Activated Platelets Upregulate ß2 Integrin Mac-1 (CD11b/CD18) on Dendritic Cells, Which Mediates Heterotypic Cell-Cell Interaction.

Recent evidence suggests interaction of platelets with dendritic cells (DCs), while the molecular mechanisms mediating this heterotypic cell cross-talk are largely unknown. We evaluated the role of integrin Mac-1 (αMß2, CD11b/CD18) on DCs as a counterreceptor for platelet glycoprotein (GP) Ibα. In a dynamic coincubation model, we observed interaction of human platelets with monocyte-derived DCs, but also that platelet activation induced a sharp increase in heterotypic cell binding. Inhibition of CD11b or GPIbα led to significant reduction of DC adhesion to platelets in vitro independent of GPIIbIIIa, which we confirmed using platelets from Glanzmann thrombasthenia patients and transgenic mouse lines on C57BL/6 background (GPIbα-/-, IL4R-GPIbα-tg, and muMac1 mice). In vivo, inhibition or genetic deletion of CD11b and GPIbα induced a significant reduction of platelet-mediated DC adhesion to the injured arterial wall. Interestingly, only intravascular antiCD11b inhibited DC recruitment, suggesting a dynamic DC-platelet interaction. Indeed, we could show that activated platelets induced CD11b upregulation on Mg2+-preactivated DCs, which was related to protein kinase B (Akt) and dependent on P-selectin and P-selectin glycoprotein ligand 1. Importantly, specific pharmacological targeting of the GPIbα-Mac-1 interaction site blocked DC-platelet interaction in vitro and in vivo. These results demonstrate that cross-talk of platelets with DCs is mediated by GPIbα and Mac-1, which is upregulated on DCs by activated platelets in a P-selectin glycoprotein ligand 1-dependent manner.

Genetic Landscape of Factor VII Deficiency: Insights from a Comprehensive Analysis of Pathogenic Variants and Their Impact on Coagulation Activity.

Congenital factor VII (FVII) deficiency is a rare genetic bleeding disorder characterized by deficient or reduced activity of coagulation FVII. It is caused by genetic variants in the F7 gene. We aimed to evaluate the rate of detection of pathogenic variants in the F7 gene in a large group of patients with FVII deficiency and investigate the correlations between the F7 genotype and FVII activity (FVII:C). Moreover, the influence of the common genetic variant rs6046: c.1238G>A; p.(Arg413Gln), designated as the M2 allele, on FVII:C was investigated. Genetic analysis of the F7 gene was performed on 704 index patients (IPs) using either direct Sanger- or next-generation sequencing. Genetic variants were detected in 390 IPs, yielding a variant detection rate (VDR) of 55%. Notably, the VDR exhibited a linear decline with increasing FVII:C levels. We identified 124 genetic variants, of which 48 were not previously reported. Overall, the frequency of the M2 allele was considerably higher in patients with mild deficiency (FVII:C > 20 IU/dl). Furthermore, IPs lacking an identified pathogenic variant exhibited a significantly higher prevalence of the M2 allele (69%) compared to IPs with a disease-causing variant (47%). These results strongly support the association of the M2 allele with decreased FVII:C levels. This study shows the utility of FVII:C as a predictive marker for identifying pathogenic variants in patients with FVII deficiency. The M2 allele contributes to the reduction of FVII:C levels, particularly in cases of mild deficiency.

The effect of recombinant versus plasma-derived von Willebrand factor on prolonged PFA closure times in ECMO patients with acquired von Willebrand syndrome - an observational study.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is applied in patients with respiratory or cardiopulmonary failure, but bleeding is a frequent complication contributing to the high mortality rates in this patient collective. A major factor predisposing patients to bleeding events is an acquired von Willebrand syndrome (aVWS). So far, specific treatment options for this phenomenon are lacking. In hereditary von Willebrand disease (VWD), treatment with recombinant or plasma-derived von Willebrand factor (rVWF or pVWF) is common practice. Closure time measured by the Platelet Function Analyser-200 (PFA-200) is an established assay to detect defects in primary hemostasis and the method is useful to monitor the effect of hemostatic therapy. The aim of this study was to assess the effect of recombinant (rVWF) vs. plasma-derived von Willebrand factor (pVWF) on closure times measured by PFA in blood obtained from ECMO patients with aVWS. METHODS: Blood was sampled from thirteen patients receiving extracorporeal membrane oxygenation and three patients with hereditary VWD. Diagnosis of aVWS was made by conventional coagulation parameters and by multimeric structure analysis. PFA analysis of blood spiked with rVWF or pVWF was performed. RESULTS: Thirteen patients receiving ECMO were recruited. Ten patients survived and three patients suffered major bleeding complications. PFA closure times in ECMO patients with aVWS spiked with rVWF were significantly shorter at all concentrations than with pVWF (e.g., rVWF vs. pVWF: 1 U/ml: 150.4 ± 21.7 s vs. 263.8 ± 11.7 s; 4 U/ml: 97.8 ± 9.8 s vs. 195.8 ± 15.4 s, p<0.001). PFA closure times were also significantly shorter in three patients with hereditary VWD treated with rVWF compared to pVWF (e.g., 1 U/ml rVWF vs. pVWF: 73.7±1.33 s vs. 231.3±43.4 s, p<0.01) CONCLUSION: In summary, this study shows that rVWF compared to pVWF more effectively reduced PFA closures times in blood samples of ECMO patients with aVWS. Higher doses of VWF are needed to normalize PFA closure time in blood samples of patients with ECMO-induced aVWS compared to hereditary VWD. These data support the use of PFA-200 to monitor hemostatic effects in a future clinical trial involving ECMO patients with aVWS.

Fibrinogen regulates lesion border-forming reactive astrocyte properties after vascular damage.

Reactive astrocytes at the border of damaged neuronal tissue organize into a barrier surrounding the fibrotic lesion core, separating this central region of inflammation and fibrosis from healthy tissue. Astrocytes are essential to form the border and for wound repair but interfere with neuronal regeneration. However, the mechanisms driving these astrocytes during central nervous system (CNS) disease are unknown. Here we show that blood-derived fibrinogen is enriched at the interface of lesion border-forming elongated astrocytes after cortical brain injury. Anticoagulant treatment depleting fibrinogen reduces astrocyte reactivity, extracellular matrix deposition and inflammation with no change in the spread of inflammation, whereas inhibiting fibrinogen conversion into fibrin did not significantly alter astrocyte reactivity, but changed the deposition of astrocyte extracellular matrix. RNA sequencing of fluorescence-activated cell sorting-isolated astrocytes of fibrinogen-depleted mice after cortical injury revealed repressed gene expression signatures associated with astrocyte reactivity, extracellular matrix deposition and immune-response regulation, as well as increased gene expression signatures associated with astrocyte metabolism and astrocyte-neuron communication. Systemic pharmacologic depletion of fibrinogen resulted in the absence of elongated, border-forming astrocytes and increased the survival of neurons in the lesion core after cortical injury. These results identify fibrinogen as a critical trigger for lesion border-forming astrocyte properties in CNS disease.

Acquired von Willebrand Syndrome and Desmopressin Resistance During Venovenous Extracorporeal Membrane Oxygenation in Patients With COVID-19: A Prospective Observational Study.

OBJECTIVES: Although COVID-19 is associated with high von Willebrand factor (vWF) parameters promoting thrombosis, venovenous extracorporeal membrane oxygenation (vvECMO) is associated with the development of acquired von Willebrand syndrome (AVWS) promoting bleeding. This study was designed to assess both the incidence and severity of AVWS in COVID-19 patients undergoing vvECMO, and the benefit of comprehensive vWF analyses. DESIGN: Prospective observational study. SETTING: ICU at a tertiary-care center. PATIENTS: Twenty-seven consecutive COVID-19 patients with acute respiratory distress syndrome (ARDS) requiring vvECMO. MEASUREMENTS AND MAIN RESULTS: Comprehensive vWF analyses (including sodium dodecyl-sulfate polyacrylamide gel electrophoresis) were performed before, during, and after vvECMO. In a subgroup of 12 patients with AVWS, effectiveness of treatment with desmopressin was assessed. The patients' mean age was 53 years (range, 23-73), 70% were male, and all had various comorbidities. Following markedly elevated vwf antigen (vWF: Ag; mean, 546% ( sd , 282]), vWF collagen binding capacity (mean, 469% [ sd , 271]), vWF activity (vWF:A; mean, 383% [ sd , 132]), and factor VIII activity (mean, 302% [ sd , 106]), and only borderline decreases in high-molecular-weight (HMW) vWF multimers before vvECMO, all of these variables decreased and HMW vWF multimers became undetectable within hours following initiation of vvECMO. All variables fully recovered within 3-38 hours after discontinuation of vvECMO. During vvECMO, decreases in the vWF:A/vWF:Ag ratio correlated with absent HMW vWF multimers. Desmopressin did not affect vWF parameters. CONCLUSIONS: In patients with COVID-19-associated ARDS, AVWS developed soon after initiation of vvECMO. The vWF:A/vWF:Ag ratio was a suitable screening test for AVWS. As desmopressin was ineffective, bleeding during vvECMO-associated AVWS should preferably be treated with concentrates containing vWF.

Endothelial cells and coagulation.

Endothelial cells form a monolayer, which lines blood vessels. They are crucially involved in maintaining blood fluidity and providing controlled vascular hemostasis at sites of injury. Thereby endothelial cells facilitate multiple mechanisms, including both procoagulant and anticoagulant, which must be kept in balance. Under physiological conditions, endothelial cells constitute a nonadhesive surface preventing activation of platelets and the coagulation cascade. Multiple fibrinolytic and antithrombotic properties act on their cell surface contributing to the maintenance of blood fluidity. These include platelet inhibition, the heparin-antithrombin III system, tissue factor pathway inhibition, thrombomodulin/protein C system, and fibrinolytic qualities. At sites of vascular damage, platelets react immediately by adhering to the exposed extracellular matrix, followed by platelet-platelet interactions to form a clot that effectively seals the injured vessel wall to prevent excessive blood loss. For solid thrombus formation, functional platelets are essential. In this process, endothelial cells serve as a support surface for formation of procoagulant complexes and clotting. This review gives an overview about the central role of the endothelium as a dynamic lining which controls the complex interplay of the coagulation system with the surrounding cells.

Acquired bleeding disorders.

Acquired bleeding disorders can accompany hematological, neoplastic, autoimmune, cardiovascular or liver diseases, but can sometimes also arise spontaneously. They can manifest as single factor deficiencies or as complex hemostatic abnormalities. This review addresses (a) acquired hemophilia A, an autoimmune disorder characterized by inhibitory autoantibodies against coagulation factor VIII; (b) acquired von Willebrand syndrome in patients with cardiovascular disorders, where shear stress abnormalities result in destruction of von Willebrand factor; and (c) liver function disorders that comprise complex changes in pro- and anti-hemostatic factors, whose clinical implications are often difficult to predict. The article provides an overview on the pathophysiology, diagnostic tests and state-of-the-art treatment strategies.

Emicizumab in children: bleeding episodes and outcome before and after transition to Emicizumab.

PURPOSE: Real-world data and study data regarding therapy with Emicizumab in pediatric cohorts with haemophilia A is scarce. Especially, data on previously untreated pediatric patients (PUPs) and minimally treated patients (MTPs) are missing. METHODS: Thirteen pediatric patients with haemophilia A and treatment with Emicizumab were retrospectively evaluated for Annual Bleeding Rates (ABR) pre-and post-Emicizumab treatment. Safety data and data on management of minor surgery as well as laboratory results were collected. Additionally, we describe the clinical features of two PUPs and one MTP that are included in our cohort. RESULTS: Median age at initiation of Emicizumab was 5.3 (range: 0.26-17.5) years, three patients were younger than one year at initiation of treatment with Emicizumab. Median follow-up time on Emicizumab was 23.8 (range: 0.7-40) months. Total ABR (p = 0.009) as well as spontaneous (p = 0.018), traumatic (p = 0.018), and joint (p = 0.027) ABR reduced significantly post-Emicizumab transition. Safety profile was favourable as only one local site reaction occurred; no cessation of treatment was necessary. Surgery was successfully performed in three patients receiving rFVlla pre- and post-surgery. Emicizumab trough levels showed a median of 43.2 µg/ml (range: 23.9-56.8) after three doses of 3 mg/kg and 51.9 µg/ml (range: 30.4-75) at first follow-up with 1.5 mg/kg. CONCLUSION: Emicizumab is safe and efficient in pediatric patients with and without inhibitors. More data on larger multicenter cohorts and especially on PUPs/MTPs are still needed.

Acquired von Willebrand syndrome in ECMO patients: A 3-year cohort study.

BACKGROUND: Bleeding is a common but possibly underreported side effect of Extracorporeal Membrane Oxygenation (ECMO). Impairment of primary hemostasis by acquired von Willebrand syndrome (aVWS) and platelet dysfunction as well as activation and consumption of plasmatic coagulation factors contribute to hemorrhage. The aim of the present cohort study of consecutively enrolled patients admitted to our ECMO center was to collect demographic, medical and laboratory data possibly associated with i) development of clinically relevant bleeding and/or ii) death during a 12-months follow-up. RESULTS: Within a 3-year period 338 white patients aged 18-89 years (median: 60; male 64.5%) were enrolled. 78 of 338 patients (23%) presented with clinical relevant bleeding symptoms. The overall death rate was 74.6% within a median time of 9 days (1-229) post intervention. Logistic-regression analysis adjusted for age and gender revealed that i) the presence of blood group O versus non-O (Odds ratio (OR)/95%CI: 1.9/1.007-3.41), ECMO duration per day (1.1/1.06-1.14), veno-venous versus veno-arterial ECMO cannulation (2.33/1.2-4.5) and the overall need for blood product administered per unit (1.02/1.016-1.028) was independenly associated with bleeding in patients suffering from aVWS. ii) Older age (increase per year) at ECMO start (1.015/1.012-1.029) and an increasing amount of blood product units were significantly related with death (1.007/1.001-1.013). Patients with veno-venous versus veno-arterial cannulation survived longer (0.48/0.24-0.94). CONCLUSION: In the present cohort study we found a clinical relevant bleeding rate of 23% in subjects with aVWS associated with blood group O, a longer ECMO duration and veno-venous cannulation.

Acquired bleeding disorders.

Acquired bleeding disorders can accompany hematological, neoplastic, autoimmune, cardiovascular or liver diseases, but can sometimes also arise spontaneously. They can manifest as single factor deficiencies or as complex hemostatic abnormalities. This review addresses (a) acquired hemophilia A, an autoimmune disorder characterized by inhibitory autoantibodies against coagulation factor VIII; (b) acquired von Willebrand syndrome in patients with cardiovascular disorders, where shear stress abnormalities result in destruction of von Willebrand factor; and (c) liver function disorders that comprise complex changes in pro- and anti-hemostatic factors, whose clinical implications are often difficult to predict. The article provides an overview on the pathophysiology, diagnostic tests and state-of-the-art treatment strategies.

Hypercoagulopathy, acquired coagulation disorders and anticoagulation before, during and after extracorporeal membrane oxygenation in COVID-19: a case series.

BACKGROUND: Thromboembolism and bleeding contribute to Coronavirus disease 2019 (COVID-19)'s morbidity and mortality and are also frequent complications of venovenous extracorporeal membrane oxygenation (vvECMO). As the interaction of the underlying pathologies caused by vvECMO in COVID-19 is barely understood, we designed this study to better differentiate coagulation disorders in COVID-19 patients before, during and after vvECMO-support. METHODS: Observational case series, six consecutive patients with Coronavirus acute respiratory distress syndrome supported with vvECMO treated in the anaesthesiologic ICU in a third level University ECMO-centre. We measured routine coagulation parameters and assessed coagulation factors. We also conducted advanced von Willebrand factor (VWF) multimer analysis, platelet aggregometry and immunological screening. RESULTS: We identified various phases of coagulation disorders: Initially, intensely activated coagulation with highly increased VWF and factor VIII activity in acute COVID-19, then severe acquired von Willebrand syndrome and platelet dysfunction during vvECMO leading to spontaneous bleeding and finally, hypercoagulopathy after vvECMO explantation. Five of six patients developed immunological abnormalities enhancing coagulation. CONCLUSIONS: Coronavirus-induced coagulopathy and bleeding disorders during vvECMO cannot be discriminated via 'routine' coagulation tests. Precise and specific analyses followed by the appropriate treatment of coagulation disorders may help us develop tailored therapeutic concepts to better manage the phases described above.

Age-Dependent Control of Collagen-Dependent Platelet Responses by Thrombospondin-1-Comparative Analysis of Platelets from Neonates, Children, Adolescents, and Adults.

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11-18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.

[The Neonatal Subgaleal Hematoma - A Neonatal Emergency]. / Das subgaleale Hämatom beim Neugeborenen ­ ein neonatologischer Notfall.

Subgaleal hematoma (SGH) is a rare complication in neonates that may lead to hemorrhagic shock due to significant blood loss into the subgaleal space. We report of two neonates who developed subgaleal hematoma with severe hemorrhagic shock and encephalopathy. In the first case of a mature female neonate, the development of the subgaleal hematoma was promoted by early-onset sepsis and delivery by vacuum extraction. The second case, of a male preterm neonate, was a complicated fetal development followed by secondary cesarean section. Both cases highlight that a subgaleal hematoma is a severe neonatal emergency. In addition to prompt treatment of the shock, therapy of the coagulopathy is essential.

Antithrombotic prophylaxis for surgery-associated venous thromboembolism risk in patients with inherited platelet disorders. The SPATA-DVT Study.

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery.

Von Willebrand factor parameters as potential biomarkers for disease activity and coronary artery lesion in patients with Kawasaki disease.

Elevated von Willebrand factor (vWF):Antigen plasma levels have been observed in conjunction with cardiovascular diseases or vasculitis. The association of Kawasaki disease, a vascular inflammatory disease and vWF:Antigen, vWF:Collagen binding activity, and vWF multimers is unknown. We therefore investigated vWF parameters in 28 patients with acute Kawasaki disease in association with disease activity and coronary artery lesions. VWF:Antigen and vWF:Collagen binding activity were assessed via enzyme-linked immunoassay. The ratio of both (vWF:Collagen binding activity and VWF:Antigen) was calculated and vWF multimeric structure analysis performed. We analyzed the association between vWF parameters and our clinical data focusing on coronary artery outcome. VWF:Antigen and vWF:Collagen binding activity levels were significantly higher in the acute than in the disease's convalescence phase, and correlated positively with CRP levels. Neither variable was associated with coronary artery lesions. The vWF:Collagen binding activity/vWF:Antigen ratio, however, was significantly decreased in patients with a coronary artery lesion (z-score > 2; N = 10; mean ratio 0.96 vs. 0.64; p = 0.031) and even more so in those with a coronary artery aneurysm (z-score > 2.5; N = 8; mean ratio 0.94 vs. 0.55; p = 0.02). In a sub-analysis, those patients with a very low ratio in the acute phase presented a persistent coronary artery aneurysm at their 1-year follow-up.Conclusion: This study suggests that comprehensive analysis of vWF parameters may help to both monitor KD inflammation and facilitate the identification of those patients carrying an increased risk for coronary artery lesion.What is Known:• Von Willebrand factor (VWF)-parameters represent surrogate markers for vascular inflammation.• Kawasaki disease is a generalized vasculitis in children, which can be complicated by coronary artery lesions.What is New:• In those Kawasaki disease patients with coronary artery lesions, the vWF:CB/vWF:Ag ratio was significantly decreased.• VWF parameters may help to identify patients at risk for coronary artery lesions.

Novel variant in HPS3 gene in a patient with Hermansky Pudlak syndrome (HPS) type 3.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder caused by defects in 10 human HPS genes, characterized by oculocutaneous albinism (OCA) and bleeding diathesis associated to platelet δ-storage pool defect (SPD). We report a case of 4-year-old boy from non-consanguineous parents with OCA and negative personal and familiar hemorrhagic history, referred to us for severe bleeding after mild trauma. His platelet function, studied by lumi-aggregometry, showed normal first wave of aggregation in response to exogenous agonists and impaired second wave with defective ATP release. This, in combination with impaired platelet δ-granules content (serotonin, ATP, ADP) and the OCA phenotype suggested the HPS diagnosis. HPS3: sequencing revealed a novel pathogenic homozygous variant (NM_032383.4:c.7>T, p.Gln3*) resulting in a premature stop codon at the amino acid 3. Moreover, our report highlights the importance of evaluating platelet function in children with OCA without bleeding diathesis to identify HPS early and prevent bleeding complications.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Novel mutation in two brothers with Hermansky Pudlak syndrome type 3.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Meanwhile, there are ten known human HPS genes, each leading to a particular clinical HPS subtype (HPS1-HPS10). We report on two Turkish brothers showing typical HPS phenotype comprising oculocutaneous albinism and bleeding symptoms. Pathological bleeding time as well as platelet aggregometry analyses revealed impaired platelet function. The brothers demonstrated absence of platelet δ-granule secretion as measured by flow cytometry. Using molecular genetic analyses, a novel homozygous 1bp-deletion in the HPS3 gene was identified in both brothers. In addition, the younger brother with HPS3 demonstrated psychomotoric retardation and cranial gliosis (magnetic resonance imaging, MRI). Array-CGH analysis revealed a de novo 0.482Mb deletion on chromosome 17 which is not present in his brother and parents. In this study, we identified a novel 1bp-deletion in the HPS3 gene causing HPS3 phenotype in two brothers. In patients with oculocutaneous albinism and increased bleeding symptoms platelet function should be analyzed. The identification of the molecular genetic defect allows the classification to a particular HPS subtype and is important for therapy and prognosis.

Identification of a new dysfunctional platelet P2Y12 receptor variant associated with bleeding diathesis.

Defects of the platelet P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP) are associated with increased bleeding risk. The study of molecular abnormalities associated with inherited qualitative defects of the P2Y12R protein is useful to unravel structure-function relationships of the receptor. We describe the case of 2 brothers, sons of first cousins, with lifelong history of abnormal bleeding, associated with dysfunctional P2Y12R and a previously undescribed missense mutation in the encoding gene. ADP (4-20 µM)-induced aggregation of patients' platelets was markedly reduced and rapidly reversible. Other agonists induced borderline-normal aggregation. Inhibition of vasodilator-stimulated phosphoprotein phosphorylation and prostaglandin E1-induced increase in cyclic adenosine monophosphate (cAMP) by ADP was impaired, whereas inhibition of cAMP increase by epinephrine was normal. [(3)H]PSB-0413, a selective P2Y12R antagonist, bound to a normal number of binding sites; however, its affinity, and that of the agonists ADP and 2-methylthio-adenosine-5'-diphosphate, was reduced. Patients' DNA showed a homozygous c.847T>A substitution that changed the codon for His-187 to Gln (p.His187Gln). Crystallographic data and molecular modeling studies indicated that His187 in transmembrane 5 is important for agonist and nucleotide antagonist binding and located in a region undergoing conformational changes. These studies delineate a region of P2Y12R required for normal function after ADP binding.

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.