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.

Novel mutation in coagulation factor VII (Carmel mutation): Identification and characterization.

BACKGROUND: Measurement of factor VII (FVII) activity does not enable prediction of bleeding tendency in individuals with inherited FVII deficiency. OBJECTIVE: To characterize the molecular and functional features of FVII in a family with FVII deficiency and correlate them with the bleeding tendency. PATIENTS/METHODS: We studied 7 family members with very low FVII activity using prothrombin time (PT), activated factor VII (FVIIa), FVII activity level, and thrombin generation. The factor 7 gene was sequenced and the mutation was analyzed by prediction software. RESULTS: The proband has very low FVII activity (0%-4%), with PT ranging between 5% to 18% depending on the tissue factor (TF) origin. Direct sequencing demonstrated a single homozygous nucleotide substitution G > A in exon 6, predicting a novel missense mutation Cys164Tyr. Three members of the family were found to be heterozygous carriers of this mutation. One of them was a compound heterozygote, carrying both the Cys164Tyr and Ala244Val mutation (linked to Arg353Gln polymorphism). Her FVII activity and antigen levels were 3%-7% and 8%, respectively. The other heterozygous carriers demonstrated FVII activity of 41%-54%, FVII antigen of 46%-66%, and FVIIa activity of 30%. FVIIa was undetectable in the homozygous and compound heterozygous subjects. Thrombin generation was normal in the presence of calcium, but no response to TF addition was observed in the homozygous proband, and a reduced response was observed in the compound heterozygous subject. CONCLUSION: The patient homozygous for the "Carmel" mutation has mild clinical manifestations despite very low FVII activity, which correlates with thrombin generation results.

Mutations in RASGRP2 gene identified in patients misdiagnosed as Glanzmann thrombasthenia patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AIM: We aimed to study the genetic cause of IPFD mimicking GT. METHODS: During 2017-2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. RESULTS: Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbß3 and αvß3, as well as normal integrin αIIbß3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DISCUSSION: Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. CONCLUSION: Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.

Pediatric immune thrombocytopenia: apoptotic markers may help in predicting the disease course.

BACKGROUND: In all, 15-30% of pediatric immune thrombocytopenia (ITP) patients will remain chronically thrombocytopenic at 1 year post diagnosis. All attempts to classify patients at diagnosis have proven unsuccessful. We hypothesized that a different pathophysiology is responsible for non-chronic versus chronic pediatric ITP. We aimed to examine differences in the apoptotic markers' presentation at diagnosis between non-chronic and chronic patients. METHODS: Blood samples were collected from 42 pediatric patients with newly diagnosed ITP prior to initiation of treatment. We incubated patients' sera with control platelets and compared the results among three research groups: healthy controls, chronic ITP, and non-chronic ITP patients. We measured apoptotic markers phosphatidylserine (PS) exposure and mitochondrial inner membrane potential (ΔΨm) by flow cytometry and the level of human apoptotic proteins by Human Apoptosis Array. RESULTS: We found increased platelet PS exposure and decreased ΔΨm in response to all ITP patients' sera compared to control subjects. Human Apoptotic Array revealed an increased expression of five apoptotic proteins: BIM, CD40, IGFBP2, P21, and SMAC, following sera incubation of non-chronic pediatric ITP patients, compared to chronic patients' sera, at diagnosis. CONCLUSIONS: Our data contribute to knowledge on apoptosis markers that may aid in predicting the prognosis of children with ITP. IMPACT: The key message of our article is that children with chronic ITP have a different apoptotic profile compared to non-chronic ITP. Addition to existing literature: This is the first study comparing apoptotic markers between children with chronic ITP to non-chronic ITP. IMPACT: Our findings indicate that, in the future, apoptotic markers may help to classify ITP patients into non-chronic versus chronic ones, at diagnosis.

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.

Integrin-mediated cell adhesion requires extracellular disulfide exchange regulated by protein disulfide isomerase.

Cell adhesion to extracellular matrix, mediated by integrin receptors, is crucial for cell survival. Receptor-ligand interaction involves conformational changes in the integrin by a mechanism not fully elucidated. In addition to several direct evidence that there is disulfide re-arrangement of integrins, we previously demonstrated a role for extracellular thiols and protein disulfide isomerase (PDI) in integrin-mediated functions using platelets as model system. Exploring the possible generality of this mechanism, we now show, using three different nucleated cells which depend on adhesion for survival, that non-penetrating blockers of free thiols inhibit α2ß1 and α5ß1 integrin-mediated adhesion and that disulfide exchange takes place in that process. Inhibiting extracellular PDI mimics thiol blocking. Transfection with WT or enzymatically inactive PDI increased their membrane expression and enhanced cell adhesion, suggesting that PDI level is a limiting factor and that the chaperone activity of the enzyme contributes to adhesion. Exogenously added PDI also enhanced adhesion, further supporting the limiting factor of the enzyme. These data indicate that: a) Dependence on ecto-sulfhydryls for integrin-mediated adhesion is not exclusive to the platelet; b) PDI is involved in integrin-mediated adhesion, catalyzing disulfide bond exchange; c) PDI enhances cell adhesion by both its oxidoreductase activity and as a chaperone.

[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.

Low Concentrations of Recombinant Factor VIIa May Improve the Impaired Thrombin Generation of Glanzmann Thrombasthenia Patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a rare bleeding disorder. The disease is caused by the lack or dysfunction of platelet membrane glycoprotein IIb/IIIa (integrin αIIbß3) which is essential for platelet aggregation. Bleeding episodes are usually managed by platelet transfusions. Recombinant activated factor VII (rFVIIa) is a common adjunct or alternative treatment option. OBJECTIVE: This article evaluates GT patients' response to increasing concentrations of rFVIIa using an ex vivo thrombin generation assay to elaborate the knowledge in which rFVIIa treats a platelet dysfunction for bleeding episodes and preoperative management. MATERIALS AND METHODS: Twenty-four GT patients in a non-bleeding state were enrolled into the study. Thrombin generation was measured in platelet-rich plasma (PRP) in the presence of 0.7 to 7.0 µg/mL rFVIIa. Clinical data of rFVIIa used to treat GT patients' bleeding episodes was collected, and patients' follow-up course was documented. RESULTS: Thrombin generation was significantly decreased in GT patients compared with controls. An individual response to rFVIIa spiking was noted in GT patients' PRP. In the majority of patients, a significant improvement in thrombin generation was already demonstrated with low concentrations (0.7 µg/mL) of rFVIIa. CONCLUSION: Thrombin generation is improved in the majority of GT patients following ex vivo spiking with rFVIIa. The magnitude of this improvement is individual and was noted at low concentrations of rFVIIa. There is a need for a prospective clinical trial to find the optimal doses or rFVIIa for treatment of GT patients.

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.

The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro.

Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain.

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.