Human CalDAG-GEFI deficiency increases bleeding and delays αIIbβ3 activation

The affinity regulation of fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) by inside-out signaling is critical for normal platelet hemostatic functions.¹  Upon agonist stimulation, activated Rap1 recruits talin to β3 cytoplasmic tail.²  At the final common step of inside-out signaling, the interaction of talin^3-5  and kindlin-3^6,7  with β3 integrin is necessary for αIIbβ3 activation.^8,9  Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule,¹⁰  and its role in calcium-dependent platelet activation has been established in CalDAG-GEFI–deficient mice.^11-14  Recently, its role in humans has also become available by the first report of the patient with CalDAG-GEFI mutation.¹⁵  Here, we report our patient with CalDAG-GEFI deficiency who presents severe bleeding symptoms. In agreement with phenotypes in CalDAG-GEFI–deficient mice, the patient’s platelets showed impaired inside-out αIIbβ3 activation, granule release, and platelet aggregation. In addition, the analysis of αIIbβ3 activation kinetics by initial velocity assay^16,17  and shear-induced thrombus formation suggests that the delayed αIIbβ3 activation is associated with severe bleeding symptoms in our patient. In sharp contrast, N-formylmethionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA)-induced neutrophil β2 integrin activation was not impaired. Our results, in conjunction with the information from genetically modified mice, clearly demonstrate the importance of CalDAG-GEFI in inside-out αIIbβ3 activation, especially rapid αIIbβ3 activation, for human platelet hemostatic functions.

The functional and genetic analysis of our patient and her parents was performed after they provided informed consent in accordance with the Declaration of Helsinki under protocols reviewed by the Osaka University ethics committees. Platelets were analyzed by aggregometer, western blotting, and flow cytometry. αIIbβ3 activation kinetics was assessed by initial velocity assay.^16,17  Sequence of CalDAG-GEFI was analyzed using Sanger sequencing. Detailed methods are included in the supplemental Methods, available on the Blood Web site.

The proband, a 16-year-old Japanese girl born from nonconsanguineous parents, has been suffering from severe bleeding tendency. Although her parents had no bleeding problems, she had severe epistaxis 3 times that required red blood cell and platelet transfusion before she was 3 years of age. Thereafter, desmopressin was administered to prevent severe bleeding, which showed some improvement in her bleeding time. However, at 14 years of age, she had massive menorrhagia with hemorrhagic shock that required red blood cell transfusion. To investigate the cause of her severe bleeding tendency, she was referred to Osaka University Hospital.

The patient constantly showed normal leukocyte count and differential. Although her platelet count and coagulation functions were normal (supplemental Table 1), her bleeding time was markedly prolonged to >30 minutes. In platelet aggregometer, her platelets aggregated poorly even with high-concentration adenosine 5′-diphosphate (ADP) stimulation (Figure 1A). Slow and impaired platelet aggregation at 1 μg/mL collagen, but relatively normal aggregation at 2 μg/mL collagen, was observed (Figure 1B). Ristocetin-induced aggregation (supplemental Figure 1) and platelet glycoprotein expression were comparable to those of healthy control platelets (supplemental Figure 2).

We then carefully performed functional analysis of αIIbβ3 and found that agonist-induced binding of PAC-1, which specifically recognizes activated αIIbβ3,¹⁸  was significantly reduced in the patient. In addition to ADP and collagen-related peptide (CRP), thrombin receptor activating peptide (PAR1-AP)- or thromboxane analog U46619-induced PAC-1 binding was also impaired (Figure 1C). In contrast, PMA- or anti-αIIbβ3–activating antibody PT25-2-induced PAC-1 binding was comparable to those of control (Figure 1D). Because released platelet granular contents act in an autocrine fashion for αIIbβ3 activation,¹⁹  the impaired granule release (supplemental Figure 3) may cause reduced αIIbβ3 activation. However, impaired PAC-1 binding by simultaneous stimulation with PAR1-AP, ADP, and U46619 suggested that inside-out αIIbβ3 activation itself was impaired (Figure 1E). Transmission electron microscopy showed no apparent abnormality in granule contents (Figure 1F), which further denied the possibility of storage pool deficiency. Collectively, these findings indicate that αIIbβ3 activation, inside-out signaling, is impaired in her platelets. Impaired αIIbβ3 activation by any agonist except for PMA stimulation suggested that CalDAG-GEFI and/or its related signaling molecules may be impaired in her platelets.¹¹  We then analyzed these critical molecules regarding αIIbβ3 activation by western blotting. In sharp contrast to the normal amounts of talin, FAK, and kindlin-3, CalDAG-GEFI was undetectable in her platelets (Figure 1G). In contrast to the first patient with dysfunctional CalDAG-GEFI due to p.Gly248Trp,¹⁵  our patient is unique because of its complete deficiency. As shown in its knockout mice, CalDAG-GEFI is essential for calcium-induced inside-out αIIbβ3 activation and hemostasis.¹¹  As expected, weaker and delayed Rap1 activation after PAR1-AP stimulation (Figure 1H) despite normal intraplatelet calcium mobilization (Figure 1I) was observed in her platelets.

Sequencing analysis revealed novel compound heterozygous mutations in CalDAG-GEFI, an A>T transition c.1178 resulting in a premature stop codon (p.Lys309X) (Figure 2A) and a 3 nucleic acids deletion (c.1331_1333delCTG), resulting in the loss of a single amino acid (p.Leu360del) (Figure 2B). The parents are heterozygous carriers of these mutations with decreased platelet CalDAG-GEFI expression (Figure 2C). No detectable CalDAG-GEFI expression by transfection of mutant CalDAG-GEFI expression vector into 293T cells confirmed that the identified novel mutations are responsible for CalDAG-GEFI deficiency in our patient (Figure 2D).

Although the inside-out αIIbβ3 activation was significantly impaired in her platelets, CalDAG-GEFI independent PAC-1 binding and platelet aggregation were still observed (Figure 1A-C). To elucidate why she has severe bleeding problems regardless of some residual αIIbβ3 activity, we determined the kinetics of αIIbβ3 activation by initial velocity assay.^16,17  In contrast to the rapid and sustained αIIbβ3 activation in control platelets, αIIbβ3 activation was delayed in patient platelets (Figure 2E), and PAC-1 binding velocity at 30 and 60 seconds after PAR1-AP stimulation was only 19% and 49% of those of control, respectively (Figure 2F). In addition, shear-induced thrombus growth on collagen was markedly impaired in our patient (Figure 2G). Although the surface coverage by adhered platelets was comparable to control, the thrombus height was significantly lower after 10 minutes of perfusion (Figure 2H). In our patient with ADP receptor P2Y₁₂ deficiency (OSP-1),²⁰  rapid and normal αIIbβ3 activation observed at 30 seconds after PAR1-AP stimulation was quickly diminished at 5 minutes (Figure 2I). As shown in our previous report, shear-induced thrombus height in OSP-1 was roughly half of control. However, that is much better than our patient with CalDAG-GEFI deficiency, which is consistent with milder bleeding tendency in OSP-1. These results obtained in a human patient with CalDAG-GEFI deficiency are consistent with the data reported in the mice model^13,21  and suggest the importance of rapid αIIbβ3 activation.

Previous studies demonstrated the reduced β2 integrin activation and impaired neutrophil functions in CalDAG-GEFI–deficient mice.²²  However, our patient has no history of frequent infections, and fMLP- or PMA-induced β2 integrin activation detected by activated β2 integrin-specific mAb24²³  was comparable between control and patient’s CalDAG-GEFI–deficient neutrophils (Figure 2J; supplemental Figure 5). Because no apparent abnormality has been reported in the first CalDAG-GEFI–mutant patient either,¹⁵  the difference by CalDAG-GEFI deficiency in neutrophil function may be a species-specific phenomenon. Further studies are needed to define the role of CalDAG-GEFI in neutrophils.

Our results confirmed the critical role of CalDAG-GEFI in human platelet inside-out αIIbβ3 activation. Although the impaired granule release and outside-in signaling also as reported previously^11,15  may contribute to her bleeding tendency, delayed αIIbβ3 activation is associated with severe bleeding problems in the patient with CalDAG-GEFI deficiency. Our findings indicate the importance of rapid αIIbβ3 activation for normal hemostatic function

During our manuscript submission, 2 additional (p.Arg113X and p.Ser381Phe) mutations responsible for CalDAG-GEFI deficiency and/or dysfunction were reported by Lozano et al.²⁴ 

The authors thank Koichi Kokame and Toshiyuki Miyata (National Cerebral and Cardiovascular Center, Suita, Japan) for help with the shear-induced thrombus formation experiment.

This work was supported by a Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Science (grant JP26461403).

Contribution: H. Kato designed and performed research, analyzed data, and wrote the paper; Y.N., D.M., and Y. Kurokawa contributed patient samples, performed experiments, and analyzed results; F.B., S.H., Y.M., and H. Kashiwagi performed experiments; Y. Kanakura and Y.T. designed and supervised the research. All coauthors reviewed, edited, and approved the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Hisashi Kato, Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; e-mail: hisashi@hp-blood.med.osaka-u.ac.jp.