Regulation of platelet numbers and sizes by signaling pathways.

Either the glycoprotein (GP) Ib deficiency or hyper-function in humans can cause macrothrombocytopenia, the molecular mechanisms of which remain unclear. Herein, the investigations for disease pathogenesis were performed in the human induced pluripotent stem cell (hiPSC) model. The hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD) were generated using CRISPR/Cas9. The GP1BA-null hiPSCs were previously derived from a Bernard-Soulier syndrome (BSS) patient. After full megakaryocyte differentiation in culture, both hiPSC mutations showed large proplatelet tips under fluorescence microscopy and yielded fewer but larger platelets compared with those of wild-type cells. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to wild-type hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and wild-type hiPSCs with a MAPK inhibitor. In conclusion, the genetically modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.

Whole exome sequencing for diagnosis of hereditary thrombocytopenia.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management.To evaluate the role of whole exome sequencing (WES) in these Pts.Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM).Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9.This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Study of Bernard-Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells.

Bernard-Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom.

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets. Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5'RACE and 3'RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the ß subunit contained 447bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αß)(4). Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC(50) of 0.25nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.