Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.

THE ABILITY OF PLATELETS to adhere to sites of vessel injury under conditions of flow is critical for hemostasis.1 The molecular basis for initial platelet adhesion after vessel disruption appears to involve the coordinate actions of 2 sets of adhesive receptors: the glycoprotein (GP) Ib-V-IX complex2,3 and the integrins α2β1 and αΙΙbβ3. The Ib-V-IX complex is composed of 4 related transmembrane GPs, ie, GPIbα, Ibβ, V, and IX, which are associated with each other in a stoichiometry of 2:2:1:2.4,5 Within this complex, Ibα and Ibβ are disulfide linked and noncovalently associated with IX.4,6 GPV is noncovalently associated with GPIb-IX,5 and surface expression of GPV is decreased in GPIb and GPIX deficiency states.7 Approximately 11,000 copies of the GPIb-IX complex are found on the surface of human platelets.4,5 This high density is consistent with its role in cell adhesion—under conditions of shear, the Ib-V-IX complex mediates reversible platelet binding to the subendothelial matrix of the vessel wall by binding to von Willebrand factor (vWF)8via sites on the Ibα chain.9 Subsequent platelet activation triggers integrin activation and irreversible binding to vWF by αΙΙbβ3.

Loss of GPIb-V-IX function causes Bernard-Soulier syndrome (BSS), a severe bleeding disorder.3,7,10 BSS is characterized by abnormal, giant circulating platelets with defective adhesion to vWF and reduced thrombin responsiveness.11 Classic BSS is associated with the loss of the entire Ib-V-IX complex.7Nonclassic presentations have also been reported in which the complex is present in normal amounts but defective in function.12,13 Mutations in the genes encoding both Ib12,13 and IX14 have been shown to cause BSS. GPV has been reported to enhance surface expression of Ib-IX in some heterologous expression systems,15,16 but not others.17 It is not known whether mutations in the gene encoding GPV will affect platelet expression of GPIb-IX and/or cause BSS.

Interestingly, GPV is a substrate for thrombin,18-21 a potent platelet activator. Thrombin binds to GPIb on human platelets,22 perhaps positioning itself to cleave an adjacent GPV molecule. These observations conjured the hypothesis that GPV might contribute to thrombin signaling.23,24 The observation that antibodies that inhibited thrombin cleavage of GPV failed to inhibit platelet activation by thrombin25suggested that GPV cleavage was not necessary for platelet activation by thrombin, and the identification of 3 distinct G protein-coupled receptors (GPCRs) for thrombin provided an alternative explanation of how platelets respond to thrombin.26-29 Nonetheless, the fact that thrombin cleavage site in GPV is conserved in the mouse, rat, and human proteins suggests that this sequence may be important for the structure or function of GPV,30 and available data do not formally exclude a contribution by GPV to thrombin signaling.

To address the role of GPV in vivo, we have generated GPV-deficient mice using gene targeting. GPV null mice developed normally and exhibited no spontaneous bleeding. GPV null platelets were normal in size and shape, responded normally to thrombin, and exhibited wild-type adhesion to mouse vWF A1 domain under shear. Thus, in the mouse, GPV is not necessary for surface expression or function of platelet Ib, for normal platelet cytoskeletal structure, or for normal responsiveness to thrombin. Our results suggest that loss of function mutations in the GPV gene are unlikely to be a cause of human BSS and support recent studies that suggest that platelet thrombin responses of shape change, aggregation, and granule secretion are mediated by protease-activated receptors (PARs).29 31 

Targeted inactivation of the GpV gene.

Two P1 bacteriophage clones that contained the GpV gene were obtained by polymerase chain reaction (PCR) screen of a mouse genomic library (Genome Systems, St Louis, MO). A 1.3-kb SalI/BamHI fragment 5′ of exon 1 and a 7.0-kbEcoRI/Xho I fragment 3′ of exon 2 were cloned into the pNTK vector32 to create the targeting vector (Fig 1A). The 5′ Sal I site was contributed by the backbone vector (pAd10SacBII). A 0.6-kbBgl II/EcoRV fragment of the GpVgene 5′ of the short arm of homology was used as a probe to identify both the wild-type and targeted alleles (Fig 1A). RF8 ES cells33 (129/SvJae) were electroporated with the targeting construct, and clones resistant to G418 and FIAU were selected and screened by Southern blot. A highly chimeric male mouse derived fromGpV+/− ES cells was bred to C57Bl/6 females to generate approximately 30 F1 GpV+/− mice. All experiments reported here were performed using the F2 offspring of these mice.

Fig. 1.

Generation of GPV-deficient mice. (A) Gene-targeting strategy. A replacement vector69 was used to substitute a neomycin phosphotransferase expression cassette (Neo) for the entireGpV gene. The wavy line represents plasmid backbone; TK, HSV thymidine kinase expression cassette. X1 and X2 represent exons 1 and 2 of the GpV gene, with the coding region shown as a white box and the 5′ and 3′ untranslated regions shown as shaded boxes. (Sal1) indicates a Sal I restriction endonuclease site found in the P1 bacteriophage vector containing the GpV gene that was used to construct the targeting vector. (B) Southern blot analysis of Bgl2-digested genomic DNA from the tails of pups derived from GpV+/− matings using 5′ flanking probe (A). Targeting removed an endogenous Bgl2 site and introduced a new Bgl2 site. The 4.2- and 5.5-kb bands correspond to wild-type and targeted alleles, respectively. (C) RT-PCR analysis of GpV+/+ andGpV−/− mouse spleen total RNA using GpV (right panel) and Par3g (left panel) primers.

Fig. 1.

Generation of GPV-deficient mice. (A) Gene-targeting strategy. A replacement vector69 was used to substitute a neomycin phosphotransferase expression cassette (Neo) for the entireGpV gene. The wavy line represents plasmid backbone; TK, HSV thymidine kinase expression cassette. X1 and X2 represent exons 1 and 2 of the GpV gene, with the coding region shown as a white box and the 5′ and 3′ untranslated regions shown as shaded boxes. (Sal1) indicates a Sal I restriction endonuclease site found in the P1 bacteriophage vector containing the GpV gene that was used to construct the targeting vector. (B) Southern blot analysis of Bgl2-digested genomic DNA from the tails of pups derived from GpV+/− matings using 5′ flanking probe (A). Targeting removed an endogenous Bgl2 site and introduced a new Bgl2 site. The 4.2- and 5.5-kb bands correspond to wild-type and targeted alleles, respectively. (C) RT-PCR analysis of GpV+/+ andGpV−/− mouse spleen total RNA using GpV (right panel) and Par3g (left panel) primers.

Close modal
Reverse transcription-PCR (RT-PCR) of mouse spleen.

Total RNA was obtained from individual mouse spleens using Trizol (GIBCO, Grand Island, NY). Two micrograms of total RNA was used as template to create first-strand cDNA using random hexamer primers and a commercially available kit per the manufacturer's instructions (Superscript; GIBCO). One microliter of the 20 μL reaction was used for PCR amplification of the GpV andPar3ggenes with 2 μmol/L primers as detailed below. ForGpV, the product size was 440 bp, the sense strand primer for PCR was 5′-TGC CTA CGA ACC TCA CAC ACA TC-3′, and the antisense primer for PCR was 5′-GCT TAA CTT GAG CCC CAA GCA G-3′. The conditions used were as follows: 94°C for 4 minutes and 72°C for 1 minute with the addition of Taq; then 94°C for 45 seconds, 60°C for 1 minute, and 72°C for 1 minute for 40 cycles; and then 72°C for 8 minutes. For Par3g, the product size was 511 bp, the sense strand primer for PCR was 5′-TCC TCA CTT GCA TGG GCA TC-3′, and the antisense primer for PCR was 5′-TCT AGG CAG CTA TTC AGG CTC CC-3′. The conditions used were as follows: 94°C for 4 minutes and 72°C for 1 minute with addition of Taq; then 94°C for 45 seconds, 58°C for 1 minute, and 72°C for 1 minute for 45 cycles; and then 72°C for 8 minutes.

Tail bleeding time.

The tail bleeding times of 45 progeny of GPV+/− matings 6 to 7 weeks of age were assayed using the technique of Dejana et al.34 The bleeding time was performed in a blinded fashion before tail cutting for genotyping by Southern blot.

Electron microscopy of murine platelets.

Platelets were fixed in 1.5% glutaraldehyde for 2 hours at 22°C in sodium cacodylate buffer, postfixed in 1% OsO4 in veronal-acetate buffer, stained with aqueous 1% uranyl acetate, dehydrated in ethyl alcohol, infiltrated with propylene oxide, and embedded in Epon (Ted Pella Inc, Redding, CA).

Flow cytometry.

For size analysis, washed platelets were fixed in 1% paraformaldehyde for 20 minutes at 4°C, washed 3 times with platelet buffer (20 mmol/L Tris-HCl, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L MgCl2, 1 mg/mL glucose, and 0.5% bovine serum albumin [BSA]) and analyzed by flow cytometry.

For measurement of GPIb-IX surface expression, washed platelets were fixed in 0.5% paraformaldehyde for 15 minutes at room temperature and then washed 3 times with phosphate-buffered saline (PBS). Platelets (5 × 107) were incubated in 0.25 mL Tyrode's buffer with BSA plus 1:1,000 (vol/vol) nonimmune rabbit serum or GPIb-IX immune serum (antiserum no. 3584; generously provided by Drs Sylvie Meyer and Beat Steiner, Hoffmann-LaRoche, Basel, Switzerland)35 for 1 hour at 4°C. Platelets were then washed once with PBS, incubated with fluorescein isothiocyanate (FITC)-goat antirabbit monoclonal antibody (Molecular Probes, Sunnyvale, CA) diluted 1:500, and analyzed by flow cytometry.36 

Platelet aggregation and secretion.

Blood was collected into citrate buffer from the inferior vena cava of pentobarbital-anesthetized mice. Blood from 3 to 4GPV−/− mice or their wild-type littermates was pooled for each platelet study. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 200g for 7 minutes. EDTA (10 mmol/L) and prostaglandin E1(PGE1; 1 μmol/L) were then added and PRP was centrifuged at 500g for 10 minutes. Platelets were then washed in platelet buffer containing 1 mmol/L EDTA and 1 μmol/L PGE1, collected by centrifugation, resuspended to an OD500 of 1.0 (∼2.5 × 108 platelets/mL) in platelet buffer lacking EDTA and PGE1, and incubated on ice for 30 minutes before use. Aggregation and secretion were measured in a Chrono-Log lumiaggregometer (Havertown, PA). Three hundred microliters of platelet suspension was added to the aggregometer chamber. Luciferase (880 U/mL), luciferin (8 μg/mL), and CaCl2 (1 mmol/L) were then added, and aggregation was followed as change in light transmission with time after addition of agonist. Results were expressed as Δlight transmission, defined as the percentage increase in light transmission over that of the unactivated platelet suspension, with 100% representing light transmission of platelet buffer alone. Platelet ATP secretion was measured as luminescence generated by platelet-released ATP compared with that of an ATP standard. Studies using botrocetin were performed in PRP diluted using platelet-poor plasma (PPP; obtained by centrifugation of the remaining blood at 1,200g for 10 minutes) to an OD500 of 1.0, with 100% light transmission representing light transmission of PPP alone. Botrocetin was obtained as a kind gift from M. Berndt (Victoria, Australia).

Platelet adhesion to murine vWF: Construction of murine vWF-A1 domain expression vector.

A region corresponding to the human vWF-A1 (475-709) was cloned from mouse genomic DNA and used to produce recombinant protein (T. Diacovo, manuscript in preparation). The murine vWF-A1 domain was subcloned into the expression vector pQE9 (Qiagen, Valencia, CA) and expressed in Escherichia coli, and the protein was purified.37 Protein concentrations were determined using the BCA method (Pierce Chemical Co, Rockford, IL). Coomassie-blue staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels showed that greater than 99% of the protein in the prep was vWF-A1 domain.

Purified recombinant mouse vWF-A1 protein (diluted to 100 μg/mL with 10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4) was loaded into microslides (rectangular glass tubes with a cross-section of 300 μm × 30 mm; H.P. Scientific, Inc, Concord, CA) by capillary action and stored overnight at 4°C. Coated microslides were subsequently rinsed and incubated with PBS containing 1% human serum albumin for 30 minutes at 37°C to block nonspecific interactions. Protein-coated microslides were secured to the stage of an inverted phase microscope (Diaphot–TMD; Nikon, Garden City, NJ), and plastic tubing was attached to each end. A uniform wall shear rate was generated by aspirating platelets through the microslide with a syringe pump (Harvard Apparatus, Holliston, MA). For attachment assays, platelets were purified by centrifugation of anticoagulated blood obtained from the retroorbital venous plexus of anesthetized mice. Platelets were washed twice in HEPES buffer (145 mmol/L NaCl, 10 mmol/L HEPES, 0.5 mmol/L Na2HPO4, 5 mmol/L KCl, 2 mmol/L MgCl2, 0.2% BSA, pH 7.4), resuspended at a concentration of 2 × 108/mL, and used within 2 hours. Platelet suspensions were drawn through microslides at shear rates of 50 or 600 s−1 for 5 minutes. The wall shear stress was calculated assuming a Newtonian fluid and a viscosity of 1.0 cP. Attached platelets were observed with phase contrast objectives and quantitated by analysis of videotape images. The number of platelets attached per unit area (0.67 mm2) was quantitated using 4 fields of view for each data point.

Generation of GPV-deficient mice.

The GpV gene was inactivated using a targeting vector that eliminated exons 1 and 2 by homologous recombination (Fig 1A). The targeted allele was detected by Southern blotting using the 5′ flanking probe shown and loss of GPV mRNA was confirmed by RT-PCR of total mouse spleen RNA (Fig 1B and C). Spleen is a hematopoietic organ in the mouse and contains megakaryocytes. GpV mRNA was detected in spleen RNA from wild-type mice but not knockout mice. Par3gmRNA, which is known to be expressed in mouse megakaryocytes,38 was detected in samples from both wild-type and knockout (Fig 1C).

GPV-deficient mice developed normally and exhibited normal hemostasis.

GpV+/− × GpV+/− matings producedGpV−/− offspring at the expected rate (eg, 31 +/+, 77 +/−, and 34 −/−); thus, there is no evidence for loss of GPV-deficient embryos during development. GPV-deficient mice were grossly normal at birth, grew like their wild-type littermates, and were fertile. They showed no evidence of spontaneous bleeding and had hematocrit levels and platelet counts indistinguishable from those of their wild-type littermates (data not shown). To test platelet function in vivo, tail bleeding times were determined on the progeny of heterozygous matings before genotyping (Fig2). GPV-deficient mice had bleeding times indistinguishable from those of their wild-type littermates, consistent with their lack of any overt bleeding.

Fig. 2.

Tail bleeding times of wild-type and GPV-deficient mice. The bleeding times of 6- to 7-week-old progeny of heterozygote matings were obtained in a blinded manner before genotyping. The results for mice subsequently identified as wild-type (+/+) and GPV-deficient (−/−) are shown.

Fig. 2.

Tail bleeding times of wild-type and GPV-deficient mice. The bleeding times of 6- to 7-week-old progeny of heterozygote matings were obtained in a blinded manner before genotyping. The results for mice subsequently identified as wild-type (+/+) and GPV-deficient (−/−) are shown.

Close modal
GPV-deficient platelets were normal in size and structure.

The morphology of platelets from GPV-deficient and wild-type mice was examined by transmission electron microscopy. Electron micrographs of wild-type and GPV-deficient mouse platelets were indistinguishable. In particular, GPV-deficient platelets showed no giantism or cytoskeletal disruption typical of BSS (Fig 3). Granule counts of 300 platelets derived from 3 distinct platelet preparations of each genotype (100 each) were also equivalent: 300 wild-type platelets contained 1,200 granules and 300 GPV-deficient platelets contained 1,174 granules. On average, both wild-type and GPV-deficient platelets had 4.0 α-granules per thin section of platelet.

Fig. 3.

Transmission electron micrograph of mouse platelets. Two major granule types are present: the -granules, which are the predominant population (a), and the serotonin-containing dense granule (d) population. Some -granules contain small tubules cut in cross-section (A; arrow, and at higher magnification in [B], arrow) that is probably vWF.67 Others contain a regular fibrillar array, which is probably partially polymerized fibrinogen (C; arrowhead). Although the majority of  granules are round, some have a tent-like shape (D) and a few are very large (E; denoted by asterisk). The platelets shown are GPV-deficient, but indistinguishable granules were also observed in the platelets of wild-type mice.

Fig. 3.

Transmission electron micrograph of mouse platelets. Two major granule types are present: the -granules, which are the predominant population (a), and the serotonin-containing dense granule (d) population. Some -granules contain small tubules cut in cross-section (A; arrow, and at higher magnification in [B], arrow) that is probably vWF.67 Others contain a regular fibrillar array, which is probably partially polymerized fibrinogen (C; arrowhead). Although the majority of  granules are round, some have a tent-like shape (D) and a few are very large (E; denoted by asterisk). The platelets shown are GPV-deficient, but indistinguishable granules were also observed in the platelets of wild-type mice.

Close modal

An independent analysis of the size of wild-type and GPV-deficient platelets was obtained by using flow cytometry to measure the forward and side scatter of light (Fig 4). GPV-deficient platelets exhibited the same range of forward and side scatter as that observed for wild-type platelets, confirming the microscopic observation that loss of GPV had no effect on platelet size or shape.

Fig. 4.

Assessment of platelet size and shape using flow cytometric analysis of wild-type (+/+) and GPV-deficient (−/−) platelets. Washed platelets were fixed and analyzed by flow cytometry for light forward and side scatter. Shown is the analysis of 10,000 platelets for each group.

Fig. 4.

Assessment of platelet size and shape using flow cytometric analysis of wild-type (+/+) and GPV-deficient (−/−) platelets. Washed platelets were fixed and analyzed by flow cytometry for light forward and side scatter. Shown is the analysis of 10,000 platelets for each group.

Close modal
Adhesion to vWF was not impaired in GPV-deficient platelets.

Platelet adhesion to vWF is dependent on GPIb function.8,39GPIb's interaction with vWF is species specific, and mouse GPIb will not bind human vWF.40 To measure the function of the mouse GPIb in wild-type and GPV-deficient mouse platelets, we therefore measured platelet adhesion to immobilized recombinant mouse vWF A1 domain. VWF-A1 domain contains unique sequences that provide a distinct binding site for GPIb.41,42 The importance of this domain with respect to vWF function is evident based on studies of type 2M vWD mutations. Patients with this genotype have mutations within the vWF-A1 domain that result in the impairment of hemostasis, but retain normal vWF multimer structure.43 44 Under conditions of both low and moderate shear, no difference in the steady-state number of adherent platelets was observed between GPV-null and wild-type platelets (Fig 5). Similarly, no difference was noted in the rate at which wild-type and GPV-deficient platelets rolled on the vWF-coated surface (data not shown). As observed for full-length vWF, the ability of recombinant vWF-A1 protein to support platelet adhesion in flow is species specific; murine, but not human, vWF-A1 bound murine platelets under flow conditions (T. Diacovo, manuscript in preparation), and no platelet adherence was noted when surfaces were not coated with vWF (not shown). Thus, GPV-deficient mouse platelets demonstrated no evidence for defective GPIb-vWF interaction in this functional assay.

Fig. 5.

Accumulation of platelets on immobilized monomeric murine vWF-A1 during flow. Washed murine platelets were infused through recombinant vWF-A1–coated microslides at a shear rate of 50 or 600 s−1. After 5 minutes of continuous flow, adherent platelets were counted in 4 different fields of view. Data are averaged from 2 experiments performed. Error bars represent the standard deviation.

Fig. 5.

Accumulation of platelets on immobilized monomeric murine vWF-A1 during flow. Washed murine platelets were infused through recombinant vWF-A1–coated microslides at a shear rate of 50 or 600 s−1. After 5 minutes of continuous flow, adherent platelets were counted in 4 different fields of view. Data are averaged from 2 experiments performed. Error bars represent the standard deviation.

Close modal
Other measures suggest normal GPIb expression in GPV-deficient platelets.

Surface expression of GPIb in wild-type and GPV-deficient platelets was also assessed by 2 other measures. First, botrocetin-mediated platelet agglutination was examined. Like ristocetin, the snake venom-derived botrocetin induces platelet agglutination in PRP by facilitating GPIb-vWF binding,45 but, unlike ristocetin, botrocetin is active on rodent platelets.46 47 Platelets were incubated with 1, 4, or 10 μg/mL botrocetin for 10 minutes with stirring, and agglutination was observed as an increase in light transmission. Representative tracings are shown in Fig 6. The percentages of maximal change in light transmission (0% equals PRP and 100% equals PPP) for wild-type versus GPV-deficient platelets were 5.7 ± 0.6 versus 6.0 ± 1.0 (mean ± SD; n = 3) at 1 μg/mL, 37 ± 6 versus 34 ± 2 at 4 μg/mL (n = 3), and 44 ± 4 versus 47 ± 4 at 10 μg/mL (n = 9), respectively. Thus, botrocetin-induced agglutination of wild-type and GPV-deficient platelets were indistinguishable, suggesting normal vWF binding of platelet GPIb in the absence of GPV.

Fig. 6.

Platelet agglutination by botrocetin. PRP were stirred and botrocetin was added at a final concentration of 10 (A), 4 (B), or 1 μg/mL (C). Agglutination was measured as the change in light transmission. +/+, wild-type platelets; −/−, GPV-deficient platelets. This experiment was replicated 3 times.

Fig. 6.

Platelet agglutination by botrocetin. PRP were stirred and botrocetin was added at a final concentration of 10 (A), 4 (B), or 1 μg/mL (C). Agglutination was measured as the change in light transmission. +/+, wild-type platelets; −/−, GPV-deficient platelets. This experiment was replicated 3 times.

Close modal

Surface expression of GPIb-IX was also measured using a polyclonal antibody to Ib-IX and flow cytometry. No difference in antibody binding was detected in wild-type versus GPV-deficient platelets. For wild-type platelets, the values for nonimmune and immune antibody binding in average fluorescence units (mean ± SD; n = 3) were 10 ± 2 and 88 ± 35, respectively. The cognate values for GPV-deficient platelets were 8 ± 1 and 97 ± 5, respectively. These data suggest normal GPIb-IX expression in the absence of GPV, consistent with the functional data on platelet rolling on vWF-coated surfaces and botrocetin-induced agglutination.

Activation of platelets by thrombin and other agonists.

To determine if thrombin cleavage of GPV contributes to activation of platelets by thrombin, we measured shape change, aggregation, and ATP secretion by wild-type and GPV null platelets in response to low (0.5 and 1 nmol/L) and high (30 nmol/L) concentrations of thrombin. Unlike PAR3 null mouse platelets28 or PAR1-inhibited human platelets,29 GPV null platelets responded to 1 nmol/L thrombin like wild-type platelets (Fig 7A). The rate and extent of shape change and aggregation by wild-type and GPV-deficient platelets in response to 0.5 nmol/L thrombin, a concentration close to the threshold for aggregation under these conditions, were also indistinguishable (n = 5 for wild-type and n = 6 for GPV-deficient platelet preparations, data not shown). In addition, the time to half-maximal secretion, a sensitive measure of thrombin signaling in platelets,29,31 did not differ between wild-type and GPV-deficient platelets stimulated with 0.5, 1, or 30 nmol/L thrombin. The mean time to half-maximal secretion in seconds for wild-type and GPV-deficient platelets, respectively, was 53.33 (n = 3) and 54.5 (n = 4) at 0.5 nmol/L thrombin, 47.25 (n = 4) and 50.67 (n = 4) at 1.0 nmol/L thrombin, and 4.8 (n = 6) and 5.0 (n = 6) at 30 nmol/L thrombin. Even at 30 nmol/L thrombin, a concentration of thrombin demonstrated to efficiently cleave GPV,48 there was no detectable difference in the rate or extent of platelet aggregation or ATP secretion (Fig 7 and data not shown). GPV-deficient platelets also responded like wild-type to the thromboxane A2 analog U46619 (10 μmol/L) and to collagen (20 μg/mL, data not shown).

Fig. 7.

Platelet activation in response to thrombin. (A) Platelet aggregation. Washed wild-type (+/+) and GPV-deficient (−/−) platelets were stirred and exposed to 1 (top) or 30 nmol/L (bottom) thrombin at 0 seconds. Results are representative of 3 experiments. (B) Platelet secretion of ATP. Washed wild-type (+/+) and GPV-deficient (−/−) platelets were exposed to 1 or 30 nmol/L thrombin and the peak ATP secretion was measured by lumiaggregometry. Data represent the mean ± SD of 4 to 6 experiments.

Fig. 7.

Platelet activation in response to thrombin. (A) Platelet aggregation. Washed wild-type (+/+) and GPV-deficient (−/−) platelets were stirred and exposed to 1 (top) or 30 nmol/L (bottom) thrombin at 0 seconds. Results are representative of 3 experiments. (B) Platelet secretion of ATP. Washed wild-type (+/+) and GPV-deficient (−/−) platelets were exposed to 1 or 30 nmol/L thrombin and the peak ATP secretion was measured by lumiaggregometry. Data represent the mean ± SD of 4 to 6 experiments.

Close modal

Since its identification as a platelet surface GP more than 20 years ago,18 GPV has been hypothesized to play 2 potential roles in hemostasis and thrombosis. It was proposed as a possible platelet thrombin receptor based on its proximity to the GPIb thrombin binding site and its susceptibility to thrombin cleavage.18,23 GPV was also postulated to be necessary for normal expression and function of GPIb based on GPV's association with Ib and IX on the platelet surface49 and its ability to enhance Ib expression in some studies.15,16 These ideas were concordant with the loss of GPV expression and vWF binding and decreased thrombin responses reported in BSS platelets7 50 and raised the important question of whether mutations in the GPV gene itself might cause BSS.

The cloning of GPV showed a type I membrane glycoprotein with 15 extracellular leucine-rich repeats (LRRs), a thrombin cleavage site just amino terminal to the transmembrane domain, and a short intracellular carboxyl tail.20,21 LRRs are also present in the extracellular domains of Ibα,51 Ibβ,52and IX.53 The functions of these presumed protein-protein interaction motifs in GPV and the other members of the Ib-V-IX complex are unknown. They may mediate interactions among Ib, V, and IX or bind to vWF or to as yet unidentified ligands. Especially intriguing is that the thrombin cleavage site of GPV is conserved in the mouse, rat, and human proteins,30 suggesting possible functional importance for this sequence and again raising the possibility that GPV might contribute to thrombin signaling.

To address the importance of GPV for Ib expression and function and for thrombin signaling, we generated GPV-deficient mice. These mice developed normally, were fertile, and had no overt bleeding phenotype. Their platelets showed normal secretion and aggregation responses to thrombin and U46619. They were normal in number and size, exhibiting none of the giant platelet morphology associated with human BSS.11 By contrast, mice lacking Ibα do have giant platelets and spontaneous hemorrhage,54 and vWF-deficient mice have a bleeding diathesis.55 The abnormal size and shape of platelets from patients with BSS were readily detected by flow cytometry56 and electron microscopy.57 These observations suggest that our results with GPV-deficient mice are unlikely to be due to insensitivity of the measurements used or to a species difference in the roles of the Ib-V-IX complex and vWF. Instead, our results show that loss of GPV is not sufficient to disrupt thrombin signaling or GPIb expression or function. The lack of a hemostatic defect and apparently normal adherence to vWF suggest that GPV is not necessary for vWF binding. This finding is consistent with recent studies comparing the function of Ib-IX and Ib-V-IX expressed heterologously.58 The normal number, size, shape, and cytoskeletal morphology of GPV-deficient platelets suggest that GPV is not required for normal thrombopoiesis or for formation of a normal platelet membrane cytoskeleton.59 

The observation that disruption of the GpV gene in mouse had no deleterious effect on platelet thrombin responses is consistent with the observation that blocking thrombin cleavage of GPV with antibodies had no effect on the thrombin activation of human platelets25 and clearly demonstrates that GPV is not necessary for thrombin-triggered platelet secretion and aggregation. Indeed, available data suggest that these responses are mediated by G protein-coupled PARs.26,28,29,38,60,61 In human platelets, PAR1 and PAR4 can mediate thrombin signaling, and inhibition of both receptors virtually ablated responsiveness to thrombin.29In mouse platelets, PAR3 and PAR4 mediate thrombin signaling.28,38 Knockout of the G-protein α subunit Gq in mice ablated platelet aggregation and secretion to thrombin,62 which is also consistent with the notion that thrombin signaling is mediated, directly or indirectly, by GPCRs. Interestingly, thrombin-triggered shape change was intact in Gq-deficient platelets. However, shape change was inhibited by blockade of PAR1 and PAR4 in human platelets, and shape change was normal in GPV-deficient platelets. Thus, available data suggest that thrombin triggers platelet shape change through PARs via a G protein other than Gq, probably G12/13.63Indeed, when Gq-deficient platelets were exposed to the PAR4-activating peptide GYPGKF, they underwent rapid shape change but not aggregation or secretion (M.L.K. and S.R.C., unpublished observations). Thus, the lack of an effect of GPV-deficiency on thrombin signaling in platelets is consistent with the model that PARs are the major mediators of this process. Whether known PARs completely account for thrombin signaling in platelets will ultimately be tested in knockout mouse models.

As noted above, the observation that the knockout of Ibα results in a BSS-like phenotype suggests that the Ib-V-IX complex plays similar roles in mouse and human. Failure to observe thrombocytopenia, giant platelets, platelet cytoskeletal abnormalities, or bleeding in GPV-deficient mice suggests that loss of function mutations in the GPV gene is unlikely to be uncovered as a cause for BSS in humans.

As an aside, it is interesting to note that, although our transmission EM studies of mouse platelets did not show differences between wild-type and GPV-deficient mouse platelets, they did show interesting differences between mouse and human platelets. When compared with human platelets,64-67 the α-granules of mouse platelets displayed more variations in shape and content (Fig 3). These variations were present in both wild-type and GPV-deficient mouse platelets.

In summary, our results with GPV-deficient mice provide strong evidence that GPV is not necessary for GPIb function or thrombin signaling and is unlikely to be a cause of BSS. It is possible that GPV plays a subtle or redundant role in the function of the Ib-V-IX complex or in thrombin signaling. Alternatively, GPV may play a role in platelet function not probed in the present study. For example, the LRRs of GPV might bind an as yet unidentified ligand(s), or GPV shed from the cell surface upon cleavage by thrombin or another protease might signal to other cells. Lastly, GPV may play a role in cells other than platelets. Indeed, GPV expression has been reported in human endothelial cells.68 The GPV-deficient mouse provides a critical reagent for probing the role of GPV in endothelial cells and for testing other hypotheses regarding GPV's function as they are generated.

The authors thank Violetta Bigornia, Ivy Hsieh, and Martine Morales for their technical assistance and Michael Berndt for his kind gift of botrocetin.

Supported in part by National Institutes of Health (NIH) Grants No. HL44907 and HL59202 and by the Daiichi Research Center, University of California, San Francisco (S.R.C.). M.L.K. was supported by NIH Grant No. HL03731-01.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Sixma
JJ
Wester
J
The hemostatic plug.
Semin Hematol
14
1977
265
2
Tschopp
TB
Weiss
HJ
Baumgartner
HR
Decreased adhesion of platelets to subendothelium in von Willebrand's disease.
J Lab Clin Med
83
1974
296
3
Nurden
AT
Caen
JP
Specific roles for platelet surface glycoproteins in platelet function.
Nature
255
1975
720
4
Berndt
MC
Gregory
C
Kabral
A
Zola
H
Fournier
D
Castaldi
PA
Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane.
Eur J Biochem
151
1985
637
5
Modderman
PW
Admiraal
LG
Sonnenberg
A
von dem Borne
AE
Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.
J Biol Chem
267
1992
364
6
Phillips
DR
Agin
PP
Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis.
J Biol Chem
252
1977
2121
7
Clemetson
KJ
McGregor
JL
James
E
Dechavanne
M
Luscher
EF
Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis.
J Clin Invest
70
1982
304
8
Savage
B
Saldivar
E
Ruggeri
ZM
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell
84
1996
289
9
Vicente
V
Houghten
RA
Ruggeri
ZM
Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding.
J Biol Chem
265
1990
274
10
Nurden
AT
Dupuis
D
Kunicki
TJ
Caen
JP
Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
J Clin Invest
67
1981
1431
11
Bernard J, Soulier J-P: Sur une nouvelle variete de dystrophie thrombocytaire-hemorragipare congenitale. Semin Hop Paris 24:3217
12
Miller
JL
Lyle
VA
Cunningham
D
Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease.
Blood
79
1992
439
13
Ware
J
Russell
SR
Marchese
P
Murata
M
Mazzucato
M
De Marco
L
Ruggeri
ZM
Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.
J Clin Invest
92
1993
1213
14
Wright
SD
Michaelides
K
Johnson
DJ
West
NC
Tuddenham
EG
Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome.
Blood
81
1993
2339
15
Calverley
DC
Yagi
M
Stray
SM
Roth
GJ
Human platelet glycoprotein V: Its role in enhancing expression of the glycoprotein Ib receptor.
Blood
86
1995
1361
16
Meyer
SC
Fox
JE
Interaction of platelet glycoprotein V with glycoprotein Ib-IX regulates expression of the glycoproteins and binding of von Willebrand factor to glycoprotein Ib-IX in transfected cells.
J Biol Chem
270
1995
14693
17
Li
CQ
Dong
JF
Lanza
F
Sanan
DA
Sae
TG
Lopez
JA
Expression of platelet glycoprotein (GP) V in heterologous cells and evidence for its association with GP Ib alpha in forming a GP Ib-IX-V complex on the cell surface.
J Biol Chem
270
1995
16302
18
Phillips
DR
Agin
PP
Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin.
Biochem Biophys Res Commun
75
1977
940
19
Mosher
DF
Vaheri
A
Choate
JJ
Gahmberg
CG
Action of thrombin on surface glycoproteins of human platelets.
Blood
53
1979
437
20
Lanza
F
Morales
M
de La Salle
C
Cazenave
JP
Clemetson
KJ
Shimomura
T
Phillips
DR
Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation.
J Biol Chem
268
1993
20801
21
Hickey
MJ
Hagen
FS
Yagi
M
Roth
GJ
Human platelet glycoprotein V: Characterization of the polypeptide and the related IB-V-IX receptor system of adhesive, leucine-rich glycoproteins.
Proc Natl Acad Sci USA
90
1993
8327
22
Okamura
T
Hasitz
M
Jamieson
GA
Platelet glycocalicin: Interaction with thrombin and role as thrombin receptor on the platelet surface.
J Biol Chem
253
1978
3435
23
Wicki
AN
Clemetson
KJ
Structure and function of platelet membrane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelets response to von Willebrand factor and thrombin.
Eur J Biochem
153
1985
1
24
McGowan
EB
Ding
A
Detwiler
TC
Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation.
J Biol Chem
258
1983
11243
25
Bienz
D
Schnippering
W
Clemetson
KJ
Glycoprotein V is not the thrombin activation receptor on human blood platelets.
Blood
68
1986
720
26
Vu
T-KH
Hung
DT
Wheaton
VI
Coughlin
SR
Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.
Cell
64
1991
1057
27
Ishihara
H
Connolly
AJ
Zeng
D
Kahn
ML
Zheng
YW
Timmons
C
Tram
T
Coughlin
SR
Protease-activated receptor 3 is a second thrombin receptor in humans.
Nature
386
1997
502
28
Kahn
ML
Zheng
YW
Huang
W
Bigornia
V
Zeng
D
Moff
S
Farese
RV
Tam
C
Coughlin
SR
A dual thrombin receptor system for platelet activation.
Nature
394
1998
690
29
Kahn
ML
Nakanishi-Matsui
M
Shapiro
MJ
Ishihara
H
Coughlin
SR
Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.
J Clin Invest
103
1999
879
30
Ravanat
C
Morales
M
Azorsa
DO
Moog
S
Schuhler
S
Grunert
P
Loew
D
Van Dorsselaer
A
Cazenave
JP
Lanza
F
Gene cloning of rat and mouse platelet glycoprotein V: Identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.
Blood
89
1997
3253
31
Kahn
ML
Zheng
Y.-W
Huang
W
Bigornia
V
Zeng
D
Moff
S
Farese
RV
Jr
Tam
C
Coughlin
SR
A dual thrombin receptor system for platelet activation.
Nature
394
1998
690
32
Mortensen
R
Production of a heterozygous mutant cell line by homologous recombination
Current Protocols in Molecular Biology.
Ausubel
FM
1993
9.16.1
Wiley
New York, NY
33
Meiner
VL
Cases
S
Myers
HM
Sande
ER
Bellosta
S
Schambelan
M
Pitas
RE
McGuire
J
Herz
J
Farese
RV
Jr
Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals.
Proc Natl Acad Sci USA
93
1996
14041
34
Dejana
E
Villa
S
de
GG
Bleeding time in rats: A comparison of different experimental conditions.
Thromb Haemost
48
1982
108
35
Meyer
S
Kresbach
G
Haring
P
Schumpp-Vonach
B
Clemetson
KJ
Hadvary
P
Steiner
B
Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.
J Biol Chem
268
1993
20555
36
Ishihara
H
Zeng
D
Connolly
AJ
Tam
C
Coughlin
SR
Antibodies to protease-activated receptor 3 inhibit activation of mouse platelets by thrombin.
Blood
91
1998
4152
37
Cruz
MA
Handin
RI
Wise
RJ
The interaction of the von Willebrand factor-A1 domain with platelet glycoprotein Ib/IX. The role of glycosylation and disulfide bonding in a monomeric recombinant A1 domain protein.
J Biol Chem
268
1993
21238
38
Ishihara
H
Connolly
AJ
Zeng
D
Kahn
ML
Zheng
YW
Timmons
C
Tram
T
Coughlin
SR
Protease-activated receptor 3 is a second thrombin receptor in humans.
Nature
386
1997
502
39
Savage
B
Almus-Jacobs
F
Ruggeri
ZM
Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow.
Cell
94
1998
657
40
Ware
J
Russell
S
Ruggeri
ZM
Cloning of the murine platelet glycoprotein Ibalpha gene highlighting species-specific platelet adhesion.
Blood Cells Mol Dis
23
1997
292
41
Handa
M
Titani
K
Holland
LZ
Roberts
JR
Ruggeri
ZM
The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments.
J Biol Chem
261
1986
12579
42
Murata
M
Ware
J
Ruggeri
ZM
Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.
J Biol Chem
266
1991
15474
43
Meyer
D
Fressinaud
E
Gaucher
C
Lavergne
JM
Hilbert
L
Ribba
AS
Jorieux
S
Mazurier
C
Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: From the patient to the gene. INSERM Network on Molecular Abnormalities in von Willebrand Disease.
Thromb Haemost
78
1997
451
44
Hillery
CA
Mancuso
DJ
Sadler
EJ
Ponder
JW
Jozwiak
MA
Christopherson
PA
Cox Gill
J
Scott
PJ
Montgomery
RR
Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.
Blood
91
1998
1572
45
Read
MS
Smith
SV
Lamb
MA
Brinkhous
KM
Role of botrocetin in platelet agglutination: Formation of an activated complex of botrocetin and von Willebrand factor.
Blood
74
1989
1031
46
Read
MS
Potter
JY
Brinkhous
KM
Venom coagglutinin for detection of von Willebrand factor activity in animal plasmas.
J Lab Clin Med
101
1983
74
47
Kuter
DJ
Gminski
D
Rosenberg
RD
Botrocetin agglutination of rat megakaryocytes: A rapid method for megakaryocyte isolation.
Exp Hematol
20
1992
1085
48
Azorsa
D
Moog
S
Ravanat
C
Schuhler
S
Follea
G
Cazenave
J
Lanza
F
Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA. Its use to follow platelet storage in transfusion.
Thromb Haemost
81
1999
131
49
Modderman
PW
Admiraal
LG
Sonnenberg
A
von dem Borne
AE
Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.
J Biol Chem
267
1992
364
50
Jamieson
GA
Okumura
T
Reduced thrombin binding and aggregation in Bernard-Soulier platelets.
J Clin Invest
61
1978
861
51
Lopez
JA
Chung
DW
Fujikawa
K
Hagen
FS
Papayannopoulou
T
Roth
GJ
Cloning of the alpha chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich alpha 2-glycoprotein.
Proc Natl Acad Sci USA
84
1987
5615
52
Lopez
JA
Chung
DW
Fujikawa
K
Hagen
FS
Davie
EW
Roth
GJ
The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.
Proc Natl Acad Sci USA
85
1988
2135
53
Hickey
MJ
Williams
SA
Roth
GJ
Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.
Proc Natl Acad Sci USA
86
1989
6773
54
Ware
J
Russell
S
Ruggeri
Z
Generation of the Bernard-Soulier syndrome phenotype by genetic disruption of the murine glycoprotein Ibα gene.
Blood
92
1998
703a
(abstr, suppl 1)
55
Denis
C
Methia
N
Frenette
PS
Rayburn
H
Ullman-Cullere
M
Hynes
RO
Wagner
DD
A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis.
Proc Natl Acad Sci USA
95
1998
9524
56
Tomer
A
Scharf
RE
McMillan
R
Ruggeri
ZM
Harker
LA
Bernard-Soulier syndrome: Quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements.
Eur J Haematol
52
1994
193
57
Maldonado
JE
Gilchrist
GS
Brigden
LP
Bowie
EJ
Ultrastructure of platelets in Bernard-Soulier syndrome.
Mayo Clin Proc
50
1975
402
58
Cranmer
SL
Ulsemer
P
Cooke
BM
Salem
HH
de la Salle
C
Lanza
F
Jackson
SP
Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.
J Biol Chem
274
1999
6097
59
Fox
JE
Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.
J Clin Invest
76
1985
1673
60
Hung
DT
Vu
TK
Wheaton
VI
Ishii
K
Coughlin
SR
Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.
J Clin Invest
89
1992
1350
61
Molino
M
Bainton
DF
Hoxie
JA
Coughlin
SR
Brass
LF
Thrombin receptors on human platelets. Initial localization and subsequent redistribution during platelet activation.
J Biol Chem
272
1997
6011
62
Offermanns
S
Toombs
CF
Hu
YH
Simon
MI
Defective platelet activation in G alpha(q)-deficient mice.
Nature
389
1997
183
63
Klages
B
Brandt
U
Simon
MI
Schultz
G
Offermanns
S
Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.
J Cell Biol
144
1999
745
64
White
JG
Clawson
CC
Overview article: Biostructure of blood platelets.
Ultrastruct Pathol
1
1980
533
65
Stenberg
PE
Shuman
MA
Levine
SP
Bainton
DF
Optimal techniques for the immunocytochemical demonstration of beta-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets.
Histochem J
16
1984
983
66
Wencel-Drake
JD
Painter
RG
Zimmerman
TS
Ginsberg
MH
Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section.
Blood
65
1985
929
67
Cramer
EM
Meyer
D
le Menn
R
Breton-Gorius
J
Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies.
Blood
66
1985
710
68
Wu
G
Essex
DW
Meloni
FJ
Takafuta
T
Fujimura
K
Konkle
BA
Shapiro
SS
Human endothelial cells in culture and in vivo express on their surface all four components of the glycoprotein Ib/IX/V complex.
Blood
90
1997
2660
69
Capecchi
MR
Altering the genome by homologous recombination.
Science
244
1989
1288

Author notes

Address reprint requests to Shaun R. Coughlin, MD, PhD, CVRI, UCSF, 505 Parnassus Ave, San Francisco, CA 94143-0130; e-mail:shaun_coughlin@quickmail.ucsf.edu.

Sign in via your Institution