American Journal of Hematology 33:261-266 (1990)

Enhanced Botrocetin-Induced Type IIB von Willebrand Factor Binding to Platelet Glycoprotein Ib Initiates Hyperagglutination of Normal Platelets K. Nishio, Y. Fujimura, K. Niinomi, Y. Takahashi, A. Yoshioka, H. Fukui, Y. Usami, K. Titani, Z.M. Ruggeri, and T.S. Zimmerman Department of Pediatrics (K.N., K.N., Y.T., A.Y., H.F.) and Blood Transfusion (Y.F.), Nara Medical College, Kashihara City, Nara, Japan; Laboratory of Biomedical Polymer Science, Institute of Comprehensive Medical Science, Fujita-Gakuen Health University, School of Medicine, Aichi, Japan (Y.U., K.T.); Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California (Z.M.R., T.S.Z.)

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutinationby von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPlb induced by these two substances have been reported. We have recently shown that the GPlb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPlb and GPlX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPlb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-inducedvWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPlb. To strengthen this concept, we have now found that heightened botrocetin-inducedtype llB vWF binding to platelet GPlb causes hyperagglutination of normal platelets. Key words: aggregation, agglutination, ristocetin

INTRODUCTION

A unique variant of von Willebrand disease (vWD), designated type IIB, was described by Ruggeri et al. [ l ] in 1980. It was characterized by the heightened interaction between IIB von Willebrand factor (vWF) and platelets in the presence of antibiotic ristocetin. Recent studies of De Marco et al. [2] have also indicated that the purified vWF of type IIB vWD directly binds to platelets with a low affinity even in the absence of ristocetin. Botrocetin, a protein partially purified from the venom of the snake Bothrops Jururucu, also induces platelet agglutination and vWF binding to platelet glycoprotein (GP) Ib, as does ristocetin [3-61. Although some differences between the platelet responses induced by ristocetin and those by botrocetin have been indicated, we have recently shown [6] that the botrocetin-dependent GPIb binding domain of vWF resides, as well as the ristocetin0 1990 Wiley-Liss, Inc.

Received for publication August 30, 1989; accepted November 16, 1989. Address reprint requests to Y. Fujimura, Department of Blood Transfusion, Nara Medical College, Kashihara City, Nara Japan. Presented in part at the XVIII Meeting of the World Federation of Hemophilia, Madrid, Spain, May 1988. This work was supported in part by a grant-in-aid of the Naito Medical Foundation to Y.F., A.Y., and K.T. and by grants-in-aid of the Japanese Ministry of Education in 1988 to H.F. and K.T. Abbreviations used: vWF, von Willebrand factor; vWD, von Willebrand disease; GP, glycoprotein; BSA, bovine serum albumin; HPLC, high-performance liquid chromatography; TBS, tris buffer saline; RIPA, ristocetin-induced platelet aggregation/agglutination; BIPA, botrocetin-induced platelet aggregationiagglutination; PRP, plateletrich plasma; PPP, platelet-poor plasma; Rcof, ristocetin cofactor; Bcof, botrocetin cofactor; MoAb, monoclonal antibody; DTE, dithioerythritol.

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dependent [7], in the 52/48 kDa tryptic fragment of vWF, which extends from amino acid residue 449 to 728 of the constituent subunit. In addition, we have provided the evidence that an anti-GPIb monoclonal antibody (AP- 1) also blocks the botrocetin-induced vWF binding to platelets. We report here that botrocetin does not initiate agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome who congenitally lacks platelet GPIWGPIX complex and GPV, whereas it does induce the heightened interaction between vWF of type IIB vWD and platelets from normal individuals. MATERIALS AND METHODS Reagents

Bothrops jararaca venom, apyrase, benzamidineHCI, ristocetin, and hirudin were purchased from Sigma Chemical Co. (St. Louis, MO). Leupeptin was supplied by Peptide Institute (Japan). Crystallized BSA was obtained from Calbiochem-Behring Corp. (La Jolla, CA). Carrier-free Na[ 12'1] was obtained from Amersham Corp. (Arlington Heights, IL). Purification of Botrocetin

Venom coagglutinin (botrocetin) was highly purified from crude Bothrops jararaca venom, as recently described [8], based on the method of Read et al. [3]. Crude venom (500 mg) was dissolved in 100 ml of 84 mM imidazole-0.15M NaCl buffer, pH 7.35, and then applied to DEAE-Sepharose CL-6B column (K 2.6 x 30 cm) equilibrated with the same buffer. After extensive washing with the buffer containing 0.1M NaCl, the column was eluted with a linear salt gradient from 0.1 to 0.7 M NaCl (each 150 ml) at 60 ml/hr. Fractions having platelet coagglutinin activity devoid of thrombin-like activity were pooled and dialyzed and then purified by size-exclusion HPLC. Further purification was achieved by hydrophobic HPLC. The details of the purification will be published elsewhere [8]. The highly purified botrocetin was dialyzed against 0.05 M Tris-HC1 0.15 M NaCl buffer, pH 7.35 (TBS), at 4°C overnight and then frozen at -70°C until used. Purification of von Willebrand Factor (vWF)

vWF was purified from cryoprecipitate (a generous gift of Japan Red Cross) as previously described [7]. Anti-vWF Monoclonal Antibody (MoAb)

Anti-vWF MoAb 2.2.9 (IgG,-k) was prepared as previously described [9]. Studies of Lopez-Fernandez et al. [9] and our preliminary results have shown that MoAb 2.2.9 reacts with all vWF multimers of different sizes and inhibits the binding of vWF to neither factor VIII,

heparin, collagen, nor platelet GPIb stimulated by ristocetin [9]. Another MoAb NMC-4 (IgG,-k) has been prepared in our laboratory [ 101 and was recently well characterized [ 121. It completely inhibits the platelet aggregation/agglutination induced by ristocetin or by botrocetin at a concentration of -10 pg/ml of purified IgG . Botrocetin Cofactor Assay (Bcof) and Platelet Aggregometry Studies

Washed platelets were prepared using BSA densitygradient centrifugation as previously described [7], and formalin-fixed platelets were prepared by overnight fixation of washed platelets with 1% formalin. Bcof was assayed utilizing formalin-fixed platelets and using an aggregometer NKK Model according to the method of Brinkhous et a]. [4]. Botrocetin-induced platelet aggregation/agglutination (BIPA) and ristocetin-induced platelet aggregation/ agglutination (RIPA) were also assayed using formalinfixed platelets from a patient with Bernard-Soulier syndrome. BIPA was studied using various concentrations of botrocetin to determine the minimum concentration needed to induce aggregation in platelet-rich plasma (PRP) of type IIA and IIB von Willebrand disease (vWD) as compared with normal PRP (PRP system). In the same way, using formalin-fixed platelets from normal individuals and plasmas of type IIA and IIB vWD, BIPA was examined (PPP system). Botrocetin-Induced vWF Binding Assay Using ['251]-labeledAnti-vWF MoAb 2.2.9 in Plasma Milieu

The assay was performed in a 125 pl of incubation volume in plastic Eppendorf tubes essentially according to the method of Schullek et al. [ l l ] as modified by Lopez-Fernandez et al. [9]. In most experiments, 20 pl formalin-fixed platelets (1.0 X 10s/ml), 5 pl inhibitor cocktail (10 mM EDTA-2Na, 10 p/ml hirudin, 20 mM benzamidine-HC1, and 10 mM leupeptin in TBS, pH 7.35), 12.5 pl of ['251]-2.2.9. (35 pg/ml), 75 p1 of plasma were mixed and incubated at 37°C for 2 hr, and then 12.5 pl botrocetin (f.c. 0-50 pg/ml) was added. After an additional 30 min incubation at room temperature, two 50 pl aliquots of the mixture were layered over 20% sucrose cushion (300 pi) in 500 pl Sarstedt microfuge tubes, and bound ligand was separated from free ligand by 5 min centrifugation at 12,OOOg. The tube tips containing bound ligand were amputated and counted in a gamma counter. Nonspecific binding was determined in the presence of type I11 vWD plasma instead of normal plasma. Speciific

Botrocetin-Induced llB vWF Binding

binding was calculated by substracting nonspecific binding from the total. Patients

A patient with Bernard-Soulier syndrome (Y.O.) has been previously described [ 131. Three patients with type IIB vWD in the same family for the present study have been also previously characterized in detail [ 141. At the time of the present study, plasma levels of VIII:C, vWF: Ag, and Rcof were 40%, 40%, and 30% for the propositus; 45%, 50%, and 30% for his brother; and 40%, 50%, and 35% for his mother, respectively. Two patients (E.S., S . K . ) with type IIA vWD and one (M.N.) with type I11 vWD were also used. At the time of the present study, plasma levels of vWF:Ag and Rcof were 50%, 50%, and

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Protein Concentration

The concentration of purified monoclonal IgG was calculated from ultraviolet absorbance at 280 nm using an extinction coefficient of E'"280 = 14.3. The concentrations of other proteins and botrocetin were measured by the method of Bradford [15] using BSA as a standard. RESULTS BlPA Using Formalin-Fixed Platelets in a Patient With Bernard-Soulier Syndrome

As shown in Figure 1, formalin-fixed platelets prepared from a patient with Bernard-Soulier syndrome (Y .O.) responded to neither the highly purified botrocetin (30 pg/ml) nor ristocetin (1 mg/ml), whereas normal platelets showed clear agglutination. Agglutination of normal platelets was completely inhibited in both instances by an anti-vWF MoAb (NMC-4), which has been reported to be a very effective inhibitor for vWF binding to platelets in the presence of ristocetin (data not shown) 1121. BlPA in Patients With Type IIA vWD and llB vWD.

PRP System. BIPA was examined using PRPs from a normal individual, from two patients with type IIA vWD, and from three patients with type IIB vWD. Platelets from a normal individual showed aggregation in a dose-response fashion over a botrocetin concentration between 0.8 and 2.1 pg/ml, whereas a much higher concentration of botrocetin (1.4-3.5 pg/ml) was required to induce the aggregation for PRPs from type IIA vWD. Platelets from type IIB vWD aggregated at such an extremely low concentration of botrocetin (0.15-0.3 pg/ml) that induced no aggregation with normal platelets. Data on a patient

Normal platelets

Fig. 1. Botrocetin- or ristocetin-induced agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome or normal individual in the presence of purified vWF. Formalin-fixed platelets (300 x 103/pl)and purified vWF (10 pg/ml) were incubated for 2 min at 37"C, and then botrocetin or ristocetin was added at a final concentration of 30 pg/ml or 1 mg/ml, respectively.

with type IIA vWD (E.S.) and propositus for type IIB vWD were shown in Figure 2. PPP system using formalin-fixed normal platelets. BIPA was determined using formalin-fixed platelets from normal individuals in the presence of plasma from normal, IIAs, or IIB vWD. In this assay system, normal plasma agglutinated platelets at a botrocetin concentration of more than 3.6 pg/ml, whereas plasma of a patient with IIA vWD (E.S.) required more than 14.4 pg/ml of botrocetin. As with the PRP system, three plasmas of patients with IIB vWD induced hyperagglutination of normal formalin-fixed platelets by the stimulus of such a relatively low concentration of botrocetin (less than 3.6 pg/ml) that induced no platelet agglutination in the presence of normal plasma (Fig. 3). Botrocetin-Induced Binding of vWF Radiolabeled With [1251] MoAb 2.2.9 to Forrnalin-Fixed Platelets

Normal vWF indirectly radiolabeled with [ 1251]MoAb 2.2.9 bound to formalin-fixed platelets from normal individuals in proportion to botrocetin concentration from 0 to 20 pg/ml. After this, a total radioactivity bound to platelets plateaued as shown in Figure 4. In the presence of plasma from a patient with type I11 vWD, the total radioactivity was unchanged with a botrocetin concen-

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type IIB vWD was incubated at 37°C for 2 min in a cuvette, and then 20 pl of partially purified botrocetin was added at a final concentration (pg/rnl) as indicated.

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patient (propositus) with type IIB vWD, and phosphate-buffered saline, pH 7.3,was incubated for 2 min at 37"C,and then 20 (*I of partially purified botrocetin was added at a final concentration (pg/ml) as indicated.

tration of 0-50 pg/ml. In addition, a well-characterized anti-vWF MoAb NMC-4 completely inhibited the binding of normal vWF radiolabeled with ['251]-MoAb 2.2.9 to formalin-fixed platelets. Based on this observation, nonspecific binding was determined by the use of plasma from a patient with type I11 vWD, and the specific binding was calculated by subtraction of the amount of nonspecific binding from the total. Indirectly radiolabeled vWF in IIB patient plasmas

showed increased binding to normal formalin-fixed platelets in the presence of botrocetin. At a lower concentration of botrocetin (2.5-30 pg/ml), the difference between normal and type IIB vWF in specific radioactivity bound to platelets was more obvious. At a higher concentration of botrocetin (30-50 pg/ml), type IIB vWF bound to platelets more than 130%-140% of normal vWF in spite of the lower level of vWF:Ag (30%40%). Type IIA vWF bound to platelets was about 30%

Botrocetin-Induced llB vWF Binding

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Fig. 4. Botrocetin-induced binding of [1251]-MoAb2.2.9-labeled vWF in plasmas to formalin-fixed platelets. [1251]MoAb 2.2.9 (35 yg/ml; 1.5 x lo9 cpm/mg), formalin-fixed platelets (1.0 x 105/pl),and 75 yl of normal or type 111 vWD plasma were incubated at 37°C for 2 hr in the presence (10 yg/ml) or absence of MoAb NMC-4. Then botrocetin was added at a final concentration of 0-50 yg/ml. After an additional 30 min incubation at room temperature, radioactivity bound to platelets was measured.

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Fig. 5. Botrocetin-induced binding of [1251]-MoAb2.2.9-labeled vWF in three type llB plasmas, ten normal plasmas, and two type HA plasmas to formalin-fixed platelets. [1251]MoAb 2.2.9 (35 yg/ml; 1.5 x lo9 cpm/rng), formalin-fixed platelets and 75 yl plasmas were incubated at 37°C for 2 hr, and then botrocetin was added at a final concentration of &50 yg/ml. After an additional 30 min incubation at room temperature, radioactivity bound to platelets was measured and expressed as amount of [1251]-MoAb2.2.9 bound to platelets.

of normal vWF. Normal vWF of ten persons bound to platelets as shown by the shaded area in Figure 5. DISCUSSION

Botrocetin, a venom coagglutinin, was partially purified from crude Bothrops Jururacu venom by the method of Read et al. [ 3 ] using anion exchange chromatography. By this procedure, coagglutinin activity was easily separated from thrombin-like activity. We used DEAESepharose CL-6B gel in this step and size exclusion and hydrophobic HPLC for further purification of the venom coagglutinin activity. Our preliminary results show that pepsin as well as CNBR treatment of the purified botrocetin resulted in the complete loss of activity in accordance with hydrolysis of the polypeptide. In addition, a reduction of the intact protein with DTE followed by s-carboxylmethylation also resulted in diminished coagglutinin activity [8]. These results suggest that the platelet coagglutinin activity resides on the protein molecule. Howard et al. [5] have suggested that PRP from a patient with Bernard-Soulier syndrome aggregates the patient platelets in response to botrocetin. As it is well known that the GPIWGPIX complex [ 161 and other glycoprotein(s) such as GPV are lacking or functionally abnormal in platelets of a patient with Bernard-Soulier syn-

drome [ 171, it may be possible that botrocetin can induce the interaction between other platelet receptor(s) and plasma protein(s). Our previous study has shown that the patient platelets (Y .O.) show a predominant decrease of the GPIWGPIX complex [ 131. Furthermore, we have also shown that an anti-GP Ib MoAb (AP- 1) [6,18] inhibits the platelet aggregation induced by either ristocetin or botrocetin in a washed platelet system, whereas antiGPIIb/IIIa MoAbs (CP-5 and CP-8) [6,19], which block vWF binding to thrombin-stimulated platelets, have no effect on aggregation. Formalin-fixed platelets obtained from a patient with Bernard-Soulier syndrome (Y .O.) did not agglutinate in the presence of purified vWF or normal plasma (data not shown) when stimulated with botrocetin as well as with ristocetin. Thus, it is likely that BIPA using formalinfixed platelets simply reflects the interaction between vWF and GP Ib. Three patients with type IIB vWD examined in the present study showed an enhanced BIPA when tested using formalin-fixed platelets from normal individuals, as seen in the patient PRP. Type IIB vWF indirectly radiolabeled by anti-vWF MoAb (MoAb 2.2.9), which shows no inhibitory effects on any vWF function, re-

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vealed a heightened binding ability to GP Ib by stimulation of botrocetin in spite of a low level of vWF:Ag ( -50%) in plasma. Thus, hyperagglutination on BIPA found in type IIB vWD patients is likely to be attributed to an enhanced botrocetin-induced type IIB vWF binding to platelets, although we do not know yet if botrocetin interacts with vWF or GP Ib.

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