Prospective study of early bereavement on psychological and behavioural cardiac risk factors.

BACKGROUND: Increasing evidence supports the role of emotional stress in the onset of cardiovascular disease. Although bereavement is a major emotional stress with both acute and more long-term features, the mechanism of its association with cardiovascular risk is not well understood, in particular because of limited studies of acute bereavement. The aim of the study was to identify psychological and behavioural changes in acute bereavement and potential modifiers of these changes. METHODS: Bereaved (n= 62) and non-bereaved individuals (n= 50) were evaluated within 2 weeks and at 6 months following loss using the Centre for Epidemiologic Studies -- Depression, Spielberger State Anxiety and Anger, Social Support Questionnaire and changes in appetite, cigarette and alcohol consumption, cortisol and lipids. RESULTS: Compared with non-bereaved, acutely bereaved had increased symptoms of depression (26.7 +/- 1.7 vs 5.9 +/- 0.7, P < 0.001), anxiety (47.4 +/- 2.0 vs 28.2 +/- 1.4, P < 0.001) and anger (median 16.0 vs 15.0, P < 0.001). Greater depressive symptoms were associated with being unprepared for the death, decreased sleep duration and younger age. Acutely, bereaved slept less than non-bereaved (5.8 +/- 0.2 vs 7.2 +/- 0.2 h, P < 0.001). Reduced sleep time was associated with increased anger and depression and decreased satisfaction with social support. Compared with the non-bereaved, the acutely bereaved had higher cortisol (median 306 vs 266, P= 0.003), reduced appetite (P < 0.001) and lower total cholesterol (median 4.9 vs 5.4, P= 0.006) and low-density lipoprotein (median 2.4 vs 2.9, P < 0.001). CONCLUSION: These results offer insight into the psychological, behavioural and physical changes that may contribute to cardiovascular risk in bereavement.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SUMMARY: Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Mean platelet diameter measurements to classify inherited thrombocytopenias.

INTRODUCTION: Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. METHODS: To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. RESULTS: Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 µm which correlated with "IT with giant platelets." Only 55% of samples expected in the "large platelet group" had MPD meeting the classification cut-off (MPD >3.2 µm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were "normal." CONCLUSION: Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.

Reduced fibrinolysis and increased fibrin generation can be detected in hypercoagulable patients using the overall hemostatic potential assay.

BACKGROUND: Routinely available coagulation assays are not capable of detecting clinically defined hypercoagulable states. A number of global coagulation assays have been developed with the potential to evaluate hypercoagulability, which predisposes to the common clinical events of arterial and venous thromboembolism (VTE). OBJECTIVES: We hypothesized that the overall hemostatic potential (OHP) assay would show abnormal fibrin generation and lysis in patients with clinically defined hypercoagulable states. METHODS: We used the OHP assay as described by Blombäck and colleagues [1,2] in 161 clinically hypercoagulable patients with arterial or VTE, pregnancy complications or autoimmune disease. Eighty patients had associated antiphospholipid antibodies (APLA). Ninety-eight normal plasma donors were tested for comparison. RESULTS: We derived three new assay parameters for correlation with hypercoagulable states: the maximum optical density, maximum slope, and delay in onset of fibrin generation. We found significantly different assay results for all patients' parameters examined when compared with controls, indicating both increased fibrin generation and reduced fibrinolysis in hypercoagulable patients. The findings were similar whether samples were collected in association with an acute thrombotic event or not. Estimated assay sensitivity for detection of a clinically defined hypercoagulable state was 96%. CONCLUSIONS: The OHP assay is a simple, inexpensive global test that is useful for assessing patients with hypercoagulable states including APLA. OHP results are significantly abnormal in hypercoagulable groups compared with controls, indicating that both increased fibrin generation and reduced fibrinolysis contribute to hypercoagulable states. The assay may ultimately assist in tailoring clinical management to patients' individual requirements.

Use of a functional assay to diagnose protein S deficiency; inappropriate testing yields equivocal results.

Inherited deficiency of protein S (PS) is a rare but accepted risk factor for venous thromboembolism. There is accumulating evidence that inherited PS deficiency may be associated with a variety of adverse obstetric events. Acquired PS deficiency may be caused by a variety of clinical states including normal pregnancy. We conducted a retrospective audit of the results of screening for PS deficiency through our reference laboratory. The majority of patients in this audit with significantly reduced (<50%) free functional PS levels had a major confounding factor likely to cause acquired PS deficiency, most frequently pregnancy. Recommendations for PS testing for the diagnosis of hereditary PS deficiency include deferring testing until at least 40 days post-partum. It appears that these recommendations are not being adhered to leading to difficulty in the interpretation of results.

Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Full-mouth tooth extraction lowers systemic inflammatory and thrombotic markers of cardiovascular risk.

Prior studies of a link between periodontal and cardiovascular disease have been limited by being predominantly observational. We used a treatment intervention model to study the relationship between periodontitis and systemic inflammatory and thrombotic cardiovascular indicators of risk. We studied 67 adults with advanced periodontitis requiring full-mouth tooth extraction. Blood samples were obtained: (1) at initial presentation, immediately prior to treatment of presenting symptoms; (2) one to two weeks later, before all teeth were removed; and (3) 12 weeks after full-mouth tooth extraction. After full-mouth tooth extraction, there was a significant decrease in C-reactive protein, plasminogen activator inhibitor-1 and fibrinogen, and white cell and platelet counts. This study shows that elimination of advanced periodontitis by full-mouth tooth extraction reduces systemic inflammatory and thrombotic markers of cardiovascular risk. Analysis of the data supports the hypothesis that treatment of periodontal disease may lower cardiovascular risk, and provides a rationale for further randomized studies.

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

A structural/functional domain on human CD36 is involved in the binding of anti-Nak(a) antibodies.

The human CD36 antigen is an integral membrane glycoprotein expressed by platelets, monocytes, endothelial cells and various tumor cell lines. CD36 acts as a receptor for thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein. Individuals possessing the Nak(a)-negative phenotype do not express CD36 and risk developing anti-CD36 isoantibodies upon blood transfusion or during pregnancy. In the present study, we have examined the interaction of an anti-Nak(a) serum with recombinantly expressed CD36. Results obtained show that five functional CD36 monoclonal antibodies (OKM5, FA6-152, L103, ESIV-C7 and 10/5) prevent the binding of the anti-Nak(a) serum whereas a single monoclonal antibody (13/10) has no effect. Consistent with this result, an epitope map of CD36 generated using cross-blocking experiments, indicates that the inhibitory monoclonal antibodies recognize closely-related epitopes whereas 13/10 reacts with a distinct CD36 determinant. Furthermore, we have demonstrated, in a recent study, that OKM5, FA6-152, L103 and 10/5 bind to the same CD36 domain defined by amino acids 155 to 183. Taken together, our results indicate that the 155-183 sequence is important for the binding of the anti-Nak(a) serum to CD36 and may represent a surface-exposed, immunogenic and presumably functional region on human CD36.

A naturally occurring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function.

We have investigated the effect of a new Leu196Pro mutation, identified in the MIDAS-like domain of the beta3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on beta3 integrin receptor function. Expression of the mutant beta3Pro196 subunit in CHO cells, either associated with recombinant human alphaIIb or alphav, resulted in normal biosynthesis of beta3 and heterodimerization with alphav or alphaIIb, but selectively interfered with alphaIIbbeta3 maturation and transport to the cell surface. Functional analysis of the beta3 mutant receptors revealed strong inhibition of alphavbeta3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125FAK phosphorylation and fibrin clot retraction, as opposed to normal alphaIIbbeta3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant aIIbbeta3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL196T motif in the beta3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in beta3 is not involved in alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen, while it is essential for alphavbeta3-mediated interaction with this ligand.

Genetic susceptibility and anti-human platelet antigen 5b alloimmunization role of HLA class II and TAP genes.

Platelet alloimmunization may result in post-transfusion purpura, and during pregnancy may cause neonatal alloimmune thrombocytopenia (NAIT), with a frequency estimated at 1.3 per 1000 live births. The risk of morbidity is significant: 20% of affected infants have neurologic sequelae and the death rate is about 10%. A better understanding of the immune response to platelet alloantigens would allow for a better definition, and thus better management of pregnant women at high risk. Limited data are available on the immune response against HPA-5b, the second most frequent antigen, after HPA-1a, implicated in NAIT. We studied HLA class II and TAP gene polymorphism in 50 women immunized against HPA-5 system antigens. Our results suggest a strong association of alloimmunization with a cluster of HLA DR molecules sharing a particular polymorphic amino acid sequence at position 69-70 (Glu-Asp encoded by GAA-GAC nucleotide sequence) of the DR beta 1 chain (RR = 2.95, RR = 5.70 when patients were homozygous for this sequence), and a negative association with the DRB1*0301 allele (2.1% vs. 28%; RR = 0.08). Furthermore, increased frequency of a TAP2 dimorphism at position 379 was observed in immunized women against the HPA-5 antigens (RR = 4.7).

Drawbacks of the MAIPA technique in characterising human antiplatelet antibodies.

Monoclonal Antibody-specific Immobilisation of Platelet Antigens (MAIPA) assays have been developed to allow the identification and characterisation of antibodies directed against platelets. A major disadvantage of the MAIPA test is the existence of false negative results. This is due to the competition between human and monoclonal antibodies (MoAb) for epitopes that are either identical or very close. In this report we used the MAIPA technique to test anti-HPA-1a alloantibodies and anti-Naka isoantibodies in conjunction with a panel of murine MoAbs directed against CD41/61 or CD36 respectively. Our data demonstrate that the choice of MoAbs used in MAIPA is very important for accurate diagnosis of clinical conditions and institution of specific therapy. Moreover, the results obtained are in favour of an heterogeneity for the recognition of the alloepitope HPA-1a and of distinguishable epitopes on GPIV.

[Fetal and neonatal immune thrombocytopenias. Study group "Mother-child immune thrombopenias"]. / Thrombopénies foetales et néonatales immunes. Le groupe d'étude "Thrombopénies immunes mère-enfant".

Neonatal thrombocytopenia has benefited from the advances achieved during the last few years in platelet immunology and foetal therapy. The major risk of the disease is cerebral haemorrhage resulting in death or neurological sequelae. Establishing the aetiological diagnosis of immune thrombocytopenia makes it possible nowadays to apply the appropriate treatment and eventually to take care of future pregnancies. Treatments in utero of foeto-maternal alloimmunization have radically altered the natural course of foetal thrombocytopenia, thereby permitting the management of pregnancies at risk. On the other hand, so far no prenatal treatment has proved to be effective against thrombocytopenia due to maternal autoimmunity.

GFI1B mutation causes a bleeding disorder with abnormal platelet function.

BACKGROUND: GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development but previously unknown to be associated with human disease. METHODS: A family with a novel bleeding disorder was identified and characterized. Genetic linkage analysis and massively parallel sequencing were used to localize the mutation causing the disease phenotype on chromosome 9. Functional studies were then performed in megakaryocytic cell lines to determine the biological effects of the mutant transcript. RESULTS: We have identified a family with an autosomal dominant bleeding disorder associated with macrothrombocytopenia, red cell anisopoikilocytosis, and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members exhibit only abnormal bleeding with surgery. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein, resulting in a reduction in platelet α-granule content and aberrant expression of key platelet proteins. CONCLUSIONS: GFI1B mutation represents a novel human bleeding disorder, and the described phenotype identifies GFI1B as a critical regulator of platelet shape, number, and function.

Heparin-induced thrombocytopenia: evaluation of IgG and IgGAM ELISA assays.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin therapy resulting from antibody production to platelet factor 4 and heparin complexes (H-PF4). METHODS: We have evaluated four enzyme-linked immunosorbent assay (ELISA)-based screening tests to identify the best assay(s) with the highest specificity but without underdiagnosis of HIT. As functional assays are difficult to perform, ELISAs are useful to provide clinicians with a timely answer. Over a 10-month period, all samples (N=107) referred to our laboratory were tested for HIT antibodies using four commercially available ELISA kits, two detecting IgG/A/M anti-H-PF4 antibodies and the other two IgG specific. RESULTS: Twenty-eight samples were positive by at least one assay; IgGAM ELISAs were found to be more sensitive with 24 samples positive by Asserachrom IgGAM and 23 by Zymutest IgGAM. Only 18 samples were positive by GTI-PF4-IgG and Zymutest IgG. The gold standard serotonin release assay (SRA) was used as a confirmation assay, and 11/28 samples tested positive. All these SRA-positive samples were positive by all four assays. None of the IgGAM-only-positive samples was found to be positive by SRA suggesting a better specificity for the IgG-only assays. CONCLUSION: Our data strongly support the use of IgG-only assays for the detection of HIT antibodies.

The association of depression with platelet activation: evidence for a treatment effect.

BACKGROUND: Depression is associated with an increased risk of cardiovascular disease (CVD). Although the mechanism is uncertain, prothrombotic and inflammatory factors may play a role. OBJECTIVES: As platelets play a key role in CVD, we determined first, whether depressed individuals had more activated platelets than non-depressed individuals and second, whether treatment of depression reduced platelet activation levels. PATIENTS/METHODS: We recruited 108 depressed outpatients and 45 control subjects all without a history of CVD. After psychological assessment, the depressed patients were offered treatment with medication and/or psychotherapy. Flow cytometric markers of platelet activation and level of depression were assessed at baseline and at 4 weeks and 6 months after treatment. RESULTS: Depression was associated with increased platelet activation with a higher number of circulating CD62p (0.76x10(9) L(-1) vs. 0.46, P=0.019) and CD63 (P=0.05) positive platelets compared with controls. Patients with depression also had more circulating platelet-leukocyte aggregates than controls (P<0.001). There was a positive correlation between the severity of depression and the level of platelet activation. Platelets from depressed patients were also hyperreactive to adenosine 5 -diphosphate (ADP) stimulation with increased CD62p and CD63 exposure (P=0.003 and 0.019, respectively). Six months of treatment resulted in a reduced number of circulating CD62p and CD63 positive platelets (29.84% and 53.38% decrease) and a 20.9% reduction in CD63 exposure after ADP activation. CONCLUSIONS: Depression is associated with increased in vivo platelet activation and resolution of depression using psychotherapy and/or medication reduces platelet activation. These findings provide insights into the link between depression and cardiovascular risk.