The disulfide-rich region of platelet glycoprotein (GP) IIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP).

Immune thrombocytopenic purpura (ITP) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets. Platelet glycoprotein (GP) IIb/IIIa is a common target for antiplatelet autoantibodies. The present studies were undertaken (1). to confirm whether the disulfide rich repeat region of GPIIIa contains target epitopes for antiplatelet antibodies in patients with ITP; (2). to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification; and (3). to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex, the vitronectin receptor, alpha(V)beta(3). Recombinant fusion proteins of four GPIIIa extracellular sequences were prepared and purified. Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti-GPIIb/IIIa antibodies with the fusion protein containing the GPIIIa sequence of residues from 468 to 691. These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of GPIIIa isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients. Smaller peptides including residues 446-501 and residues 593-691 each reacted with only 5 of the 24 patient sera; furthermore all but 3 of these interactions were very weak. Visualization of the conformation of the extracellular portion of alpha(V)beta(3) reveals the location of the 222-residue antigenic GPIIIa (beta(3)) peptide 'B' at the immediately extracellular region of the protein that includes a beta-tail domain and several integrin-EGF domains. In summary, predictions of hydrophilicity, surface accessibility and antigenicity and the three dimensional structure of the beta(3) integrin correlate with autoantibody binding to a recombinant GPIIIa peptide 'B' containing residues 468-691.

Pathophysiology of immune thrombocytopenic purpura.

In 1951, the young hematologist in training, Dr. William Harrington, infused himself with plasma from a patient with immune thrombocytopenic purpura (ITP). He rapidly developed severe, but transient, thrombocytopenia and was at risk for serious hemorrhage. Thus, the humoral autoimmune cause of ITP was established. Since 1953, when Dr. Harrington's in vivo studies ended, in vitro investigations have aimed to determine the molecular and cellular details of immune-mediated platelet destruction.

Home treatment of mild to moderate bleeding episodes using recombinant factor VIIa (Novoseven) in haemophiliacs with inhibitors.

OBJECTIVE: To assess the safety and efficacy of a fixed dose of recombinant activated factor VII (rFVIIa; NovoSeven) in the home setting for mild to moderately severe joint, muscle; and mucocutaneous bleeding episodes in patients with haemophilia A or B with inhibitors. DESIGN: Multicentre, open-label, single arm, phase III study of one year duration. METHODS; Patients or their caregivers administered up to three doses of rFVIIa (90 microg/kg i.v.) at 3 h intervals within 8 h of the onset of a mild to moderate bleeding episode. Once the subject considered that rFVIIa had been "effective" with regard to haemostasis (after 1-3 injections), one further (maintenance) dose of rFVIIa was administered. RESULTS: Of 60 patients enrolled, 56 experienced at least one bleed, and 46 completed the one year study. 614 of 877 bleeds (70%) were evaluable according to protocol definitions. Haemostasis was rated as "effective" in 92% (566/614) of evaluable bleeds after a mean of 2.2 injections. For successfully treated episodes, the time from onset of bleeding until administration of the first injection was 1.1+/-2.0 h (mean+/-SD). Twenty-four hours after initial successful response, haemostasis was reported as having been maintained in 95% of cases. Efficacy was comparable for muscle, joint and target joint, and mucocutaneous bleeding episodes. In an intent-to-treat analysis of all 877 bleeding events, efficacy outcomes were equivalent to the evaluable bleeds, with an effective response in 88% of treated episodes. Treatment-related adverse events occurred in 32 (3% of all) bleeding episodes and consisted of re-bleeds/new bleeds in more than 50% (18/32) of these events. A single episode of superficial thrombophlebitis was the only thrombotic complication encountered, and there were no patient withdrawals due to adverse events. Development of FVII(a) antibodies could not be detected, and hypersensitivity reactions to rFVIIa were not reported. CONCLUSION: rFVIIa is effective and well tolerated when used in the home setting to treat mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors.

Cerebral vasculopathy and neurologic sequelae in infants with cervicofacial hemangioma: report of eight patients.

PURPOSE: To determine the association of cerebral arterial anomalies and progressive cerebral arterial occlusive disease in infants with facial hemangiomas. MATERIALS AND METHODS: The cases of eight infants (seven girls and one boy) with the diagnosis of cervicofacial hemangioma and intracranial arterial anomalies were reviewed retrospectively. Findings from clinical and imaging examinations--including cranial computed tomography, magnetic resonance imaging and angiography, and catheter angiography--were evaluated. Serial imaging findings were studied to document progressive intracranial vascular changes. RESULTS: Five patients had additional associated congenital anomalies. Seven were treated with corticosteroids, interferon alfa-2a, or both. Progressive cerebrovascular occlusive changes were documented in four of the seven patients with serial imaging findings. Four other patients (all treated pharmacologically) had MR imaging documentation of cerebral infarction, and all had consistent, acquired neurologic symptoms. CONCLUSION: Intracranial arterial anomalies can coexist with cervicofacial hemangioma. Aneurysmal and occlusive changes are potentially progressive and can result in cerebral infarction. A causative association between occlusive cerebrovascular disease and pharmacologic treatment has not been excluded.

Giant platelets with abnormal surface glycoproteins: a new familial disorder associated with mitral valve insufficiency.

PURPOSE: To study the platelets from a family with a new form of inherited giant platelet disorder. PATIENTS AND METHODS: Two siblings exhibited a hemorrhagic disorder characterized by moderate thrombocytopenia, giant platelets, and markedly prolonged bleeding time. The parents had no discernible platelet defect. Both children also developed mitral regurgitation requiring medication, and one underwent surgical replacement at the age of 3 years. RESULTS: The mean platelet size was greater than 20 microm3. Direct measurements of the two major axes of each of 12 platelets on electron micrographs revealed a range of 2x4 to 4x6 microm. Electron microscopy did not demonstrate any abnormality of granule content. The platelets agglutinated normally with ristocetin and aggregated normally with collagen. However, the aggregation was slightly slower than normal with ADP, epinephrine, and Na arachidonate. Two-dimensional unreduced versus reduced SDS-polyacrylamide gel electrophoresis of surface radioiodinated platelet glycoproteins revealed absence of proteins Ia, Ic, and IIa in both affected children, whereas GP Ib, IIb, and IIIa appeared normal. The 2D gels of platelet glycoproteins from both parents were identical to controls. Western blots demonstrated that GP Ic, Ic', Ib, and Ia/IIa were present. CONCLUSIONS: This disorder represents a new syndrome of thrombocytopenia with giant platelets distinct from Bernard-Soulier, Montreal giant platelets, Swiss cheese platelets, May-Hegglin anomaly, and other previously described syndromes.

Platelet autoantibodies in immune thrombocytopenic purpura.

In summary, the search for a useful clinical laboratory diagnostic assay for the antiplatelet antibodies has been long and difficult. Measurement of platelet associated IgG (PAIgG) has been disappointing as a way to detect autoantibodies. This is primarily due to the fact that platelets normally contain IgG in their alpha granules in an amount that varies with plasma IgG levels and age of the platelets. Furthermore, the amounts of platelet associated IgG is affected by the presence of circulating immune complexes, platelet activation, and drug dependent antibodies. The newer, platelet antigen capture techniques are promising, but further testing will be needed to confirm their value to the clinician. Methods that allow incubation of patient serum or plasma with intact platelets (MAIPA and immunobead) have greater sensitivity than techniques in which the patient antibody is tested against previously isolated platelet glycoproteins. These assays are currently available in a only a limited number of platelet immunology laboratories. Platelet autoantibodies are directed against a number of glycoprotein antigens on the platelet surface. Most studies have shown that anti GPIIb/IIIa antibodies are the most common, although antibodies against GPIb/IX and other targets are frequently detected. Many patients have multiple antiplatelet antibodies circulating simultaneously. The clinical significance of antibodies with different specificity is under investigation. The precise epitopes on GPIIIa that bind antiplatelet autoantibodies have been studied to a limited extent. Some investigators report that the vast majority of platelet antigens are conformation dependent, being destroyed by treatment with EDTA (separation of GPIIb and GPIIIa) or denaturation with detergents. Others report sequence specific peptide antigens. Further investigation promises to better define the targets for platelet autoantibodies; improved clinical management of patients with ITP is the long term goal of these studies.

Sympathetic block during spinal anesthesia in volunteers using lidocaine, tetracaine, and bupivacaine.

BACKGROUND AND OBJECTIVES: Spinal anesthesia to high thoracic dermatomes is alleged to result in almost complete block of all sympathetic efferent nerves. To examine the degree of sympathectomy during spinal anesthesia, the sympathetic response to a cold pressor test (CPT) applied to unblocked dermatomes before and during spinal anesthesia was measured with use of three different local anesthetics. METHODS: Twelve healthy volunteers were studied in a randomized and double-blind fashion on three separate occasions. In random order, each volunteer received approximately equipotent spinal doses of tetracaine 15 mg, bupivacaine 15 mg, and lidocaine 100 mg in hyperbaric solutions. Prior to and 30 minutes after spinal injection of local anesthetic, a CPT was applied for 2 minutes, and changes from baseline resting conditions in five physiologic variables were measured. RESULTS: The CPT 1 given before anesthetic administration resulted in an increase in heart rate, mean arterial pressure, cardiac index, and plasma concentrations of norepinephrine and epinephrine. Spinal anesthesia to a median level of T3 resulted in a decrease in mean arterial pressure by 10-12% but did not significantly decrease the other variables. Spinal anesthesia did not change the increase in heart rate or cardiac index in response to the second CPT, but the increase in mean arterial pressure was attenuated compared to the CPT before anesthesia. No increase in norepinephrine or epinephrine concentration was observed during the CPT given during spinal anesthesia. There was no significant relationship between level of analgesia and sympathetic response to stress. CONCLUSIONS: Spinal anesthesia with hyperbaric solutions of tetracaine 15 mg, bupivacaine 15 mg, and lidocaine 100 mg attenuated sympathetic function but did not produce complete sympathectomy. The effects were independent of the local anesthetic used.

Safety and efficacy of low-dose intravenous immune globulin (IVIG) treatment for infants and children with immune thrombocytopenic purpura. Low-Dose IVIG Study Group.

PURPOSE: This report presents pooled data from two multicenter studies conducted to assess the efficacy, safety, and tolerance of lower-dose intravenous immune globulin (IVIG) regimens of 250 mg/kg/day, 400 mg/kg/day, and 500 mg/kg/day for 2 days, compared to an established higher-dose regimen of 1 g/kg/day for 2 days, in children with immune thrombocytopenic purpura (ITP). PATIENTS AND METHODS: A total of 24 children received IVIG (Gammar i.v.). In Study 1, 10 centers enrolled 12 children between 5 and 12 years old who received IVIG at either 400 mg/kg/day or 1 g/kg/day for 2 days. In Study 2, five centers enrolled 12 infants and children younger than 5 years old who received IVIG at 250 mg/kg/day or 500 mg/kg/day for 2 days. Both studies were prospective and randomized. RESULTS: IVIG treatment was effective (platelets increased at least 30,000/cu mm over baseline) in 94% (16 of 17) of the evaluable patients in the low-dosage group. Platelet increases occurred rapidly: by 48 hours, total platelet counts ranged from 32,000/cu mm to 256,000/cu mm, and peak platelet counts reached 38,000/cu mm to 551,000/cu mm. Adverse events (AEs) were most often mild, lasted less than 3 hours, and were usually those typically associated with immunoglobulin administration-headache, nausea, vomiting, and fever. There were two serious AEs-an anaphylactoid reaction in one patient in the 400 mg/kg group and aseptic meningitis in one patient in the 1 g/kg high-dosage group. Both patients recovered without sequelae and were responders. Although the incidence of AEs varied by dosage groups, this difference was not significant. However, the incidence of AEs was affected by age. AEs were significantly lower in patients younger than 5 years of age. CONCLUSIONS: In this small, randomized trial, low-dose IVIG in 2-day regimens of 250, 400, or 500 mg/kg/day rapidly reversed thrombocytopenia just as effectively as 1 g/kg/day in infants and young children with ITP. Lower-dosage regimens are safe and well-tolerated; the incidence of AEs is lower in children younger than 5 years of age.

Hyperbaric dye solution distribution characteristics after pencil-point needle injection in a spinal cord model.

BACKGROUND: The flow-rate limiting and directional characteristics of caudally directed microcatheters, which lead to intrathecal maldistribution of hyperbaric 5% lidocaine, are believed to have contributed to at least 11 cases of cauda equina syndrome. The authors investigated the distribution characteristics of hyperbaric dye solutions via caudally directed side port needles at various rates of injection in a spinal cord model to determine the potential for maldistribution. METHODS: Using a digital video image processing technique, we injected a hyperbaric solution of phthalocyanine blue dye through caudally directed side-port needles into a supinely oriented transparent spinal canal model filled with simulated cerebrospinal fluid. Injections via commonly used spinal needles (24-gauge and 25-gauge Sprotte, and 25-gauge and 27-gauge Whitacre) were recorded using five injection rates (2, 4, 6, 8, and 16 ml/min). RESULTS: For all needles tested, injection rate had a significant effect on the peak dye concentration (P < 0.0001). Injection rates > or = 6 ml/min (2 ml/20 s) resulted in peak dye concentrations of less than 168 mg/1 (extrapolated concentration of 1% lidocaine). Injection via the 24-gauge Sprotte needle, which has a larger orifice area and internal diameter, resulted in significantly lower peak dye concentrations than via the smaller Whitacre needles tested (P < 0.05). CONCLUSIONS: Sacral maldistribution could be minimized by using injection rates > or = 6 ml/min (2 ml/20 s), for all of the side-port spinal needles used in this model study. When very slow injection rates (2 ml/min) are used, peak dye concentrations varied inversely and significantly with needle internal diameter and orifice area.

International study to compare antigen-specific methods used for the measurement of antiplatelet autoantibodies.

Platelet-associated and plasma autoantibodies against platelet glycoproteins (GP) have been demonstrated in patients with autoimmune thrombocytopenia (AITP) using various methods. Eight laboratories in seven countries participated in this international study to evaluate the interlaboratory agreement using glycoprotein-specific immunoassays for these autoantibodies. The participating laboratories received blind samples of frozen washed platelets and plasma from 22 normal donors and 22 AITP patients. Platelet-associated and plasma autoantibodies against GPIIb-IIIa and GPIb-IX were measured by MAIPA, immunobead assay or modified antigen capture assay. Of the control samples, 96.0% and 97.2% of all results for platelet-associated and plasma autoantibodies to GPIIb-IIIa/ GPIb-IX, respectively, were negative. The mean variation coefficient of the control samples of platelet-associated and plasma autoantibodies was 89.5% (range 11.1-272.9%) and 46.5% (range 21.0-78.0%), respectively. In 20/22 patient samples, platelet-associated autoantibodies to either glycoprotein were noted by at least two laboratories. The mean degree of agreement in these samples was 74.0%. There was a significant correlation in the individual antibody measurements between all laboratories (Kendall coefficient of concordance 0.60 and 0.38, P < 0.001; Spearman rank order test, range of correlation coefficient 52.3-94.0% and 42.2-85.0%, P < 0.05, for anti-GPIIb-IIIa and anti-GPIb-IX, respectively). In contrast, plasma autoantibodies to either glycoprotein were noted by at least two laboratories in only 13/22 patient samples. Moreover, the degree of agreement was poor (50.1%) and a significant correlation was noted between only six pairs of laboratories. We conclude that methods used in this study yield good interlaboratory agreement in measuring platelet-associated autoantibodies against GPIIb-IIIa and GPIb-IX. In contrast, poor agreement was found in detecting plasma autoantibodies to the same glycoproteins.

Varicella-associated thrombocytopenia: autoantibodies against platelet surface glycoprotein V.

Varicella zoster infection in children can be complicated by acute idiopathic thrombocytopenic purpura (ITP). To determine the etiologic mechanism of this thrombocytopenia, we studied three children with clinically diagnosed varicella infection. Immunoblot analysis of these patients' anti-platelet antibodies identified a unique band at 85 kD. Characterization of this protein revealed that it was platelet surface glycoprotein V (GPV) because it was not affected by a disulfide bond reduction but was cleaved by thrombin. Bernard-Soulier syndrome (BSS) platelets deficient in GPIb-IX and GPV did not react with the sera from our varicella-infected study patients. There was no apparent cross-reactivity between anti-varicella antibody and patients' anti-GPV Ig. We report here the first cases of GPV as the target antigen in autoimmune thrombocytopenia.

Reticulated platelet values in normal and thrombocytopenic neonates.

OBJECTIVES: Reticulated platelets (RPs) are newly synthesized platelets with increased ribonucleic acid content. The percentage of RPs is elevated in adults with thrombocytopenia as a result of increased platelet destruction. The objectives of this study were to determine normal RP values in neonates at birth and to determine whether neonates with thrombocytopenia as a result of increased platelet destruction have an increased percentage of RPs. STUDY DESIGN: The RP percentages were measured at birth in 89 neonates without thrombocytopenia in three gestational age groups (<30,30 to 36, and >36 weeks), six neonates with immune thrombocytopenia, and one neonate with thrombocytopenia as a result of decreased platelet production. RESULTS: The RP percentages in neonates without thrombocytopenia >36 weeks and 30 to 36 weeks of gestation were 4.0% +/- 2.4% (mean +/- SD) and 4.6% +/- 1.7%, respectively, similar to values reported in healthy adults. Neonates younger than 30 weeks of gestation had significantly higher RP percentages (8.8% +/- 5.1%) than older neonates (p

Local densities orthogonal to beta-sheet amide planes: patterns of packing in globular proteins.

We have investigated the efficiency of packing by calculating intramolecular packing density above and below peptide planes of internal beta-pleated sheet residues in five globular proteins. The orientation of interest was chosen to allow study of regions that are approximately perpendicular to the faces of beta-pleated sheets. In these locations, nonbonded van der Waals packing interactions predominate over hydrogen bonding and solvent interactions. We observed considerable variability in packing densities within these regions, confirming that the interior packing of a protein does not result in uniform occupation of the available space. Patterns of fluctuation in packing density suggest that the regular backbone-to-backbone network of hydrogen bonds is not likely to be interrupted to maximize van der Waals interactions. However, high-density packing tends to occur toward the ends of beta-structure strands where hydrogen bonds are more likely to involve nonpolar side-chain groups or solvent molecules. These features result in internal protein folding with a central low-density core surrounded by a higher-density subsurface shell, consistent with our previous calculations regarding overall protein packing density.

Nonoperative management of a splenic tear in a Jehovah's Witness with hemophilia.

Splenic laceration, the most common visceral lesion following blunt abdominal trauma, can be treated in a nonoperative fashion in only a select group of stable patients with minimal injury. We report a unique case of life-threatening splenic trauma in a Jehovah's Witness with hemophilia that was successfully managed without surgery.

The frequency of homozygous deletion of a developmentally regulated Vh gene (Humhv3005) is increased in patients with chronic idiopathic thrombocytopenic purpura.

Little is known of the genetic factors that may contribute to the development of chronic idiopathic thrombocytopenic purpura (cITP). We have previously shown that a developmentally regulated Vh gene (Humhv3005) is absent in 10/41 (24%) of patients with systemic lupus erythematosus while it is absent in only 7/88 (8%) of normal controls. This finding suggests that a homozygous deletion of an Ig variable (V) gene may alter the immune system and thus predispose the host to an autoimmune disorder. We have analyzed the same gene in 44 patients with cITP and found that Humhv3005 and like genes were absent in a higher percentage of patients (14 of 44, 31.8%) than they were absent in either normals (7/88, 8%, p = 0.002) or thrombocytopenic patients without cITP (6/53, 11.3%, p = 0.042); the hv3005 deletion frequency in the latter group did not differ from that in normals (P = 0.74). These data suggest that deletions of Humhv3005 and/or highly homologous Vh genes may predispose individuals to the development of cITP, and may contribute toward production of pathogenic antiplatelet antibodies.

Transient neurologic deficit after spinal anesthesia: local anesthetic maldistribution with pencil point needles?

Recent reports of transient neurologic deficits have raised concern about the potential toxicity of single-dose spinal 5% lidocaine in 7.5% dextrose. Two cases of volunteers who experienced minor local sensory deficits after slow (60 s) injections of 2 mL 5% lidocaine via Whitacre needles are described. One case was a result of a double injection because of a "failed" block. It seemed possible that the neurologic deficit in these cases resulted from neurotoxicity associated with maldistribution of local anesthetic. Using an in vitro spinal model, we investigated drug distribution resulting from injections through side-port spinal needles to determine whether the use of these needles could result in high local concentrations of hyperbaric solutions. A spinal canal model was fabricated using human magnetic resonance measurements. The model was placed in a surgical supine position and filled with lactated Ringer's solution to simulate the specific gravity of cerebral spinal fluid at 22 degrees C. A hyperbaric solution of phthalocyanine blue dye and dextrose (SG 1.042), simulating the anesthetic, was injected through three different needles (27-gauge 4 11/16-in. Whitacre, 25-gauge 3 1/2-in. Whitacre, 25-gauge 3 1/2-in. Quincke). Triplicate injections were done at rapid (2 mL/10 s) and slow (2 mL/60 s) rates, with needle side ports oriented in a sacral and cephalad direction. At slow rates of injection, using 27- or 25-gauge sacrally directed Whitacre needles, injections showed evidence of maldistribution with extrapolated peak sacral lidocaine concentrations reaching 2.0%. In contrast, distribution after slow injection through sacrally directed Quincke needles was uniform.(ABSTRACT TRUNCATED AT 250 WORDS)

Does spinal anesthesia result in a more complete sympathetic block than that from epidural anesthesia?

BACKGROUND: Spinal and epidural injection of local anesthetics are used to produce sympathetic block to diagnose and treat certain chronic pain syndromes. It is not clear whether either form of regional anesthesia produces a complete sympathetic block. Spinal anesthesia using tetracaine has been reported to produce a decrease in plasma catecholamine concentrations. This has not been demonstrated for epidural anesthesia in humans with level of anesthesia below C8. One possible explanation is that spinal anesthesia results in a more complete sympathetic block than epidural anesthesia. To examine this question, a cross-over study was performed in young, healthy volunteers. METHODS: Ten subjects underwent both spinal and epidural anesthesia with lidocaine (plain) on the same day with complete recovery between blocks. By random assignment, spinal anesthesia and epidural anesthesia were induced via lumbar injection. Before and 30 min after local anesthetic injection, a cold pressor test (CPT) was performed. Blood was obtained to determine epinephrine and norepinephrine plasma concentrations at four stages: (1) 20 min after placing peripheral catheters, (2) at the end of a 2-min CPT (before conduction block), (3) 30 min after injection of epidural or spinal lidocaine, and (4) at the end of a second CPT (during anesthesia). Mean arterial pressure, heart rate, noninvasive cardiac index, and analgesia to pin-prick were monitored. RESULTS: Neither spinal nor epidural anesthesia changed baseline resting values of catecholamines or any hemodynamic variable, except heart rate, which was slightly decreased during spinal anesthesia. Median level of analgesia was T4 during spinal and T3 during epidural anesthesia. CPT before conduction block reliably increased heart rate, mean arterial pressure, cardiac index, epinephrine, and norepinephrine. Conduction block attenuated the increase in response to CPT only in mean arterial pressure (spinal and epidural) and cardiac index (spinal only). Neither technique blocked the increase in heart rate, norepinephrine, or epinephrine to CPT. CONCLUSIONS: Spinal anesthesia did not result in a more complete attenuation of the sympathetic response to a CPT than did epidural anesthesia. In response to the CPT, spinal anesthesia blocked the increase in cardiac index, and epidural anesthesia resulted in a decrease in total peripheral resistance compared to the pre-anesthesia state. The differences between the techniques are not significant and are of uncertain clinical implications.