Platelets Of Patients Research Articles

Treatment of immune thrombocytopenia associated with interferon therapy of hepatitis C with the anti-CD20 monoclonal antibody, rituximab

This case report describes a patient with hepatitis C virus infection responding to pegylated INF/ribaviron therapy, who developed immune thrombocytopenia. The severe thrombocytopenia failed to resolve with cessation of the peg-IFN/ribaviron. Because of rising hepatitis C virus RNA levels and evidence of rising serum transaminases, the patient was treated with rituximab, anti-CD20 humanized monoclonal antibody. After treatment with rituximab, the patient's platelet count normalized and the patient was able to resume the pegylated IFN/ribaviron. The patient's hepatitis C virus RNA levels decreased, and the serum transaminases normalized without impairment of hepatic function or recurrence of the thrombocytopenia.

Characterization of a Heritable Bleeding Disorder in a Horse: Reduce PS-Flip Leads to Progressive Dysfunction in the Common Pathway of Platelet Coagulation.

A 3 year old Thoroughbred horse presented to the Veterinary Teaching Hospital with a three week history of bleeding. Clinical laboratory findings showed normal complete blood count including a normal platelet count. In addition the horse had normal prothrombin and partial thromboplastin times, as well as normal coagulation factors. These data suggested a defect in platelet function. The common pathway of the coagulation cascade occurs at the platelet surface when phosphatidylserine (PS) is flipped to the outer leaflet of the platelet membrane, providing binding sites for the prothrombinase complex that catalyzes the production of thrombin, which carries out the proteolytic activation of fibrinogen. We characterized the capacity of horse's platelets to carry out each of these steps. PS-flip in the patient's platelets was induced using several physiological agonists and found to be significantly reduced after stimulation with thrombin (0.1U/ml). The PS was detected by labeling platelets with FITC-annexin V, and measuring FITC intensity by flow cytometry. In comparison to control platelets, approximately 50% of the patient's platelets were positive for FITC-annexin V after stimulation with thrombin. The fluorescent intensity of the label on the patient's positive platelets was 40% that of controls. The reduced PS on the patient's platelets represents a potential loss of prothrombinase binding sites. Using a prothombinase assay based on the hydrolysis the chromogenic substrate S-2238, we determined the amount of thrombin produced by the patient's platelets over time. In response to thrombin (0.1U/ml), the patient's platelets produced thrombin at a rate of 0.023±0.002 NIH-U/min/1 X 108 platelets, which was significantly less than control platelets, 0.053±0.004 NIH-U/min/1 X 108 platelets. Neither resting platelets nor the thrombin used to stimulate them produced appreciable hydrolysis of S-2238. We correlated this reduced prothrombinase activity with the capacity of the patient's platelets bind FITC-fibrinogen. After stimulation with thrombin (0.1U/ml), control platelets form platelet microaggregates that are intensely labeled with FITC-fibrinogen and can be observed by flow cytometry. The patient's platelets do not form platelet microaggregates and the FITC-fibrinogen labeling is only 20% that of control platelets. This reduced FITC-fibrinogen labeling is not due to a defect in the function of the a2bb3a integrin, because the patient's platelets bind normal amounts of FITC-fibrinogen in response to ADP. In this regard, the affected horse is unlike patients with Glannzmann's thrombasthenia, who cannot bind fibrinogen. Instead, the dysfunction is more closely related to individuals with Scott syndrome, who cannot produce the PS surface that is required for assembly of the prothrombinase and ultimately activation of fibrinogen. Ongoing studies using the techniques described above demonstrate one affected and one normal progeny from the original patient.

Decreased Platelet Expression of MYL9 (Myosin Regulatory Light Chain Polypeptide) and Other Genes with Platelets Dysfunction and CBFA2 Mutation: Insights from Platelet Expression Profiling.

We have previously reported (Blood , 87:1368, 1996) a patient with a mild thrombocytopenia, and markedly abnormal aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and activation of GPIIb-IIIa complex. We recently showed (Blood 2004, 103: 948–954) in this patient a heterozygous mutation in hematopoietic transcription factor CBFA2 (Core Binding factor A2, also called AML1) and decreased platelet PKC-theta. We postulated that platelet expression profiling would provide insights into the pathophysiological mechanisms and gene expression changes resulting from a CBFA2 mutation. We performed platelet expression profiling in 4 control subjects (6 profiles, 2 individuals profiled twice) and the patient (profiled twice, 10 months apart) using the Affymetrix microarray Human U133A and B GeneChips (total ~44000 probe sets). ~10,000 probe sets (genes and ESTs) are expressed in human platelets. Iterative comparisons of data from each individual platelet profiles had correlation coefficients (r) > 0.85. Using GeneSpring® analytical software we selected genes that showed at least 1 present call in the 8 profiles, and p<0.05 showing a significant difference between the 2 groups. Relative expression values <0.5 and >2 were considered as significant changes. In the patient, 298 probe sets were significantly down regulated and 29 significantly upregulated. We focused on genes showing a > 5-fold decrease (fold change, fc < = 0.20). There was a striking ~75 fold decrease (fc = 0.013; p=0.00022) in myosin regulatory light chain polypeptide (MYL9 gene). Other down-regulated genes included calcium binding protein 5 (CABP5, fc = 0.02; a protein with similarity to calmodulin), Na/K/Ca exchanger member 3 (SLC24A3, fc = 0.11), b3-endonexin (ITGB3BP, fc = 0.01), platelet factor 4 variant 1 (PF4V1, fc = 0.03), chemokine CXCL5 (ENA-78) (fc = 0.20), tropomyosin I (alpha) (TPM1, fc = 0.13) and arachidonate 12-lipoxygenase (ALOX-12, fc = 0.22). The expression array findings were validated by decreased platelet mRNA levels on real time PCR or by immunoblotting (platelet PF4). Several down-regulated genes are directly relevant to the patient's platelet defect. Agonist-stimulated MLC phosphorylation is impaired in patient platelets. The strikingly decreased expression of MYL9 with normal expression of MLC kinase (MLCK, by array and real time PCR) suggests that the blunted myosin phosphorylation arises from decreased MYL9 than due to decreased MLCK. Myosin light chains play a role in diverse processes including in cell contractility, mobility and cytokinesis, and are relevant to impaired platelet function and production. The decreased expression of the Na/K/Ca exchanger and b3-endonexin 3 (both implicated in activation of GPIIb-IIIa) provide mechanisms for the diminished GPIIb-IIIa activation. Chemokine CXCL5 and PF4V1 are expressed in megakaryocytes, and the genes are colocalized to “chemokine cluster” on chromosome 4. 12-lipoxygenase, a major platelet enzyme, regulates arachidonic acid metabolism and production of the 12-hydroxy eicosanoids. This is the first proof of concept that platelet expression profiling can be applied to obtain new insights into the molecular basis of inherited platelet dysfunction. Further studies will provide new information on the genes regulated by CBFA2, and on mechanisms regulating platelet production and activation.

Response to High Dose (HD) Imatinib Therapy in Relapsed c-kit+ Acute Myeloid Leukemia (AML).

BACKGROUND. c-kit is a receptor tyrosine kinase for the ligand stem cell factor and a member of the PDGF receptor family. c-kit positivity, as defined by the presence of CD117 by flow cytometry (FC) in > 20% blasts, is detected in up to 90% of patients with AML, and is a potential target for imatinib, a selective tyrosine kinase inhibitor of c-kit as well as of BCR-ABL, PDGFR, and ARG. Previous reports of treatment with single-agent imatinib at 400 mg/day showed no response in AML (Cortes et al, Cancer 2003), while up to 24% of patients treated with 600 mg/day responded (Kindler et al, Blood 2003, Abstract 3242). Little information is available on the response in AML to imatinib as a single agent at 800 mg/day.CASE AND DISCUSSION. A 42-year old Caucasian male with relapsed c-kit expressing AML with del(9q), concurrent secondary high-grade (HG) glioma and few remaining therapeutic options presented to our service. He was initially diagnosed with primary oligodendrioglioma in January 2001 and underwent subtotal tumor resection, followed by whole brain radiation therapy, followed by 6 cycles of procarbazine, CCNU, and vincristine (PCV) chemotherapy, and entered complete remission (CR). In December 2002, he was diagnosed with AML M2 with del(9q) and was treated with cytosine arabinoside (Ara-C) and daunorubicin (7+3), followed by 3 cycles of consolidation with HD Ara-C, and entered CR. In March 2004, he had recurrent abnormal magnetic resonance imaging of the brain and HG malignant glioma was diagnosed by stereotactic biopsy. He was started on temozolamide but was unable to tolerate treatment due to pancytopenia, and a bone marrow (BM) at another institution disclosed relapsed AML with 75% blasts. Upon presentation to our institution, a BM examination showed > 90% blasts, 53% of which expressed CD117 positivity by FC. Imatinib therapy was initiated at a dose of 400 mg/day and escalated to 800 mg/day (HD) after lack of response. (Table 1.) The peripheral blood (PB) white blood cell count (WBC) peaked on Day 9 of treatment with imatinib and declined thereafter. On Day 21, blasts were undetectable in the PB and there was a proportional increase of PB neutrophils (PMN). By Day 25, the BM blasts decreased to 50% and only 12% blasts showed positivity for CD117. The patient experienced severe muscle/bone pains at the start of imatinib therapy, which resolved within five days. Response to HD imatinib was also accompanied by dramatic decline in serum LDH. The patient's platelet (PLT) count improved to a peak of 70k on Day 14, declined thereafter, and the patient required PLT transfusion support by Day 31. The patient also received dexamethasone 4 mg q6h for his HG glioma, which remained stable during this time of observation.CONCLUSION. Our case demonstrates that HD imatinib 800 mg/day has significant preferential activity against the CD117+ AML blasts, is active in a shorter time span than previously reported at lower doses, and should be studied further in combination with other agents in AML. The patient's HG glioma may have been partially stabilized by imatinib as PDGFR overexpression has been reported in secondary glioblastomas.Table 1.Imatinib Dose, mg/dayDayWBC x 10-3/uLPMN, %PB Blast, %BM Blast,%BM Blasts CD117 +, %PLT x 10-3/uL-−71.8415> 905334400117.5321--28600759.6540--50800871.0921--418009100.0930--318001411.02225--70800216.3610--31800254.0490–3501213

Myelodysplastic Syndrome Due to Somatic GATA1 Mutation.

Inappropriate hematopoietic differentiation resulting in trilineage dysplasia is a diagnostic hallmark of myelodysplastic syndrome (MDS). Understanding the molecular basis of MDS will not only shed insight into this common hematological disease, but also Acute Myeloid Leukemia (as MDS often progresses to AML) and normal hematopoiesis. The genetic basis of MDS is unclear and in particular the role of deregulated transcription factor activity, which is often seen in AML, has not been clearly defined. For the first time we describe a novel somatic GATA1 mutation in a 13-year-old boy with MDS who has 10-year history of anemia, neutropenia, moderate thrombocytopenia, bone pain and hepatomegaly. Bone marrow biopsy demonstrated trilineage dysplasia with a normal karyotype of bone marrow mononuclear cells. Sequence analysis showed an A to G mutation in the X-linked GATA1 gene at residue 598 in exon 4 in platelets and bone marrow mononuclear cell cDNA. The mutation was also detected in genomic DNA from purified myeloid progenitors but not peripheral blood leucocytes. This discrepancy may be due to selection of peripheral blood leucocytes that do not carry the GATA1 mutation. The mutation was also not seen in skin fibroblasts. The predicted amino acid substitution E200G is located in a highly conserved residue at the beginning of the N-terminal zinc finger (Nf). This did not perturb DNA binding to GATA sites by EMSA analysis nor reduce interaction with FOG1. Importantly, immunoblot analysis of the patient's platelets showed strongly reduced protein levels of GATA1, normal FOG1 and increased RUNX1. The index case had an increased Ivy bleeding time (>15 min) and a significant prolongation of the PFA-100 occlusion time (collagen/ADP and collagen/epinephrine). Platelet ATP secretion and platelet aggregation (epinephrine, ADP and U46) were decreased compared to control platelets. In contrast to patients with an inherited GATA1 mutation, the platelet phenotype in the MDS patient is completely different. Platelets from the patient are smaller in size (MPV 8.6fL) and electron microscopy revealed a markedly reduced number of dense granules. His platelets have a normal GPIbα expression and normal ristocetin-induced platelet agglutination. The previously described patients with inherited Nf GATA1 mutations (D218G GATA1 Blood,2001,98,85) have giant platelets with characteristic membrane complexes and reduced GPIbα expression with abnormal ristocetin-induced platelet agglutination. Further study of the index patient will provide important detailed insight into GATA1 function and help unravel how perturbed activity of key myeloid transcription factors results in MDS.

Clinical Trial of Paricalcitol in Myelodysplastic Syndrome.

Myelodysplastic syndrome is often a pernicious disorder associated with pancytopenia in the elderly. Therapeutic approaches need to balance their toxicities versus the side-effects of the disease. 1,25(OH)2-vitamin-D3 inhibits proliferation and induces differentiation of leukemic cells in vitro. Small clinical trials have shown slight efficacy in MDS. Hypercalcemia prevents the administration of doses of this seco-steroid, which have been shown to be effective in vitro. This has provided a stimulus to identify vitamin D analogs that have anti-leukemic activity with minimal hypercalcemic effects. Paricalcitol (19-nor-1,25(OH)2D2, Zemplar) has been approved by the FDA for the treatment of secondary hyperparathyroidism; the drug is unique because it has little hypercalcemic potential; but in vitro, it has strong antileukemic effects. We conducted a clinical trial of oral paricalcitol to twelve MDS patients whose disease varied between an IPSS of low to high. Therapy began at 8 μg per day and increased at two week intervals until serum calcium was slightly above normal level; at which point, dose was decreased by 4-8 μg qd. The amount of paricalcitol taken varied between 8 μg qod to 54 μg qd (average 16 μg qd). We confirmed that the drug was having biologic activity in vivo by examining a target of the activated vitamin D3 receptor, 1,25-(OH)2-vitamin-D3-24(OH)ase mRNA. Each patient had prominent induction of this transcript in his or her white blood cells. Furthermore, in selected patients serum paricalcitol was measured and confirmed to be prominently present. The drug was well tolerated in all patients. Two of the 12 patients showed a clinical response. One patient's platelet counts rose from 50,000 to 120,000/ul blood over 5 weeks; however, the patient succumbed to a fatal fungal infection. The second patient responded by a decrease in RBC transfusions associated with a rise in his hemoglobin, which lasted for about 5 months. Eventually, his hemoglobin began to fall, and erythropoietin therapy was substituted for paricalcitol. In summary, high doses of paricalcitol were well tolerated in all patients. Two patients had a partial clinical response. In general, paricalcitol given as a single agent to individuals with MDS is not therapeutically very efficacious; further trials should examine it in combination with other approaches.

Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura

Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura

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To the Editor: Dr Barton suggests that the etiologic agent of our patient's pneumonia may have contributed to her hemolytic anemia, and that Mycoplasma pneumoniae and Streptococcus pneumoniae have been associated with hemolytic anemia. Although we are well aware of the association between M pneumoniae and hemolytic anemia, we do not find any reference in the English literature of S pneumoniae being associated with hemolytic anemia independent of hemolytic uremic syndrome (HUS). We are also well aware of pneumococcus-associated HUS with the vast majority of such patients having had pneumonia. However, our patient's laboratory data and clinical course are inconsistent with a diagnosis of HUS. Although in HUS the degree of thrombocytopenia and renal dysfunction may vary and may not correlate with the severity of hemolysis, some degree of thrombocytopenia is expected.1.Corrigan Jr., J.J Boineau F.G Hemolytic-uremic syndrome.Pediatr Rev. 2001; 22: 365-369PubMed Google Scholar If our patient had progressive hemolysis as a result of HUS from the time of admission, thrombocytopenia and renal dysfunction are expected, if not at admission, then at least by the seventh day when she had severe anemia with cardiorespiratory arrest. On admission, our patient's platelet count was normal, and the urinalysis revealed no blood and no red blood cells. The platelet count during cardiopulmonary arrest was 194,000/μL. Immediately after the cardiopulmonary arrest, her serum blood urea nitrogen and creatinine levels were 16 mg/dL and 1.0 mg/dL, respectively. The serum creatinine normalized within three days. The brief abnormalities in renal function and the transient thrombocytopenia that followed her arrest are likely the result of multi-organ dysfunction secondary to prolonged ischemia. In addition, indirect hyperbilirubinemia is frequently noted in patients with HUS.1.Corrigan Jr., J.J Boineau F.G Hemolytic-uremic syndrome.Pediatr Rev. 2001; 22: 365-369PubMed Google Scholar Our patient's serum bilirubin after her arrest was 3.1 mg/dL with a direct component of 2.1 mg/dL, inconsistent with chronic hemolysis. Cefuroxime-induced hemolysis is the most fitting diagnosis based on our patient's laboratory data and clinical course.

Pseudohyperkalemia during the onset of critical illness

In everyday practice we observe a difference between the potassium levels measured by routine biochemistry and by the gas analysis machine. This is expected since the former measures serum potassium while the later measures plasma potassium. The serum/plasma potassium concentration difference is related to the patient's platelet count and increases to statistically significant levels in thrombocytaemia. We measured the serum/plasma potassium difference of ICU patient within the first 6 hours of their admission and compared it with the difference measured in chronically severely ill (uremic) patients, as well as a group of healthy volunteers. We measured the serum potassium (SKA), plasma potassium (PKA), serum/plasma potassium difference (SPdif-A = SKA – PKA), age and platelet (PLT) count in 50 ICU patients (Group A). They had been previously healthy and had suffered a major catastrophe (road traffic accident, gastrointestinal bleed, major trauma) less than 6 hours from the measurements. We also measured the serum potassium (SKB), plasma potassium (PKB), serum/plasma potassium difference (SPdif-B = SKB – PKB), age and PLT count in 31 chronic uremic patients on renal replacement therapy (Group B). The patients had come for treatment as scheduled, with no evidence of any other acute disease. The same variables (SKC, PKC, SPdif-C) were measured in 20 healthy volunteers (Group C). Blood samples were obtained from the radial artery. Serum potassium was collected in a Vacutainer tube, Plasma potassium in a syringe treated with heparin, and platelet counts were measured in EDTA-treated plasma samples. Potassium level were measured using the same equipment.

Compound heterozygosity for a novel nine-nucleotide deletion and the Asn45Ser missense mutation in the glycoprotein IX gene in a patient with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder due to quantitative or qualitative abnormalities in the platelet glycoprotein (GP) Ib/IX/V complex, the major von Willebrand factor receptor. The complex comprises four subunits, each encoded by a separate gene. Several mutations have been described for each of the subunits, except for GPV, as a cause of BSS. We describe here the genetic basis of the disorder in a child with BSS. Flow-cytometric analysis of the patient's platelets showed a markedly reduced surface expression of all three glycoproteins of the GPIb/IX/V complex. DNA sequencing analysis showed the patient to be a compound heterozygote for two mutations in the GPIX gene, a novel nine-nucleotide deletion starting at position 1952 of the gene that changes asparagine 86 for alanine and eliminates amino acids 87, 88, and 89 (arginine, threonine, and proline) and a previously reported point mutation that changes the codon asparagine (AAC) for serine (AGC) at residue 45. Her mother was heterozygous for the Asn45Ser mutation, and her father, for the nine-nucleotide deletion. Our findings suggest that the additive effects of both mutations in the GPIX gene are responsible for the BSS phenotype of the patient.

Case Report: Induction of T-cell Tolerance in a Patient with Idiopathic Thrombocytopenic Purpura by Single Injection of Humanized Monoclonal Antibody to CD40 Ligand

Normal B cells can be induced to express immune costimulatory molecules by activated T cells, and activated CD4 T cells can express CD40 ligand, a molecule that can engage CD40 on the B-cell surface. CD40–CD40 ligand interaction plays an important role in the pathology of certain autoimmune diseases. We report a patient with chronic idiopathic thrombocyopenic purpura (ITP) who was effectively treated with a single injection of humanized monoclonal antibody to CD40 ligand (E6040). The patient was refractory to steroid therapy, and had baseline platelet counts below 30×109/l during the 3-month period before antibody treatment. The patient's platelet counts have increased to more than 100×109/l long-term after E6040 administration. Platelet-associated IgG was decreased with thrombocytosis. Compared with the initial period of E6040 administration, the number of anti-GPIIb/IIIa antibody-producing B cells decreased, and proliferative response of autoreactive T cells to GPIIb/IIIa was also improved. A single injection of humanized monoclonal antibody to CD40 ligand may induce T-cell tolerance in patients with ITP.

An Unusual Case of Thrombocytopenia and Leukopenia Associated with Diltiazem and Acyclovir

Objective: To describe the case of a patient who developed thrombocytopenia associated with diltiazem and leukopenia associated with diltiazem or acyclovir. Case Summary: An 81-year-old white man was admitted for management of chronic obstructive pulmonary disease (COPD). On admission, his white blood cell (WBC) count and platelet count were within normal limits. Admission medications included metoprolol, which was replaced on day 3 with diltiazem due to concern that it might exacerbate the COPD. The patient's platelet count at the initiation of diltiazem therapy was 180 × 103/mm3. The next day the platelet count was 232 × 103/mm3, but decreased to 182 × 103/mm3 on day 5 and continued to decline through day 30, at which time it reached a nadir of 10 × 103/mm3. By then, several causes of thrombocytopenia, including disseminated intravascular coagulation and thrombotic thrombocytopenic purpura, had been ruled out. A hematology consult suggested diltiazem as the cause of thrombocytopenia, and it was discontinued. The platelet count began to rise 8 days after discontinuation of diltiazem and returned to normal values over a period of 5 days. On day 15, acyclovir was initiated for an oral herpes simplex virus–1 infection. The WBC count on day 20 was 8.7 × 103/mm3, and it continued to decline daily. On day 24, the value was 2.5 × 103/mm3, at which time acyclovir was discontinued. The WBC count continued to decline for 6 days, reaching a nadir of 0.6 × 103/mm3, at which point filgrastim was initiated; the WBC count gradually returned to normal over the next 5 days. Conclusions: Myelosuppression is a rare complication of diltiazem and acyclovir therapy. Patients using these medications should be monitored for signs and symptoms of myelosuppression.

Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding.

We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 microM ADP, similar to normal platelets with blocked P2Y(12) receptor. The response to 20 microM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y(12) antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E(1) in the patient's platelets, even though the number and affinity of 2-methylthioadenosine 5'-[(33)P]diphosphate-binding sites was normal. Analysis of the patient's P2Y(12) gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y(12) as compared with wild-type receptor. These studies delineate a region of P2Y(12) required for normal function after ADP binding.

Childhood Thrombocytopenia Associated with Graves Disease is Distinct from Idiopathic Thrombocytopenic Purpura

The authors report a case of thrombocytopenia in a 10-year-old Chinese girl who was also found to have Graves disease 6 months later. The clinical, hematologic, and bone marrow features were indistinguishable from chronic idiopathic thrombocytopenic purpura, but the patient's platelet counts were normalized concurrently with the control of thyrotoxicosis with carbimazole treatment. Childhood thrombocytopenia associated with autoimmune thyroid disease is a rarely reported condition and its management differs from that of idiopathic thrombocytopenic purpura.

Long-term results of initial and repeated partial splenic embolization for the treatment of chronic idiopathic thrombocytopenic purpura.

Although splenectomy is a standard surgical treatment for chronic idiopathic thrombocytopenic purpura, partial splenic embolization is another treatment option. We retrospectively studied the long-term results of initial and repeated partial splenic embolization. Thirty-nine patients, 15 men and 24 women, underwent initial embolization; 12 of the 39 underwent a repeated embolization. The therapeutic effects of the initial and repeated embolization were classified as a complete response if the patient's platelet count rose to more than 10 x 10(4)/ micro L without medication 1 year after the initial or repeated embolization, as a partial response if the platlet count reached 5-10 x 10(4)/ micro L under the same circumstances, or as no response. Twenty patients (51%) responded to the initial embolization (complete response in 11 and partial response in nine). No significant differences were found between those patients who responded to the treatment (responders) and those who did not respond to the treatment (nonresponders) in age, sex, lowest platelet counts, and steroid response before embolization. Peak platelet response was significantly higher in the responders (p = 0.029). One of the 11 complete responders and five of the nine partial responders relapsed after a median follow-up period of 34 months (range, 15-23 months) and underwent repeated embolization, resulting in complete response in one patient, partial response in the remaining four patients, and no response in one patient. However, in the six nonresponders (to the initial embolism), repeated embolization elicited a partial response in only one patient. The remission rate of 51% was maintained by means of repeated embolization for a median follow-up period of 76 months after the initial embolization. Partial splenic embolization combined with repeated embolization may be an effective alternative to splenectomy in the treatment of chronic idiopathic thrombocytopenic purpura.

Platelet spherocytosis: a new bleeding disorder.

The present study evaluated a child with thrombocytopenia and a rare congenital bleeding disorder associated with platelet spherocytosis. Differential interference phase contrast microscopy (DIC) revealed that his platelets were spherical in form. Examination of thin sections in the electron microscope showed that his platelets were nearly devoid of microtubules (MT) and microtubule coils (MTC) that support the discoid shape in 100% of normal platelets. Immunofluorescence with a monoclonal antibody to tubulin, the precursor protein of MT, revealed bright rings in normal cells and diffuse fluorescence of patient platelets. The brightness of the fluorescence emitted by patient platelets was comparable to the diffuse fluorescence of normal platelets after chilling to dissolve intact MT, suggesting normal and patient cells contained comparable amounts of tubulin. Exposure of patient platelets to Taxol, an agent that stabilizes and induces MT, caused MT formation in 82% of patient platelets and MTC development in 11%, resulting in their conversion to discs. Glycoproteins GPIIb/IIIa and GPIb were present on patient platelets, and the cells contained normal numbers of dense bodies, ruling out storage pool disease. The patient's platelets adhered to and spread normally on glass but failed to undergo rapid, irreversible aggregation when stirred with agents that produced a complete response in normal discoid platelets, even when the patient's platelets were concentrated. The poor response of spherical platelets was associated with failure to become irregular and extend long filopodia. Thus, the spherical shape of patient platelets may contribute to the thrombocytopenia and to the clinical bleeding symptoms.

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

AbstractThis article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of β3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa–transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.

Medical Management of a Large Aortic Thrombus in a Young Woman With Essential Thrombocythemia

Medical Management of a Large Aortic Thrombus in a Young Woman With Essential Thrombocythemia

Evidence for Immune Etiology in Clozapine-Induced Thrombocytopenia of 40 Months' Duration: A Case Report

Platelet dysfunction and thrombocytopenia rarely occur during clozapine therapy, but constitute an important source of morbidity and mortality if they are not detected and therapy is discontinued. The manufacturer recommends discontinuing clozapine when the platelet count falls below 100,000/muL and resuming therapy when the count returns to within normal range (150,000-450,000/muL). If thrombocytopenia recurs, clozapine should be permanently discontinued. The authors report a rare case of long-term thrombocytopenia persisting 40 months postclozapine treatment. In addition, increased in vitro platelet [14C]serotonin release was observed in the presence of the drug, suggesting an immune-related cause for the thrombocytopenia.

Surface expression of glycoprotein Ibα is dependent on glycoprotein Ibβ: evidence from a novel mutation causing Bernard-Soulier syndrome

Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary  IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.