Enhanced ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib in patients with steroid-responsive nephrotic syndrome

To elucidate the mechanism of enhanced ristocetin-induced platelet aggregation (RIPA) in steroid-responsive nephrotic syndrome (SRNS), plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RCof) were examined in 6 patients and the amount of ristocetin-induced vWF binding to platelets was determined. At the initial or relapse stage, the plasma vWF:Ag level was 415 +/- 137% and the RCof level was 364 +/- 117%. The ratio of RCof/vWF:Ag was 0.90 +/- 0.15 and no abnormalities of vWF:Ag multimers were observed, indicating that neither functional nor structural abnormalities were present in patient's plasma. The amount of ristocetin-induced normal vWF binding to nephrotic washed platelets, when ristocetin was used at concentrations of 0.5, 0.75, and 1.0 mg/ml, was 152-163% above the binding to normal platelets. In addition, nephrotic washed platelets resuspended in either normal or nephrotic plasma aggregated at a low concentration of ristocetin (0.75 mg/ml) which did not induce aggregation of normal platelets. In accordance with these observations, the decrease of Alcian blue 8GX binding to platelets, reflecting diminished surface negative charge, was also observed. These results appear to indicate that the plasma vWF level and the altered surface-negative charge in platelets both contribute to heightened vWF binding to GPIb, thus lowering the ristocetin concentration required for RIPA in SRNS.     

Thrombin decreases von Willebrand factor binding to platelet glycoprotein Ib

Thrombin is a physiological agonist that promotes platelet aggregation and secretion. In this study we observed that thrombin can also inhibit a function of platelets related to primary hemostasis. Platelet stimulation by thrombin decreased the binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib and decreased ristocetin-induced agglutination, in vitro reactions that correlate with initial platelet adhesion to the vessel wall. Binding of the monoclonal antibody API to GP Ib was also decreased. Cytoskeletal participation in the change of GP Ib was suggested because pretreatment of platelets with cytochalasin to prevent actin filament formation prevented the thrombin-induced decreases in vWF binding. API binding, and ristocetin-induced agglutination. Measurement of GP Ib in detergent extracts by electroimmunoassay demonstrated no loss after thrombin stimulation. Electroimmunoassay also demonstrated that the API epitope of GP Ib on intact thrombin-treated platelets was accessible for complete digestion by chymotrypsin. Therefore GP Ib was neither released from the platelet surface nor internalized by thrombin treatment. A previously recognized effect of thrombin is its induction of receptor sites on platelet surface GP IIb-IIIa for contact-promoting proteins, including vWF that are involved in the platelet spreading and aggregation that follow adhesion. Therefore the action on GP Ib may combine with the effect on GP IIb-IIIa to shift platelet reactivity from GP Ib-vWF-mediated initial contact with the vessel wall to GP IIb-IIIa-mediated spreading and aggregation.     

Enhanced botrocetin-induced type IIB von Willebrand factor binding to platelet glycoprotein Ib initiates hyperagglutination of normal platelets

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by 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 GPIb induced by these two substances have been reported. We have recently shown that the GPIb 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 GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.     

Functional analysis of a type IIB von Willebrand disease missense mutation: increased binding of large von Willebrand factor multimers to platelets.

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.     

Antiplatelet glycoprotein Ib monoclonal antibody (OP-F1) totally abolishes ristocetin-induced von Willebrand factor binding, but has minimal effect on the botrocetin-induced binding.

We describe here a new antiplatelet glycoprotein (GP) Ib monoclonal antibody (MoAb) designated OP-F1 (IgG1 kappa). Both OP-F1 and a well-characterized anti-GPIb MoAb, AP-1, totally abolished ristocetin-induced von Willebrand factor (vWF) binding to platelets and desialylated vWF binding to platelets at an IgG concentration of 2-8 micrograms/ml. AP-1 also blocked snake venom botrocetin-induced vWF binding at a similar IgG concentration, whereas OP-F1 had a minimal effect on botrocetin-induced binding. At a higher IgG concentration (150 micrograms/ml), OP-F1 inhibited botrocetin-induced binding by 50%. AP-1 (IgG) did not interfere with binding of [125I]OP-F1 (IgG) to platelets. Thus, the epitope involved in the binding of OP-F1 or AP-1 appears to be quite different. These results suggest that the vWF binding site(s) on the GPIb molecule generated by these inducers is in close proximity but not completely identical.     

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.     

Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.     

Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.     

Evidence from limited proteolysis of a ristocetin-induced conformational change in human von Willebrand factor that promotes its binding to platelet glycoprotein Ib-IX-V

von Willebrand factor (VWF) does not normally interact with platelets in the bloodstream. Binding to exposed vascular subendothelium, however, enables VWF to interact with the platelet glycoprotein Ib-IX-V complex (GP Ib-IX-V). This change in function may reflect a change in its conformation. Ristocetin also promotes interaction of VWF with GP Ib-IX-V; it thus provides a model for changes in VWF conformation and function that may occur in vivo. The fluid-phase conformation of VWF was evaluated from its susceptibility to proteolytic digestion. Ristocetin markedly altered the pattern of VWF digestion by trypsin, increasing the prevalence of two major proteolytic fragments (109 and 160 kDa), and decreasing that of four fragments (130, 145, 181 and 199 kDa). Vancomycin, a structurally related antibiotic, did not affect the digestion pattern. However, it partially reversed the ristocetin-induced change in digestion. Changes in prevalence of five of the tryptic fragments of VWF with ristocetin and vancomycin correlated closely with changes in VWF binding to GP Ib-IX-V. Heparin also partially inhibited the ristocetin-induced changes in tryptic digestion of VWF. These observations suggest that ristocetin may modulate VWF conformation in such a way as to expose its GP Ib-binding domain and enable it to interact with the platelet. Such modulation also exposes a cryptic site (or sites) for proteolytic cleavage by trypsin.     

von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin.

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin-induced platelet activation is a potential mechanism for regulating platelet adhesivity.     

Polymorphisms of platelet membrane glycoprotein Ib alpha and plasma von Willebrand factor antigen in coronary artery disease.

To examine the relationships of two polymorphisms of platelet glycoprotein (GP) Ib alpha and coronary artery diseases (CAD) in Japanese patients, we conducted a case-control study with 158 Japanese patients and 169 control subjects. The frequencies of HPA-2 polymorphism and the variable number of tandem repeat (VNTR) polymorphisms in the macroglycopeptide region did not significantly differ between CAD patients and control subjects. The polymorphisms of GPIb alpha were not associated with the number of affected vessels in CAD patients. When patients with acute coronary syndrome only were analyzed, the frequencies of the two polymorphisms of GPIb alpha showed no significant difference. Although plasma von Willebrand antigen (vWF:Ag) levels in patients were significantly higher than in controls, no association between vWF concentration and GPIb genotypes was observed. In patient groups with higher or lower vWF:Ag concentrations, no increase in the frequencies of Met145 or larger VNTR polymorphisms was seen in either group. Our findings indicate that no association exists between the frequencies of the two polymorphisms of GPIb alpha and CAD.     

N1421K mutation in the glycoprotein Ib binding domain impairs ristocetin- and botrocetin-mediated binding of von Willebrand factor to platelets

von Willebrand disease (VWD) is a common inheritable bleeding disorder caused by deficiency of von Willebrand Factor (VWF), which is involved in platelet adhesion and aggregation. We report a family consisting of three patients with VWD characterized by an apparently normal multimeric pattern, moderately decreased plasma factor VIII (FVIII) and VWF levels, and disproportionately low-plasma VWF:RCo levels. The patients were found to be heterozygous for the novel N1421K mutation, caused by a 4263C > G transversion in exon 28 of the VWF gene coding for the A1 domain. Botrocetin- and ristocetin-mediated binding of plasma VWF to GPIb were reduced in the patients. In vitro mutagenesis and expression in COS-7 cells confirmed the impairment of the mutant in botrocetin- and ristocetin-mediated VWF binding to GPIb. VWF collagen binding capacity was unaffected in plasma from the heterozygous individuals as well as in medium from transfected COS-7 cells. Our findings indicate that the N1421K substitution in the VWF affects the GPIb binding site or a recognition element by a conformational change of the A1 domain.     

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

Structural elements influencing von Willebrand factor (vWF) binding affinity for platelet glycoprotein Ib within a dispase-digested vWF fragment

We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.     

Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5' end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.     

Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor

Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.     

Structural determinants within platelet glycoprotein Ibalpha involved in its binding to von Willebrand factor

Platelet adhesion to subendothelial structures upon injury to a vessel wall is one of the first steps in a sequence of reactions critical for the formation of a haemostatic plug, or in diseased vessels for the development of an arterial thrombus. This adhesion process is mediated by an interaction between the glycoprotein (GP) Ib-V-IX complex on the platelet surface with von Willebrand Factor (vWF), associated with collagen on the subendothelial surface. After this initial adhesion, platelets will activate, resulting in recruitment of additional platelets and adherence to each other to form the platelet plug or developing thrombus. Several studies to date have attempted to identify the regions of the GPIb-V-IX complex that are critical for binding to vWF. The vWF binding site is contained in the 45 kDa N-terminal domain of the GPIbalpha chain. This N-terminal domain is characterized by a structural motif consisting of 7 leucine-rich repeats (LRRs), followed by a double disulphide-bonded loop and an anionic sulphated region. This review summarizes recent research efforts elucidating the characteristics of the GPIb-vWF interaction. Potential mechanisms that regulate the GPIb-vWF function are discussed, and advances in identifying functional sequences within GPIba involved in the binding to vWF are reviewed.