We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the ␤ 3 integrin and a P53L mutation in glycoprotein (GP) Ib␣. We show that GPIb␣-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The ␤ 3 -H723 causes constitutive, albeit partial, activation of the ␣ IIb ␤ 3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the ␣ IIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO ␣ IIb ␤ 3 -H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, ␣ IIb ␤ 3 -H723, but not wild-type ␣ IIb ␤ 3 , generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34 ؉ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the ␣ IIb ␤ 3 -H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree. IntroductionInherited thrombocytopenias are a rare group of diseases with a wide spectrum of clinical phenotypes. Because of their rare occurrence, patients with an inherited low platelet count may be misdiagnosed with autoimmune thrombocytopenia (ITP) and receive inappropriate therapy. 1,2 Among the inherited thrombocytopenias, macrothrombocytopenia constitutes a subgroup in which Bernard-Soulier Syndrome (BSS), caused by mutations in the GP1BA, GP1BB, and GP9 genes, is the most common one, with large platelets and severe bleeding. 3 The molecular mechanisms of some of the rarer syndromes which are accompanied by macrothrombocytopenia have also been elucidated. Mutations in the myosin heavy-chain protein (MYH9) were identified in groups of patients with a spectrum of platelet disorders, such as the MayHegglin anomaly and the Epstein, Fechtner, and Sebastian syndromes. 4,5 The mode of inheritance of these disorders is generally autosomal recessive, but autosomal-dominant forms of a BSS-like disorder caused by nonsynonymous single-nucleotide polymorphisms (nsSNPs) in the GP1BA gene have also been reported. 6 More recently, mutations in the transcription factor GATA1 were defined as the cause of X-linked macrothrombocytopenia. 7 The most frequent autosomal-recessive platelet bleeding disorder is Glanzmann thrombasthenia (GT), which is caused by mutations in the ITGA2B or ITGB3 genes that encode for the integrin ␣ IIb ␤ 3 . This integrin, also named platelet glycoprotein (GP) IIbIIIa, is the the most abundantly expressed platelet membrane glycoprotein, 8 and its role is pivotal for platelet function. 9,10 Qualitative and quantitative defects...

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin ␣IIb␤3 occurs exclusively through the synergistic ␥ -411sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen ␥ 400 -411 sequence and the RGD motif in the molecular events leading to ligand-induced ␣IIb␤3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the ␥ -411sequence (fragment D), and CHO cells expressing resting wild type (␣IIb␤3wt), constitutively active (␣IIb␤3T562N), or non-functional (␣IIb␤3D119Y) receptors. Our data provide evidence that the ␥ 400 -411 site by itself is able to initiate ␣IIb␤3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the ␤3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the ␤3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, ␣IIb␤3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinaseindependent and are inhibited by the Src antagonist PP2.Plasma fibrinogen is one of the most abundant soluble adhesion molecules present in blood vessels and serves as a ligand to a variety of vascular cells, including platelets, endothelial cells, and monocytes. Fibrinogen is primarily involved in the maintenance of hemostasis by mediating platelet aggregation, clot formation, and wound healing. In addition, together with thrombin-converted insoluble fibrin, fibrinogen also functions as a component of the extracellular matrix in non-hemostatic normal or pathological processes promoting placenta development, angiogenesis, atherosclerosis, metastasis, as well as a variety of vascular and renal diseases (1). Both fibrinogen and fibrin expose multiple interacting sites that serve as adhesion motifs for vascular cell receptors. Undoubtedly, the first and best characterized of these binding sites are those interacting with the ␤3 integrins, the platelet-specific ␣IIb␤3 fibrinogen receptor (2), and the ␣v␤3 vitronectin receptor (3).Human fibrinogen contains three putative ␤3 integrin binding sites, two RGD motifs within the A␣ chain, A␣ 95-98 (RGDF) and A␣ [572][573][574][575] (RGDS) (4), and a non-RGD dodecapeptide sequence in the ␥ chain (C-terminal ␥ 400 -411 ) (5). Although fibrinogen ...

Summary. Background: We have recently reported a novel mutation in the b 3 subunit of the platelet fibrinogen receptor (a IIb b 3 D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated a IIb b 3 D723H or a IIb b 3 D723A salt bridge mutants, but not fully activated a IIb b 3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actindriven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of a IIb b 3 , the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34 + -derived megakaryocyte proplatelets. Mutant a IIb b 3 D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wildtype a IIb b 3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO a IIb b 3 D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO a IIb b 3 D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.

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, a IIb b 3 T562N mediated cell adhesion (40.5 ± 3.8 cells/field), while a IIb b 3 wt did not (5.3 ± 1.4 cells/ field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 lM fibrinogen c-chain dodecapeptide [HHLGGAK-QAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 lM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by a IIb b 3 T562N. Conclusions: Our data show that a IIb b 3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.