Hagit Hauschner

A 13‐bp deletion in αIIb gene is a founder mutation that predominates in Palestinian‐Arab patients with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of αIIbβ3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13‐bp deletion in the αIIb gene that causes in‐frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13‐bp deletion, and determined the mechanism by which the 13‐bp deletion abolishes αIIbβ3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13‐bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300–600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective αIIbβ3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in αIIb gene, TT1616‐7 deletion in β3 gene, and IVS14: −3C → G in β3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of β3 and was expressed as dysfunctional αIIbβ3. None of 15 unrelated Jordanian patients carried any of the described mutations.

Deleterious mutation in the FYB gene is associated with congenital autosomal recessive small-platelet thrombocytopenia.

The FYB gene encodes adhesion and degranulation‐promoting adaptor protein (ADAP), a hematopoietic‐specific protein involved in platelet activation, cell motility and proliferation, and integrin‐mediated cell adhesion. No ADAP‐related diseases have been described in humans, but ADAP‐deficient mice have mild thrombocytopenia and increased rebleeding from tail wounds.

A unique interaction between αIIb and β3 in the head region is essential for outside-in signaling–related functions of αIIbβ3 integrin

The main interface of the 2 subunits of platelet integrin alphaIIbbeta3 comprises the beta-propeller domain of alphaIIb and the betaA domain of beta3. In the center of the beta-propeller, several aromatic residues interact by cation-pi and hydrophobic bonds with Arg261 of betaA. In this study, we substituted alphaIIb-Trp110 or beta3-Arg261 by residues abundant in other alpha or beta subunits at corresponding locations and expressed them in baby hamster kidney cells along with normal beta3 or alphaIIb, respectively. These mutant cells displayed normal surface expression and fibrinogen binding but grossly impaired outside-in signaling-related functions: adhesion to immobilized fibrinogen, cell spreading, focal adhesion kinase phosphorylation, clot retraction, and reduced alphaIIbbeta3 stability in EDTA (ethylenediaminetetraacetic acid). Expression of mutants with substitutions of Arg261 in beta3 by alanine or lysine with normal alphav yielded normal surface expression of alphavbeta3 and soluble fibrinogen binding as well as normal outside-in signaling-related functions, contrasting findings for alphaIIbbeta3. Structural analysis of alphaIIbbeta3 and alphavbeta3 revealed that alphavbeta3 has several strong interactions between alphav and beta3 subunits that are missing in alphaIIbbeta3. Together, these findings indicate that the interaction between Trp110 of alphaIIb and Arg261 of beta3 is critical for alphaIIbbeta3 integrity and outside-in signaling-related functions.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the β-propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary.  Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ3. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ3 was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β3. Although the αIIbβ3 was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ3 crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ3. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ3 surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ3, αVβ3 harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ3 to calcium chelation compared with αIIbβ3. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ3. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

Persistent neonatal thrombocytopenia can be caused by IgA antiplatelet antibodies in breast milk of immune thrombocytopenic mothers

Immune thrombocytopenia (ITP) in pregnant women can cause neonatal thrombocytopenia by transport of antiplatelet autoantibodies across the placenta. Usually, an infants platelet count normalizes within 2 months. We observed neonatal thrombocytopenia that persisted more than 4 months and disappeared following discontinuation of breastfeeding. The aim of our study was to discern whether breast milk of ITP mothers contained antiplatelet antibodies causing persistent thrombocytopenia. We collected milk samples from 3 groups of women: ITP group, 7 women who had ITP during pregnancy; R-ITP group, 6 women who recovered from ITP before pregnancy; and 9 healthy controls. We found increased levels of antiplatelet antibodies of the immunoglobulin A type in the milk of ITP patients compared with the other 2 groups. Similar increase was demonstrated for antibodies binding to αIIbβ3 expressed in cultured cells. Thus, transfer of antiplatelet antibodies from ITP mothers by breastfeeding can be associated with persistent neonatal thrombocytopenia.

Model of trauma-induced coagulopathy including hemodilution, fibrinolysis, acidosis and hypothermia: Impact on blood coagulation and platelet function.

BACKGROUND Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease. METHODS A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s−1 using Impact-R device. RESULTS Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function. CONCLUSION The study revealed a differential effect of TIC constituents—hemodilution, hypothermia, fibrinolysis, and acidosis—on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy. LEVEL Of EVIDENCE A new model of TIC is created. Contribution of various constituents to pathogenesis of TIC and their interactions are evaluated.

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary.  Background: The cytoplasmic tails of αIIb and β3 regulate essential αIIbβ3 functions. We previously described a variant Glanzmann thrombasthenia mutation in the β3 cytoplasmic tail, IVS14: −3C>G, which causes a frameshift with an extension of β3 by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of αIIbβ3 from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) αIIb as follows: β3‐742stop, a truncated mutant to evaluate the effect of deleted residues; β3‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β3‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of αIIbβ3 of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the αIIbβ3 mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β3 tail is important for maintaining αIIbβ3 in a resting conformation.

Role of heterotrimeric G proteins in platelet activation and clot formation in platelets treated with integrin αIIbβ3 inhibitor

Abstract Mechanisms of platelet activation are triggered by thrombin, adenosine diphosphate (ADP), epinephrine, thromboxane A2, and other soluble agonists which induce signaling via heterotrimeric Gαq, Gαi, and Gα12/13 proteins. We have undertaken a study addressing the contribution of these G proteins to platelet activation and clot formation in the presence of eptifibatide, thus excluding outside-in signaling provided by integrin αIIbβ3–fibrinogen engagement. Selective and combined activation of the G proteins was achieved by using combinations of platelet agonists and inhibitors. Platelet activation in platelet-rich plasma was evaluated by P-selectin expression using flow cytometry. Contribution of platelets to whole blood clotting was assessed by rotation thromboelastometry (ROTEM). Selective signaling of Gαq or Gαi but not Gα12/13 promoted P-selectin expression. Further enhancement of P-selectin expression was achieved by ADP-induced combined signaling of Gαq and Gαi, and to more extent by U46619 at high concentration (1.5 μM) induced combined signaling of Gαq and Gα12/13 while maximal P-selectin expression was achieved by thrombin receptor-activating peptide (TRAP)-induced combined signaling of Gαq, Gαi, and Gα12/13. In ROTEM, selective activation of Gαq, Gαi, or Gα12/13 failed to affect blood clotting. Combined signaling of Gαq and Gαi or Gαq and Gα12/13 or all three G proteins shortened the clotting time and stimulated clot strength. Pretreatment of platelets with acetylsalicylic acid did not change the effect of ADP but inhibited the effect of TRAP. Signaling of Gαq and Gα12/13 triggered by U46619 also stimulated clot formation. Combined signaling of either Gαq and Gαi or Gαq and Gα12/13 is sufficient to stimulate maximal platelet activation and enhanced clot formation in platelets treated with inhibitor of integrin αIIbβ3. It could be suggested that outside-in signaling is not necessarily required to fulfill these platelet functions.

Platelet factor XI: intracellular localization and mRNA splicing following platelet activation

BACKGROUND The structure and function of platelet factor XI (FXI) protein and the presence of F11 mRNA in platelets are controversial. Although platelets are anucleated cells they contain spliceosome components and pre-mRNAs. Three platelet proteins have been demonstrated to be spliced upon platelet activation. OBJECTIVE To determine whether FXI is also spliced upon activation and to discern the localization of FXI in platelets. METHODS Localization of FXI in platelets was assessed by confocal immunofluorescence staining. ELISA, chromogenic assay and western blot analyses were used to measure antigen levels, activity levels and size of FXI in platelets, respectively. Splicing patterns of F11 mRNA were assessed in three states of platelet activation: activated platelets, resting platelets and αIIbβ3-integrin activated platelets. RESULTS Platelet FXI was exhibited in platelet granules. Activated platelets exhibited higher levels of mature F11 mRNA and protein and lower levels of F11 pre-mRNA compared to resting or αIIbβ3-integrin activated platelets. CONCLUSIONS We confirmed the presence of FXI in platelets and showed that it is localized in granules but is not restricted to the same α-granule subtype as von-Willebrand factor and p-selectin. Our study also shows that F11 is present in platelets as pre-mRNA and is spliced upon platelet activation.

Acquired thrombotic thrombocytopenic purpura in pregnancy: The role of placental and breast-milk mediated transfer of ADAMTS13-autoantibodies

To the editor: Acquired thrombotic thrombocytopenic purpura (TTP) is an acute, life threatening thrombotic microangiopathy resulting from autoantibodymediated inhibition of ADAMTS13 (ADisintegrin AndMetalloproteinase with a Thrombospondin type 1 motif, member 13), the key enzyme involved in the cleavage of ultra-large von Willebrand factor multimers [1]. Approximately 5–10% of TTP cases are encountered in the setting of pregnancy, with associated adverse maternal and fetal outcomes [1]. Herein we describe a case of acquired TTP in a pregnant patient and depict the role of anti-ADAMTS13 antibodies in the fetus.