Platelet-Type von Willebrand Disease: Toward an Improved Understanding of the “Sticky Situation”

Identification of p.W246L As a Novel Mutation in the GP1BA Gene Responsible for Platelet-Type von Willebrand Disease

We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al,[1] wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein—a receptor for von Willebrand factor (VWF)—and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide.

Despite being a rare bleeding disorder, PT-VWD represents a significant challenging clinical problem as it may cause life-threatening bleeding, if not appropriately treated, particularly in situations related to surgeries and childbirth. The clinical diagnosis challenge stems from the close similarity of PT-VWD to the more common bleeding disorder, type 2B VWD.[2] [3] The discrimination and correct diagnosis can only be made after carefully assessing less commonly performed laboratory tests,[3] [4] [5] [6] [7] [8] and confirmed only after DNA analysis of the binding regions in the two genes VWF and GP1BA [9] has been performed.

The report by Woods et al[1] highlights some important issues that add to our understanding of this rare but potentially life-threatening bleeding disorder. First, the patient presented with severe bleeding symptoms (rather than mild/moderate bleeding symptoms, and unlike previously reported cases) while also showing other typical laboratory phenotypic data known in this disease such as macrothrombocytopenia, mild spontaneous platelet aggregation, absence from plasma of high-molecular-weight VWF multimers, positive ristocetin-induced platelet aggregation (RIPA) at 0.3 and 0.4 mg/mL, VWF ristocetin cofactor (VWF:RCo) < 10 IU/dL, and VWF:RCo to VWF antigen (VWF:Ag) ratio of less than 0.2. The authors initiated the diagnosis via meticulous laboratory assessment, including RIPA mixing tests and cryoprecipitate challenge tests, and ultimately providing comprehensive laboratory discrimination from type 2B VWD. In addition, they confirmed their diagnosis by DNA analysis, by revealing a c.3805 G>T GP1BA gene mutation that predicted the protein change Try246Leu. This mutation was absent in the unaffected mother and also in 100 healthy control subjects.

Second, in that report,[1] an attempt was made to quantify the bleeding symptoms in a patient with PT-VWD (bleeding score of 13). The knowledge about variation in bleeding symptoms and the ability to objectively assess these symptoms have proven to be important in management of bleeding disorders and can help predict disease outcome and also aid in treatment.[10] The only other such attempt was in a small case series reported recently. This series showed considerable variability in bleeding severity among PT-VWD that is independent of age or gender. The phenotypic variability in type 2B VWD has been reviewed in relation to various mutations and different patients' cohorts.[12] However, a systematic analysis of PT-VWD phenotype and the genotype–phenotype relationship remains to be investigated.

Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD.[1] VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel–Palade bodies (endothelial cells). Upon release and under physiologic pH, the VWF multimers and VWFpp dissociate and are secreted in 1:1 stoichiometric amounts.[13] Studies have shown a regulatory role for VWFpp as an intramolecular chaperone for the mature VWF protein and an aid to its storage and multimerization.[14] [15] The VWFpp circulates in the plasma for a short time, with a half-life of approximately 2 to 3 hours and plasma levels of approximately 1 μg/mL, whereas multimeric VWF circulates with a half-life of approximately 8 to 12 hours and plasma levels of approximately 10 μg/mL.[16] [17] Recent studies have shown that a fraction of VWF and VWFpp remains associated in the circulation in a functional interaction that is precisely between the VWFpp and the D′D3 domain of VWF. This interaction was thought to reduce the accessibility of the VWF-A1 domain for platelet GPIbα under hydrodynamic shear conditions, and subsequently reduce VWF proteolysis by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motifs, number 13).[18] It is possible that this regulatory role of VWFpp becomes more pronounced in PT-VWD, where VWF half-life in circulation is reduced. A shorter survival indicated by an increased VWFpp:VWF ratio in patients with type 2B VWD was reported, where an enhanced affinity of the abnormal VWF to normal platelet GPIbα is observed.[19] In their report,[1] Woods et al showed that the VWFpp and VWFpp:VWF ratio were unexpectedly normal in PT-VWD Try246Leu mutation. This may indicate that the clearance and survival of VWF may be two different mechanisms in patients with PT-VWD.

Fourth, Woods et al[1] have also adopted the newly recommended nomenclature for the description of the GP1BA sequence variant based on the human genome variation society (http://www.hgvs.org/mutnomen/).[20] [21] The discrepancy between the old and new number is due to the fact that GP1BA gene has 48 nucleotides within its coding sequence (exon 2) that codes for a 16 amino acids signal peptide. The old GP1BA gene and GPIbα protein nomenclature, historically used in the literature, started numbering the gene from the transcription (TSS) start site “G” and the protein from the amino acid His; the first amino acid in the mature protein (and not from the first amino acid Met in the polypeptide chain). Since the mature protein starts 16 amino acids downstream (due to the signal peptide), then based on this nomenclature, 16 amino acids need to be added to the old numbers to fulfill the new recommendation.[21] Accordingly, Try246Leu would be Try230Leu in the old nomenclature. To avoid confusion and to facilitate data interpretation in the context of previous publications, we will use the old numbering system while providing [Table 1] that illustrates all previously reported PT-VWD mutations, using both the old and new numbering system. At times, for clarity, we will also provide the new number in brackets.

Table 1 GP1BA gene mutations' old and new nomenclature

Common nomenclature	Nucleotide number		Amino acid number		References
		Conventional – 42		Conventional + 16
	Conventional (G at TSS as +1)	Recommended (A of ATG as +1)	Conventional (His as +1)	Recommended (Met as +1)
Trp230Leu	c. 3847 G > T	c. 3805 G > T	Trp230Leu	p.Trp246Leu	Woods et al, 2013[1]
Gly233Val	c.788 G > T	c.746 G > T	Gly233Val	p.Gly249Val	Miller et al, 1991[22]
Gly233Ser	c.788 G > A	c.746 G > A	Gly233Ser	p.Gly249Ser	Matsubara et al, 2003[24] Favaloro et al, 2007[7]
Asp235Tyr	c.793G > T	c.752 G > T	Asp235Tyr	p.Asp251Tyr	Enayat et al, 2012[25]
Met239Val	c.806 A > G	c.764 A > G	Met 239 Val	p.Met 255 Val	Takahashi et al, 1981[33] Moriki et al, 1997[23]
27bp del macroglycopeptide region	c.1348_1356 del	c.1326_1334 del	420_428 del	p.436_444 del	Othman et al, 2005[26]

Abbreviation: TSS, transcription start site.

Note: All previously reported GPIbα gene mutations and the predicted amino acid substitutions as described in the historically used conventional style as well as in the newly recommended style.[21]

Five PT-VWD mutations (four missense[7] [22] [23] [24] [25] and one deletion[26]) have been identified in the GP1BA gene so far. All except one (27 bp deletion in the macroglycopeptide region) are single point mutations and are located within the VWF binding region (first 200 amino acids of the amino-terminal end of the protein), more precisely within a short amino acid stretch between 233 and 239. The newly identified mutation Try230Leu (Try246Leu) is located within the binding region, a little upstream of this stretch but exists in a strongly conserved position.

Historically, functional characterization of naturally occurring or artificially induced PT-VWD mutations has used a heterologous system based on Chinese Hamster Ovary (CHO) cells expressing the β and IX chains of the GPIb/IX complex and a subsequent measurement of VWF binding to cells expressing a wild-type versus a mutant functional GPIbα. Although this system was fairly efficient to verify the gain-of-function nature of most of those reported mutations, it has several limitations. The description of the crystal structure of the N-terminal domain of GPIbα[27] in 2002 followed by the GPIbα-VWF-A1 domain complex[28] [29] have provided the molecular details of GPIbα binding to VWF and a framework for understanding the effects of the hereditary mutations, although the precise mechanism of action remains to be defined. The GPIbα receptor structure has the shape of a hand with the leucine-rich repeats (LRRs) forming the palm and a protruding loop termed the R-loop representing the thumb. The positively charged VWFA1 domain binds GPIbα via contacts with the R-loop (also termed β-switch) and the negatively charged LRRs. Several GPIbα structures are available and surveying those structures have shown that a distinct conformation predominates for the R-loop when GPIbα is not bound to VWF-A1 and that the thumb-like R-loop folds back toward the LRRs in a more compact triangular shape, which cannot interact with VWF-A1. [Fig. 1A] illustrates the structure of this compact form with residues affected by PT-VWD colored purple. Interactions within this structure are illustrated as green-dotted lines including a salt bridge between Asp235 and Lys237. Hydrophobic contacts between Met239 and side chains from Trp230, Val236, and Phe201 are also observed.

Fig. 1 The compact triangular conformation of the R-loop is disrupted in PT-VWD: (A) The GPIb R-loop is shown forming a compact structure with a short turn of 3₁₀ helix and β-turn (pdb:1P8V). Residues affected by PT-VWD mutations are shown in purple with the β-turn hydrogen bond and the D235-K237 salt bridge shown as green-dotted lines. The newly characterized PT-VWD residue Trp230Leu (Trp246Leu) may act by disrupting the interactions formed by Trp230 in the R-loop including the hydrogen bond to Gln232 and packing against Met239. (B) The R-loop with mutant Leu230 modeled. (C) An illustration of the GPIbα-VWFA1 complex crystal structure. The positions of the residues Trp230 and Asp235 are shown as spheres colored purple. Residue Trp230 is similar to Met239 in that it does not contact VWF, whereas Asp235 makes direct contacts with the VWFA1 domain shown as green-dotted lines. PT-VWD, platelet-type von Willebrand disease.

All PT-VWD missense mutations essentially result in a supersticky GPIbα protein that binds VWF without stimulation or shear stress. The mechanism of action proposed for the PT-VWD mutations Met239Val and Gly233Val was that they increase the affinity for VWF by stabilizing the R-loop in an extended β-hairpin structure through introducing a Cβ-branched residue.[23] [30] The recombinant expression of these mutations in heterologous cells demonstrated a higher affinity between the receptor and ligand.[23] [31] Following this, a third change involved the 233 residue, that is, a Gly233Ser mutation.[7] [24] [32] This residue was also tested artificially by site-directed mutagenesis changing the residue into Ser and Val.[31] [32] The fourth mutation was Asp235Tyr.[25] Notably, this residue is located at the tip of the R-loop and its mechanism of action could not be explained by stabilization of the extended β-hairpin structure. We thus proposed that this and other PT-VWD mutations could act by reducing the stability of the compact triangular form of the R-loop. Thus, the Asp235Tyr mutation acts by disrupting the salt bridge normally existing between Lys237 and Asp235 and thus favoring the high affinity form of the receptor.[25] Consistent with this hypothesis are site-directed mutagenesis studies involving a change of Lys at 237 to Val, which also resulted in a gain of function phenotype characterized in the CHO cell system.[30]

Using the GPIbα crystal structure, we examined the newly reported mutation Trp230Leu (Trp246Leu) by Woods et al.[1] [Fig. 1A] shows the position of Trp230 within the unliganded GPIbα R-loop structure illustrating that it forms a hydrogen bond to the Gln232 side chain and also packs against the side chain of Met239. The mutation to Leu would mean no hydrogen bond could be formed to Gln232 and the shorter side chain could not extend to pack against Met239 ([Fig. 1B]). [Fig. 1C] shows the position of Trp230 residue in the structure of the GPIbα-VWFA1 complex. The position of the amino acid is such that (unlike Asp235) it does not contact the VWFA1 domain but is closer to the GPIbα-LRRs (like Met239). The mutation to Leu does not introduce a short branched chain amino acid–like Val and is hence another example of a mutation that cannot act by stabilization of the extended β-hairpin structure. However, this mutation does result in the loss of the hydrogen bond to Gln232 and the packing interaction with Met239 that the larger Trp230 side chain provides, and hence the mechanism here could involve destabilization of the structure shown in [Fig. 1A].

Although the short amino acid stretch 233 to 239 in the GPIbα protein was considered the sticky spot within the first 200 amino acids of the amino terminal region involved in VWF binding, the report from Woods et al[1] has described a new gain-of-function mutation Trp230Leu (Trp246Leu) outside this stretch and indicates that there is probably more to uncover with respect to other GPIbα protein spots that are critical in the VWF interactions. The article by Woods et al[1] also illustrates the value of studying naturally occurring mutations to assist the understanding of platelet protein function.

Examination of crystal structures provides a framework for forming hypotheses relating to the “sticky” mode of action of PT-VWD missense mutations that then needs to be further tested by protein expression studies, structural characterization of the mutants, and kinetic analysis in functional assays. Reporting more patients with this rare disease is likely to provide more information to increase the knowledge about GPIbα-VWF interactions and the unique role of this protein in hemostasis.

• References

• 1 Woods AI, Sanchez-Luceros A, Bermejo E , et al. Identification of p.W246L as a novel mutation in the GP1BA gene responsible for platelet-type von Willebrand disease [In Focus Article]. Semin Thromb Hemost 2014; 40 (2) 151-160
• 2 Othman M. Platelet-type von Willebrand disease and type 2B von Willebrand disease: a story of nonidentical twins when two different genetic abnormalities evolve into similar phenotypes. Semin Thromb Hemost 2007; 33 (8) 780-786
• 3 Favaloro EJ. Phenotypic identification of platelet-type von Willebrand disease and its discrimination from type 2B von Willebrand disease: a question of 2B or not 2B? A story of nonidentical twins? Or two sides of a multidenominational or multifaceted primary-hemostasis coin?. Semin Thromb Hemost 2008; 34 (1) 113-127
• 4 Favaloro EJ. 2B or not 2B? Differential identification of type 2B, versus pseudo-von Willebrand disease. Br J Haematol 2006; 135 (1) 141-142 , author reply 143
• 5 Favaloro EJ, Bonar R, Meiring M, Street A, Marsden K ; RCPA QAP in Haematology. 2B or not 2B? Disparate discrimination of functional VWF discordance using different assay panels or methodologies may lead to success or failure in the early identification of type 2B VWD. Thromb Haemost 2007; 98 (2) 346-358
• 6 Favaloro EJ, Koutts J. 2B or not 2B? Masquerading as von Willebrand disease? Journal of thrombosis and haemostasis. J Thromb Haemost 2012; 10 (2) 317-319
• 7 Favaloro EJ, Patterson D, Denholm A , et al. Differential identification of a rare form of platelet-type (pseudo-) von Willebrand disease (VWD) from Type 2B VWD using a simplified ristocetin-induced-platelet-agglutination mixing assay and confirmed by genetic analysis. Br J Haematol 2007; 139 (4) 623-626
• 8 Giannini S, Cecchetti L, Mezzasoma AM, Gresele P. Diagnosis of platelet-type von Willebrand disease by flow cytometry. Haematologica 2010; 95 (6) 1021-1024
• 9 Hamilton A, Ozelo M, Leggo J , et al. Frequency of platelet type versus type 2B von Willebrand disease. An international registry-based study. Thromb Haemost 2011; 105 (3) 501-508
• 10 Rydz N, James PD. The evolution and value of bleeding assessment tools. J Thromb Haemost 2012; 10 (11) 2223-2229
• 11 Kaur H, Scovil S, McEacern K, James P, Othman M. International systematic study for assessment of bleeding phenotype in platelet type von Willebrand disease. Paper presented at: 26th Congress of the International Society on Thrombosis and Haemostasis; June 29–July 4, 2013; Amsterdam, The Netherlands
• 12 Othman M, Favaloro EJ. Genetics of type 2B von Willebrand disease: “true 2B,” “tricky 2B,” or “not 2B.” What are the modifiers of the phenotype?. Semin Thromb Hemost 2008; 34 (6) 520-531
• 13 Wagner DD, Fay PJ, Sporn LA, Sinha S, Lawrence SO, Marder VJ. Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells. Proc Natl Acad Sci U S A 1987; 84 (7) 1955-1959
• 14 Haberichter SL, Budde U, Obser T, Schneppenheim S, Wermes C, Schneppenheim R. The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF. Blood 2010; 115 (22) 4580-4587
• 15 Haberichter SL, Jacobi P, Montgomery RR. Critical independent regions in the VWF propeptide and mature VWF that enable normal VWF storage. Blood 2003; 101 (4) 1384-1391
• 16 Montgomery RR, Zimmerman TS. von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease. J Clin Invest 1978; 61 (6) 1498-1507
• 17 Borchiellini A, Fijnvandraat K, ten Cate JW , et al. Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1-deamino-8-D-arginine vasopressin in humans. Blood 1996; 88 (8) 2951-2958
• 18 Madabhushi SR, Shang C, Dayananda KM , et al. von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion. Blood 2012; 119 (20) 4769-4778
• 19 Casonato A, Gallinaro L, Cattini MG , et al. Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia. Haematologica 2010; 95 (8) 1366-1372
• 20 Othman M. Differential identification of PT-VWD from type 2B VWD and GP1BA nomenclature issues. Br J Haematol 2008; 142 (2) 312-314 , author reply 314–315
• 21 Goodeve AC, Reitsma PH, McVey JH ; Working Group on Nomenclature of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Nomenclature of genetic variants in hemostasis. J Thromb Haemost 2011; 9 (4) 852-855
• 22 Miller JL, Cunningham D, Lyle VA, Finch CN. Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease. Proc Natl Acad Sci U S A 1991; 88 (11) 4761-4765
• 23 Moriki T, Murata M, Kitaguchi T , et al. Expression and functional characterization of an abnormal platelet membrane glycoprotein Ib alpha (Met239 —> Val) reported in patients with platelet-type von Willebrand disease. Blood 1997; 90 (2) 698-705
• 24 Matsubara Y, Murata M, Sugita K, Ikeda Y. Identification of a novel point mutation in platelet glycoprotein Ibalpha, Gly to Ser at residue 233, in a Japanese family with platelet-type von brand disease. J Thromb Haemost 2003; 1 (10) 2198-2205
• 25 Enayat S, Ravanbod S, Rassoulzadegan M , et al. A novel D235Y mutation in the GP1BA gene enhances platelet interaction with von Willebrand factor in an Iranian family with platelet-type von Willebrand disease. Thromb Haemost 2012; 108 (5) 946-954
• 26 Othman M, Notley C, Lavender FL , et al. Identification and functional characterization of a novel 27-bp deletion in the macroglycopeptide-coding region of the GPIBA gene resulting in platelet-type von Willebrand disease. Blood 2005; 105 (11) 4330-4336
• 27 Uff S, Clemetson JM, Harrison T, Clemetson KJ, Emsley J. Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation. J Biol Chem 2002; 277 (38) 35657-35663
• 28 Dumas JJ, Kumar R, McDonagh T , et al. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibalpha complex reveals conformation differences with a complex bearing von Willebrand disease mutations. J Biol Chem 2004; 279 (22) 23327-23334
• 29 Huizinga EG, Tsuji S, Romijn RA , et al. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain. Science 2002; 297 (5584) 1176-1179
• 30 Dong J, Schade AJ, Romo GM , et al. Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions. J Biol Chem 2000; 275 (36) 27663-27670
• 31 Murata M, Russell SR, Ruggeri ZM, Ware J. Expression of the phenotypic abnormality of platelet-type von Willebrand disease in a recombinant glycoprotein Ib alpha fragment. J Clin Invest 1993; 91 (5) 2133-2137
• 32 Nurden P, Lanza F, Bonnafous-Faurie C, Nurden A. A second report of platelet-type von Willebrand disease with a Gly233Ser mutation in the GPIBA gene. Thromb Haemost 2007; 97 (2) 319-321
• 33 Takahashi H, Nagayama R, Hattori A, Ihzumi T, Tsukada T, Shibata A. Von Willebrand disease associated with familial thrombocytopenia and increased ristocetin-induced platelet aggregation. Am J Hematol 1981; 10 (1) 89-99