet activation (Shattil (it al, 1985).

* Present address: Cardiovascular Department, Sandoa AG, CH4002 Basel, Switzerland. Correspondence: Professor R. M. Hardisty, Katharine Dormandy Hacmophilia Centre and Haemostasis Unit, Royal Free Hospital, Pond Street, Hampstead. London N W 3 2QG.

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T. E. Warkentin, M . /. Powling and R. M . Hurdisty

MATEKIALS AND METHODS Antibodies. FITC-conjugated rabbit anti-human fibrinogen, anti-human Clq and anti-human C3c were purchased from Dakopatts, Glostrup, Denmark. When incubated for 15-20 min with citrated whole blood at up to twice the concentration used for the fibrinogen binding assay, the antifibrinogen had no inhibitory effect on ADP-induced aggregation, measured by the counting of single platelets (Cheeseman et al. 1984). PACl, a murine IgM monoclonal antibody which binds to GP IIb-IIIa only in its activated state, was kindly donated by Dr Sanford J. Shattil. Philadelphia, and was biotinylated by incubating 500 pg of antibody in 500 pI of phosphatebuffered saline (PBS) for 4 h at room temperature with 8 pl of sulpho-N-hydroxysuccinimide biotin ester (1 mg/ml in water) and 1 drop of 1 mol/l Na2C03.final pH 9.0. The biotinylated protein was dialysed overnight against PBS, pH 7.2. and was stored at - 70°C until use. RFGP37, a murine IgG, monoclonal antibody to platelet GPIb, was raised at the Koyal Free Hospital School of Medicine by Dr Alison Goodall. Phycoerythrin (PE)-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories, Inc., and from Becton Dickinson Ltd, Oxford, U.K., and PEconjugated goat anti-mouse immunoglobulin from Sera-lab. The murine monoclonal antibody IV.3. against the Fcg receptor IT (p40) (Rosenfeld et ul. 198 S), was purchased from Medarex Inc.. West Lebanon, New Hampshire. Preparation of blood samples for flow cytometrg. Blood was obtained from normal volunteers and patients by clean venepuncture. using a 2 I-gauge butterfly needle, and mixed with one-ninth volume of 3.8%) trisodium citrate. Within about 1 min, 5 pl volumes of whole blood (containing about 1-2 x 10" platelets) were added to round-bottomed polystyrene tubes containing 50 p1 HEPES buffer (10 mmol/l HEPES. 1 4 5 mmol/l NaCI, 5 mmol/l KCI, 1 mmol/l MgS04, pH 7.4). and 5 pl of an optimal concentration (1.2 5 g/l) of the FITC-antifibrinogen antibody. with or without 5 pl of ADP. After initial mixing, the tubes were left in the dark at room temperature without further agitation for 30 min after the addition of the blood, when the reaction was stopped and the samples prepared for flow cytometry by dilution with 0 . 5 ml of filtered buffered saline, pH 7.4, containing 0 . 2 % glutaraldehyde or formaldehyde. In some experiments the tubes also contained 5 pI of KFGP37 (ascitic fluid, 1: 1000). and 5 pl of PE-conjugated goat anti-mouse immunoglobulin ( I : 2 ) was added after 1 5 min, to identify the platelets by their binding of the GP-Ib antibody. In experiments designed to determine the optimal amount of FITC-antifibrinogen and the effect of fibrinogen-antifibrinogen immune complexes, varying amounts of antibody were added to an equivalent volume ( 3 pl) of leucocyte-poor PRP instead of 5 pl whole blood, in the presence or absence of a saturating concentration of IV.3, the monoclonal antibody against the Fcy receptor. The leucocyte-poor PRP was obtained by removing a small volume of the uppermost layer after centrifugation of whole blood at 1 5 0 g, and was used in these experiments in order to obviate binding of a n indeterminate amount of the Fcy receptor antibody to leucocytes. In

separate experiments on the quantitation of platelet Fcy receptors by radioimmunoassay, no difference was found in the binding curves of samples obtained from the top and bottom layers of PKP prepared in this way. For double labelling experiments with PAC1, the tubes also contained 5 pl of a saturating concentration (200 pg/ml) of biotinylated PAC1; 5 pl of PE-streptavidin was added after 15 min and incubation continued for a further 1 5 min before fixation-dilution. For the determination of the time course of fibrinogen binding, a series of tubes containing 50 pl HEPES buffer, 5 pl whole blood and 5 p1 antifibrinogen was incubated for 2 0 min before dilution with the fixative, ADP being added at intervals of 2 0 min to 30 s before the final fixation-dilution. Repeated flow cytometric analysis of these samples over the subsequent 60 min showed that no further increase in binding of the antibody occurred, even in those tubes which had been exposed to ADP for only 30 s before fixationdilution, confirming that this procedure induced a stable endpoint. Flow cytometric anulysis. Samples were analysed in either a Kecton Dickinson FACScan or a Coulter EPICS Profile flow cytometer. Both instruments were calibrated for fluorescence and light scatter daily using the manufacturers' standard beads. FITC fluorescence was detected using a 530 nm band pass filter, and phycoerythrin fluorescence with a 585 nm filter. Light scatter and fluorescence data were obtained with gain settings in the logarithmic mode. The platelet population

immune

FSC Fig 1 . Side scatter versus forward scatter of platelets and fibrinogenantifibrinogen immune complexes. The relative positions of platelets, red cells and debris are indicated. Also shown is a region of highly fluorescent particles which formed only when certain proportions of whole blood or PRP and antifibrinogen werc used (see text), suggesting the formation of immune complexes when antigenantibody equivalence was approached (SSC: side scatter; FSC. forward scatter).

Whole Blood Meusurenient of Fibririogen Binding was identified on the basis of its forward and side scatter profile, which distinguished it clearly from both small particulate debris and red cells. In double labelling experiments. a t least 98% of the particles within this population bound RFGP37, the antibody to GPIb. In some experiments an additional highly fluorescent population could be identified, with greater side scatter but slightly lower forward scatter than the platelets (Fig 1). This population was also seen when platelet-poor plasma was substituted for whole blood, and was more pronounced when the proportion of FITC-antifibrinogen to whole blood or PRP was increased, resulting in smaller antigen excess (see below): it appeared to represent immune complexes between the antifibrinogen and plasma fibrinogen or fibrin monomer. For analysis, a gate was set around the platelet population and 5000-1 0 000 cells analysed for FITC fluorescence as a n index of binding of the antifibrinogen antibody, and thus of fibrinogen binding to the platelet membrane. In double labelling experiments, PE fluorescence was analysed to measure the stimultaneous binding of PAC1 to the same platelet population. Results were expressed either as mean fluorescence intensity of the whole platelet population or as the percentage of antibody-positive platelets, defined as those with a fluorescence intensity exceeding that of 95-98')/, of unstimulated platelets. RESIJLTS 'ritrafion of FITC-~iitifibrino(l~n: c $ k t of inrmunc compkx forimtion Fig 2 shows the results of a n experiment in which 3 pl leucocyte-poor PKP was incubated with various amounts of FITC-antifibrinogen in the presence or absence of ADP ( 10 pmol/ll. Near-maximal ADP-induced fibrinogen binding. without evidence of immune complex formation, is reached

1.3 2.5

12.5

6.3

18.8

25.0

Amount of Fluorescein Antifibrinogen Added ( in pg ) ~

Fig 2 . Per cent platelet activation in 3 pl volumes of PRP versus amount of FITC-antifibrinogen added, in the presence (closed circles) or absence (open circles) of 1 0 PM ADP. Immune complex formation was quantitated by determining the percentage of events falling within a gate set around the immune complex population (Fig 1): < 5 y $ ++, 5-10%; 10-20%: >20%.

+.

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a t 6.3 pg antibody, but increasing immune complex formation, as seen by flow cytometry, is observed as the antibody concentration is further increased. The formation of immune complexes has no apparent further effect on fibrinogen binding in the presence of this concentration of AUP. but is associated with a very marked increase in binding by unstimulated platelets, presumably due to direct activation through the platelet Fc receptors. This 'background' fibrinogen binding a t high antibody concentrations in the absence of AUP was greatly reduced in the presence of a n excess of the Pcy receptor antibody IV.3 (e.g. from 74'%,to 1 7%with 2 5 pg C-antifibrinogen),but IV. 3 had no effect on AIIP-induced fibrinogen binding. These results are consistent with the system being in antigen excess, immune complexes forming increasingly as equivalence is approached, and causing platelet activation through Fc receptor binding in these conditions. The proportion of 6.3 pg ( 2 . 5 pl) antifibrinogen to 3 pI PKP (or 5 pl whole blood) appears to be optimal. in that it gives high specific fluorescence of activated platelets in the absence of significant immune complex formation. The findings with the Fcy receptor antibody in the presence of ADP also show that the rabbit antifibrinogen itself was not binding directly to stimulated platelets via its Fc terminus. rather than through its specific binding to fibrinogen. Immune complex formation also occurred when FITC-conjugated anti-C1 q or antiX3c was substituted for antifibrinogen at the same concentrations, but was not accompanied by increased fluorescence of the platelets on activatioii with ADP; this further confirms the specificity of binding of the antifibrinogen. We are a t a loss to explain the greater formation of immune complexes. a t the higher antifibrinogen concentrations. in ADP-stimulated than in unstimulated PKP: it seems unlikely that the relatively small proportion of fibrinogen bound to the platelets would alter the ratio of soluble fibrinogen tu antifibrinogen sufficiently to produce such a definite effect. Whatever the reason for this observation. however. these experiments serve not only to demonstrate the activation of platelets by immune complexes. but to define optinial concentrations of antibody at which no significant immune complex formation occurs. ADP-inducPd fibrinogm binding Fig 3 shows a typical series of fluorescence histograms of platelets in normal whole blood, incubated for 3 0 min with FITC-antifibrinogen without AUP (front histogram) and in the presence of increasing concentrations of ADP. As the ADP concentration is increased, so also are both the proportion of platelets binding the fluorescent antibody and the inem fluorescence intensity of the whole population. Mean doseeresponse curves for AIIP. expressed as the percentage of antibody-positive platelets. are shown in Fig 4. Half maximal binding occurs a t about 0.4 pmol/l ADP. but about 1 5% of platelets appear not to bind greater amounts of fibrinogen than unstimulated platelets, even a t 80 pnol/l. When smaller volumes of blood ( 1 or 2 pl) were incubated with the same amount of antifibrinogen. higher mean fluorescence intensities (MFI) were observed, and over 9 5'%, of platelets became positive when stimulated by AUP at

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T. E. Warkentin, M . I. Powling and R. M . Hardistg

Fig 3. Log fluorescence histograms of platelets in 5 pl volumes of whole blood incubated for 30 min with FITCantifibrinogen and 5 p1 of ADP at the following final concentrations ( p ~front . to back): nil. 0.08. 0.2, 0.8. 2.0. 4.0. 8.0. 40.0.

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Fig 4. Mean log dose response curve (&SEM: n = 5-10) for ADP. showing the percentage of normal platelets binding FITC-antifibrinogen during 30 min incubation.

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Fig 5. Time course of fibrinogen binding to normal platelets incubated with 10 p~ ADP. Closed circles, percentage of positive cells: open circles, mean fluorescence intensity (log scale) of whole platelet population. Means SEM: n = 3 .

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Pig 6. Time course of fibrinogen binding induced by 10 p~ ADP in a patient with Glanzmann's thrombasthenia ( 0 ) . her father ( O ) , mother ( 0 )and three siblings ( W . A, A ) . Vertical bars represent the means fSEM of three normal subjects. GP IIb-IIIa binding assays gave the following results (10' copies per platelet): 0 , 2.4: 0 . 25.7: H.48.2: A, 32.5; A , 36.1; normal range, 4 3 4 ) + 2 . 5 (SBM).

10 pmol/l or above. This might partly have reflected the higher proportion of plasma fibrinogen bound by the fluorescent antibody in these conditions. leading to greater specific fluorescence of that bound to the platelets, but more probably represented additional stimulation of the platelets by fibrinogen-antifibrinogen immune complexes formed in conditions more nearly approaching equivalence (see above). From the fact that 100 pg of fibrinogen were required to reach equivalence with 1 ml of antibody (manufacturers' information), it can be calculated that 5 j11 of a l : 2 dilution of antibody (6.3 p g ) was sufficient to bind about 2.5-50/, of the fibrinogen in 5 p1 whole blood, or 12.5-2 5% of that in 1 p1. The use of smaller volumes of blood also resulted in significant fibrinogen binding to platelets unstimulated by ADP, evidently as a result of platelet activation by the immune complexes. in conformity with the titration results reported above. The time course of fibrinogen binding in response to 10 pmol/l ADP (Fig 5) is shown both as the percentage of positive cells and as the MFI (measured on a log scale) of the whole platelet population. The two parameters increase roughly in parallel, though the MFI. representing the average number of molecules of fibrinogen bound per platelet, continues to increase after the proportion of platelets binding more than the unstimulated sample has reached a maximum. A minor population of platelets thus appears to bind relatively little fibrinogen in response to ADP, while the majority bind it progressively over a 2 0 min period. It should be noted that under the conditions of this experiment, in which the time of incubation of blood with the antibody was kept constant a t 2 0 min, the ADP being added at various times before the end of this period, most of the labelling of the

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Anti-fibrinogen fluorescence Fig 7. Log fluorescencehistograms showing binding to plateiets of PACl (upper panel) and antifibrinogen (lowerpanel). 5 pI volumes of blood in 5 0 p1 HEPES buffer were incubated for 3 0 min with 5 pl 100 p~ ADP. 5 pl FITC-antifibrinogen. 1 .2 5 mg/rnl, and 5 pl biotinPACl at the following concentrations: A, no PACI; B, 1 0 0 pg/ml: C, 200 pg/ml: D. 450 pg/ml; E. 900 p g h l . 5 pl of PE-streptavidin was added for the last 1 5 min of incubation.

plasma fibrinogen occurred before it was bound to the platelets.

Whole blood fibrinogen binding in t;lnnzmnn's thromhnsthenin Time courses of ADP-induced fibrinogen binding, expressed as MFI, are shown in Fig 6 for a patient with severe thrombasthenia. her parents and her three siblings. Negligible binding was seen in the patient's blood and subnormal values, consistent with heterozygosity, in both parents and two of the siblings, in close agreement with the results of binding assays for GP IIb-IIla carried out independently by Dr S. J. Machin, who kindly referred the family for investigation. In the remaining sibling both GP Ilb-IIIa and fibrinogen binding were within the normal range.

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T. E. Warkentin, M . J. Powling and R. M . Hardisty

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Fig 8 . Binding ofantifibrinogcn(FLI ) and PACl (E'L.2) to unstimulated platclcts (left-handhistogram)and to platclets incubated for 3 0 inin with 4 prnolll ADP (right-hand histogram).The percentages in each quadrant are indicated: in this experiment, the proportion of platelets binding antifibrinogcn (quadrants 2 and 4) incrcased on stimulation from 6% to 57'%,,and of those binding PACl (quadrants 1 and 2) from 2% to 68%.

Correlation of PAC1 nndfibrinogm binding PACl and fibrinogen each competitively inhibit the binding oftheother(Shatti1etaZ. 1 9 8 5 , 1986).For thepurposesofthe present investigation. in which platelets were doubly labelled with PACl and antifibrinogen. it was established that the addition of biotin-PAC1 at a near-saturating concentration ( 2 0 0 pg/ml) had little inhibitory effect on fibrinogen binding, as determined by the binding of FITC-antifibrinogen ( I .2 5 mg/ml) to platelets (Fig 7). Using the antibodies at these

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concentrations, there was a close correlation between the percentages of platelets which bound each of them in response to any given stimulus. An example of PACl and fibrinogen binding in response to 4 pmol/l ADP is shown in Fig 8: 93% (quadrant 3 ) bind neither antibody in the resting state, but after stimulation. 16% (quadrant 1 ) bind PACl alone, 5% (quadrant 4) bind antifibrinogen alone, and 52% (quadrant 2 ) bind both antibodies. A correlation curve of responses to ADP concentrations ranging from 0.01 to 100 LLM(in the presence and absence of 0.01-100 pmol/l PGE,) is shown in Fig 9. The shape of this curve shows that a higher proportion of platelets bind PACl than fibrinogen in response to minimal activation, though over 90% of the cells are capable of binding both when maximally activated. This may suggest that greater stimulation is required for the binding of fibrinogen to its receptor than for the induction of the receptor itself. A similar relationship between PAC1 and fibrinogen binding was observed in response to adrenaline (not shown).

40

60

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cells binding Antifibrinogen

Fig 9. Correlation betwcen percentagesofplateletsbinding PACl and antifibrinogen in response to stimulation with 0.01-100 ~ L ADP M in the presence or absence of 0.01 -1 00 p~ PGEl.

The binding of ADP and other agonists to their receptors on the platelet membrane rapidly induces the expression of binding sites for fibrinogen and other adhesive proteins on the integrin GP IIb-IIIa, and so leads to fibrinogen binding and platelet aggregation. Fibrinogen binding assays may therefore be ofvalue not only in the investigation of structural and functional abnormalities of GP IIb-IIIa and in the study of interactions between platelet activation mechanisms in vitro. but as a n index of early platelet activation in vivo. For the first two of these purposes, isotopic methods using washed platelet suspensions have the advantage of permitting quantitation of the number of fibrinogen molecules bound under

Whole Blood Measurenzent of Fibrinogen Binding carefully controlled conditions. though they provide no information about heterogeneity of binding within a given platelet population, such as may be obtained by flow cytometric methods. If fibrinogen binding is to be used as a n index of in-vivo platelet activation, however, it is essential as far as possible to avoid any further activation after drawing the blood, so that methods involving the isolation of platelets, or even centrifugation to obtain PRP, are inappropriate. In the method described here, 5 it1 volumes of whole blood are diluted tenfold for assay within a minute or two of venepuncture, as described by Shattil r t a1 (19 8 7) for other activationdependent antibodies: this minimizes handling artefacts and has the additional advantage of allowing the investigation of neonatal or thrombocytopenic samples. or of the effect of a wide range of agonists on very small volumes of blood. We have found the method suitable for the detection of small proportions of activated platelets in the circulation (unpublished results), but the present paper is confined to its validation as a means of studying fibrinogen binding to platelet receptors in vitro. Despite the presence in whole blood of an approximately 100-fold excess of plasma to platelet-bound fibrinogen, requiring the use of a system involving a large antigen excess, we have been able to establish in several ways that plateletbound fibrinogen can be reliably measured by means of a polyclonal FITC-antifibrinogen. Thus binding of the antifibrinogen closely parallels binding of P A C l , though the latter seems to be a somewhat more sensitive marker of minor degrees of in vitro platelet activation: antibody binding was closely correlated with GP IIb-IIIa expression in members o f a family with Glanxmann's thrombasthenia; and antibody binding induced by ADP was not inhibited by blocking the platelet Fc receptors. Interference by fibrinogen-antifibrinogen complexes could have been avoided by the use of Fab fragments instead of whole antibody, but we found it possible to avoid stimulation of the plateletes via the Fc receptors by the use of optimal proportions of whole antibody to blood. Fibrinogen-antifibrinogen complexes might theoretically also have a different affinity for the GP IIb-IIIb receptor from that of fibrinogen alone: in this case, since only a small proportion of the plasma fibrinogen is bound by the antibody in this system, binding of the FITC-labelled moiety might not be representative of the whole. This cannot be formally tested in a whole-blood system, but n o difference in binding was observed whether ADP was added before or after incubation of the blood with the antibody: this suggests that platelets bind fibrinogen equally well before and after its reaction with the antibody and that the antibody does not discriminate between platelet-bound and free plasma fibrinogen. Jackson &+ Jennings (1 989) have recently described a flow cytometric method for the determination of fibrinogen binding which resembles the one described herc in the use of a FITC-conjrigated polyclonal antifibrinogen antibody, but differs in that the platelets were studied after the preparation of platelet-rich plasma and its dilution in autologous plateletpoor plasma. These time-consuming procedures may themselves result in a variable degree of platelet activation, and so make the method less reliable for the detection of in-vivo changes. The dilution step was found to be necessary for the

393

avoidance of platelet aggregation. but this is achieved in the present method by simply diluting the whole blood in buffered saline. Like Jackson & Jennings (1989). we found that a minor population of normal platelets appeared to bind little or no fibrinogen even when maximally stimulated by ADP. They speculated that the inability to respond might be related either to platelet age or to damage during collection and processing, but we have found that almost all the platelets are capable of binding fibrinogen when the proportion of antifibrinogen to blood is increased. This, however, also results in the formation of immune complexes, recognizable by their characteristic light scatter profile and intense fluorescence. which themselves contribute to platelet activation. Although we have identified optimal reaction conditions to minimize the influence of these complexes in normal blood samples. these would not necessarily obtain in samples seriously deficient in plasma fibrinogen. It also remains possible that platelet activation artefacts might be caused by the formation of smaller immune complexes. invisible by flow cytometry. This is unlikely. however, since ADP-induced fibrinogen binding is not reduced in the presence of the Fcy receptor anti body. Monoclonal antibodies have recently been raised which react with surface-bound but not with soluble fibrinogen (Zamarron et ($1. 1989: Abrams et al. 19YO), and Abrams et d ( 1 9 90) have used one of these to measure platelet-bound fibrinogen in a whole-blood system very similar to the one described here. While such antibodies have the advantage of specificity. and presumably do not form immune complexes with plasma fibrinogen. a polyclonal antibody might theoretically be prepared with a higher stoichiometric binding ratio to fibrinogen, resulting in greater sensitivitiy a t low levels of platelet activation. This could ofcourse only beestablished by a direct comparison of the monoclonal a n d polyclonal reagents; meanwhile it may be noted that we have found a very similar dose-response relationship between ADP concentration and fibrinogen binding to that reported by Abrams ~t nl (1 YYO), providing further validation of the method. and additional evidence against interference by immune complex formation. ACKNOW1,EDGMRNTS

We are indebted to Dr Peter 1. Powers for the use of laboratory facilities. to Joseph P. Seminerio. Varida Incretolli and Janine Poirier for technical assistance. and to Ilr John Kelton for helpful discussion. KEFEKENCES Abrams. C.S., Ellison, N.. Kudzynski. A,%.& Shattil. S.1. ( I 9 9 0 ) Direct detection oi activated platelcts and platelet-dcrivedmicroparticles in humans. Blood. 75, 128-1 38. Bennett. J.S. & Vilaire. G. ( 1 979) I:xposurc of platelet fibrinogen receptors by ADP and epinephrine.]oirrriril 01C'linicol Irtvc,stiqatiorl. 64, 1393-1401. Cheeseman, J.E.. Mills, S.P. ti Hardisty. K.M. ( 1 984) Platelet aggregometry on whole blood: the use of the ELT X/ds blood cell

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counter in the investigation of bleeding disorders. Clinical and Laboratory Haematology, 6, 265-272. Jackson. S.W. & jennings. L.K. (1989) Heterogcneity of fibrinogen receptor expression on platelets activated in normal plasma with ADP: analysis by flow cytometry. British journal of Haematology, 72, 707-714. Kasahara. K., Takagi, J., Sekiya. F.. Inada. Y. & Saito. Y. (1987) Analysis of distribution of receptors among platelets by flow cytometry. Thrombosis Research, 45, 763-770. Marguerie. G.A.. Plow, E.F. & Edgington, T.S. (1979) Human platelets possess an inducible and saturable receptor specific for fibrinogen. Journal of Biological Chemistry. 254, 5357-5363. Peerschke, E.I.B. [ 198 5) The platelet fibrinogen receptor. Seminurs in Haematology, 22, 241-259. Rosenfeld. S.I.,Looney, R.J.. Leddy, J.P.,Phipps, D.C., Abraham, G.N. &Anderson,C.L. (1985) Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000-molecular-weight mem-

brane protein shared by monocytes. Journal ofClinical Investigation, 76,2317-2322. Shattil, S.J.. Cunningham, M. & Hoxie, 1.A. (1987) Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood. 70, 307-31 5. Shattil. S.1.. Hoxie, J.A.. Cunningham. M. & Brass, L.F. (1985) Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. journal of Biological Chemistry, 260, 11107-11114. Shattil, S.J., Motulsky, H.J., Insel. P.A., Flaherty, L. & Brass, L.F. (1986) Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine. Blood. 68, 1224-1231. Zamarron, C., Ginsberg, M.H. & Plow, E.F. (1989) Receptor induced binding sites (RIBS) are exposed in fibrinogen as a consequence of its interaction with platelets. (Abstract). Blood. 74, (Suppl. 1). 208a.

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation.

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FI...
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