Positive Platelets Research Articles

Heparin-induced thrombocytopenia occurring after surgical treatment of atrial myxoma: A case report

Heparin-induced thrombocytopenia (HIT) is an acquired, prothrombotic disorder, caused by antibodies to a complex of heparin and platelet factor 4 (PF4) that activates platelets, resulting in the release of procoagulant microparticles, thrombocytopenia occurrence, generation of thrombin, and frequent thromboses. We present a case of severe HIT in a 68-year-old female, which occurred after cardiosurgery of the left atrial myxoma with the aim to point out the importance of differential diagnosis of thrombocytopenia in patients recently exposed to heparin. Platelet count dropped on the eleventh postoperative day, six days after unfractioned heparin and enoxaparine threatment, to 4x10(9)/I.The correct diagnosis failed to be made at first. Since thrombocytopenia remained refractory to a corticosteroid treatment and platelet transfusion, the patient was hospitalized on the 13th postoperative day at the Institute of Haematology. The diagnosis of HIT was confirmed with the high-probability clinical score (4T's) and strongly positive anti-heparin-PF4 (PaGIA) test as well as positive platelet aggregation test. The treatment started with a smaller therapeutic doses of danaparoid than recommended of 750 U intravenous bolus and was followed by continuous infusions of 100 U per 1 h and intravenous gammaglobulins in full dosage for four days. The platelet count started to rise on the third day and it was completely normalized on the 5th day of the therapy. Treatment of severe HIT with small doses of danaparoid supplemented by intravenous gamma globulin was successful. Additional awareness of heparin-induced thrombocytopenia is needed, especially of HIT in differential diagnosis of thrombocytopenia in patients recently exposed to heparin.

Bonne pratique et valeur diagnostique de la biologie : hémostase-hématologie

Inherited bleeding disorders are potentially causes of menorrhagia and must be investigated if no specific cause is identified. The reported prevalence of inherited bleeding disorders is high in women with menorrhagia compared to the general population. The most frequent disorders reported are von Willebrand's disease and Factor XI deficiency. Menorrhagia is, also, a frequent finding in women with congenital bleeding disorders. Morever, menorrhagia represents the major cause of iron-deficiency anemia among women of reproductive age. Primary evaluation for an underlying disorder of hemostasis in a woman wih menorrhagia is a focused history for familiy and personal history of bleeding symptoms and a complete blood cell count. This will rule out thrombocytopenic bleeding and also assesses for the degree, if any, of anemia. Those women with a positive screen and normal platelet count should be evaluated with laboratory investigation including prothrombin time, activated partial thromboplastin time, factor VIII, VWF ristocetin cofactor and antigen. If initial hemostasis testing above is normal, then further hemostasis testing can be considered, especially in terms of platelet aggregation, in a multidisciplinary clinic.

Apoptosis in Platelets from Pediatric Patients with Acute Immune Thrombocytopenic Purpura (ITP) Is Ameliorated by IV Ig.

Interaction of auto-antibodies with platelet surface glycoproteins (GP) has been implicated in the accelerated clearance of platelets in ITP. It was recently shown, using a murine model, that the ITP induced by injection of anti-GPIIb is associated with apoptotic-like processes in platelets. These apoptotic-like processes in platelets were similar to those observed during apoptosis in nucleated cells: activation of caspase-3 (aCASP3), loss of mitochondrial inner transmembrane potential and externalisation of phosphatidylserine (PS). Administration of IVIg, that ameliorates the ITP in this model, also inhibited aCASP3 and PS externalization (Leytin et al., 2006, Br J Haematol ; 133:78; Song et al., 2003, Blood ; 101:3708). Whether these observations in mice are applicable to patients with ITP had not been investigated. Thus, the aim of our study was to investigate whether platelets from paediatric patients with acute ITP show an enhancement of apoptotic-like processes. Further, we examined whether the increase in platelet count in response to IVIg in these patients is accompanied by an inhibition of apoptotic-like processes in platelets. Children with clinical and laboratory diagnosis of acute ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a paediatric bleeding score for ITP at the time of diagnosis and after IVIg therapy. Blood samples were obtained at these time points for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. Specifically, platelet aCASP3 and PS externalisation were measured in citrated platelet-rich plasma. Platelets were identified as CD42 positive events; aCASP3 was measured as % platelets with bound FLICA-FITC fluorescence; and PS exposure as % platelets with bound annexin A5-FITC fluorescence. The fraction of young, reticulated platelets (RP) was analysed using thiazole orange. All patients (median age 7.5, n = 12) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3. 10 patients had platelet counts below 10'000/micro l, and 2 had platelets counts over 10'000/micro l. ITP patients had increased levels of platelets with aCASP3 (n = 11) and PS exposure (n = 9) compared with healthy control children (see Figure) and increased reticulated platelets (15.6 ± 2.7 %, n = 10; controls: 1.2 ± 0.8, n = 11). The 10 patients with platelet counts below 10,000/micro l were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose). All patients showed a rise in platelet counts to above 20'000/micro l and amelioration of bleeding symptoms 24 – 72 hours after IVIg administration. Concomitantly, platelet aCASP3 and PS exposure decreased (see Figure), as did RP, to 3.8 ± 1.5 % (n = 9). One patient with an initial platelet count of 18,000/micro l was not treated. This patient showed 5% positive aCASP3 platelets, 3.5 % platelets with PS exposure and 3.5% RP, similar to controls; one month later, the platelet count increased spontaneously to 60'000/micro l with 10% aCASP positive platelets, 4% platelets with PS exposure and 3 % RP. One patient was lost to follow-up. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP3 and PS externalisation in paediatric acute ITP, similar to the reported murine model. Consistent with acute ITP, platelet counts were low and levels of RP were high; after administration of IVIg, platelet counts increased whereas levels of RP decreased. IVIg administration also led to a decrease of aCASP3 and PS exposure in the patients' platelets. Thus, amelioration of acute ITP in children by IVIg is accompanied by inhibition of enhanced apoptosis-like events in platelets. [Display omitted]

Dramatic Efficacy of Sequential Bypassing Agent Treatment for a Life-Threatening Bleeding in An Infant with Severe Hemophilia and Inhibitor

In hemophiliacs the treatment of bleeding in infancy is more difficult than in adults and children due to insufficient knowledge in pharmacokinetics. Moreover the clinical response to coagulation concentrates is known to have not only inter-patients but also inter-bleeding episodes variability and only scarce experience is available. We report an experience in a one-month old infant managed with a successful sequential therapy.Severe hemophilia A was diagnosed at day 4 of life because of a cephalhematoma without intra cerebral hemorrhage (ICH) and severe anemia (Hb: 4 g/dl). Packed red blood cells (RBC) were given, FVIII concentrate was infused daily during 12 days and the hematoma resolved. At day 50 recurrent seizures led to the diagnosis of subdural haemorrhage, in the absence of any trauma. Anti FVIII inhibitor was detected (while negative 10 days before). Treatment with recombinant FVIIa (rFVIIa) was started at 1.2 mg (200μg/kg) every 2 hours and then progressively decreased. Outcome was good. To avoid ICH recurrence because FVIII inhibitor titer>30 BU, rFVIIa prophylactic treatment was decided and a central venous access device (CVAD) was inserted into the left internal jugular vein. No abnormal blood loss was observed during this procedure and a compressive bandage on CVAD was immediately performed. Collapse and respiratory distress developed within 24 hours. Chest X-ray showed a complete left hemothorax. Hemoglobin(Hb) level dropped to 5.2 g/dl. RBC infusion was required and rFVIIa dose was increased to 2.4 mg then alternately 1.2 mg and 2.4 mg every 2 hours until hemodynamic stabilization. Pleural aspiration yielded only 25 ml bloody fluid. Twelve hours later the Hb level fell again to 7 g/dl and RBC were again infused. Recombinant FVIIa treatment was stopped and activated prothrombin complex concentrate (aPCC) was started at a dose of 500 units (83u/kg) every 6 hours. After 2 doses of aPCC the Hb level fell again requiring new RBC infusion. A sequential therapy was decided with alternate rFVIIa 1.2 mg and aPCC 500 units every 4 hours. and, the hemodynamic parameters and Hb level stabilized. Pleural aspiration yielded 190 ml. The infant's clinical status improved dramatically with the disappearance of the hemothorax. This alternative treatment was maintained for 20 hours with good tolerance according to frequent monitoring of platelet count, D-dimers and fibrinogen. A more usual treatment (aPCC 500 units tid) was prescribed but after the first 2 infusions fibrinogen level fell to 0.9 g/l, with positive D-dimers and platelet count dropped to 36 109/l. The therapy was decreased to 500 units bid and the biological parameters quickly improved. The baby's clinical status was excellent and one day after, therapy was switched to rFVIIa 1.2 mg every 6 hours. The outcome was excellent.Conclusion: This report evidences differential response to the same treatment according to the type of bleeding in the same patient with inhibitor. After inhibitor development, for ICH, where r FVIIa treatment is well known to be efficient, the patient's response was very good at usual doses for this age. For the second episode (hemothorax) where life was threatened at several instances the escalating and sequential treatment schedule shows that sometimes very aggressive therapy is required despite high risks of thrombosis and disseminated intravascular coagulation.

Cross Reactivity of Heparin-Induced PF4 Antibodies with Heparin- Bound IL-8.

Background: Heparin-Induced Thrombocytopenia (HIT) is caused by polyclonal antibodies, formed to conformational neoepitopes exposed in platelet factor 4 (PF4) bound to heparin (H). HIT antibodies are commonly measured by one of several commercially available ELISAs. The literature reports cases of clinical HIT and/or positive HIT antibody-mediated platelet activation tests in patients without antibodies detectable in PF4 based ELISAs, and suggests that antibodies to other chemokines, such as interleukin-8 (IL-8), may be involved. The current study was undertaken to characterize the frequency of such antibodies in a population of consecutive serum specimens submitted for HIT diagnosis by 14C-serotonin release assay (SRA), and also in a group of retrospectively selected SRA-positive specimens.Methods: Specimens (n=90) analyzed by SRA between March and July 2008 were subsequently tested by PF4 Enhanced ELISA (GTI, Waukesha, WI), and the Zymutest HIA ELISA (Hyphen BioMed, Neuville sur Oise, France). An additional ELISA was carried out on the specimens using the Zymutest heparin-bonded plate coated with purified, recombinant human interleukin-8 (IL-8) (72 aa form, Prospec, Rehovot, Israel). The second group consisted of archived specimens (n=32) that tested positive by SRA but negative by GTI HAT ELISA; these specimens were subsequently tested by the Zymutest HIA and the H:IL-8 ELISA.Results: Recombinant IL-8 (1μg/well) added to heparin-bound ELISA plates was recognized by anti-IL-8 (BD Biosciences), but not anti-PF4 (Leinco Technologies, Inc.) antibodies and was not displaced from the plate by addition of PF4 from normal serum at the dilution (1:100) used in the ELISA. Of the 90 consecutive specimens, 21 (23.3%) tested positive in both the GTI and the Zymutest HIA tests; 15 of the 90 (16.6%) tested positive by one but not both of the PF4-based ELISAs (Table 1). In this group of 90 specimens, 17 (18.9%) had OD > 0.500 on the heparin-bound IL-8 plate. Only 3 of the specimens bound to the H:IL-8 exclusively, while 14 were also positive in the GTI and/or Zymutest HIA ELISA. Thus, of the 36 specimens that tested positive in one or both of the PF-4-based ELISAs, 14 (38.9%) had antibodies that also targeted heparin-bound IL-8. Ten of the 90 specimens tested positive in the SRA. Six of SRA-positives had PF4-specific antibodies that did not cross react with H:IL-8, 3 had H:IL-8 reactive antibodies in addition to PF4 reactive antibodies, and 1 SRA positive specimen tested negative in all ELISAs.Table 1. Comparison of GTI HAT vs Zymutest HIA results: n = 90 consecutive patientsNumbers in parentheses indicate how many specimens in each subgroup contained antibodies that bound to H:IL-8.Zymutest HIA+−Total+21 (11)9 (1)30GTI HAT−6 (2)54 (3)60Total276390Among the set of 32 specimens that tested positive by SRA and negative by GTI ELISA, 8 tested positive in the Zymutest ELISA, including 4 that had OD > 0.500 on the H:IL-8 plate. The 24 specimens that tested negative by both HIT ELISAs ranged in percent serotonin release (tested with 0.1 U/ml H) between 21–84% with a median of 45.6%, and did not contain antibodies that bound the H:IL-8 plate.Conclusions: The data agree with other studies that show differences between results of commercially available HIT ELISA kits. The study also shows that over one third of specimens that test positive in the HIT ELISAs contain antibodies that are not specific for PF4, but also recognize heparin-bound IL-8. These results do not prove that the antibodies that bind PF4 targets are the same ones that bind IL-8. However, the significant degree of homology (40%) between the peptides suggests that the same antibodies may cross react with either target. The relevance of these antibodies is as yet unclear. Only 3% of specimens bound H:IL-8 plates but not PF4 plates, and these were not associated with SRA activity. Also HIT ELISA negative specimens with SRA activity, which showed only moderate percent serotonin release, did not contain H:IL-8-binding antibodies. The study did not find evidence of a role for H:IL-8 antibodies in HIT-related platelet activation tests.

Circulating Microparticles, Plasma Protein C and Free Protein S Levels in Children with Sickle Cell Anemia.

Microparticles (MP) originate from blebbing and shedding from cell membrane surfaces in physiological and pathological conditions. Increased levels are generated by a number of mechanisms including platelet activation, vascular endothelial damage, thrombin activity, C5b-9 activation, and PF4-heparin-antibody interaction. Increased circulating MP have been described in patients with sickle cell anemia (SCA). Elevated monocyte-derived MP expressing tissue factor have been reported in patients in crisis. The lack of a standardised method for MP quantification remains problematic. We measured MP (numbers and functional markers), protein C and free protein S, in a large cohort of pediatric patients to investigate the role of MP in SCA and their relation to protein C and free protein S plasma levels. One hundred and eleven children of sub-Saharan African ethnicity with SCA (hemoglobin (Hb) SS) were studied: 51 without previous history of crisis in steady state (mean age 5.3 years); 15 in crisis (9 chest crises, 6 other, mean age 5.4 years); 30 on hydroxyurea (mean age 7.2 years); 15 on transfusion therapy (mean age 6.5 years); 17 children of sub-Saharan African ethnicity of similar age (mean age 4.6 years) were used as control group (Hb AA). MP were analyzed by flow cytometry, according to Biró et al ( J Thromb Haemost. 2004; 2(10):1842–51), using Annexin V and antibodies against, CD61, CD42a, CD62P (P-selectin), CD235a, CD14, CD142 (tissue factor), CD201 (endothelial protein C receptor or EPCR), CD62E (E-selectin), CD36 (thrombospondin or TSP-1), CD47 (TSP-1 receptor), CD31 PECAM (platelet-endothelial cellular adhesion marker), CD144 (VE-cadherin). Protein C (chromogenic) and free protein S (latex based assay) were measured in all subjects. Correlation was measured by Pearson Rank test, and comparisons between groups were analyzed by Mann-Whitney test. Total MP AV were lower in crisis (1.26 × 106/ml; 0.56–2.44 × 106) and steady state (1.35 × 106/ml; 0.71– 3.0 × 106) compared to transfusion (4.33 × 106/ml; 1.6–9.2 × 106p<0.01). Protein C levels were significantly lower in crisis (median 0.52 IU/ml; interquartile range 0.43–0.62) compared to all other groups: HbAA (0.72 IU/ml; 0.66–0.82, p<0.001); hydroxyurea (0.67 IU/ml; 0.58–0.77, p<0.001); steady state (0.63 IU/ml; 0.54–0.70, p<0.05) and transfusion (0.60 IU/ml; 0.54–0.70, p<0.05). In addition levels were significantly reduced in steady state (0.63 IU/ml; 0.54–0.70) compared to HbAA (0.72 IU/ml; 0.66–0.80, p<0.01). Protein S levels were significantly higher in HbAA (0.85 IU/ml; 0.72–0.97) compared with crisis (0.49 IU/ml; 0.42–0.64, p<0.001), hydroxyurea (0.65 IU/ml; 0.56–0.74, p<0.01), and transfusion (0.59 IU/ml; 0.47–0.71, p<0.01). There was also a significant difference in crisis patients compared to steady state (0.49 IU/ml; 0.42–0.64 v 0.68 IU/ml; 0.58–0.79, p<0.05). There was high correlation (R>0.9, p<0.05) between total numbers of Annexin V positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p<0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p<0.001). %MP CD201 was higher in crisis (11.6%) compared to HbAA (3.2%, p<0.05); %MP CD144 was higher in crisis (7.6%) compared to transfusion (2.1%, p<0.05); %CD14 (0.77%) was higher in crisis compared to transfusion (0.0%, p<0.05) and steady state (0.0%, p<0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared to the rest (40%); %MP CD235a was higher in crisis (17.9%) compared to transfusion (8.9%), hydroxurea (8.7%) and steady state (9.9%, p<0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. These studies indicate that there are significantly lower levels of protein C and free protein S in children with sickle cell crisis. In addition there are significantly lower numbers of circulating platelet MP in steady state and crisis patients; however in crisis a significantly higher percentage of MP express markers of endothelial and vascular damage, and of red cell origin. Among these are composite hybrid microparticles expressing markers of more than one cell type, probably brought about by severe vascular stress and close contact of various circulating cell types with vascular endothelium.

Idiopathic Non-Cirrhotic Intrahepatic Portal Hypertension Is Associated with Sustained ADAMTS13 Deficiency.

Idiopathic non-cirrhotic portal hypertension (NCPH) is caused by obliterative venopathy affecting the terminal radicals of the hepatic portal vein. It has been associated with areas of low socio-economic development. We observed a long history of thrombocytopenia and association with enteric disorders in a Caucasian population presenting with NCPH and postulated that the underlying mechanism could be deficiency of ADAMTS13, leading to the circulation of ultra large molecular weight von Willebrand factor (VWF) multimers and consequent platelet aggregate formation, causing occlusion of hepatic portal venules. ADAMTS13 activity was measured in all patients by a collagen binding assay, while antigen and IgG antibody were measured using ELISA kits (American Diagnostica Inc), normal ranges: 66–126%, 485–1242ng/ml and <11AU/ml, respectively. Plasma ADAMTS13 activity and antigen levels were measured in 14 patients with NCPH and 25 control patients with miscellaneous liver diseases associated with portal hypertension and Model for End Stage Liver Disease (MELD) scores indicating similar disease severity, median 12 (IQR 10–14) and 11 (8–16), for NCPH cases and controls. The platelet count was reduced in 13/14 NCPH cases median 69 x109/L (IQR 40–97) and 123 (85–193) in cases and controls, but did not correlate with MELD score or ADAMTS13 activity. ADAMTS13 activity was very low in 13/14 NCPH cases (median 12% (IQR <5–20)) and significantly lower than controls (59% (4–84), p<0.0001). In 4 cases, serial samples over several months revealed consistently low ADAMTS13 levels. There was a corresponding reduction in ADAMTS13 antigen, median 238ng/ml (IQR 186–361) and 584 (442–831), although NCPH cases showed a small excess of antigen over activity (antigen:activity ratio: median 25.7 (IQR 11.3–32.6) and 9.4 (8.1–13.8), in cases and controls, p<0.001), suggesting that an abnormal molecular species could be present. An inhibitor was not detected, although 10 cases and 15 controls had IgG anti-ADAMTS13: median levels 14.8AU/ml (8.8–20.0) and 13.4 (9.0–22.6), but these IgG levels did not correlate with activity. Non-inhibitory IgG antibodies to ADAMTS13 have been previously observed in certain other clinical groups (eg. coronary syndromes, sepsis, etc.). In NCPH, there was no correlation between ADAMTS13 level and either VWF antigen or VWF collagen binding activity. Increased very high molecular weight VWF multimers were observed in 4/11 NCPH cases and 0/22 controls. CD31 positive platelet aggregates were observed in the hepatic vessels of 7/20 (35%) NCPH cases and 1/15 (7%) controls. Our results suggest that sustained deficiency of ADAMTS13 associated with thrombocytopenia is characteristic of NCPH. It differs from other hepatic pathologies, where the decrease in ADAMTS13 has been reported to correlate with the severity of liver disease. NCPH also differs from thrombotic thrombocytopenic purpura because there is a chronic disease process, there is an absence of obvious red cell fragments and the primary organ affected is the liver. Since the predominant plasma source of ADAMTS13 appears to be hepatic stellate cells, the portal microcirculation is most at risk from pathological interactions of VWF and platelets if enteric factors further deplete circulating levels of ADAMTS13. The cause of the decreased ADAMTS13 remains to be elucidated, but could be due to decreased expression and synthesis, immune depletion (IgM and IgA anti-ADAMTS13 have not been investigated), or consumption in the circulation.

Drug Dependent Immune Destruction of Human Platelets in the NOD/SCID Mouse.

Animal models have been of limited value for studying survival and immune destruction of human blood cells because of xenoantibodies that mediate rapid elimination of the transfused cells. The NOD/SCID mouse is a genetic variant that lacks immunoglobulins, including xenoantibodies, and may therefore be useful for studying survival and immune destruction of human blood cells (Newman, PJ et al. J Thromb Haemost. 2007; 5 Suppl 1:305–9). Human platelets, infused into the retroorbital plexus of NOD/SCID mice survive about three days, nearly as long as mouse platelets themselves. Injection of IgG from a patient with quinine-induced immune thrombocytopenia (TP) and a newly developed quinine-dependent murine monoclonal antibody (mAb) were without effect on platelet survival. However, both antibodies caused rapid platelet clearance after IP injection of quinine. 500 μg, but not 50 μg, of IgG from serum containing an anti-HPA-1a (PlA1) antibody also caused rapid clearance of HPA-1a positive platelets. These findings indicate that the NOD/SCID mouse can be valuable for the study of immune clearance of human platelets and perhaps other unresolved problems in transfusion medicine. However, it is not known whether mice recognize cells opsonized with human IgG or mouse IgG equally well. To examine this, we compared the survival of human platelets sensitized with 1) mAb 7E3 (IgG1) specific for an epitope in the beta A domain of GPIIIa or 2) c7E3 (from Centocor Inc), which recognizes the same epitope but possesses a human IgG1 Fc. In titration studies, we found that about four times as much c7E3 as 7E3 is needed to produce equivalent clearance of human platelets, indicating that mouse IgG is a more efficient opsonin than human IgG. However, this difference should not limit use of the model for studies of antibody-mediated blood cell destruction, since mg quantities of IgG can easily be isolated from small amounts (0.1– 0.2 ml) of human serum.

Abstract 5466: In Vivo Molecular Imaging Revealed Chronic Inflammation and Increased Adhesion Molecules in Obese Adipose Tissue in Mice

Metabolic syndrome is a major risk factor of cardiovascular events, and obese visceral adipose tissue remodeling and malfunctioning based on chronic inflammation plays a central role. To assess dynamic multi-cellular interplay, a novel ex vivo (a–c) and in vivo (d–f) adipose tissue imaging method was developed. We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese adipose (c,arrow). In addition, we found increased leukocyte-platelet-endothelial cell interactions in the micro-circulation of obese visceral adipose that were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation (e,f). Both macrophages and endothelial cells showed increased adhesion molecules including ICAM-1 and Selectin families, and P-selectin positive platelets were increased locally in obese adipose. Up-regulated expression of adhesion molecules on multiple cell types suggests their increased interactions contribute to local activation of inflammatory processes within visceral obese adipose tissue. Interestingly, the heightened leukocyte-platelet-endothelial interactions were not observed in obese subcutaneous fat pads. Our results demonstrated the power of our imaging technique to analyze complex inflammatory cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral adipose tissue obesity is an inflammatory disease.

Abstract 4008: PSGL-1-Expressing CD4 T Cells are Enriched in Culprit Coronary Artery Lesion of Acute Coronary Syndrome

Background: Recently, several evidences on the crucial role of adhesion molecules in the development of atherosclerosis and plaque instability have been reported. While expression of VCAM-1, ICAM-1 and L-selectin has been consistently observed in atherosclerotic plaques it is still uncertain how adhesion molecules on T cells contribute to the incidence of acute coronary syndrome (ACS). In this study, we examined whether adhesion molecules on T cells in ACS have a significant role in the plaque stability and prone to cause ACS. Methods and Results: Fresh CD4 T cells were isolated from the peripheral blood of 76 ACS patients (AMI=35, UAP=41) and 74 age-matched controls (NC). CD69, an activation marker of T cells, was strongly expressed on CD4 T cells from ACS than from NC by FACS (P&lt;0.0001). CD4 T cells from ACS highly expressed p-selectin glycoprotein ligand-1 (PSGL-1) and integrin β (CD18), but not L-selectin by FACS (P &lt; 0.03, P &lt; 0.01, n.s., respectively). Soluble PSGL-1 (sPSGL-1) levels in plasma were lower in ACS patients than in NC (P=0.0001), which correlated negatively with the PSGL-1 expression on CD4 T cells (R=0.405, P&lt;0.02). We further investigated the thrombus-aspirating device samples (n=14) and fresh CD4 T cells derived from both the coronary artery and peripheral blood from the each same patient with ACS. CD4 T cells from the coronary artery strongly expressed PSGL-1 (P&lt;0.002), but not integrin β (CD18) and L-selectin by FACS. Finally, PSGL-1 was expressed on T cells, but not on CD68 positive macrophage, MPO positive neutrophil, or CD41 positive platelets in the thrombus-aspirating device samples. Conclusions: From these results, we demonstrated that PSGL-1-expressing CD4 T cells are enriched in the culprit coronary artery lesion of ACS, contributing to the acceleration of plaque instability and occurrence of ACS.

Platelet C4d Is Associated With Acute Ischemic Stroke and Stroke Severity

Platelets bearing complement C4d were recently reported to be 99% specific for a diagnosis of systemic lupus erythematosus (SLE) and associated with neuropsychiatric lupus. We compared the prevalence of platelet C4d and investigated the clinical associations of platelet C4d in patients with acute ischemic stroke. We recruited 80 patients hospitalized for acute ischemic stroke. Stroke severity was measured by the National Institutes of Health Stroke Scale (NIH-SS). Infarct volume was determined by MRI. Platelet C4d was measured by flow cytometry. Mean age was 57.9 years (range: 24.6 to 86.8 years), 58% were male, and 91% were white. Eight patients (10%) with acute ischemic stroke were platelet C4d-positive, which was significantly higher in prevalence compared to healthy controls (0%, P<0.0001) and non-SLE patients with immune/inflammatory disease (2%, P=0.004). The median NIH-SS score and infarct volume for acute stroke patients were 6 (interquartile range [IQR]: 2 to 13) and 3.4 cc (IQR: 1.1 to 16.6), respectively. Platelet C4d-positive patients were more likely to have a severe stroke compared to those with negative platelet C4d (NIH-SS median: 17.5 versus 5, P=0.003). Positive platelet C4d was independently associated with stroke severity (P=0.03) after controlling for age, anticardiolipin antibody (aCL) status, and total anterior circulation of stroke involvement, and also with infarct volume (P=0.005) after controlling for age, aCL status, and old stroke by MRI. Platelet C4d is associated with severe acute ischemic stroke. Platelet C4d may be a biomarker as well as pathogenic clue that links cerebrovascular inflammation and thrombosis.

Role of anti‐Naka antibody, monocytes and platelets in the development of transfusion‐related acute lung injury

Transfusion-related acute lung injury (TRALI) is one of the most serious side-effects of transfusion. We report here the first two cases of TRALI caused by anti-Nak(a) (anti-CD36) antibody from a single blood donor. The aim of this study was to clarify the role of the anti-Nak(a) antibody in TRALI development. Human lung microvascular endothelial cells were co-cultured with Nak(a)-positive monocytes and Nak(a)-positive platelets together with serum prepared from blood products of a TRALI-caused anti-Nak(a) antibody-carrying donor. Expressions of leukotriene B(4) (LTB(4)) and tumour necrosis factor alpha (TNF-alpha) in the co-culture supernatants were determined. The expressions of LTB(4) and TNF-alpha were found to be markedly increased, particularly in the presence of both Nak(a)-positive monocytes and platelets. The expressions of these mediators were almost completed within 4 h after the initiation of co-culture. Monocyte contribution seemed to be stronger than that of platelets. In the absence of human lung microvascular endothelial, no significant LTB(4) or TNF-alpha release was observed. Anti-Nak(a) antibody may be strongly implicated in lung microvascular endothelial dysfunctions that lead to TRALI in a monocyte- and platelet-dependent manner.

A comparison of direct thrombin inhibitors in the treatment of heparin-induced thrombocytopenia: a single institution experience

Heparin-Induced Thrombocytopenia (HIT), if left untreated, can lead to thrombocytopenia, thromboembolic complications and even death. Two thrombin inhibitors, lepirudin and argatroban, have been shown to improve clinical outcomes compared to historical controls in the management of HIT. The purpose of this retrospective study was to compare the effects of lepirudin and argatroban in the management of HIT. Adult subjects with a positive anti-heparin platelet factor 4 (PF4) antibody test and >50% decrease in platelet count during the first 30 days of admission over a period of 2 years were included in the study. Patient demographics, platelet counts, choice of antithrombin therapy, occurrence of thrombosis, length of hospital stay, and date and cause of death, if applicable, were collected for each patient. Eighty-two patients met inclusion criteria: 41 patients did not receive any thrombin inhibitors after the diagnosis of HIT, 24 patients received lepirudin and 17 patients received argatroban. Subjects treated with a thrombin inhibitor were more likely to experience platelet count recovery (87.5% for the lepirudin group and 82.4% for the argatroban group) compared to those who did not receive antithrombin therapy (51.2%) after the diagnosis of HIT was made (P < 0.001). The thrombosis rate for subjects who did not receive antithrombin therapy after the diagnosis of HIT was 26.8%, compared to 8.3% for the lepirudin group and 5.9% for the argatroban group (P < 0.01). The incidence of death was also higher in the group of subjects that did not receive antithrombin therapy (48.8%) compared with the lepirudin group (16.7%) or the argatroban group (23.5%), P < 0.01. Our findings suggest that thrombin inhibitors can improve the outcomes of patients with HIT by decreasing the incidence of morbidity and mortality relating to HIT. No significant difference could be determined in outcomes between argatroban and lepirudin therapy.

Massive Right Atrial Thrombus Due to Heparin-Induced Thrombocytopenia Type II

2 weeks after elective coronary artery bypass grafting, a 61-year-old male patient was admitted to the intensive care unit with severe acute dyspnea. The initial postoperative clinical course had been uneventful, however, at postoperative day 6, the patient had suffered a right hemispheric transient ischemic attack. Thus, high-dose heparin therapy was started and activated partial thromboplastin time carefully monitored within therapeutic levels. Since paroxysmal atrial fibrillation was present, a thromboembolic event was initially suspected. Transesophageal echocardiography revealed small residues of a thrombus within the left atrial appendage (Figure 1). Interestingly, a highly mobile massive right atrial (RA) thrombus was detected (Figure 2) which was in agreement with the CT scan demonstrating multiple pulmonary embolisms of the right pulmonary artery. Laboratory analysis revealed novel thrombocytopenia (27 platelets/nl) as compared to a normal preoperative platelet count (198 platelets/nl). These findings were consistent with the diagnosis of heparin-induced thrombocytopenia type II (HIT II). Therefore, heparin was immediately discontinued and replaced by the direct thrombin inhibitor lepirudin. The suspected HIT II was confirmed by positive heparin-induced platelet aggregation (HIPA test). Within 36 h after the cessation of heparin, the platelet count recovered (91 platelets/nl) without any further thromboembolic events. Subsequent transthoracic echocardiography demonstrated increasing mobility of the massive RA thrombus partially prolapsing into the right ventricle (Figure 3) with threatening fatal occlusion of the right ventricular outflow tract. The patient was immediately transferred to cardiosurgery for successful removal of the massive round-shaped RA thrombus. In summary, HIT II caused thromboem1 Department of Medicine II, Cardiology, Asklepios Klinik St. Georg, Hamburg, Germany.

Effects of different inner face of human venous blood container on platelet activation

Objective To study the effects of different inner face status of human venous blood container on platelet activation. Methods The plastic( polyethene terephthalate,PET) and glass tubes were coated with polyalkyleneoxide modified polydimethylsiloxane(L722). The contact angles of L722-coated glass and L722- coated PET tubes, glass tubes, PET tubes, silane coupling agent-coated glass tubes and polypropylene (PP) tubes were analyzed respectively. The blood were drawn into above tubes, and then incubated in a roller bottle at room temperature for 10-60 mira The marker of activated platelete, CD62p, was detected by flow cytometry(FCM). Results The inner face contact angle of the blood collection tubes with different material and surface treatment reflected platelet activation to a certain extent, but was not linear. The percent age of CD62p positive platelets in L722-modified PET tubes reduced from (37.4 ±14. 8) % to(21.9 ± 12. 4) %. The platelet activation by glass tubes was (54.5 ± 18.6 ) %, markedly more than PET tubes. While membrane formed, the platelet activation by glass tubes decreased remarkably, and the percentage of CD62p positive platelet in silane coupling agent-coated glass tubes were ( 28. 3 ± 8.2 ) %,markedly less than that of the L722-coated glass tubes. The platelet activation by PET-based material ectally modified with L722 was obviously less than L722-coated glass tubes and the percentage of CD62p positive platelet in silane coupling agent-coated glass tubes were ( 41.5 ± 15.9 ) % and ( 22. 0 ± 12. 8 ) %,respectively. The time course of platelet activation by different tubes showed that the platelet activation by L722-coated PET tubes and polypropylene tubes in 60 rain was not significantly different from the results in30 mitt Conclusions The diverse surface energy status induced by different material and surface treatment of blood collection tubes have obvious effects on the activation of platelets. The silicic oil surface treatment can effectively improve the blood compatibility of blood collection tubes. CD62p detectod by FCM is a sensitive marker for the evaluation of the activation of platelets induced by the material of blood collection tubes. It is of importance in the establishment of surface treatment model of blood container and screening out the material of clinical application. Key words: Blood specimen collection; Diagnostic equipment; Platelet activation; P-selectin ; Biocompatible materials

Platelet associated IgG and immune thrombocytopenia in lymphoproliferative and autoimmune disorders.

Thrombocytopenia is frequently encountered in patients with lymphoproliferative disorders (LPD) and systemic erythromatosus (SLE) and to a lesser extent in association with other diseases such as rheumatoid arthritis (RA), pernicious anaemia (PA) and autoimmune haemolytic anaemia (AIHA). This report attempts to document the incidence of thrombocytopenia in these disorders, other than that overtly due to malignant infiltration or marrow suppression by drugs and to demonstrate, that in a significant proportion antibody mediated immune destruction of platelets can be confirmed by positive platelet antibody tests. Platelet associated IgG (PAIgG) was measured in all patients by a quantitative enzyme linked assay. Platelet antibodies were found in 11 of 24 (46%) thrombocytopenic patients with LPD, 10 of 16 (62%) patients with SLE and thrombocytopenia, and in all patients with RA and PA who had low platelet counts at the time of study. In addition, elevated PAIgG levels were found in the following non-thrombocytopenic patients: 9 of 43 (21%) patients with LPD, 2 of 12 (17%) with SLE, 2 of 12 (17%) with AIHA, 2 of 39 (5%) with PA and 5 of 61 (8%) patients with RA. The nature and the role of raised PAIgG levels in diseases other than autoimmune thrombocytopenia is controversial. Our reasons for interpreting these as true platelet autoantibodies in this selected group of disorders and the clinical implications of our results are discussed.

Morphological and functional platelet abnormalities in Berkeley sickle cell mice

Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37 ± 3.2 vs. 27 ± 1.4, mean ± SD; p < 0.001), in association with moderate thrombocytopenia (505 ± 49 × 10 3/μl vs. 1151 ± 162 × 10 3/μl; p < 0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell–Jolly bodies and “pocked” erythrocytes ( p < 0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5 ± 1% vs. 1 ± 1%; p < 0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages.Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Comparison of the effects of desflurane and sevoflurane on the expression of platelet surface glycoproteins in unstimulated and adenosine diphosphate-induced platelets in vitro ⋆

Study Objective To compare the effects of one minimum alveolar concentration (MAC) desflurane and sevoflurane on the expression of CD42b (glycoprotein [GP] Ib), CD41 (GPIIb), CD61 (GPIIIa), CD62P (P-selectin), and CD63 in both unstimulated and adenosine diphosphate (ADP)–stimulated platelets in vitro. Setting University laboratory. Subjects 15 healthy volunteers. Interventions Platelet-rich plasma was obtained and divided into three groups: platelet-rich plasma exposed to air (group 1); air plus one MAC desflurane (6% vol; group 2), and air plus one MAC sevoflurane (2% vol; group 3), for 40 minutes. Percentage of antigen-positive cells (% +) mean channel fluorescence (MCF Σ), and index of platelet activation for positive platelets (IPA +) as expression markers for GPIb, GPIIb, GPIIIa, P-selectin, and CD63, were measured. Measurements and Main Results In unstimulated platelets, expression markers for GPIIb and GPIIIa were significantly lower in groups 2 and 3 than group 1 ( P < 0.001). P-selectin expression markers were significantly higher in group 2 than in group 1 or group 3 ( P < 0.016). CD63 expression markers were significantly lower in group 3 than group 1 ( P < 0.016). In ADP-stimulated platelets, expression markers for all glycoproteins were significantly higher in all groups. Conclusion Neither one MAC desflurane nor sevoflurane showed any significant change in ADP-stimulated platelets compared with the control group.

Platelet P‐selectin Expression in Patients With Sickle Cell Disease Who Undergo Apheresis

Activated platelets have been identified in patients with sickle cell disease. However, the association of platelet P-selectin expression and automated red cell exchange procedures in these patients is not well known. We hypothesized that altered whole platelet P-selectin expression is associated with automated red cell exchange. Flow cytometric quantification of platelet P-selectin expression was carried out in 23 patients with sickle cell disease before and after automated red cell exchange. P-selectin expression was quantified as a binding index for platelet P-selectin (the percentage of positive platelets multiplied by the mean fluorescence of positive platelets). The patients were divided into two groups: individuals with painful vaso-occlusive crises (four women and five men; group 1) and those in a steady state (six women and eight men; group 2). The 33 exchange procedures were evaluated prospectively and used acid-citrate-dextrose A solution (whole blood to anticoagulant ratio = 14:1). Platelet P-selectin expression did not significantly change after automated red cell exchange. Clinical factors such as the volume of replacement fluid and the citrate infusion rate did not correlate with postapheresis platelet P-selectin expression. In addition, the association of platelet P-selectin expression and automated red cell exchange was independent of other laboratory factors (hematocrit level, hemoglobin S level, platelet count, and nitric oxide level). Finally, the difference between the study groups regarding platelet P-selectin expression before and after apheresis was insignificant. In conclusion, automated red cell exchange procedures do not induce platelet P-selectin expression in patients with sickle cell disease in the steady state or in vaso-occlusive crisis.