Inhibition of LDHA to induce eEF2 release enhances thrombocytopoiesis.

Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet-specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell-derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.

The role of AGK in thrombocytopoiesis and possible therapeutic strategies.

Abnormal megakaryocyte development and platelet production lead to thrombocytopenia or thrombocythemia and increase the risk of hemorrhage or thrombosis. Acylglycerol kinase (AGK) is a mitochondrial membrane kinase that catalyzes the formation of phosphatidic acid and lysophosphatidic acid. Mutation of AGK has been described as the major cause of Sengers syndrome, and the patients with Sengers syndrome have been reported to exhibit thrombocytopenia. In this study, we found that megakaryocyte/platelet-specific AGK-deficient mice developed thrombocytopenia and splenomegaly, mainly caused by inefficient bone marrow thrombocytopoiesis and excessive extramedullary hematopoiesis, but not by apoptosis of circulating platelets. It has been reported that the G126E mutation arrests the kinase activity of AGK. The AGK G126E mutation did not affect peripheral platelet counts or megakaryocyte differentiation, suggesting that the involvement of AGK in megakaryocyte development and platelet biogenesis was not dependent on its kinase activity. The Mpl/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat3) pathway is the major signaling pathway regulating megakaryocyte development. Our study confirmed that AGK can bind to JAK2 in megakaryocytes/platelets. More interestingly, we found that the JAK2 V617F mutation dramatically enhanced the binding of AGK to JAK2 and greatly facilitated JAK2/Stat3 signaling in megakaryocytes/platelets in response to thrombopoietin. We also found that the JAK2 JAK homology 2 domain peptide YGVCF617CGDENI enhanced the binding of AGK to JAK2 and that cell-permeable peptides containing YGVCF617CGDENI sequences accelerated proplatelet formation. Therefore, our study reveals critical roles of AGK in megakaryocyte differentiation and platelet biogenesis and suggests that targeting the interaction between AGK and JAK2 may be a novel strategy for the treatment of thrombocytopenia or thrombocythemia.

A Homozygotic Mutation in KDSR may Cause Keratinization Disorders and Thrombocytopenia: A Case Report.

Pathogenic mutations in 3-keto-dihydrosphingosine reductase (KDSR) gene are associated with keratinization disorders and impaired platelet function. However, no case with both homozygotic mutation of KDSR and hepatic hemangioendothelioma has ever been reported due to its low prevalence. Here we report a seven months old Chinese boy with a homozygotic missense mutation in KDSR and both of his parents carry a same heterozygous mutation. He was born with thick plate-like scales overlying erythrodermic skin, but the skin symptoms were resolved spontaneously over the first month of his birth. He was also diagnosed with hepatic hemangioendothelioma at birth and accepted a resection surgery at 2 months old. At birth, his platelet count was severely low (10-20×109/L) with recurrent skin and gingival bleeding. Meanwhile, he suffered a mild normocytic, normochromic anemia with normal iron and hematinic levels. The anemia spontaneously recovered over the first 6 months, while the platelet count keeped at a low level (4-20×109/L). Treatment with corticosteroids, immunoglobulin or thrombopoietin was all suboptimal.

Purinergic ecto-enzymes participate in the thromboregulation in acute in mice infection by Trypanosoma cruzi.

Coagulation disorders have been described in Chagas disease with thrombocytopenia as an important event. Several mechanisms may be related to this pathogenesis, such as enzymes of the purinergic system, purine, and receptors involved in the regulation and modulation of physiological events related to hemostasis. Therefore, the aim of this study was to evaluate the activities of E-NTPDase, E-5'nucleotidase, and ecto-adenosine deaminase (E-ADA) in platelets of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. Mice of infected group were intraperitoneally inoculated with 104 trypomastigotes of T. cruzi (strain Y). On day 12 post-infection (PI), blood samples were collected for quantitation and separation of platelets. A significant reduction in the number of platelets of infected mice (P < 0.05) was observed. The activities of E-NTPDase (ATP and ADP substrates), E-5'nucleotidase, and E-ADA in platelets increased significantly (P < 0.05) in mice infected by T. cruzi compared with uninfected animals. A negative correlation (P < 0.01)was observed between the number of platelets and ATP hydrolysis (r = -0.64), and ADP hydrolysis (r = -0.69) by E-NTPDase. Therefore, there is a response from the purinergic system activating ecto-enzymes in platelets of mice T. cruzi infected, as a compensatory effect of thrombocytopenia.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

OBJECTIVE: The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). APPROACH AND RESULTS: HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. CONCLUSIONS: Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Three-year efficacy, safety, and survival findings from COMFORT-II, a phase 3 study comparing ruxolitinib with best available therapy for myelofibrosis.

Ruxolitinib is a potent Janus kinase (JAK)1/JAK2 inhibitor that has demonstrated rapid reductions in splenomegaly and marked improvement in disease-related symptoms and quality of life in patients with myelofibrosis (MF). The present analysis reports the 3-year follow-up (median, 151 weeks) of the efficacy and safety of Controlled Myelofibrosis Study With Oral Janus-associated Kinase (JAK) Inhibitor Treatment-II (the COMFORT-II Trial), comparing ruxolitinib with the best available therapy (BAT) in 219 patients with intermediate-2 and high-risk MF. In the ruxolitinib arm, with continued therapy, spleen volume reductions of ≥35% by magnetic resonance imaging (equivalent to approximately 50% reduction by palpation) were sustained for at least 144 weeks, with the probability of 50% (95% confidence interval [CI], 36-63) among patients achieving such degree of response. At the time of this analysis, 45% of the patients randomized to ruxolitinib remained on treatment. Ruxolitinib continues to be well tolerated. Anemia and thrombocytopenia were the main toxicities, but they were generally manageable, improved over time, and rarely led to treatment discontinuation (1% and 3.6% of patients, respectively). No single nonhematologic adverse event led to definitive ruxolitinib discontinuation in more than 1 patient. Additionally, patients randomized to ruxolitinib showed longer overall survival than those randomized to BAT (hazard ratio, 0.48; 95% CI, 0.28-0.85; log-rank test, P = .009).

Protein kinase C δ deficiency enhances megakaryopoiesis and recovery from thrombocytopenia.

OBJECTIVE: We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. APPROACH AND RESULTS: Using PKCδ(-/-) and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ(-/-) mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ(-/-) megakaryocytes compared with wild-type. Finally, PKCδ(-/-) mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. CONCLUSIONS: These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.

G6PC3 Deficiency: Primary Immune Deficiency Beyond Just Neutropenia.

Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency was recently defined as a new severe congenital neutropenia subgroup remarkable with congenital heart defects, urogenital malformations, endocrine abnormalities, and prominent superficial veins. Here, we report 3 patients with G6PC3 deficiency presenting with recurrent diarrhea, failure to thrive, and sinopulmonary infections leading to bronchiectasis. In patient I and II, a combined immune deficiency was suspected due to early-onset disease with lymphopenia, neutropenia, and thrombocytopenia, along with variable reductions in lymphocyte subpopulations and favorable response to intravenous γ-globulin therapy. Apart from neutropenia, all 3 patients had intermittent thrombocytopenia, anemia, and lymphopenia. All patients had failure to thrive and some of the classic syndromic features of G6PC3 deficiency, including cardiac abnormalities and visibility of superficial veins in all, endocrinologic problems in PI and PIII, and urogenital abnormalities in PII. Our experience suggests that a diagnosis of congenital neutropenia due to G6PC3 may not be as straightforward in such patients with combined lymphopenia and thrombocytopenia. A high index of suspicion and the other syndromic features of G6PC3 were clues to diagnosis. Screening of all combined immune deficiencies with neutropenia may help to uncover the whole spectra of G6PC3 deficiency.

Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis.

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.

Successful liver transplantation in a patient with splanchnic vein thrombosis and pulmonary embolism due to polycythemia vera with Jak2v617f mutation and heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatment resulting in a severe acquired thrombophilic condition with an associated mortality of about 10 %. We report the first case of successful urgent liver transplantation (LT) in a patient with end-stage liver disease due to a Budd-Chiari syndrome, portal vein thrombosis and pulmonary embolism due to acquired thrombophilia associated to polycythemia vera carrying JAK2V617F gene mutation and HIT in the acute phase. Lepirudin was used to provide anticoagulation in the LT perioperative period that was performed without haemorrhagic and thrombotic complications despite the donor received heparin during liver explantation.

Contributions of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet production in immune thrombocytopenia.

Recent in vitro studies provide evidence for autoantibody-induced suppression of megakaryocytopoiesis and show a reduction in megakaryocyte production and maturation in the presence of immune thrombocytopenia (ITP) plasma. Here, we present CD34(+) cells from healthy umbilical cord blood mononuclear cells cultured in medium containing thrombopoietin, stem cell factor, interleukin-3, and 10% plasma from either ITP patients or healthy subjects. The quantity, quality, and apoptosis of megakaryocytes were measured. We observed that most ITP plasma boosted megakaryocyte quantity but impaired quality, resulting in significantly less polyploidy cells (N ≥ 4) and platelet release. In these megakaryocytes, we found a lower percentage of cell apoptosis, a lower expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and a higher expression of Bcl-xL. Furthermore, there was a decrease of sTRAIL in ITP plasma and in cell culture supernatants of this group compared with the control group. Our findings suggest that decreased apoptosis of megakaryocytes also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production. The abnormal expression of sTRAIL in plasma and TRAIL and Bcl-xL in megakaryocytes may play a role in the pathogenesis of impaired megakaryocyte apoptosis in ITP.

Decreased indoleamine 2,3-dioxygenase expression in dendritic cells and role of indoleamine 2,3-dioxygenase-expressing dendritic cells in immune thrombocytopenia.

Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) can induce or maintain peripheral immune tolerance. Impaired IDO-mediated tryptophan catabolism has been observed in autoimmune diseases. In order to investigate the effects of IDO-mediated tryptophan catabolism and IDO-expressing DCs in immune thrombocytopenia, the concentrations of kynurenine were detected by high-pressure liquid chromatography. The expressions of IDO were analyzed by flow cytometry and western blot analysis. The effects of IDO(+) DCs stimulated with CTLA-4-Ig on T cells proliferation and activation, lymphocyte apoptosis, and Tregs were measured by flow cytometry. We found that the expression of IDO in DCs of immune thrombocytopenia (ITP) patients was significantly decreased. CTLA-4-Ig significantly increased the expression of functional IDO in DCs of ITP patients. IDO(+) DCs stimulated with CTLA-4-Ig suppressed T cells proliferation and activation, promoted lymphocyte apoptosis, and increased the percentage of Tregs. These results suggest that decreased IDO expression in DCs may play a critical role in ITP. CTLA-4-Ig successfully corrected the disorder of IDO expression in ITP. IDO(+) DCs stimulated with CTLA-4-Ig inhibited immune responses by an IDO-dependent mechanism. Increasing the expression and activity of IDO in DCs might be a promising therapeutic approach for ITP.

Platelet protein kinase C-theta deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1.

OBJECTIVE: Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C- (PKC-, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC- in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. METHODS AND RESULTS: In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region -1225 to -1056 bp containing a RUNX1 consensus site ACCGCA at -1088 to -1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC- protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC- protein were decreased by short interfering RNA knockdown of RUNX1. CONCLUSIONS: Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC- deficiency associated with RUNX1 haplodeficiency.

DNA methyltransferase 3B gene promoter and interleukin-1 receptor antagonist polymorphisms in childhood immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP) (C46359T) in DNA methyltransferase 3B (DNMT3B) gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 in 32 children (17 boys) with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution of DNMT3B polymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06-3.94). In case of IL-1 Ra polymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02-6.50). Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913 DNMT3B SNP as well as IL-1 Ra VNTR polymorphism may contribute to the susceptibility to ITP.

Influence of methylene tetrahydrofolate reductase polymorphisms and coadministration of antimetabolites on toxicity after high dose methotrexate.

BACKGROUND AND OBJECTIVE: Through interruption of maintenance treatment with 6-mercaptopurine (6MP), toxicity after high-dose methotrexate (HDMTX) may compromise the efficiency of the treatment of children with acute lymphocytic leukaemia (ALL). We investigated the influence of polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) gene and coadministration of antimetabolites on post-HDMTX toxicity. METHODS: Toxicity was retrospectively analysed after 656 HDMTX courses administered to 88 paediatric ALL patients at a single treatment centre. RESULTS: High-dose methotrexate with high-intensity co-treatment (6MP 75 mg/m(2)/d + MTX 20 mg/m(2)/wk) was found associated with increased odds of haematological toxicity (OR's: 3.47-7.88; P's: <0.001), hepatic toxicity (OR = 6.91; P < 0.001), hospitalization with fever (OR = 2.2; P = 0.004) and interruption maintenance treatment (OR = 15.9; P < 0.001) compared to HDMTX with low-intensity co-treatment (6MP 25 mg/m(2)/d). Addition of cytarabine to the low-intensity co-treatment increased the odds of neutropenia (OR = 3.51; P = 0.002), thrombocytopenia (OR = 6.56; P < 0.001), hepatic toxicity (OR = 3.84; P = 0.012) and interruption of maintenance treatment (OR = 4.25; P = 0.002). Alterations in 6MP dose were associated with significant changes in toxicity. Dose reduction reduced the odds of haematological toxicity (OR's: 0.22-0.34; P's: <0.001-0.020), while dose increase increased the odds of haematological toxicity (OR's: 2.72-7.42; P's: 0.006-0.027), fever (OR = 2.65; P = 0.037) and interruption of maintenance treatment (OR = 3.04; P = 0.032). No convincing associations were found between the MTHFR C677T or A1298C polymorphisms and toxicity. CONCLUSION: Our findings demonstrate that toxicity after HDMTX is influenced by coadministrated antimetabolites, and modifiable by alterations in 6MP dose. Prevention of toxicity related withdrawals through 6MP dose reduction could be a way of increasing total dose intensity.

Evidence of jak2 val617phe positive essential thrombocythemia with splanchnic thrombosis during estroprogestinic treatment.

The discovery of the Janus kinase 2 Val617Phe mutation has brought new insights into the development of myeloproliferative disorders; however, the pathogenesis of essential thrombocythemia and its related thrombotic complications has not been completely understood. Although the Janus kinase 2 Val617Phe mutation confirms the initially suspected clonal character of the disease, factors influencing clonal transformation and expansion in the bone marrow have not been fully detected. Furthermore, patients affected by essential thrombocythemia who are carriers of the Janus kinase 2 Val617Phe mutation show a higher incidence of venous thromboembolism both before, and at the time of diagnosis, compared with noncarriers, and recent evidence of splanchnic and cerebral vein thrombosis in carriers of the Janus kinase 2 Val617Phe mutation has been reported. The intake of oral contraceptives is a strong and independent risk factor for venous thromboembolism. In addition, in-vitro tests showed both an altered primary haemostatic plug formation and enhanced platelet aggregation in patients taking such drugs. Little is known, though, about the influence of steroid hormones on both megakaryopoiesis and platelet function in patients with the Janus kinase 2 Val617Phe mutation. Herewith, we report the case of a 30-year-old woman who took a third generation oral contraceptive for 5 months and developed an essential thrombocythemia with spleno-portal axis and superior mesenteric vein thrombosis. She was found to carry the kinase gene Janus kinase 2 mutation.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.