Scand J Haematol(l979) 22, 267-276

A Platelet Defect in a Patient with Eosinophilic Leukaemia: Absent Ristocetin-Induced Platelet Aggregation Associated with a Reduced Platelet Sialic Acid Content J. L. WAUTIER, H. SOUCHON, D. DUPUIS,J. P. CAEN& A. T. NURDEN HGpital Lariboisi;re, Paris, France

Platelets from a patient with eosinophilic leukaemia were not aggregated by ristocetin. The defect was not corrected by normal human plasma and was due t o a platelet abnormality. The patient’s platelets also showed a diminished sensitivity to aggregation by bovine factor Vlll,,wB~.The defect was not associated with a prolonged bleeding time. N o abnormalities were detected in ADP, collagen or thrombin-induced platelet aggregation. Biochemical studies showed that the platelets were deficient in sialic acid. This deficiency was associated with a reduced staining for glycoprotein I following SDS-polyacrylamide gel electrophoresis. The results suggest an acquired platelet surface abnormality. K e y words: bovine factor VllIv,yB. - eosinophilic leukaemia - glycoprotein I - platelet aggregation - ristocetin - sialic acid

Accepted for publication June 2, 1978 Correspondence to: Dr. I. L. Wautier, Unit6 150 de I’Inserm, HGpital Lariboisitre, 2, rue Ambroise Part, 75475 Pans Cedex 10, France

The two classical haemostatic disorders where ristocetin-induced platelet aggregation is quantitatively reduced or absent are von Willebrand’s disease and the BernardSoulier syndrome. The functional abnormalities in von Willebrand’s disease appear due to either a severe reduction in the plasma von Willebrand protein level or to functional deficiencies within the molecule (see Hoyer 1976). In the Bernard-Soulier syndrome, where no such plasma abnormalities have been reported, a platelet defect involving surface glycoproteins appears responsible (Evensen et al 1974, Caen et al 1976). The defect in Bernard-Soulier platelets is further manifested by their lack of

aggregation by bovine fibrinogen preparations (Bithell 1972, Caen et a1 1976), the active component of which is bovine factor VIIIy,yB.(Bottechia & Vermylen 1976). We now report a further example of an absent ristocetin-induced platelet aggregation resulting from a platelet defect. This probably acquired abnormality occurred in a patient with chronic eosinophilic leukaemia. Functional and biochemical studies will be described which underline the unusual specificity of this newly discovered platelet defect which differed from that observed in the Bernard-Soulier syndrome in that it was not associated with a prolonged bleeding time.

0036-553X/79/030267-10 $02.50/0 .@ 1979 Munksgaard, Copenhagen

268

WAUTIER, SOUCHON, DUPUIS, CAEN & NURDEN

bovine thrombin (Stago) 0.2-0.6 units; collagen (Stago) 5, 10 and 12 pg; ristocetin (Lundebeck) Case report. The patient (LeT)was 52 years old 0.5-2.5 mg and bovine fibrinogen (Kabi) 0.8 and when his disease was first detected in 1974. The 1.6 mg. All reagents were prepared in physiologinitial symptoms were asthenia, weight loss and ical saline and the given concentrations are their splenomegaly. A routine haematological examina- final concentratiodml in the aggregometer tube. tion gave the following results: RBC 4.4 x 10l2/l, All aggregations were performed at a final plateH b 13.3 d d l , WBC 33.5 x 109A with 90 % eosino- let count of 2 x 10Vml. philic polymorphonuclear Ieuucocytes. Other clinical data (Wautier et al 1976) was consistent with Preparation of washed platelet suspensions. Blood the diagnosis of chronic eosinophilic leukaemia. was taken into a modified hypercitrated antiDuring the following 3 years the leucocyte count coagulant containing 2.73 % citric acid, 4.48 % increased t o 50 x W/l. The patient recently devel- trisodium citrate and 2.0 % glucose (1 vol. antioped a cardiac failure diagnosed as an endo- coagulant : 9 vol. blood). PRP was prepared by myocardial fibrosis and cerebral vascular throm- centrifugation at 150 g for 15 min. The platelets were sedimented a t 3000 g for 15 min and rebosis. suspended in washing buffer which consisted of Haemostatic tests. The bleeding time (Ivy test), 0.01 M Tris HCI pH 7.4 containing 0.15 M NaCl plasma factor VIII procoagulant activity and and 1 mM EDTA (12 ml buffer/100 ml PRP). The other haemostatic tests were determined using concentrated platelet suspension was centrifuged standard procedures as previously described by at 2800 g for 15 min in transparent conical plastic Sultan et al (1974). Factor VIII related antigen tubes-contaminating red cells and white cells was measured by the Laurel1 technique using a (including eosinophils) sedimented t o the base specific antiserum against human factor VIII. Von of the cone. The overlying platelets were reWillebrand factor (ristocetin cofactor) activity suspended and the procedure repeated until the was measured using gel filtered platelets (see be- platelet suspension was free of contaminating cells low) which were subsequently washed and re- as assessed under the phase contrast microscope. suspended in buffer as described by Sultan et al Control and patient samples were treated identically. The platelets were further washed twice (1974). with equivalent PRP volumes of washing buffer. They were finally resuspended a t 2 x W/ml in Platelet size. Patient and control human blood the washing buffer to which had been added as was obtained by venepuncture and taken directly protease inhibitors 0.4 mM phenylmethyl sulfonyl into 3.8 % trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 150 g fluoride (Sigma) and 0.5 mM NCB-glutamyl tyrofor 10 min. Platelet vol. measurements were made sine (Sigma) (Nachman et al 1973). Approximately using a Model B Coulter Counter with a 70 p M 50-60 % of the platelets in the PRP were reaperture tube attached t o a multichannel analyzer covered in the final suspensions, n o differences wtre noted between the percentage recovery of as described by Kahn et a1 (1975). the patient or control platelets. MATERIAL AND METHODS

Platelet aggregation. Citrated PRP was prepared as described above. Gel filtered platelets (GFP) were isolated according t o the method of LevyToledano et a1 (1972), the buffer used consisted of 0.14 M NaCI, 0.025 M Tris, 0.055 M glucose, 0.056 M KCI, 1 x 10-4 M MgClz and 6 x 10-6 M CaC12 buffered to p H 7.2 with N HCI. Aggregation was performed in a Labintec aggregometer at 37O C for 3 min. Aliquots (0.3 ml) of GF P were mixed with 0.1 ml GF P buffer or control or patient platelet-poor plasma (PPP). The following aggregating agents were employed: ADP (Sigma, sodium salt grade 1) 0.6,1.2 and 2.5 pM;

Sialic acid determination. 1 ml aliquots of washed platelets were frozen and thawed before being incubated a t 80° C for 1 h in the presence of 0.05 M H2W4. The released sialic acid was determined using the thiobarbituric acid assay of Aminoff (1961). The optical densities were read a t both 532 and 549 nm and adjusted for the presence of 532 nm adsorbing impurities (Warren 1959). Identical washed platelet samples were solubilised at 37O C for 30 min in the presence of 1 M NaOH and their protein content determined (Lowry et al 1951). The results were calculated using N-acetyl-

ACQUIRED PLATELET SIALIC ACID DEFIClENCY

269

TABLE 1 Haemostatic data ~~

I

Control range

Test Bleeding time (min)

Patient 7 (5-7) 180 (150-220) 12 (12-13) 55 (53-62) 340 (300-370) 100 (8@120) 80 (70-130) 110 (100-130)

Q9 200-300

Platelet count (x 10Vpl) Plasma prothrombin time (s)

12- 14

Partial prothrombin time (s)

54- 65 200-400

Plasma fibrinogen level (mg/dl) Factor VIII coagulant activity ( d d l )

65-150

Factor VIII related antigen ( d d l )

65-124

Von Willebrand factor activity (u/dl)

60-140

The control data was obtained from 70 determinations. The values for the patient are the mean (range in parentheses) of at least 6 determinations for each test.

neuraminic acid (Sigma, Type IV) and crystallized bovine serum albumin (Sigma) as standards. SDS-polyacrylamide gel electrophoresis (SDSP A G E ) . Aliquots of the washed platelet suspensions were incubated at 100°C for 5 min in the presence of 2 % w/v SDS. After cooling, the samples were dialysed overnight against 0.2 % SDS and their protein concentration determined (Lowry et al 1951). Aliquots containing 400 pg protein were diluted t o 0.15 ml with 0.2 % SDS. The samples were then further incubated for 5 min at looo C in the presence of 0.0625 M Tris HCl (pH 6.8) containing 2 % SDS and 5 % v/v 2-mercaptoethanol. SDS-PAGE was performed according t o the procedure of Laemmli (1970), 10 x 0.5 cm 7 % polyacrylamide (0.192 % bis) separating gels were employed. Following electrophoresis the gels were fixed in an isopropanol-containing solution (Fairbanks et al 1971), stained by the periodate-Schiff reaction (Zacharius et al 1969) and densitometrically scanned at 546 nm using an ISCO Model UA4 absorbance monitor. Platelet-leucocyte interaction. Eosinophilic polymorphonuclear leucocytes were obtained from the patient by leukapheresis. The cells were collected

t

0 .-v)

* m

'O0l

Figure 1. Ristocetin-induced aggregation of gelfiltered platelets. Ristocetin (2.5 mg/ml) was added to suspensions of (a) control GFP, (b) patient 0.1 ml patient PPP, GFP, (c) control G F P (d) control G F P 0.1 ml control PPP and 0.1 ml control PPP. The re(e) patient G F P sults are representative of 6 experiments.

+

+

+

WAUTIER, SOUCHON, DUPUIS, CAEN & NURDEN polymorphonuclear leucocytes, were added to control G F P t o give leucocyte : platelet ratios of 1 : 40 and 1 : 10 immediately prior to platelet aggregation tests.

2 100L .-0cn .-cn

E

& # A W ' W

cn C

*

= a

50-

RESULTS

/pF

Haemostatic data. The patient's bleeding A time and plasma prothrombin time were well within the normal range (Table 1). No abnormalities were detected in the plasma levels of factor VIII procoagulant activity, the factor VIII related antigen or the von 1 min Willebrand factor (ristocetin cofactor) acFigure 2. Bovine fibrinogen (containing bovine tivity. The plasma fibrinogen concentration factor VIIIvwF) - induced platelet aggregation. was normal. The platelet count was on the Bovine fibrinogen at 0.8 mg/ml (a, b) and 1.6 mg/ low side of the normal range. .c

.-JU J

/

-

ml (c, d) was added tQ suspensions of control (a, c) and patient (b, d) GFP. The results are representative of 6 experiments. on ACD anticoagulant and sedimented in plastic conical tubes (12 ml vol.) at 200 g for 10 min. The leucocytes were carefully resuspended in cold G F P buffer leaving behind a small pellet of contaminating erythrocytes which had pelleted t o the base of the tube. This process was repeated and the isolated leucocytes, which consisted of 99 %

Platelet size. An electron microscope study (results not shown) revealed no abnormalities in platelet morphology, with the platelet size and shape being similar to those of normal platelets. On one occasion the vol. distribution curve of the patient's platelets in citrated PRP was determined using a Model B Coulter Counter. The calculated

C

d

P f

e

a b

I

t4-P t , lm i, n Figure 3. Collagen, ADP and thrombin-induced platelet aggregation. Collagen (10 pg), ADP (1.2 pM) or thrombin (0.4 units) was added to suspensions of control (a, c, e) and patient (b, d, f) GFP. The ADP and collagen induced platelet aggregation was performed in the presence of 0.1 ml control PPP.

27 1

ACQUIRED PLATELET SIALlC ACID DEFIClENCY TABLE 2 Sialic acid and protein content of LeT platelets Subject LeT Control

I

Protein (mg/109 platelets)

I

1.64 (1.53-1.70) 2.02 (1.95-2.05)

Sialic acid oig/IO9 platelets)

10.3 ( 9.6-11.0) 15.5 (14.3-17.8)

1

(fcg/mg protein) 6.3 ( 6 . 2 4 4 ) 7.7 (7.3-8.8)

The sialic acid and protein content of the platelets were determined as described in the methods. Results are expressed as the mean (range in parentheses) of assays performed on washed LeT and control platelet suspensions isolated on 4 (pg sialic acid/109 platelets) or 3 (mg proteidl09 platelets, ,ug sialic acid/mg platelet protein) separate occasions.

mean volume of the LeT platelets was 7.0 fl which was the identical vol. obtained as the mean of the corresponding values for the platelets of 16 controls. Platelet aggregation Ristocetin. Preliminary studies using PRP showed that ristocetin induced no aggregation of the LeT platelets even at high ristocetiii concentrations (2.5 mg/ml). Further studies were performed using gel-filtered platelets (Figure 1). Ristocetin-induced no aggregation of the LeT GFP either alone or in the presence of patient or normal human plasma. Control GFP were normally aggregated in the presence of patient PPP thus ruling out the presence of an acquired plasma inhibitor. The residual eosinophil contamination of the LeT GFP was assessed as not more than 25 eosinophils per lo5 platelets. To rule out the possibility that eosinophils themselves were capable of inhibiting ristocetin-induced platelet aggregation, polymorphonuclear leucocytes (99 % eosinophils) were isolated from the patient and added to control GFP to give leucocyte : platelet ratio of 1 : 40 prior to the addition of normal human plasma and ristocetin (2.5 mg/ml). No significant inhibition of the ristocetin-induced platelet aggregation was observed.

Bovine factor VZZZT-ll.F.When high concentrations (1.6 mg/ml) of a bovine fibrinogen preparation (containing bovine factor VIIIr,TB as the aggregating factor) were added to the LeT GFP aggregation occurred but the response was delayed and the extent of the aggregation reduced (Figure 2). At a lower concentration (0.8 mg/ml) no aggregation was induced in the LeT GFP by the bovine factor VIIIy,vF. ADP, collagen and thrombin. No abnormalities were detected in either ADP (0.6, 1.2 and 2.5 pM) or collagen (5, 10 and 12 pg) induced aggregation of the LeT GFP performed in the presence of either patient or control PPP. Similarly the LeT GFP were normally aggregated by thrombin (0.2, 0.4 and 0.6 units) this study being performed without addition of PPP. These findings are illustrated for one concentration of each reagent in Figure 3. Platelet siafic acid and protein content. The sialic acid content of LeT platelets has been determined using washed platelet suspensions isolated on four different occasions (Table 2). Expressed as p g sialic acid per lo9 platelets the mean value for the LeT platelets was 33 % decreased compared with that for the control platelets isolated

272

WAUTIER, SOUCHON, DUPUIS, CAEN & NURDEN

under the same conditions. In the initial determination the results were solely calculated in terms of the platelet count. Subsequently we also determined the protein concentration of the LeT platelets. The LeT platelet protein content was also found to be decreased, by an average of 19 % compared with the corresponding controls.When the platelet sialic acid concentration was expressed as pg sialic acid per mg protein, an average decrease of 17 % was observed for the LeT platelets. The absence of eosinophils in the LeTwashed platelet suspensions was confirmed by an identical 549 : 532 nm absorbance ratio in the butanol extracts of the LeT and control platelet suspensions following the thiobarbituric acid assay. An increased 532 nm absorbance would be expected (Warren 1959) if nucleated cells were present in the sample. Platelet glycoprotein profiles. Aliquots of washed LeT and control platelet suspensions were solubilized by 2 % SDS and constituent disulfide bonds reduced by the addition of 5 % 2-mercaptoethanol. The solubilized platelets were subjected to SDSPAGE using the discontinuous SDS-trisglycine buffer system of Laemmli (1970). A typical glycoprotein profile for normal human platelets is illustrated in Figure 4. The major PAS-staining membrane glycoproteins (GP Ib, IIb, IIIa and IIIb) are labelled according to the nomenclature of Clemetson et a1 (1977). Also labelled are glycocalicin, which is a peripheral surfaceorientated and labile fraction of glycoprotein I (Okumura & Jamieson 1976a); and the thrombin sensitive protein (TSP) a granule-localized glycoprotein released from washed platelets by thrombin (Hagen et a1 1977). The details of the methods used for

Ib

I

+ Glycocalicin

I

0.2

,

0.4

Relative

,

,

I

I

)

0.8

0.6

Mobility

Figure 4. Densitmetric profiles of the glycopre teins of washed control and LeT platelets following SDSPAGE. Aliquots (400 p g protein) of SDSsolubilised and 2-mercaptoethanol reduced washed, platelets were subjected to SDSPAGE on 7 % polyacrylamide gels. The electrophoretic system of Laemmli (1970) was employed. The glycoproteins were located by the periodate-Schiff reaction. Electrophoresis was from left to right and the gel origin is marked (+).

the precise identification of these glycoproteins following SDS-PAGE using the Laemmli system will be given elsewhere (Nurden & Dupuis, in preparation). No qualitative glycoprotein abnormalities were observed for the LeT platelets although a decreased total carbohydrate staining intensity within the gel was apparent. This was particularly noticeable in the glycoprotein I (GP Ib glycocalicin) region, an observation which was consistent for four different preparations of

+

ACQUIRED PLATELET SIALIC ACID DEFICIENCY

washed LeT platelets. On the last occasion studied an internal standard (20 p g fetuin) was added to both the solubilized LeT and control platelet samples prior to the electrophoresis to allow the accurate quantification of the staining intensities of analogous bands as described by George (1976). On this occasion the staining intensity of LeT platelet glycoprotein I was found to be 30 % reduced compared to the corresponding value of the control platelet glycoprotein I band. None of the other bands was reduced in staining intensity. No abnormalities were detected in the polypeptide profiles of the LeT platelets. DISCUSSION

The present study describes the absence of aggregation by ristocetin of the platelets of a patient with eosinophilic leukaemia. Normal levels of factor VIII related antigen and von Willebrand factor (ristocetin cofactor) activity were located in the patient's plasma. The latter finding, performed using washed control human platelets, suggested that the LeT plasma von Willebrand protein was capable of normally supporting ristocetin-induced platelet aggregation. No inhibition of ristocetin-induced aggregation of control gel-filtered platelets was induced by the LeT plasma showing the absence of a plasma inhibitor and the inability of ristocetin to aggregate the LeT gel-filtered platelets was not corrected by the addition of normal human plasma. All evidence therefore pointed to the presence of a platelet abnormality. This appeared to be a specific defect as no abnormality was detected in platelet aggregation induced by the physiological aggregation-inducing agents tested (ADP, collagen, thrombin). Bernard-Soulier platelets are not aggreScand J Haematol(l979) 22

273

gated by ristocetin as a result of a platelet defect (Howard et a1 1973, Caen & LevyToledano 1973). A low platelet sialic acid content in this syndrome (Grottum & Solum 1969, Evenson et a1 1974) has been shown to be associated with severe molecular deficiencies of specific surface glycoproteins (Nurden & Caen 1975, Caen et a1 1976). The platelets of 4 Bernard-Soulier patients have recently been examined in our laboratory using the SDS-PAGE procedure employed in the present study (Nurden & Caen 1979). A consistent and specific finding was the severe reduction in the staining intensity of the glycoprotein I band (membrane G P Ib glycocalicin). Studies using an alloantibody isolated from the plasma of a polytransfused Bernard-Soulier patient have strongly suggested that the absence of ristocetin-induced platelet aggregation observed in the Bernard-Soulier syndrome is directly related to the platelet glycoprotein deficiency (Tobelem et a1 1976, Degos et a1 1978). In view of the above observations a glycoprotein abnormality was suspected in the LeT platelets. The LeT platelets were shown to have a low sialic acid content. Expressed as p g sialic acid per lo9 platelets the average value for the patients platelets was 33 % lower than that obtained for the control platelets isolated and studied under the same conditions. Furthermore the level of LeT platelet sialic acid (10.3 j18/109 platelets) was considerably beneath the lowest value in the range (15.9-26.6 pg/109 platelets) previously obtained in our laboratory during a study of 28 normal human donors (Nurden & Caen 1974). The low sialic acid content could not be accounted for by a reduced platelet size, the LeT platelet volume as calculated using a Coulter Counter was normal. Furthermore no platelet morpho-

+

18

274

WAUTIER, SOUCHON, DUPUIS, CAEN & NURDEN

logical abnormalities were detected by electron microscopy. On each of the four occasions the patient’s platelets were studied a consistent finding was a reduced periodate-Schiff staining intensity of the glycoprotein I band located following SDS-PAGE. However, the severity of the reduction (estimated as being 30 % on the last occasion studied) was much less than that observed in the Bernard-Soulier syndrome. Of the major glycoprotein I constituents at least glycocalicin is rich in sialic acid (Okumura & Jamieson 1976b). The reduced staining for carbohydrate of this band therefore correlates well with the low LeT platelet sialic acid content. It is interesting that alterations in the periodate-SchB staining intensity of the glycoproteins have been observed for the platelets of patients with other types of leukaemia (Vainer & Bussel 1977, Bolin et a1 1978) although no studies on platelet aggregation by ristocetin were performed. The ristocetin-platelet interaction proceeds through an initial agglutination of the platelets, as indicated by the ability of ristocetin to agglutinate formalin-fixed platelets (Grant et a1 1976). The agglutination appears to result from the binding of factor VIIIvwBto the platelets in the presence of the ristocetin (Zucker et a1 1977). The total lack of optical density change observed following the addition of the ristocetin to the LeT GFP indicated a lack of the initial agglutination and therefore a platelet surface defect. Several possible explanations may be suggested to explain the LeT platelet insensitivity to ristocetin. Kuwahara (1977) inhibited ristocetin-induced aggregation of guinea pig platelets by borate which forms complexes with carbohydrate groupings. A surface carbohydrate abnormality in the LeT platelets

could involve a specific receptor essential for ristocetin-induced platelet aggregation. However, it should be noted that this is unlikely to uniquely involve sialic acid groupings as neuraminidase treatment of human platelets does not induce a loss of platelet sensitivity to ristocetin (Greenberg et a1 1975). Okumura & Jamieson (1976~) competitively inhibited ristocetin-induced human platelet aggregation with purified glycocalicin. It is therefore also possible that the concentration of this glycoprotein on the LeT platelet surface is below that necessary for ristocetin-induced aggregation to occur. Finally Grant et a1 (1976) showed that ristocetin-induced aggregation of normal human platelets could be inhibited by ADP. These authors suggested that this was a result of an ADP-induced rearrangement of platelet surface groupings. This finding implies that the platelet surface topography is most sensitive to change. Therefore a further possibility is that the platelet receptor involved in ristocetin-induced aggregation is no longer accessible on the LeT platelet surface, although it should be noted that the LeT platelets were not refractory to ADP. Okumura & Jamieson (1976~) also showed that glycocalicin inhibited platelet aggregation by thrombin. As a result these authors suggested that glycocalicin may be a receptor for both ristocetin and thrombin on the platelet surface. In the current study the absence of LeT platelet aggregation by ristocetin was not associated with significant changes in platelet aggregation by thrombin. This suggests that the defective grouping responsible for the lack of ristocetininduced LeT platelet aggregation is not involved in platelet aggregation induced by thrombin although it does not rule out the possibility that different parts of the same

ACQUIRED PLATELET SlALlC ACID DEFICIENCY

or different glycocalicin molecules may be involved in the two processes. The results with the presently studied patient show that a lack of ristocetin-induced platelet aggregation can occur in the absence of detectable abnormalities in the plasma factor VIIIV\VF,and in the absence of the type of severe molecular glycoprotein deficiencies located in Bernard-Soulier platelets. It is interesting that the inability of ristocetin to aggregate the LeT platelets was accompanied by their reduced sensitivity to aggregation by bovine factor VIIIvlvB7but was not accompanied by a detectable change in the patient’s bleeding time. These observations may reflect the d s e r e n t degrees of sensitivity of these tests to modifications in the platelet surface glycoprotein topography. ACKNOWLEDGEMENT We would like to thank D. Saint-Dizier, Institut de Recherches Servier, Suresnes, France, for performing the platelet volume measurements. This work was supported by INSERM contract number C.R.L. 78.5.128.1 and DGRST contract number 77.7.0243. REFERENCES Aminoff D (1961) Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 81, 384-90. Bithell T C , Parekh S J & Storn R R (1972) Platelet function in the Bernard-Soulier syndrome. A n n N Y Acad Sci 201, 14540. Bolin R B, Okumura T & Jamieson G A (1977) Changes in distribution of platelet membrane glycoproteins in patients with myeloproliferative disorders. A m J Haematol 3, 63-71. Bottechia D & Vermylen J (1976) Factor VIII and human platelet aggregation. I. Evidence that the platelet aggregating activity of bovine factor VIII is a property of its carrier protein subunit. Br J Haematol 34, 303-11.

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Caen J & Levy-Toledano S (1973) Interaction between platelets and von Willebrand factor provides a new scheme for primary hemostasis. Nature New Biol 244, 15940. Caen J P, Nurden A T, Jeanneau C, Michel H, Tobelem G, Levy-Toledano S, Sultan Y, Valensi F & Bernard J (1976) Bernard-Soulier syndrome: a new platelet glycoprotein abnormality, Its relationship with platelet adhesion to subendothelium and with the factor VIII von Willebrand protein. J Lab Clin M e d 87, 58696. Clemetson K J, Pfueller S L, Liischer E F & Jenkins C S P (1977) Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography. Biochim Biophys Acta 464, 493-508. Degos L, Tobelem G, Lethielleux P, Levy-Toledano S, Caen J & Colombani J (1977) A molecular defect in platelets from patients with Bernard-Soulier syndrome. Blood 50, 899-905. Evensen S A, Solum N 0, Grottum K A & Hovig T (1974) Familial bleeding disorder with a moderate thrombocytopenia and giant blood platelets. Scand J Haematol 13, 203-14. Fairbanks G, Steck T L & Wallach D F H (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-16. Grant R A, Zucker M B & McPherson J (1976) ADP-induced inhibition of von Willebrand factor mediated platelet agglutination. Am J Physiol 230, 1406-10. George J N (1976) Platelet membrane glycoproteins: alteration during storage of human platelet concentrates. Thrombosis Research 8, 71924. Greenberg J, Packham M A, Cazenave J P, Reimers H-J & Mustard J F (1975) Effects on platelet function of removal of platelet sialic acid by neuraminidase. Lab Invest 32, 476-84. Grottum K A & Solum N 0 (1969) Congenital thrombocytopenia with giant platelets: a defect in the platelet membrane. Br J Haematol 16, 277-90. Hagen I, Olsen T & Solum N 0 (1976) Studies on subcellular fractions of human platelets by the lactoperoxidase-iodination technique. Biochini Biophys Acta 455, 214-55. Howard M A, Hutton R A & Hardisty R M (1973) Hereditary giant platelet syndrome: a disorder

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glycocalicin. I. Orientation of glycoproteins of the human platelet surface. J Biol Chem 251,

5944-49. Okumura T & Jamieson G A (1976b) Platelet glycocalicin. 11. Purification and characterisation. J Biol Chem 251, 5950-55. Okumura T & Jamieson G A (1976~)Platelet glycocalicin: a single receptor for platelet aggregation induced by thrombin or ristocetin. Thrombosis Research 8, 701-06. Sultan Y, Bernal-Hoyos E, Levy-Toledano S, Jeanneau C & Caen J P (1974) Dominant inherited familial factor VIII deficiency associated with thrombocytopathic thrombocytopenia. Pathol Biol 22, 27-36. Tobelem G, Levy-Toledano S, Bredoux R, Michel H, Nurden A T, Caen J P & Degos L (1976) New approach to determination of specific functions of platelet membrane sites. Nature 263,

427-29. Vainer H & Bussel A (1977)Altered platelet surface glycoproteins in chronic myeloid leukemia. Int J Cancer 19, 143-50. Warren L (1956) The thiobarbituric acid assay of sialic acids. J Biol Chem 234, 1971-75. Wautier J L, Dupuy E, Michel H & Caen J P (1976) Anomalie plaquettaire dans un syndrome my6loprolif6ratif B tosinophiles. Nouv Presse Med 5, 704-06. Zacharius R M, Zell T E, Morrison J M & Woodlock J J (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30, 148-52. Zucker M B, Kim S J , McPherson J & Grant R A Binding of factor VIII to platelets in the presence of ristocetin. Br J Haematol 35, 535-49.