SEARCH

SEARCH BY CITATION

Keywords:

  • thrombi;
  • fibrin;
  • Glanzmann's thrombasthenia;
  • perfusion chamber;
  • αIIbβ3 integrin

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

Summary. To explore the possible role of a residual or variant αIIbβ3 integrin (αIIbβ3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4·5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly αIIbβ3IIbβ3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no αIIbβ3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-αIIbβ3 antithrombotic agents.

The integrin αIIbβ3[initially termed glycoprotein (GP)IIb/IIIa] is involved in cell adhesion processes and in platelet aggregation by its ability to carry the receptor for fibrinogen (Nurden & Caen, 1974, 1975; Nachman & Leung, 1982; Hynes, 1992; Shattil et al, 1998). αIIbβ3 abnormalities are responsible for Glanzmann's thrombasthenia (GT) which is characterized by a prolonged bleeding time, absence of platelet aggregation to all agonists (collagen, epinephrine, ADP or thrombin), absent or delayed clot retraction, and a reduced level or absence of αIIbβ3 whereas aggregation to ristocetin is normal. Caen et al (1966) proposed a classification for GT. In type I, clot retraction and intraplatelet fibrinogen were absent, while in type II clot retraction was only delayed and intraplatelet fibrinogen slightly decreased. Following the description of an abnormal protein pattern in GT by Nurden & Caen (1974), it was demonstrated that the level of αIIbβ3 was < 5% in type I and about 10–20% in type II. In subsequent reports (Caen et al, 1983; Ginsberg et al, 1986; Nurden et al, 1987; Caen, 1989; George et al, 1990; Newman et al, 1991; Lanza et al, 1992; Caen & Rosa, 1995; Nurden, 1999), variant or type III thrombasthenia were described and characterized by normal or nearly normal levels of αIIbβ3 but with functional abnormalities. Although GT patients often exhibit a tendency to life-long bleeding, we previously reported a severe proximal deep vein thrombosis in a patient with variant GT (Gruel et al, 1997). In the present study, the possible role of a residual αIIbβ3 in platelet adhesion and thrombogenesis was explored using a new model of perfusion chamber in which human type III collagen is exposed to flowing native non-anticoagulated blood (Andréet al, 1996). In the systems used to date, perfusion was performed on everted arteries in an annular perfusion chamber with exposure to large amounts of anticoagulated blood (Tschopp et al, 1975; Baumgartner et al, 1977; Sakariassen et al, 1986; Lawrence & Gralnick, 1987; Weiss et al, 1991, 1993) or, in a few experiments, to non-anticoagulated blood (Weiss et al, 1986, 1987). Recently, parallel-plate chambers, in which immobilized collagen was exposed to non-anticoagulated blood, were introduced (Barstad et al, 1996).

The new capillary perfusion chamber used in this study has three major advantages compared with the others: (1) the chamber is circular, mimicking vessel morphology; (2) it only requires very small amounts of blood; and, above all, (3) it does not need anticoagulants and thus reflects physiological conditions.

Thus, for the first time, blood from patients with different subtypes of GT was studied in this model.

Patients. Blood from six normal subjects and five GT patients was intravenously drawn directly through the perfusion chamber as described below. Each patient gave informed consent to participate in the study, as required by French Legislation. GT patients were classified using functional tests (platelet aggregation, clot retraction, determination of platelet fibrinogen content) and platelet membrane αIIbβ3 measurement (by flow cytometry using a panel of specific monoclonal antibodies) (Gruel et al, 1986). In all the patients, the platelet count and plasma fibrinogen level were within the normal range.

Patients 1 and 2 had a variant form of GT. Patient 1 corresponds to patient 50 in the large series reported some years ago (George et al, 1990); he had very mild haemorrhages but exhibited an extensive severe limb venous thrombosis after a long plane journey (Gruel et al, 1997). The molecular abnormality was identified as a Ser 752[RIGHTWARDS ARROW] Pro mutation in the cytoplasmic domain of the integrin β3 subunit, leading to a defective inside-out activation of αIIbβ3 (Chen et al, 1992, 1994) and to a defective αIIbβ3-mediated outside-in signalling (Chen et al, 1992, 1994). Patient 2 was reported as patient 48 by George et al (1990), and she had moderate haemorrhages characterized during childhood by epistaxis and then by gingival bleeding. Platelet transfusions were required after she delivered her three children and after a post-traumatic perihepatic haematoma. In this patient, the αIIbβ3 complex was unstable (Nurden et al, 1987) related to an Arg214[RIGHTWARDS ARROW]Trp mutation in the β3 integrin domain which is involved in the Ca2+-dependent binding to αIIb (Djaffar & Rosa, 1993); a mutation already described as the Strasbourg I variant (Lanza et al, 1992).

Patient 3, with type II GT, was also previously reported as patient 41 in the original series (George et al, 1990). This patient had a chronic moderate haemorrhagic syndrome, but previously required several transfusions, in particular, for severe post-partum haemorrhages. This woman was a compound heterozygote for the β3 gene, combining two novel mutations, a stop codon (Arg 216) and a Cys 598 Tyr substitution in mature β3 (Schlegel et al, 1999).

Daughters of the type II GT patient (patient 3) and of the type III GT patient (patient 1) were also studied. They were heterozygous for the disorder as demonstrated by a level of αIIbβ3 at about 50% the normal level. They exhibited no haemorrhagic manifestations and their functional tests (platelet aggregations, clot retraction) were normal.

Two patients with type I GT (patients 4 and 5) were studied. Patient 4 has been previously reported as patient 12a in the series published by George et al (1990). In this patient, the disease was very mild because he had almost no haemorrhages. The molecular abnormality involved is still unknown. Patient 5 corresponds to patient 1b of the above series (George et al, 1990). In this patient, haemorrhages were more frequent and platelet transfusions were sometimes required, especially for birth delivery. The molecular abnormality was characterized as an alteration of the β3 gene, with a large insertion in intron 8, responsible for defective splicing of this intron and instability of the abnormal mRNA (Djaffar et al, 1993).

Platelet adhesion and thrombus formation in an ex vivo cylindrical perfusion chamber. The model used has already been described (Andréet al, 1996). Briefly, native non-anticoagulated blood from a vein in the forearm was drawn straight from a 19-gauge butterfly infusion set (Venisystems, Abbot Laboratories, Ireland). This set was connected to the chamber through a T-piece shunt with silastic tubing already filled with rinsing buffer (130 nmol/l NaCl, 2 mmol/l KCl, 12 mmol/l NaHCO3, 2·5 mmol/l CaCl2, 2H2O, 0·9 mmol/l MgCl2 and 5 mmol/l glucose, pH 7·4) at 37°C. Blood was perfused at a shear rate of 1600/s (corresponding to moderately stenosed arterial shear rate) through a human type III collagen-coated glass cylindrical chamber (radius: 0·315 mm). Blood perfusion lasted for 4·5 min at a blood flow rate of 2·36 ml/min fixed by a roller pump (2120 Varioperpex® II Pump; LKB Bromma, Sweden) positioned distally to the chamber (Andréet al, 1996). Human type III collagen (Sigma Chimie, L'Isle d'Abeau Chesnes, France) was used to coat the capillaries to a final density of about 2·6 µg/cm2. At the end of the perfusion, chambers were rinsed, fixed, post-fixed and double-embedded in epon as previously described (Andréet al, 1996). Semi-thin cross-sections of the embedded preparation were cut 5 mm downstream of the proximal end of the cylinder and stained (0·01% toluidine blue and 0·01% fuchsin) for light microscope computer-assisted morphometry. The microscope view of the thrombotic deposits was displayed on a colour video monitor (HL series; Microvitec, France) at a final magnification of × 1400 by a video camera (3CCD; Sony, France) fitted onto the photographic lightpath of the microscope (Axioplan; Zeiss, France). Thrombotic deposits were automatically recorded and contrasted by a colour effect generator (NS 15000). Data were managed with the lucie program (Microvision, Evry, France) on a PC 486Dx33 computer (Elonex, France). The percentage of the inner chamber surface coverage with platelets and/or fibrin (i.e. % adhesion), and the average thrombus volume per unit area (µm3/µm2) were determined according to Sakariassen et al (1988). The presence of fibrin was characterized morphometrically as already reported (Weiss et al, 1986) and the fibrillar structure was clearly visualized using the Mallory staining.

The immunohistochemistry for fibrin and VWF. After removing the epon as previously described (Le Charpentier et al, 1981), immunohistochemistry for fibrin was performed after trypsinization (0·01% for 10 min) and using an indirect ABC-peroxidase method revealed by 3–3′ diaminobenzidine (DAB; Vector, Burlinghame, CA, USA) as chromogene. Mouse monoclonal anti-human fibrin antibody (DD3B6 clone; American Diagnostica, Andrésy, France) was diluted to 1/25. VWF immunohistochemistry was performed by the indirect alkaline phosphatase anti-alkaline phosphatase (APAAP) method, revealed with fast-red TR salt (Dako APAAP kit, system 40, K670; Dako, Glostrup, Denmark) as chromogen. Polyclonal rabbit anti-human VWF (A082 clone; Dako) diluted to 1/25 was used. For both labels, omission of primary antibody was used as a negative control.

Normal subjects

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

Perfusion of blood from normal subjects for 4·5 min through the collagen type III-coated cylindrical chamber at a shear rate of 1600/s induced the formation of many thrombi, covering almost 60% of the inner surface (Table I). They were large, mushroom shaped with a narrow base, mainly composed of densely aggregated platelets and occasional round platelets that were more loosely attached on the top of the thrombi (Fig 1A). A thin fibrin meshwork was observed at the base of thrombi (Fig 1A) and its fibrin nature was confirmed by immunochemistry. The labelling for VWF was also very strong at the base of thrombi and more diffuse inside the thrombi (Fig 2A).

Table I.  Platelet interaction with type III collagen in Glanzmann's thrombasthenia (GT).
 Adhesion percentageMean thrombus volume µm3/µm2
  • *

    Percentages of mean thrombus volume versus normal subjects.

Normal subjects (n = 6)66 ± 311·4 ± 0·9
Variant GT
 Patient 11004·9 (43%)*
 Patient 21003·7 (32%)*
Type II GT
 Patient 31005·6 (49%)*
 Heterozygous daughter of patient 16912·7 (100%)*
 Heterozygous daughter of patient 37212·8 (100%)*
Type I GT
 Patient 41000
 Patient 51000
image

Figure 1. Collagen-induced platelet deposition at 1600/s after 4·5 min perfusion with non-anticoagulated blood (0·01% toluidine blue and 0·01% fuchsin, original magnification ×800). Thrombi from a normal subject (A) and a heterozygous type II GT patient (E) are similar. They are mushroom shaped with narrow base and small fibrin deposits (arrowhead). They are mostly composed of packed platelets with irregular and undefined outline, nevertheless oval- or round-shaped platelets can be seen on the top of thrombi. Thrombi from variant GT (B) and type II GT (C) patients exhibit the same histological features. They are small to medium sized, composed of a core with densely aggregated platelets surrounded by many round-shaped platelets. Moreover, there is a thick fibrin network between and around thrombi and at the collagen coating (arrowhead). In a type I GT patient (D), there is no thrombus formation. Collagen is covered by a monolayer of platelets without any visible fibrin network.

Download figure to PowerPoint

image

Figure 2. Immunohistochemistry for VWF of collagen-induced platelet depositions at 1600/s after 4·5 min perfusion with non-anticoagulated blood (APAAP/Fast-red, original magnification ×800). In normal subjects (A), variant GT (B) and type II GT patients (C), VWF is seen at the interphase between platelet thrombi and collagen coating ([RIGHTWARDS ARROW]), inside the thrombi (black arrowhead) and fibrin network (open arrowhead). In type I GT, VWF is present in a thin monolayer between the platelets and collagen coating (D).

Download figure to PowerPoint

Patients with variant and type II Glanzmann's thrombasthenia

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

In patients with variant GT (patients 1 and 2) or type II GT (patient 3), medium sized thrombi (respectively 32%, 43% and 49% of the normal values) were surprisingly observed, with a maximal adhesion as they covered the entire inner surface (Table I and Fig 1B and C). These thrombi were composed of a core with densely aggregated platelets surrounded by many round-shaped platelets and connected by a fibrin meshwork. This fibrin meshwork was very thick, surrounded all the thrombi and was also noted on the collagen coating (Figs 1B and C, and 3). VWF was located within the thrombi, between platelet aggregates, and between the collagen coating and anchored platelet aggregates (Fig 2B and C).

image

Figure 3. Immunohistochemistry for fibrin of collagen-induced platelet depositions at 1600/s after 4·5 min perfusion with non-anticoagulated blood in a GT variant patient (ABC-peroxydase/DAB, original magnification ×800). In type 2 and variant GT (shown), a strong labelling is confirmed, and is seen surrounding and between platelet aggregates (arrowhead).

Download figure to PowerPoint

Patients with type I Glanzmann's thrombasthenia

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

Blood from two patients with type I GT (patients 4 and 5) did not induce any platelet thrombus formation in either case (Fig 1D) compared with the normal controls (Fig 1A). However, platelet–collagen adhesion exceeded the average adhesion observed in blood from the normal subjects (about 66%), as it was maximal [covering the entire surface in both patients (Table I)]. The platelets formed a monolayer on the collagen coating, without detectable fibrin deposition. They were round, only showed contact with the collagen surface and never spread, as opposed to most platelets from normal subjects (Fig 1D). VWF was seen between the platelet monolayer and collagen coating (Fig 2D).

Heterozygous daughters of Glanzmann's thrombasthenia patients

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

Blood from the heterozygous daughters of the type II GT patient (patient 3) (Fig 1E) and the variant GT patient (patient 1) formed thrombi in the perfusion chamber. These thrombi were of a normal size, identical to the thrombi of normal subjects and platelet–collagen adhesion was also in the normal range (Table I). Morphologically and immunohistochemically, no difference was noted between the thrombi of the two heterozygous subjects and those of normal subjects. They were mushroom shaped, without or with minor fibrin deposits at their base, and were composed of irregular packed platelets, with occasional round platelets located on the surface of thrombi. Labelling for VWF was strong at the seating and also present within the thrombi.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

In normal conditions, platelets adhere to the subendothelium, particularly to collagen, after vessel wall injury (Sakariassen et al, 1987). Nowadays, it is assumed that this adhesion first involves platelet GPIb–IX via VWF, especially when shear rates are high (Ruggeri, 1997), then subsequently platelet GPIaIIa and GPVI in a two-step mechanism (Barnes et al, 1998). Platelets then aggregate to form thrombi after fibrinogen binding to their αIIbβ3 integrin receptor (Nurden et al, 1999). In vitro platelet aggregation and in vivo thrombi formation are thus null in patients with type I GT, who completely lack αIIbβ3. Nevertheless, it is not known whether in vitro platelet aggregation is also absent in classical type II and type III (variant) disease; data are available on the in vivo thrombi formation capacity in these patients. To address this point, we used an ex vivo model of a cylindrical perfusion chamber, in which human type III collagen is exposed to non-anticoagulated blood as under physiological conditions. This chamber is circular, like a vessel wall. Owing to the small amount of blood required, it can be used in the clinical investigation of patients with bleeding disorders. In normal subjects, we observed many mushroom-shaped large thrombi with an average platelet adhesion of 66% and a moderate fibrin meshwork. VWF was present at the base of the thrombi and within the thrombi. In patients with type II and variant GT, platelet–collagen adhesion was maximal and we surprisingly observed the formation of medium-sized thrombi, together with a thick meshwork of fibrin around the platelet aggregates. A strong labelling for VWF was detected both inside thrombi and fibrin network. The heterozygous daughters of the patients with type II or variant GT had normal platelet adhesion and normal thrombus formation. In contrast, in patients with type I GT, we confirmed that there was neither thrombi formation nor fibrin meshwork as already reported (Sakariassen et al, 1986; Weiss et al, 1986, 1987, 1991; Nurden et al, 1999), although platelet collagen adhesion was maximal and underlined by a positive thin staining for VWF. Furthermore, we noticed that type I GT platelets showed no signs of spreading, possibly reflecting the lack of β3-mediated outside/inside platelet signalling, which in normal platelets induces cytoskeletal protein activation, filopodia emission and platelet spreading (for review see Fox, 2001).

Our data, and those of pharmacological studies using the same model (Andréet al, 1996), stress the crucial role of αIIbβ3 in platelet spreading and thrombi formation. Enhanced platelet adhesion, which was observed in all the GT patients, might be due to the failure of platelets to aggregate and the subsequent availability of the other glycoproteins, such as GPIb–IX, GPIa–IIa and GPVI, to adhere to collagen. Of note, the formation of thrombi of medium size with a thick fibrin meshwork in type II and variant GT was very surprising and has never been reported before. To date, most investigators used everted segments of de-endothelialized rabbit aorta or human umbilical artery, which were exposed in an annular perfusion chamber intended to recirculate anticoagulated blood (Baumgartner, 1973; Tschopp et al, 1975; Sakariassen et al, 1986; Weiss et al, 1993). In these previous studies, the thrombi formation was probably prevented by anticoagulation, which inhibits fibrin formation and can affect mechanisms involved in thrombotic pathways. Moreover, all the results obtained previously with citrated blood from patients with type I or type II GT were always pooled, thus masking the type II response (Weiss et al, 1986, 1991). Nevertheless, a slight increase in fibrin was reported in a perfusion chamber using non-anticoagulated blood from a pool of patients with type I or II GT (Weiss et al, 1986; Kirchhofer et al, 1995). Furthermore, to our knowledge, thrombogenesis has never been studied in variant GT which is very rare. In this cylindrical perfusion chamber, the fibrin-dependent thrombogenic responses from patients with type II or variant GT, whatever their mutations, were identical. Thus, classical type II GT might be due to a qualitative abnormality of the αIIbβ3 complex, leading to a very reduced expression of this complex, while in classical type III GT the qualitative abnormality of the complex is accompanied by a normal or subnormal amount. Recently, several patients with classical type II GT and a qualitative abnormality either on the αIIb gene (Wilcox et al, 1995; Grimaldi et al, 1998) or on β3 (Grimaldi et al, 1996; Jackson et al, 1998; Ward et al, 2000; Fullard et al, 2001; Morel-Kopp et al, 2001; Ruiz et al, 2001), as in patient 3 (Schlegel et al, 1999), have been reported. The original classification might be modified towards a new classification as we proposed recently (Bellucci & Caen, 2002). The new type 1 GT would correspond to the classical type I while the new type 2 GT would include the type 2a (classical type II) and the type 2b (classical type III). The unexpected presence of fibrin meshwork in the thrombi from our classical type II and variant patients could be due to the fact that fibrin formation is not negligible in this cylindrical perfusion chamber, perhaps produced at least partly from fibrinogen released from alpha granules; it might be less important in type I GT, as the failure of platelets to form thrombi may result in the maintenance of an efficient blood flow that might reduce fibrin formation, and also allow any fibrin and thrombin formed to be removed from the capillary tube. We noticed that thrombin generation in platelet-rich plasma from our variant or type II GT patients, according to the method of Reverter et al (1996), was normal or subnormal but not increased (data not shown), in agreement with the data of other GT patients (Reverter et al, 1996). Thus, the strong fibrin meshwork observed in these GT patients might not necessarily originate from an increased platelet procoagulant activity. But type II GT platelets with a much-reduced amount of αIIbβ3 exhibited reduced fibrinogen binding (Lee et al, 1981), which was nil in our variant GT patients (Caen et al, 1983; Nurden et al, 1987). Thus, in type II and variant GT, this defective fibrinogen binding might favour either an increase in platelet–fibrin interaction or the stabilization of bound fibrin. In agreement with this hypothesis, Niewiarowski et al (1981) had reported that polymerized fibrin was incorporated into both type II GT and normal platelets. Among the possible binding sites for fibrin, studies with mutant fibrinogen peptide ligands raised the possibility that fibrinogen and fibrin bind to different or overlapping sites on αIIbβ3 (Rooney et al, 1996); β3 integrin was then shown to bind fibrin by either the αIIbβ3 or αvβ3 complex (Katagiri et al, 1995), and subsequently β1 integrin was also shown to allow platelet adhesion to fibrin (Tanoue et al, 1999). In our patients, clot retraction was normal in patient 1 and present, although reduced, in our type II GT patients and patient 2, confirming a positive reaction of platelets with fibrin. Very recently, a new variant GT patient with a Leu262Pro mutation in the integrin β3 was reported (Ward et al, 2000). In this patient, by the expression of the mutant β3 integrin in human embryonal kidney 293 cells, it was possible to directly show that the abnormal αIIbβ3 complex was able to bind fibrin but not fibrinogen (Ward et al, 2000). On the other hand, the strong labelling of VWF detected in our model, together with the fibrin network, both inside and between the thrombi, argues for a role for VWF in the interaction of platelets with polymerized fibrin, as previously shown by Loscalzo et al (1986). This interaction, which involves platelet GPIb, was well demonstrated in vitro in platelet-rich plasma by Beguin et al (1999), and does amplify platelet procoagulant activity and thrombin generation. Finally, in the flow conditions studied in our model, platelet interaction of type II and variant patients with fibrin could first involve αIIbβ3 or αvβ3, as it has not been observed in type I GT patients lacking αIIbβ3, which could be amplified by the interaction of fibrin-bound VWF with platelet GPIb. The precise molecular mechanisms remain to be defined in further studies.

Taken together, our data underline a new distinction between classical type I GT on one hand and classical type II and type III GT on the other, and argue for a new classification based on molecular biology, as mentioned above. In the new type 1 GT (corresponding to the classical type I GT), there is no αIIbβ3 complex and no ex vivo thrombi formation; in the new type 2 GT (corresponding to the classical types II and III GT), the αIIbβ3 complex is present but qualitatively abnormal, and our data show the presence of ex vivo fibrin-mediated thrombi formation.

These data should be taken into consideration in future clinical studies when searching for correlation with the intensity of haemorrhagic manifestations; in addition, they could have pharmacological applications with the development of new anti-αIIbβ3 antithrombotic drugs which could interfere with the function of the complex, thus mimicking new type 2 forms of the disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References

We thank all the technicians from the pathological anatomy laboratory (Lariboisière Hospital) for their skilful technical assistance, and Mrs Aline Pruvot and Miss Aurélie Waernessyckle for their excellent secretarial work.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Normal subjects
  6. Patients with variant and type II Glanzmann's thrombasthenia
  7. Patients with type I Glanzmann's thrombasthenia
  8. Heterozygous daughters of Glanzmann's thrombasthenia patients
  9. Discussion
  10. Acknowledgments
  11. References
  • André, P., Arbeille, B., Drouet, V., Hainaud, P., Bal dit Sollier, C., Caen, J.-P. & Drouet, L.-O. (1996) Optimal antagonism of GPIIb/IIIa favors platelet adhesion by inhibiting thrombus growth: an ex-vivo capillary perfusion chamber study in the guinea pig. Arteriosclerosis Thrombosis and Vascular Biology, 16, 5663.
  • Barnes, M.-J., Knight, C.-G. & Farndale, R.-W. (1998) The collagen–platelet interaction. Current Opinion in Hematology, 5, 314320.
  • Barstad, R.-M., Orvim, U., Hamers, M.-J., Tjonnfjord, G.-E., Brosstad, F.-R. & Sakariassen, K.-S. (1996) Reduced effect of aspirin on thrombus formation at high shear rates and disturbed laminar blood flow. Thrombosis and Haemostasis, 75, 827832.
  • Baumgartner, H.-R. (1973) The role of blood flow in platelet adhesion, fibrin deposition and formation of mural thrombi. Microvascular Research, 5, 167179.
  • Baumgartner, H.-R., Tschopp, T.-B. & Weiss, H.-J. (1977) Platelet interaction with collagen fibrils in flowing blood. Impaired adhesion–aggregation in bleeding disorders. A comparison with subendothelium. Thrombosis and Haemostasis, 37, 1728.
  • Beguin, S., Kumar, R., Keularts, I., Seligsohn, U., Coller, B.-S. & Hemker, H.-C. (1999) Fibrin-dependent platelet procoagulant activity requires GPIb receptors and von Willebrand factor. Blood, 93, 564570.
  • Bellucci, S. & Caen, J. (2002) Molecular basis of Glanzmann's Thrombasthenia and current strategies in treatment. Blood Reviews (in press).
  • Caen, J.-P. (1989) Glanzmann's thrombasthenia. Baillieres' Clinical Haematology, 2, 609625.
  • Caen, J.-P. & Rosa, J.-P. (1995) Platelet–vessel wall interaction: from the bedside to molecules. Thrombosis and Haemostasis, 74, 1824.
  • Caen, J.-P., Castaldi, P.-A., Leclerc, J.-C., Inceman, S., Larrieu, M.-J., Probst, M. & Bernard, J. (1966) Congenital bleeding disorders with long bleeding time and normal platelet count. I. Glanzmann's thrombasthenia (report of 15 patients). American Journal of Medicine, 41, 426.
  • Caen, J.-P., Rosa, J.-P., Soria, C. & Boizard, B. (1983) Variant de thrombasthénie Paris-I Lariboisière, anomalie moléculaire du complexe glycoprotéinique plaquettaire IIb–IIIa. Comptes-Rendus de L'académie des Sciences, 296, 479481.
  • Chen, Y.-P., Djaffar, I., Pidard, D., Steiner, B., Cieutat, A.-M., Caen, J.-P. & Rosa, J.-P. (1992) Ser-752 [RIGHTWARDS ARROW] Pro mutation in the cytoplasmic domain of integrin β3 subunit and defective activation of platelet integrin αIIbβ3 (glycoprotein IIb–IIIa) in a variant of Glanzmann thrombasthenia. Proceedings of National Academy of Sciences of the United States of America, 89, 1016910173.
  • Chen, Y.-P., O'Toole, T.-E., Ylanne, J., Rosa, J.-P. & Ginsberg, M.-H. (1994) A point mutation in the integrin β3 cytoplasmic domain (S-752 [RIGHTWARDS ARROW] P) impairs bidirectional signaling through αIIbβ3 (platelet glycoprotein IIb–IIIa). Blood, 84, 18571865.
  • Djaffar, I. & Rosa, J.-P. (1993) A second case of variant of Glanzmann's thrombasthenia due to substitution of platelet GPIIIa (integrin β3) Arg 214 by Trp. Human Molecular Genetics, 2, 21792180.
  • Djaffar, I., Caen, J.-P. & Rosa, J.-P. (1993) A large alteration in the human platelet glycoprotein IIIa (integrin β3) gene associated with Glanzmann's thrombasthenia. Human Molecular Genetics, 2, 21832185.
  • Fox, J.E. (2001) Cytoskeletal proteins and platelet signaling. Thrombosis and Haemostasis, 86, 198213.
  • Fullard, J., Murphy, R., O'Neill, S., Moran, N., Cottridge, B. & Fitzerald, D.J. (2001) A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand. British Journal of Haematology, 115, 131139.
  • George, J.-N., Caen, J.-P. & Nurden, A.-T. (1990) Glanzmann's Thrombasthenia: the spectrum of clinical disease. Blood, 75, 13831395.
  • Ginsberg, M.-H., Lightsey, A., Kunicki, T.-J., Kaufman, A., Marguerie, G. & Plow, E.-F. (1986) Divalent cation regulation of the surface orientation of platelet membrane glycoprotein IIb. Correlation with fibrinogen binding function and definition of a novel variant of Glanzmann's thrombasthenia. Journal of Clinical Investigation, 78, 11031111.
  • Grimaldi, C.M., Chen, F., Scudder, L.E., Coller, B.S. & French, D.L. (1996) A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (β3) in a Chinese patient with Glanzmann's thrombasthenia. Blood, 88, 16661675.
  • Grimaldi, C.M., Chen, F., Wu, C., Weiss, H.J., Coller, B.S. & French, D.L. (1998) Glycoprotein IIb Leu 214Pro mutation produces Glanzmann thrombasthenia with both quantitative and qualitative abnormalities in GPIIb/IIIa. Blood, 91, 15621571.
  • Gruel, Y., Boizard, B., Daffos, F., Forestier, F., Caen, J. & Wautier, J.-L. (1986) Determination of platelet antigens and glycoproteins in the human fetus. Blood, 68, 488492.
  • Gruel, Y., Pacouret, G., Bellucci, S. & Caen, J.-P. (1997) Severe proximal deep vein thrombosis in a Glanzmann thrombasthenia variant successfully treated with a low molecular weight heparin. Blood, 90, 888890.
  • Hynes, R.-O. (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69, 1125.
  • Jackson, D.E., White, M.M., Jennings, L.K. & Newman, P.J. (1998) A Series 162 [RIGHTWARDS ARROW] Leu mutation within glycoprotein (GP)IIIa (integrin β3) results in an unstable αIIbβ3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia. Thrombosis and Haemostasis, 80, 4248.
  • Katagiri, Y., Hiroyama, T., Akamatsu, N., Suzuki, H., Yamazaki, H. & Tanoue, K. (1995) Involvement of alpha v beta 3 integrin in mediating fibrin gel retraction. Journal of Biological Chemistry, 270, 17851790.
  • Kirchhofer, D., Tschopp, T.B., Steiner, B. & Baumgartner, H.-R. (1995) Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood. Blood, 86, 38153822.
  • Lanza, F., Stierlé, A., Fournier, D., Morales, M., André, G., Nurden, A.-T. & Cazenave, J.-P. (1992) A new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb–IIIa complexes and a glycoprotein IIIa 214Arg [RIGHTWARDS ARROW]214Trp mutation. Journal of Clinical Investigation, 89, 19952004.
  • Lawrence, J.-B. & Gralnick, H.-R. (1987) Monoclonal antibodies to the glycoprotein IIb–IIIa epitopes involved in adhesive protein binding: effects on platelet spreading and ultrastructure on human arterial subendothelium. Journal of Laboratory and Clinical Medicine, 109, 495503.
  • Le Charpentier, Y., Galian, A., Gaste, A. & Herbe, L. (1981) L′etude en immunofluorescence des biopsies jejunales (incluses en resine epoxy) au cours de la maladie des chaines alpha. Annales de Pathologie, 1, 160162 .
  • Lee, H., Nurden, A.-T., Thomaidis, A. & Caen, J.-P. (1981) Relationship between fibrinogen binding and the platelet glycoprotein deficiencies in Glanzmann's thrombasthenia type I and II. British Journal of Haematology, 48, 4757.
  • Loscalzo, J., Inbal, A. & Handin, R.I. (1986) Von Willebrand protein facilitates platelet incorporation into polymerizing fibrin. Journal of Clinical Investigation, 78, 11121119.
  • Morel-Kopp, M.C., Melchior, C., Chen, P., Ammerlaan, W., Lecompte, T., Kaplan, C. & Kieffer, N. (2001) A naturally occuring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function. Thrombosis and Haemostasis, 86, 14251434.
  • Nachman, R.-L. & Leung, L.-L. (1982) Complex formation of platelet membrane glycoprotein IIbIIIa with fibrinogen. Journal of Clinical Investigation, 69, 263269.
  • Newman, P.-J., Seligsohn, U., Lyman, S. & Coller, B.-S. (1991) The molecular genetic basis of Glanzmannn thrombasthenia in the Iraqi-Jewish and Arab populations in Israel. Proceedings of National Academy of Sciences of the United States of America, 88, 31603164.
  • Niewiarowski, S., Levy-toledano, S. & Caen, J.-P. (1981) Platelet interaction with polymerizing fibrin in Glanzmann's thrombasthenia. Thrombosis Research, 23, 457463.
  • Nurden, A.T. (1999) Inherited abnormalities of platelets. Thrombosis and Haemostasis, 82, 468480.
  • Nurden, A.-T. & Caen, J.-P. (1974) An abnormal glycoprotein pattern in three cases of Glanzmann' thrombasthenia. British Journal of Haematology, 28, 253260.
  • Nurden, A.-T. & Caen, J.-P. (1975) Specific roles for platelet surface glycoproteins in platelet function. Nature, 255, 720722.
  • Nurden, A.-T., Rosa, J.-P., Fournier, D., Legrand, C., Didry, D., Parquet, A. & Pidard, D. (1987) A variant of Glanzmann's thrombasthenia with abnormal glycoprotein IIb–IIIa complexes in the platelet membrane. Journal of Clinical Investigation, 79, 962969.
  • Nurden, A.-T., Poujol, C., Durrieu-Jais, C. & Nurden, P. (1999) Platelet glycoprotein IIb/IIIa inhibitors: basic and clinical aspects. Arteriosclerosis Thrombosis and Vascular Biology, 19, 28352840.
  • Reverter, J.-C., Béguin, S., Kessels, H., Kumar, R., Hemker, H.-C. & Coller, B.-S. (1996) Inhibition of Platelet-mediated, tissue factor induced thrombin generation by the mouse/human chimeric 7E3 antibody. Journal of Clinical Investigation, 98, 863874.
  • Rooney, M.M., Parise, L.V. & Lord, S.T. (1996) Dissecting clot retraction and platelet aggregation. Journal of Biological Chemistry, 271, 85538555.
  • Ruggeri, Z.-M. (1997) Mechanisms initiating platelet thrombus formation. Thrombosis and Haemostasis, 78, 611616.
  • Ruiz, C., Liu, C.Y., Sun, Q.H., Sigaud-Fiks, M., Fressinaud, E., Muller, J.Y., Nurden, P., Nurden, A.T., Newman, P.J. & Valentin, N. (2001) A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb–IIIa (αIIbβ3) integrin locked in a high-affinity state and a Glanzmann thrombasthenia-like phenotype. Blood, 98, 24322441.
  • Sakariassen, K.-S., Nievelstein, P.-F.-E.-M., Coller, B.-S. & Sixma, J.-J. (1986) The role of platelet membrane glycoproteins Ib and IIb–IIIa in platelet adherence to human artery subendothelium. British Journal of Haematology, 63, 681691.
  • Sakariassen, K.-S., Fressinaud, E., Girma, J.-P., Meyer, D. & Baumgartner, H.-R. (1987) Role of platelet membrane glycoproteins and von Willebrand factor in adhesion of platelets to subendothelium and collagen. Annals of the New York Academy of Sciences, 516, 5265.
  • Sakariassen, K.-S., Kuhn, H., Muggli, R. & Baumgartner, H.-R. (1988) Growth and stability of thrombi in flowing citrated blood: assessment of platelet–surface interactions with computer-assisted morphometry. Thrombosis and Haemostasis, 60, 392398.
  • Schlegel, N., Chen, P., Binard, S., Maisonneuve, L., Rosa, J.-P., Kieffer, N. & Caen, J.-P. (1999) Type II Glanzmann thrombasthenia (GT) in a compound heterozygote for the glycoprotein (GP) IIIa gene associating two novel mutations, a stop codon (Arg 216) and a Cys598Tyr substitution in mature GPIIIa. Blood, 94, 451a.
  • Shattil, S.-J., Kashiwagi, H. & Pampori, N. (1998) Integrin signaling: the platelet paradigm. Blood, 91, 26452657.
  • Tanoue, K., Baba, I., Akamatsu, N., Aoki, K., Arai, M., Takahashi, K. & Suzuki, H. (1999) Platelet adhesion to fibrin is mediated by β1 as well as β3 integrins. Thrombosis and Haemostasis, s250, 784a.
  • Tschopp, T.-B., Weiss, H.-J. & Baumgartner, H.-R. (1975) Interaction of thrombasthenic platelets with subendothelium: normal adhesion, absent aggregation. Experientia, 31, 113116.
  • Ward, C.-M., Kestin, A.-S. & Newman, P.-J. (2000) A Leu262Pro mutation in the integrin beta(3) subunit results in an alpha(IIb)–beta(3) complex that binds fibrin but not fibrinogen. Blood, 96, 161169.
  • Weiss, H.-J., Turitto, V.-T. & Baumgartner, H.-R. (1986) Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb–IIIa, Ib, and storage granules. Blood, 67, 322330.
  • Weiss, H.-J., Baumgartner, H.-R. & Turitto, V.-T. (1987) Regulation of platelet–fibrin thrombi on subendothelium. Annals of the New York Academy of Sciences, 516, 380397.
  • Weiss, H.-J., Turitto, V.-T. & Baumgartner, H.-R. (1991) Further evidence that glycoprotein IIb–IIIa mediates platelet spreading on subendothelium. Thrombosis and Haemostasis, 65, 202205.
  • Weiss, H.-J., Hoffmann, T., Yoshioka, A. & Ruggeri, Z.-M. (1993) Evidence that the arg1744 gly1745 asp1746 sequence in the GPIIb–IIIa-binding domain of von Willebrand factor is involved in platelet adhesion and thrombus formation on subendothelium. Journal of Laboratory Clinical Medicine, 122, 324332.
  • Wilcox, D.A., Paddock, C.M., Lyman, S., Gill, J.C. & Newman, P.J. (1995) Glanzmann thrombasthenia resulting from a single amino acid substitution between the second and third calcium-binding domain of GPIIb. Journal of Clinical Investigation, 95, 15531560.