Application of a clinical decision rule and laboratory assays in pediatrics: Adult heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is rare among pediatric patients. The diagnosis of HIT depends upon clinical decision tools to assess its pretest probability, supported by laboratory evidence of anti-platelet factor 4 (anti-PF4)/heparin antibodies. AIMS: To compare the use of the 4Ts score clinical decision tool, clinical characteristics, and laboratory findings between pediatric and adult patients with suspected HIT. METHODS: We compiled all pediatric patients in our center for whom HIT testing was performed during the years 2015-2021. These were compared with a cohort of consecutive adult patients. Laboratory diagnosis of HIT was performed with particle gel immunoassay (PaGIA) as screening test and confirmed by an automated latex-enhanced immunoturbidimetric assay (LIA) and/or by functional flow cytometry assay (FCA). RESULTS: The cohort included 34 children (under 18 years) and 105 adults. Adults mostly received heparins for thromboembolism prophylaxis and treatment (72.4%, n = 76), and were more frequently treated with low-molecular-weight heparin (LMWH). Children were mostly exposed during cardiopulmonary bypass and extracorporeal membrane oxygenation (ECMO, 61.8%, n = 21), and were more frequently treated with unfractionated heparin (UFH). Compared with adults, children had significantly higher 4Ts scores. Nevertheless, adults had a slightly higher rate of a positive diagnosis of HIT. Six out of 16 adults with confirmed HIT presented with thrombosis (37.5%), whereas all three pediatric patients with HIT presented with thrombosis (p = .087). CONCLUSIONS: 4Ts scores are higher in children compared with adult patients for whom laboratory tests for HIT were obtained. A potentially higher incidence of thrombosis in children with HIT may be attributable to the severity of underlying illness.

Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets.

Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P-selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live-cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP-selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.

[PLATELETS FUNCTION IN A DROP OF BLOOD: FLOW CYTOMETRY ANALYSIS COMPARED TO PLATELET AGGREGATION].

INTRODUCTION: Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders, but requires large amounts of blood samples and normal platelet count. OBJECTIVES: To compare flow cytometric (FC) platelet function testing to standard LTA in the general population, in patients treated with anti-platelets drugs and in term and preterm neonates. METHODS: Platelet function was assessed with LTA and FC using PAC1 binding and p-selectin expression, as platelet activation markers, in response to agonist activation. A comparison between LTA and FC was performed in a Clopidogrel treated patient, before and after (24 and 72 hours) loading the drug. The platelet activation markers PAC1 and p-selectin, were compared in umbilical cord blood samples of in-term and preterm neonates. RESULTS: ADP-induced platelet aggregation was comparable to p-selectin expression assayed by FC (r=0.79-0.86) as measured before and after Clopidogrel loading. Both tests showed good response to Clopidogrel in 72 hours but not in 24 hours after its loading. Preterm cord blood platelets showed decreased ADP-induced activation in both activation markers: PAC1 and p-selectin, but only p-selectin reached statistical significance. We identified possible platelet activation markers in response to commonly used agonists' stimulation for FC analysis. CONCLUSIONS: FC analysis of platelet function has added value in the diagnosis of impaired platelet function and anti-platelet drug response. Using FC enables us to test platelet function in minimal blood volume and regardless of platelet count. Identification of the unique activation marker for each agonist is prerequisite for FC analysis of platelet function.

Sensitivity to Deviance and to Dissimilarity: Basic Cognitive Processes Under Activation of the Behavioral Immune System.

Throughout evolutionary history, pathogens have imposed strong selection pressures on humans. To minimize humans' exposure to pathogens, a behavioral immune system that promotes the detection and avoidance of disease-connoting cues has evolved. Although most pathogens cannot be discerned by our sensory organs, they produce discernable changes in their environment. As a result, a common denominator of many disease-connoting cues is morphological deviance-figurative disparity from what is normal, visual dissimilarity to the prototype stored in memory. Drawing on an evolutionary rationale, we examine the hypothesis that activation of the behavioral immune system renders people more sensitive to morphological deviance and more prone to perceive dissimilarities between stimuli. In Study 1 ( N = 343), participants who scored higher on disgust sensitivity demonstrated greater differentiation between normal and disfigured faces, reflecting greater sensitivity to morphological deviance in the bodily domain. In Study 2 ( N = 109), participants who were primed with pathogen threat demonstrated greater differentiation between perfect and imperfect geometrical shapes, reflecting greater sensitivity to morphological deviance even in stimuli that have nothing to do with health or disease. In Study 3 ( N = 621), participants who scored higher on disgust sensitivity perceived pairs of neutral pictures as less similar (i.e., more dissimilar) to each other. Literature on the relations to social deviance and implications for social perception and for social behavior is discussed.

A Proposed Role of Surfactant in Platelet Function and Treatment of Pulmonary Hemorrhage in Preterm and Term Infants.

BACKGROUND: We evaluated the effect of surfactant on platelet function as a potential contributing mechanism to the pathogenesis of pulmonary hemorrhage (PHEM) in term and preterm infants. METHODS: Cord blood samples were collected from neonates following delivery. Complete blood count and platelet function were measured using a cone and platelet analyzer (CPA). Increasing surfactant concentrations were added to platelets in vitro, and the adhesion molecule P-selectin and the monoclonal antibody PAC-1 were evaluated following platelet activation by flow cytometry. RESULTS: Forty-one infants (11 preterm and 30 term) were studied. CPA revealed a significant decrease in the average size of the aggregates and in platelet adhesion when surfactant was added. In term infants, the addition of surfactant to native platelets yielded an increased binding capacity of PAC-1 but did not affect P-selectin expression. In preterm infants, platelet activation with adenosine diphosphate in the presence of a high surfactant concentration (0.5 mg/mL) resulted in increased PAC-1 binding and no change in P-selectin expression. CONCLUSIONS: The platelets of preterm infants are less active (hyporesponsive) than those of term infants, both in their native state as well as after stimulation with various agonists. Surfactant may play an important role in treating PHEM in preterm infants.

From thrombasthenia to next generation thrombocytopenia: Neonatal alloimmune thrombocytopenia induced by maternal Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder of platelet function caused by mutations in the genes coding for integrin αIIbß3. The aim of this study was to examine the outcome of newborns of GT mothers, with emphasis on thrombocytopenia and bleeding manifestations and their relation to maternal antiplatelet antibodies. PROCEDURE: Medical files of all female patients with GT treated in a single tertiary center from 1999 to 2017 were searched for details on pregnancy and birth. The medical files of their newborns were retrieved, and data on the postnatal course were collected. RESULTS: Nine babies were born to five patients with GT at our center during the study period. Three of the nine newborns had severe thrombocytopenia, and all three were offspring of GT mothers who were positive for antiplatelet antibodies. CONCLUSION: Pregnant GT patients should be examined for platelet antibodies. Assessment and management protocols (including treatment with intravenous immunoglobulins) for fetal and neonatal alloimmune thrombocytopenia should be considered.

A novel approach using ancillary tests to guide treatment of Glanzmann thrombasthenia patients undergoing surgical procedures.

BACKGROUND: Glanzmann thrombasthenia (GT) is a disorder of platelet function. Standard therapy includes platelet transfusions, which may be hampered by antiplatelet antibodies. AIMS: To assess potential correlation between bleeding and number of active platelets in GT patients undergoing surgery. Clinical peri- operative patients' hemostasis was compared with flow cytometry analysis (FC), and whole blood clot formation. METHODS: GT patients undergoing surgery were included. Blood counts, platelet activation studies, FC and rotational thromboelastography (ROTEM) were performed as ancillary tests to estimate the effectiveness of treatment. RESULTS: A total of 4 GT patients undergoing 5 surgeries were included. Consecutive FC analysis following platelet transfusions showed gradual decrease of donor platelets with a nadir of 3280 platelets in patients who experienced no post procedural bleeding following minor procedures. After major surgery, bleeding occurred when donor platelets decreased to 2600-4280. Decline in donor platelets was associated with reduced clot firmness as noted by ROTEM. CONCLUSION: Results suggest that very low number of active donor platelets may suffice to achieve proper hemostasis in certain procedures. Our study points to the potential role of consecutive FC examinations to demonstrate the number of donor platelets as an ancillary tool for decision making in GT patients undergoing surgery.

SMYD1 is the underlying gene for the AnWj-negative blood group phenotype.

BACKGROUND: AnWj is a high-incidence blood group antigen associated with three clinical disorders: lymphoid malignancies, immunologic disorders, and autoimmune hemolytic anemia. The aim of this study was to determine the genetic basis of an inherited AnWj-negative phenotype. METHODS: We identified a consanguineous family with two AnWj-negative siblings and 4 additional AnWj-negative individuals without known familial relationship to the index family. We performed exome sequencing in search for rare homozygous variants shared by the two AnWj-negative siblings of the index family and searched for these variants in the four non-related AnWj-negative individuals. RESULTS: Exome sequencing revealed seven candidate genes that showed complete segregation in the index family and for which the two AnWj-negative siblings were homozygous. However, the four additional non-related AnWj-negative subjects were homozygous for only one of these variants, rs114851602 (R320Q) in the SMYD1 gene. Considering the frequency of the minor allele, the chance of randomly finding 4 consecutive such individuals is 2.56 × 10-18 . CONCLUSION: We present genetic and statistical evidence that the R320Q substitution in SMYD1 underlies an inherited form of the AnWj-negative blood group phenotype. The mechanism by which the mutation leads to this phenotype remains to be determined.

Treatment tailoring for factor V deficient patients and perioperative management using global hemostatic coagulation assays.

INTRODUCTION: Congenital factor V deficiency (FVD) is a rare bleeding disorder with an estimated incidence of 1 in 1000,000 in the general population. Since the common coagulation tests do not correlate with the bleeding tendency there is an unmet need to predict FVD patients' bleeding hazard prior to surgical interventions. AIM: To optimize treatment prior to surgical interventions, using global coagulation assays, thrombin generation (TG) and rotating thromboelastogram (ROTEM). METHODS: Our cohort included 5 patients with FVD, 4 severe and one mild. Two of them underwent TG and ROTEM prior to surgical interventions, including ex vivo spiking assays using bypass agents and platelets spiking. RESULTS: All five patients exhibited prolonged PT and PTT, non-dependent on their bleeding tendency. Patient 1, who demonstrated severe bleeding phenotype, underwent surgery treated by combination of APCC (FEIBA) and platelet transfusion. Therapy was guided by global tests (TG as well as ROTEM) results. During the pre and post-operative period neither excessive bleeding nor any thrombosis was noted. In contrast, TG and ROTEM analysis of patient 4 has lead us to perform the surgery without any blood products' support. Indeed, the patient did not encounter any bleeding. CONCLUSION: Global coagulation assays may be useful ancillary tools guiding treatment decisions in FVD patients undergoing surgical procedures.

A Novel Compound Targeting Protease Receptor 1 Activators for the Treatment of Glioblastoma.

Data from human biopsies, in-vitro and in-vivo models, strongly supports the role of thrombin, and its protease-activated receptor (PAR1) in the pathology and progression of glioblastoma (GBM), a high-grade glial tumor. Activation of PAR1 by thrombin stimulates vasogenic edema, tumor adhesion and tumor growth. We here present a novel six amino acid chloromethyl-ketone compound (SIXAC) which specifically inhibits PAR1 proteolytic activation and counteracts the over-activation of PAR1 by tumor generated thrombin. SIXAC effects were demonstrated in-vitro utilizing 3 cell-lines, including the highly malignant CNS-1 cell-line which was also used as a model for GBM in-vivo. The in-vitro effects of SIXAC on proliferation rate, invasion and thrombin activity were measured by XTT, wound healing, colony formation and fluorescent assays, respectively. The effect of SIXAC on GBM in-vivo was assessed by measuring tumor and edema size as quantified by MRI imaging, by survival follow-up and brain histopathology. SIXAC was found in-vitro to inhibit thrombin-activity generated by CNS-1 cells (IC50 = 5 × 10-11M) and significantly decrease proliferation rate (p < 0.03) invasion (p = 0.02) and colony formation (p = 0.03) of these cells. In the CNS-1 GBM rat animal model SIXAC was found to reduce edema volume ratio (8.8 ± 1.9 vs. 4.9 ± 1, p < 0.04) and increase median survival (16 vs. 18.5 days, p < 0.02 by Log rank Mental-Cox test). These results strengthen the important role of thrombin/PAR1 pathway in glioblastoma progression and suggest SIXAC as a novel therapeutic tool for this fatal disease.

Quantification of specific T and B cells immunological markers in children with chronic and transient ITP.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is characterized by a transient (nonchronic) or permanent (chronic) decline in the number of platelets. Predicting the course of ITP, at the time of diagnosis, is of importance. Here we studied at diagnosis, clinical and immunological parameters in order to distinguish between different courses. The latter included the measure of new B and T cells using quantification of kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs), respectively. METHODS: Blood samples were collected from 44 children with a clinical diagnosis of ITP. Real-time PCR was performed in order to quantify the number of copies of TREC and KREC followed by collection of clinical data from medical files. The children were retrospectively divided into two groups: chronic and nonchronic. RESULTS: Twenty-four patients (54%) were classified as nonchronic ITP and 20 patients (46%) were classified as chronic ITP. We confirmed some clinical parameters (e.g., gender, age) but not others (e.g., preceding infection, level of thrombocytopenia) that distinguish patients with chronic and nonchronic course. While KREC quantification was similar in patients regardless the outcome of their disease, it was significantly higher than the level of controls (P < 0.05). TREC quantification was not different between patients and controls. CONCLUSIONS: KREC but not TREC levels are different in patients comparing to controls, pointing to an overreaction of B-cell development as a role in the pathogenesis of ITP. These results may shed more lights on the immune mechanism of ITP.

A man-made disease: Fetal neonatal alloimmune thrombocytopenia due to incompatibility between oocyte donor and gestational mother.

The incompatibility causing fetal and neonatal alloimmune thrombocytopenia (FNAIT) results from a fetus inheriting a paternal human platelet antigen (HPA), which is different from the maternal HPA. We present a unique case of FNAIT in a pregnancy involving an oocyte recipient mother with Turner syndrome. This is the first report of FNAIT in which the suggested mechanism involves antibodies produced by a gestational mother against the incompatible HPA of the oocyte donor.

Spinal Epidural Hematoma Following Cupping Glass Treatment in an Infant With Hemophilia A.

A 6 months old infant, diagnosed with a rare mutation causing severe hemophilia A, presented with spinal epidural hematoma. Parents later admitted the infant had glass cupping therapy performed within 2 weeks of the onset of symptoms. The rare mutation, rare bleeding complication, and the eventual course of therapy applied in this case will be discussed in our case report.

The role of protein disulfide isomerase in the post-ligation phase of ß3 integrin-dependent cell adhesion.

INTRODUCTION: Protein disulfide isomerase (PDI) catalyzes disulfide bond exchange. It is crucial for integrin-mediated platelet adhesion and aggregation and disulfide bond exchange is necessary for αIIbß3 and αvß3 activation. However, the role of disulfide bond exchange and PDI in the post-ligation phase of αIIbß3 and αvß3 mediated cell adhesion has yet to be determined. METHODS: To investigate a possible such role, we expressed wild type (WT) human αIIb and either WT human ß3, or ß3 harboring single or double cysteine to serine substitutions disrupting Cys473-Cys503 or Cys523-Cys544 bonds, in baby hamster kidney (BHK) cells, leading to expression of both human αIIbß3 and a chimeric hamster/human αvß3. Adhesion to fibrinogen-coated wells was studied in the presence or absence of bacitracin, a PDI inhibitor, with and without an αvß3 blocker. RESULTS: Flow cytometry showed WT and mutant αIIbß3 expression in BHK cells and indicated that mutated αIIbß3 receptors were constitutively active while WT αIIbß3 was inactive. Both αIIbß3 and αvß3 integrins, WT and mutants, mediated adhesion to fibrinogen as shown by reduced but still substantial adhesion following treatment with the αvß3 blocker. Mutated αIIbß3 integrins disrupted in the Cys523-Cys544 bond still depended on PDI for adhesion as shown by the inhibitory effect of bacitracin in the presence of the αvß3 blocker. Mutated integrins disrupted in the Cys473-Cys503 bond showed a similar trend. CONCLUSIONS: PDI-mediated disulfide bond exchange plays a pivotal role in the post-ligation phase of αIIbß3-mediated adhesion to fibrinogen, while this step in αvß3-mediated adhesion is independent of disulfide exchange.

Evaluation of platelet response to different clopidogrel dosing regimens in patients with acute coronary syndrome in clinical practice.

High-post clopidogrel platelet reactivity in acute coronary syndrome (ACS) patients is associated with adverse outcomes and may be related to clopidogrel dosing. Clinical studies evaluating different clopidogrel doses have resulted in conflicting conclusions. Clopidogrel dosing regimens have evolved over time, enabling us to evaluate platelet reactivity in real-life ACS patients undergoing percutaneous coronary intervention and treated with three different clopidogrel doses. Platelet reactivity was assessed with light transmitted aggregometry on the third day post clopidogrel loading in 404 consecutive ACS patients. Of them, 198 were treated with a standard regimen (300 mg loading, 75 mg/day maintenance dose), 95 with a high loading regimen (600 mg loading, 75 mg/day maintenance dose) and 111 with a high loading/high maintenance regimen (600 mg loading, 150 mg/day maintenance). Compared with the standard regimen, the high loading regimen resulted in significantly lower mean platelet reactivity to adenosine diphosphate (ADP) with a lower proportion of patients exhibiting clopidogrel non-responsiveness (11% vs. 28%, p = 0.004). Compared with the high loading regimen, the high loading/high maintenance regimen resulted in significantly lower mean platelet reactivity to ADP, but without a further drop in the number of non-responders (8.1% vs. 11%, p = 0.16). In conclusion, greater overall inhibition can be achieved with higher loading and maintenance doses in ACS patients. However, despite high clopidogrel doses, a sizable proportion of patients remained "resistant" to the effects of clopidogrel.

Disulfide bond exchanges in integrins αIIbß3 and αvß3 are required for activation and post-ligation signaling during clot retraction.

BACKGROUND: Integrin αIIbß3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbß3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbß3 and αvß3 activation. METHODS: In order to investigate the role of integrin activation and disulfide bond exchange during αIIbß3- and αvß3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human ß3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. RESULTS: Cells expressed WT or mutated human αIIbß3 as well as chimeric hamster/human αvß3. The αIIbß3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvß3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvß3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. CONCLUSIONS: Both αIIbß3 and αvß3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbß3, but not of αvß3 enhances clot retraction. Both αIIbß3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.

Surfactant impairs coagulation in-vitro: a risk factor for pulmonary hemorrhage?

BACKGROUND: Pulmonary hemorrhage (PHEM) complicates the hospital course of 3-5% of preterm infants with respiratory distress syndrome (RDS), and bears a high mortality rate. Impaired thrombin generation and poor clot formation in premature neonates affect PHEM severity. OBJECTIVES: We evaluated the impact of surfactant upon in-vitro clot formation in order to assess the role of surfactant in the pathogenesis of PHEM. METHODS: Blood samples were obtained from healthy volunteers for measuring complete blood count, PT, PTT, and platelet function. Surfactant at increasing concentrations was added to blood samples, and whole blood clotting assays were performed using rotation thromboelastogram (ROTEM®, Pentapharm Munich, Germany) and whole blood platelet adhesion and aggregation (Impact-R®, Diamed, Switzerland). RESULTS: The mean PT level increased from 10.05 ± 033 to 11.64 ± 0.85 sec (p=0.06) in the presence of surfactant. Platelet aggregation with the agonists adenosine diphosphate and epinephrine significantly decreased with escalating surfactant concentration (p<0.001). Adhesion, manifested by surface coverage (SC), significantly decreased with increasing surfactant concentration: mean SC 9.25 ± 2.96 compared to 6.1 ± 0.96 and 0.05 ± 0.058 with 0/0.1/5mg/ml surfactant, respectively, p<0.001 Whole blood ROTEM studies showed a trend towards lengthening of clotting time with increased surfactant concentration and lower clot strength. CONCLUSION: The presence of surfactant impairs coagulation in-vitro. The risk of PHEM may therefore be greater in extremely premature infants. Future studies are required to assess the clinical significance and relevance of our preliminary findings.

Antiplatelet effect of thienopyridine (clopidogrel or prasugrel) pretreatment in patients undergoing primary percutaneous intervention for ST elevation myocardial infarction.

Although previous retrospective studies have suggested the clinical benefits of clopidogrel pretreatment in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI), the antiplatelet effect of thienopyridines during a narrow door-to-balloon time frame has not been evaluated. Seventy-nine consecutive patients with STEMI were treated with either 600 mg of clopidogrel (n = 49) or 60 mg of prasugrel (n = 30) loading on admission. All patients underwent PPCI with a door-to-balloon time of 48 ± 20 minutes. Adenosine diphosphate (ADP)-induced platelet aggregation (PA) was determined by light transmission aggregometry before thienopyridine loading, at PPCI, and after 72 hours. Baseline ADP-induced PA was comparable in clopidogrel- and prasugrel-treated patients (79 ± 10% vs 76 ± 9%, p = 0.2). Although ADP-induced PA was reduced significantly in both clopidogrel- and prasugrel-treated patients (p <0.01 for both), it was significantly lesser in prasugrel-treated patients (63 ± 18% vs 74 ± 12%, p = 0.002). Yet, <50% of the prasugrel-treated patients achieved adequate platelet inhibition (ADP-induced PA <70%) at PPCI. Prasugrel-treated patients, compared with clopidogrel-treated patients, were more likely to have Thrombolysis In Myocardial Infarction myocardial perfusion grade of ≥2 (79% vs 49%, p = 0.01), lower Thrombolysis In Myocardial Infarction frame count (10.2 ± 5.7 vs 13.6 ± 7.2, p = 0.03), and a numerically greater incidence of early ST-segment resolution >50% (26 of 30 [87%] vs 35 of 49 [71%], p = 0.1), suggesting better myocardial reperfusion. In conclusion, overall, prasugrel compared with clopidogrel pretreatment resulted in greater platelet inhibition at PPCI, but even with prasugrel, only <50% of the patients achieved early adequate platelet response.

The impact of genetic and environmental factors on homocysteine levels in preterm neonates.

BACKGROUND: Hyperhomocysteinemia may be associated with vascular complications in adults. Whereas pediatric thrombosis risk peaks in neonates, data on homocysteine (Hcy) levels assessed in term and preterm infants during the perinatal period are scarce. In the present study, we aimed to establish Hcy reference values for preterm infants and study their potential associations with the early post-natal health status. Plasma Hcy and hematocrit levels and MTHFR polymorphisms (C677T and A1298C substitution) were studied in a large cohort of preterm infants in a tertiary referral medical center during an 18-month period. Data were collected on maternal history and delivery as well as on post-natal complications. RESULTS: The study cohort included 167 infants whose mean gestational age was 30.98 ± 2.34 weeks (range: 26-36 weeks), mean birth weight 1327.6 ± 327 g, and mean Hcy level 7.99 ± 3.27 (range: 2.2-21.2) µmol/L. Maternal intake of folic acid was inversely associated with the babies' Hcy levels (P = 0.0001). Increased Hcy levels positively correlated with birth weight, gestational age (P < 0.005), total number of pregnancies (P = 0.012), and presence of MTHFR polymorphism. Higher Hcy levels were associated with feeding (P = 0.008), especially total parenteral nutrition (P = 0.0001). There was no correlation between Hcy levels and any vascular post-natal complications. CONCLUSIONS: During their post-natal hospitalization, preterm infants may have relatively high, that is, within the adult normal range, Hcy levels which are influenced by genetic and environmental factors. Despite the fact that no correlation was found between Hcy levels and post-natal complications, these associations should be further studied.

Unique disulfide bonds in epidermal growth factor (EGF) domains of ß3 affect structure and function of αIIbß3 and αvß3 integrins in different manner.

The ß3 subunit of αIIbß3 and αvß3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbß3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbß3 and αvß3. We created ß3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbß3 and αvß3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbß3 and αvß3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbß3 and αvß3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbß3. Activation of C523S/C544S αvß3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ß3 in αIIbß3 but not in αvß3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ß3 differently affect the function of αIIbß3 and αvß3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbß3 and αvß3 and for Cys-523-Cys-544 only in αvß3.