Platelet-specific Glycoprotein Research Articles

High Frequency of Genetic Variants Influencing Diagnosis and Bleeding Pattern in Patients with Presumed Primary Immune Mediated Thrombocytopenia (ITP)

Introduction Immune mediated thrombocytopenia (ITP) is a platelet disorder in which the immune system attacks and destroys the body's own platelets. Mechanisms leading to low platelet count in ITP are multifactorial, involving both increased peripheral platelet destruction and decreased platelet production. This is caused by autoantibodies targeting megakaryocytes and platelet-specific glycoproteins, as well as cytotoxic T cells directly acting on platelets. Although antibodies in some cases can be found in patients with ITP, there is no well-established diagnostic test and the diagnosis is thus, one of exclusion. While most cases of ITP resolve within 3 months with or without treatment, some patients do not respond to conventional treatment and develop a refractory ITP. It is likely that in the group of patients diagnosed with refractory ITP, some does not have an immune mediated thrombocytopenia, but other causes of low counts (PLC)s. With the development of high throughput sequencing and the increasing knowledge of inherited bleeding disorders, the possibility to identify the underlying cause of disease in these patients is steadily growing. In ITP, there is a high degree of interindividual differences in bleeding rates, where some patients have no bleeding symptoms despite of unmeasurable PLC, whereas other patients show severe bleeding symptoms at PLC > 30 x10 9 /L. The underlying etiology of these discrepancies is currently unknown and complicates the clinical care of these patients. We hypothesize that genetic defects influence the bleeding pattern in true ITP patients as well as being one of the underlying causes of thrombocytopenia in patients misdiagnosed with “refractory ITP”. The aim of this pilot study was to investigate whether upfront genetic screening of patients suspected of ITP could be a valuable diagnostic tool to determine presence of inherited thrombocytopenia, secondary thrombocytopenia and genetic variants that might affect bleeding pattern. Method Patients were included after informed consent following the declaration of Helsinki. We performed whole genome sequencing (WGS) in patients diagnosed with presumed ITP at the Skåne University Hospital, Sweden. Whole genome sequencing was performed at the Center for Genomic Medicine at Rigshospitalet, Copenhagen. An in-silico gene panel consisting of 113 genes known to be associated with bleeding disorders as well as 86 genes associated with ITP (Using the VarSeq gene prioritisation algorithm phorank) was analysed and pathogenicity was classified according to the ACMG/AMP guidelines. Patients' medical records were examined, laboratory work up was performed and clinical data regarding demographics and information regarding bleeding was collected. A final evaluation of susceptible variants took place at multiple disciplinary team conferences. Results Twenty-one patients were enrolled (9 women, 12 men, 17 were adults and 4 children). Mean age of adult patients was 50,5 years and mean age of paediatric patients was 11,5 years. Out of 21 patients, 7 were shown to have genetic variants with relevance to their disease. All variants were found in adult patients. Two patients had variants related to inherited thrombocytopenia (X-linked thrombocytopenia(WAS) and monoallelic Bernard-Soulier Syndrome(GP1BB)), 4 had variants in genes related to secondary ITP (3 with variants associated with common variable immunodeficiency (NFKB1, TNFRSF13B) and 1 with systemic lupus erythematosus (TREX1)) and 1 had a variant associated with platelet dysfunction (platelet delta storage pool deficiency (UNC13D)). Only the variant associated with X-linked thrombocytopenia was previously described (class 5) whereas the remaining 6 variants were initially classified as variants of unknown significance (VUS). Functional work-up and co-segregation studies indicated them as probably pathogenic (class 4) but confirming studies are still pending. Conclusion In this pilot study, 33% of patients with presumed primary ITP had variants with relevance to their clinical phenotype, and we were able to diagnose 1 patient with an inherited bleeding disorder (X-linked thrombocytopenia). Whole genome sequencing seems to be a useful diagnostic tool in the work up of patients with thrombocytopenia where ITP is suspected to ensure the diagnosis and could also add important information when assessing bleeding risk in individual patients.

Galectin functions in cancer-associated inflammation and thrombosis.

Galectins are carbohydrate-binding proteins that regulate many cellular functions including proliferation, adhesion, migration, and phagocytosis. Increasing experimental and clinical evidence indicates that galectins influence many steps of cancer development by inducing the recruitment of immune cells to the inflammatory sites and modulating the effector function of neutrophils, monocytes, and lymphocytes. Recent studies described that different isoforms of galectins can induce platelet adhesion, aggregation, and granule release through the interaction with platelet-specific glycoproteins and integrins. Patients with cancer and/or deep-venous thrombosis have increased levels of galectins in the vasculature, suggesting that these proteins could be important contributors to cancer-associated inflammation and thrombosis. In this review, we summarize the pathological role of galectins in inflammatory and thrombotic events, influencing tumor progression and metastasis. We also discuss the potential of anti-cancer therapies targeting galectins in the pathological context of cancer-associated inflammation and thrombosis.

Treatment of drug-induced immune thrombocytopenias.

Several therapeutic agents can cause thrombocytopenia by either immune-mediated or non-immune-mediated mechanisms. Non-immune-mediated thrombocytopenia is due to direct toxicity of drug molecules to platelets or megakaryocytes. Immune-mediated thrombocytopenia, on the other hand, involves the formation of antibodies that react to platelet-specific glycoprotein complexes, as in classic drug-induced immune thrombocytopenia (DITP), or to platelet factor 4, as in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT). Clinical signs include a rapid drop in platelet count, bleeding or thrombosis. Since the patient's condition can deteriorate rapidly, prompt diagnosis and management are critical. However, the necessary diagnostic tests are only available in specialized laboratories. Therefore, the most demanding step in treatment is to identify the agent responsible for thrombocytopenia, which often proves difficult because many patients are taking multiple medications and have comorbidities that can themselves also cause thrombocytopenia. While DITP is commonly associated with an increased risk of bleeding, HIT and VITT have a high mortality rate due to the high incidence of thromboembolic complications. A structured approach to drug-associated thrombocytopenia/thrombosis can lead to successful treatment and a lower mortality rate. In addition to describing the treatment of DITP, HIT, VITT, and vaccine-associated immune thrombocytopenia, this review also provides the pathophysiological and clinical information necessary for correct patient management.

Thrombocyte Inhibition Restores Protective Immunity to Mycobacterial Infection in Zebrafish.

Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

Soluble glycoprotein VI, a specific marker of platelet activation is increased in the plasma of subjects with seropositive rheumatoid arthritis.

ObjectivesAnti-citrullinated protein antibodies (ACPA) have been shown to cause platelet activation in vitro, through the low-affinity immunoglobulin G (IgG) receptor (FcγRIIa) on platelets. Platelet activation via engagement of FcγRIIa results in proteolytic cleavage and shedding of platelet specific glycoprotein VI (GPVI) which can be detected in the plasma as soluble GPVI (sGPVI). We hypothesized that plasma levels of sGPVI would be increased among patients with seropositive RA as a consequence of antibody-induced platelet activation and GPVI shedding.MethodsSamples from 84 patients with RA (65 seropositive and 19 seronegative) and 67 healthy controls were collected prospectively and analysed for sGPVI using a standardised ELISA.ResultsPatients with seropositive RA had significantly higher levels of sGPVI compared to seronegative RA and controls. Median (IQR) sGPVI levels were 4.2 ng/ml (3.2, 8.0) in seropositve RA, 2.2 ng/ml (1.5, 3.5) in seronegative RA and 2.2 ng/ml (1.6, 3.4) in controls (p<0.0001). sGPVI levels correlated with ACPA titres (r = 0.32, p = 0.0026) and with RF titres (r = 0.48, p<0.0001).ConclusionPlasma sGPVI, a specific marker of platelet activation is increased among patients with seropositive RA.

Regulation of platelet activation and thrombus formation by reactive oxygen species

Reactive oxygen species (ROS) are generated within activated platelets and play an important role in regulating platelet responses to collagen and collagen-mediated thrombus formation. As a major collagen receptor, platelet-specific glycoprotein (GP)VI is a member of the immunoglobulin (Ig) superfamily, with two extracellular Ig domains, a mucin domain, a transmembrane domain and a cytoplasmic tail. GPVI forms a functional complex with the Fc receptor γ-chain (FcRγ) that, following receptor dimerization, signals via an intracellular immunoreceptor tyrosine-based activation motif (ITAM), leading to rapid activation of Src family kinase signaling pathways. Our previous studies demonstrated that an unpaired thiol in the cytoplasmic tail of GPVI undergoes rapid oxidation to form GPVI homodimers in response to ligand binding, indicating an oxidative submembranous environment in platelets after GPVI stimulation. Using a redox-sensitive fluorescent dye (H2DCF-DA) in a flow cytometric assay to measure changes in intracellular ROS, we showed generation of ROS downstream of GPVI consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation. In this review, we will discuss recent findings on the regulation of platelet function by ROS, focusing on GPVI-dependent platelet activation and thrombus formation.

Antibodies against complement-regulatory proteins on platelets in immune thrombocytopenia

In immune thrombocytopenia (ITP), antibodies reacting with platelet membrane glycoproteins (GP) mediate premature platelet cleavage, resulting in thrombocytopenia and therefore a risk of bleeding. These antibodies may induce complement activation, thus mediating complement-induced platelet destruction. In this study, we investigated the possibility of an additional complement-related pathogenic mechanism, where antibodies against the complement-regulatory factors CD55 and CD59 may directly interfere with normal complement function. CD55 downregulates both the classic and the alternative activation pathways, while CD59 blocks the formation of the membrane attack complex; both proteins are present on platelets and may therefore be targets of autoantibodies.Using the simultaneous analysis of specific platelet antibodies (SASPA) assay, we found that in some cases of immune-mediated thrombocytopenia, anti-CD55 and -CD59 antibodies are detectable in patients’ sera and/or on their autologous platelets in combination with antibodies against platelet-specific GP. Although antibodies against CD55 and CD59 seem to be a rare phenomenon, this finding may have clinical relevance due to the availability of highly effective therapeutics targeting the complement system.

P123 HPA typing with LinkS[formula omitted]q™, a real-time PCR detection system

The interaction of membrane-bound platelet-specific glycoproteins (GP) with the extracellular matrix plays a significant role in hemostasis. Human Platelet Antigens (HPA) are glycoproteins expressed on platelet membranes. Polymorphisms in Human Platelet Antigens (HPA) can stimulate production of antibodies in recipients of transfused platelets from donors with different HPAs. Platelet incompatibility is associated with various forms of thrombopenias, post-transfusion purpura and other blood disorders. HPA typing is typically performed with serological techniques that are currently being replaced by molecular methods. Linkage Biosciences has developed and validated a test consisting of 24 reactions that identifies both variants of the 12 relevant SNPs located within HPA genes. Of these 24 variants, 18 alleles have been shown to produce alloantigens. The LinkS e ¯ q system overcomes the major challenges of HPA molecular typing by providing a robust and automated approach resulting in increased laboratory productivity and decreased turn-around time. The analysis is mediated by SureTyper™ software which generates rapid typing results. With less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, LinkS e ¯ q uses state of the art real-time PCR detection to provide complete molecular genotyping results in approximately 90 minutes. Further, since amplified products are never handled, the risk of laboratory contamination is reduced. The LinkS e ¯ q system can provide a simple, effective and robust method for determining the HPA typing profile of DNA samples.

Leukoreduced red blood cell transfusions do not induce platelet glycoprotein antibodies in patients with sickle cell disease.

Alloimmunization to red blood cell (RBC) antigens after transfusion is well described in patients with sickle cell disease (SCD). We recently demonstrated that leukocyte-reduced RBC transfusions appeared to induce human leukocyte antigen (HLA) antibodies in some children with SCD; now, we hypothesize that residual platelets contained in transfused RBC products may lead to platelet glycoprotein antibody formation. A cross-sectional study was conducted among never pregnant pediatric patients with SCD who either had received many RBC transfusions or had never received any transfusions. Serum was tested for antibodies to platelet-specific glycoproteins using a commercial enzyme immunoassay. Platelet-specific glycoprotein antibodies were found in 12 of 90 patients (13%) in the transfused group versus 5 of 24 patients (21%) in the never transfused group (p = 0.35). The prevalence of antibodies as well as the median standardized optical density for these two groups was not significantly different for any of the studied platelet glycoprotein antigens. There was no association with the presence of platelet-specific glycoprotein antibodies with either RBC or HLA antibodies. Leukocyte-reduced RBC transfusions do not appear to induce platelet-specific glycoprotein antibodies. The positive platelet-specific glycoprotein antibody results from this study may represent platelet autoantibodies, platelet alloantibodies, or false-positive reactions. A better understanding of the immunobiology of patients with SCD at baseline and after blood product exposure may help improve future transfusion and transplantation.

Advances in regulation of platelet receptors glycoprotein Ib and IV

Platelet has been known to play a critical role in thrombosis and hemostasis. Increasing evidence from studies on bleeding or coagulation disorders demonstrates that platelet specific membrane receptor glycoprotein (GP)Ⅵ and GPⅠb subunit of GPⅠb-Ⅸ-Ⅴ complex are the main receptors regulating platelets' function (thrombosis and hemostasis). And abnormal expression or function of GPⅠb and GPⅥ are associated with many bleeding or coagulation disorders, suggesting the importance of these two receptors. Understanding the mechanisms of the regulation of GPⅠb and GPⅥ will be beneficial in the evaluation or prediction of the risk of thrombosis or bleeding in patients, which might provide guidance on the clinical treatment. This article reviews literatures on the structure, function and regulation of platelet receptors GPⅠb and GPⅥ, as well as their clinical significance in human diseases. Key words: Platelet membrane glycoprotein; Platelet activation; Hydrolysis

Desialylation Induces Apoptosis and Phagocytosis of Platelets in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction:Prolonged isolated thrombocytopenia (PIT) represents a significant complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is associated with an adverse patient prognosis and higher transplant-related mortality owing to a higher risk of infection events, severe (grades 3 to 4) acute graft-versus-host disease (GVHD) and chronic GVHD. PIT is defined as a peripheral platelet count less than 100×109/L without sustained anemia or leukopenia for more than 3 months after allo-HSCT (Zhang X, et al. Biol Blood Marrow Transplant, 2011). However, the underlying mechanisms remain unclear. Different kinds of functional glycoproteins are expressed on the platelet surface, with sialic acid residues at the end of their glycan. Desialylation of platelet glycoproteins has been found to be associated with accelerated platelet clearance in refrigerated platelets (Gerard Jansen AJ, et al. Blood 2012). Platelet-specific glycoprotein GPIbα,the functional subunit of the von Willebrand factor receptor, was the majorly desialylated glycoprotein; NEU1, one of the four human sialidases, was the enzyme that catalyzed GPIbα desialylation. However, few studies have focused on this mechanism in patients suffering PIT after allo-HSCT. In this study, we hypothesized that desialylation on platelet surfaces is associated with PIT after allo-HSCT. The mechanisms participating in this process may include GPIbα clustering, platelet apoptosis and phagocytosis by macrophages.Patients and methods:Blood samples were collected 90 days after allo-HSCT from 70 patients with PIT. Samples from post-transplantation patients who have normal platelet counts were taken as controls. Sialylation and desialylation were measured by detecting specific lectins via flow cytometry. Human sialidase expression was determined by immunofluorescence, flow cytometry and reverse transcription PCR. Platelet apoptosis markers were measured by flow cytometry, and macrophages stimulated from THP-1 cells were used in the phagocytosis assay.Results:We tested sialic acid residues and the desialylation markers, including β-galactose and β-N-Acetyl glucosamine, on the platelet surface, and found that platelets from PIT patients had significantly higher desialylation levels. Serum sialic acid levels were measured, and the results showed higher levels in PIT patients. Further, NEU1 was found to be over-expressed on the surface of platelets from PIT patients, and was found to be the enzyme that catalyzed the platelet surface desialylation. To further reveal the mechanism that lead to PIT, we proved that GPIbα was the desialylated glycoprotein on platelets from PIT patients. We found that GPIbα desialylation and clustering in PIT patients induced changes in the expression of Bcl-2 family protein, as a 2-fold increase in active Bax expression and a similar decrease in Bcl-XL expression were observed. Depolarization of the inner mitochondria membrane was augmented in desialylated platelets from PIT patients, indicating increased platelet apoptosis. Moreover, macrophages stimulated from the THP-1 cell line preferred to phagocytose desialylated platelets from PIT patients in vitro; this process could be blocked by the sialidase inhibitor, DANA. In the in vitrostudy, we found that dexamethasone led to a 32% decrease in phagocytosis, whereas oseltamivir, an antiviral medicine that can block sialidase from influenza virus, could also partially function on human sialidase and protect 43% of platelets from phagocytosis.In conclusion, our results demonstrate that desialylation played a role in the mechanism of prolonged isolated thrombocytopenia after allo-HSCT, most likely through platelet apoptosis induction and increased phagocytosis by macrophages in the peripheral circulation. Dexamethasone and oseltamivir could decrease platelet apoptosis and inhibit platelet phagocytosis in vitro, implying a novel potential method for treating PIT after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Biological role of prolyl 3-hydroxylation in type IV collagen

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

Circulating levels of soluble EMMPRIN (CD147) correlate with levels of soluble glycoprotein VI in human plasma

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147), which binds to the platelet-specific collagen receptor glycoprotein (GP) VI, is expressed in a range of cell types including platelets and leukocytes, and has been implicated in neoplastic disease and atherosclerotic coronary disease. Both CD147 and GPVI can be shed from cell membranes and detected in plasma. However, while the relationship between soluble CD147 (sCD147), soluble GPVI (sGPVI) and standard markers of platelet activation has received little attention, such analysis may help reveal pathways mediating release of sCD147. We investigated the relationship between sCD147 and platelet markers including sGPVI, soluble and platelet-bound CD62P (P-selectin), active αIIbβ3 (assessed by PAC-1 binding) and platelet CD147 in 25 patients with stable angina pectoris (SAP), 13 patients with no coronary artery disease (CAD) and 10 healthy donors. Plasma levels of sCD147 significantly correlated with sGPVI (r = 0.46, p = .004), but did not correlate with any other platelet markers examined. Linear regression analysis identified that sCD147 levels could be predicted by sGPVI levels (β = .445, p = 0.003) and age (β = 0.304, p = 0.038), but were independent of potential clinical confounders such as CAD, diabetes and medication usage. As sCD147 strongly correlates with platelet-specific sGPVI, a common platelet source and/or mechanism of release may contribute to sCD147 levels in vivo.

Abnormal platelet kinetics are detected before the occurrence of thrombocytopaenia in HBV‐related liver disease

Thrombocytopaenia is a frequent feature in patients with HBV-related liver disease. Its underlying mechanism is not fully understood. Multiple factors might contribute to the development of thrombocytopaenia. In this study, we investigated the reticulated platelets (RP), glycocalicin (GC), serum thrombopoietin (TPO) and platelet glycoprotein (GP) in different stages of the disease. One hundred and fourteen patients with HBV-related liver disease (30 with chronic hepatitis B (CHB), 20 patients in Child A without thrombocytopaenia, 19 patients in Child A with thrombocytopaenia, 45 in Child B/C with thrombocytopaenia) and 25 normal controls (NC) were enrolled. Liver cirrhosis (LC) was classified according to modified Child-Turcotte-Pugh (CTP) score. Serum TPO levels and GC were measured by ELISA. RP and platelet glycoprotein (GP) expression were detected by flow cytometry. The TPO levels of patients with LC were significantly lower than that of controls, even in patients of Child A without thrombocytopaenia group. Serum TPO level was positively correlated (r = 0.65, p < 0.01) with serum albumin in Child B/C group. Both the RP percentages and the glycocalicin index (GCI) levels were significantly higher in patients groups including CHB and Child A without thrombocytopaenia than that of normal controls. A negative correlation existed in HBV DNA copies and the GPs% in patients with CHB and Child A without thrombocytopaenia. Abnormal platelet production, destruction and platelet-specific glycoproteins levels were detected before the occurrence of thrombocytopaenia in HBV-related liver disease, indicating that multiple mechanisms might play roles in thrombocytopaenia in HBV-infected patients.

665. Intraosseous Delivery of Lentiviral Vectors Expressing Factor VIII under the Control of the Platelet-Specific Glycoprotein 1bα Promoter Leads to Long-Term Correction of Bleeding in Hemophilia A Mice

665. Intraosseous Delivery of Lentiviral Vectors Expressing Factor VIII under the Control of the Platelet-Specific Glycoprotein 1bα Promoter Leads to Long-Term Correction of Bleeding in Hemophilia A Mice

Alloantibodies to human platelet glycoprotein antigens (HPA) and HLA class 1 in a cross section of Nigerian antenatal women

The prevalence of antibodies to human platelet antigens (HPA) and human leukocyte antigens (HLA) class 1 antigens among Nigerian pregnant women has not been reported in our country. This study was therefore aimed at screening the obstetric population for evidence of alloimmunization due to human platelet and HLA class 1 antigens. One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP 11b/111a 12 (8.3%), GP 1a/11a 35 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9 (6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-HPA-1a,-3a,-4a (4.2%), anti-HPA-1b,-3b,-4a (4.2%), anti-HPA-30 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%). A high prevalence rate of human platelet arid cytotoxic antibodies has been observed in our obstetric population. There is need to establish platelet serology laboratory for the proper antenatal and postnatal management of pregnant mothers in this region.

Survey of HPA and HLA class 1 antibodies in Nigerian antenatal women (169.5)

Abstract Background: The prevalence of antibodies to HPA and HLA class 1 antigens among Nigerian pregnant women has not been reported in our country. Aims: To screen for platelet specific and HLA class 1 antibody among pregnant women. Methods: One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. Results: The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP IIb/IIIa 12 (8.3%), GP Ia/IIa 30 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9(6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-H PA-1a,-3a,-4a (4.2%), anti-H PA-1b,-3b,-4a (4.2%), anti-H PA- 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%).). Conclusion: A high prevalence rate of human platelet and cytotoxic antibodies has been observed in our obstetric population.

Contribution of flow cytometry to the diagnosis and differential diagnosis of thrombocytopenia1

Abstract Thrombocytopenia is commonly found in patients with apparent bleeding tendency, and in the laboratory evaluation of patients with other disorders or in clinically healthy individuals. Identification of the underlying cause is therefore crucial. There are several different laboratory tests available for this purpose. Flow cytometry employs very small sample volumes, which is of particular interest in patients with severe thrombocytopenia or in pediatric samples. Thus, the immunological determination of platelet numbers by flow cytometry is recommended as the reference method to achieve an exact quantification of very low platelet numbers, which could influence therapeutic decisions. In addition, flow cytometric analysis of immature platelets plays a role in the differentiation of increased platelet destruction and decreased platelet synthesis. As this parameter can be measured by current hematology analyzers, it will become more important in routine use. Another field for flow cytometry is the investigation of immunologically mediated thrombocytopenias. These can be caused by antibodies against platelet-specific glycoproteins such as autoimmune thrombocytopenia or by antibodies against heparin-platelet factor 4 complexes such as heparin-induced thrombocytopenia. Moreover, flow cytometric assays are used for the diagnosis of rare congenital thrombocytopenias, where they are either able to confirm a certain diagnosis, e.g., Bernard-Soulier syndrome, or at least establish a basis for further analyses. The contribution of flow cytometry to the investigation of lymphoma and leukemia, which regularly cause thrombocytopenia by bone marrow infiltration, is not discussed in this article.

Strategies to Enhance the Efficacy of Platelet-Derived Factor (F) VIII: Studies with Inactivation Resistant FVIII (IR8) and Canine FVIII in Hemophilia A Mice.

Abstract 3496Poster Board III-433Hemophilia A is the most common, inherited severe bleeding diathesis and is due to deficient FVIII. We and others have shown that targeted delivery of ectopic human B-domainless (hBD) FVIII from within platelet α-granules is effective at improving clotting in several injury models using FVIIInull mice. Importantly, platelet-derived (p) hBDFVIII is ∼100-fold more effective than comparable levels of plasma hFVIII against circulating inhibitors, a significant problem in the hemophilia A population. However, we have also shown in both a cuticular injury model and an in situ cremaster laser arteriole/venule injury model that there are certain limitations to pFVIII therapy, especially concerns of clot stability and risks of clot embolization. In the present study, we examined the efficacy of platelet-delivered inactivation resistant FVIII (IR8), resistant to inactivation by thrombin and activated protein C, and canine (c) BDFVIII, a species of FVIII that has 3-5-fold greater specific activity than hBDFVIII. Both FVIIIs were expressed in platelets using both a transgenic mouse approach and lentiviral gene therapy, and compared to similarly expressed phBDFVIII mice. Multiple founder lines of mice were generated for pIR8 and pcBDFVIII using the platelet-specific glycoprotein Ibα proximal promoter to drive platelet-specific expression. Lentiviral studies involved transducing FVIIInull murine hematopoietic cells with an HIV-1 based self-inactivating lentivirus encoding the previously mentioned FVIIIs driven by either a ubiquitin promoter or by the platelet-specific platelet factor 4 (PF4) proximal promoter; lethally irradiated FVIIInull mice were rescued by injection of virally transduced bone marrow. FVIII antigenic levels in both platelet lysates and releasates were measured, and free, plasma FVIII levels were detected by a FVIII ELISA. Maximum pIR8 levels achieved were comparable to those seen with hBDFVIII in spite of the fact that IR8 binds poorly to von Willebrand factor (vWF), supporting prior observations that pFVIII is stored in α-granules independent of the presence of vWF in α-granules. Surprisingly, maximal pcBDFVIII levels were 1/3rd of those of phBDFVIII. The basis for this observation is presently being determined. The ability of the pFVIII to correct hemophilia A in vivo was tested by a FeCl3 carotid artery injury model and a cuticular bleeding model. In addition, laser injury studies to cremaster arterioles and venules are on-going. FeCl3 carotid artery and cuticular studies demonstrated that both pIR8 and pcBDFVIII were more potent than phBDFVIII in improving outcome in FVIIInull mice. For pcBDFVIII, this occurred despite the low levels of pcBDFVIII. In the cremaster laser injury model, lentiviral-based gene therapy using the three FVIII variants, markedly improved clot formation compared to FVIIInull mice, but further studies are needed to define whether a specific FVIII variant is particularly efficacious. Prior studies of plasma IR8 correction using an adenoviral delivery system showed no improved outcome relative to hBDFVIII. We propose that this difference for IR8 between plasma and platelet expression is due to IR8 binding vWF poorly and that vWF binding is important for maintaining plasma FVIII levels. The higher specific activity of pcBDFVIII appears to more than compensate for its lower level in circulating platelets. We now intend to define the molecular basis for the greater efficacy of pIR8 and pcBDFVIII compared to phBDFVIII and to use these insights to further optimize the efficacy and safety of this delivery strategy in the care of patients with hemophilia A. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Drug Inhibition of GPIb.

Abstract SCI-38The platelet-specific glycoprotein (GP)Ib, member of the GPIb/IX/V complex, is the main receptor for von Willebrand Factor (VWF), but also for a growing list of other ligands such as thrombin, coagulation factor XI and XII, Mac-1 and P-selectin. For VWF and GPIb to interact, VWF needs to undergo a conformational change. In the circulation, this is induced by shear forces that stretch VWF, especially when bound onto collagen at vascular lesions. As the contribution of VWF to platelet adhesion becomes more important with increasing shear forces, the idea emerged that inhibition of the GPIb-VWF interaction could actually result in an antithrombotic effect targeted to high shear arterioles or stenosed arteries, leaving hemostasis virtually unaffected in vessel beds with slower circulation. And in the mean time, a growing amount of evidence suggests that this could lead to a lower bleeding risk than what is seen with currently available antithrombotic agents. Several compounds have been tested in the past such as fragments of VWF, snake venoms and antibodies. We developed an anti-human GPIb monoclonal antibody, 6B4, that competes directly for the binding site of VWF. The humanized 6B4 Fab-fragment effectively prevented repetitive occlusions of the damaged and stenosed femoral artery in a baboon thrombosis model. Interestingly, only little prolongation of the template bleeding time and minimal increase in blood loss from a standardized incision was observed. This apparent larger therapeutic window of GPIb inhibitors could be particularly useful in ischemic stroke, where the utility of current platelet inhibitors and anticoagulants is significantly counterbalanced by the risk of intracerebral bleeding complications. Recent promising results in a mouse model of cerebral ischemia/reperfusion indeed show a significantly less severe stroke outcome using a GPIb-inhibitor, either administered before or, importantly, after cerebral artery occlusion. No increase in intracerebral hemorrhages was detected, in contrast to what is seen with e.g. GPIIb/IIIa inhibition. Finally, as antibodies may be useful for acute treatments but not for chronic applications, the identification of a GPIb-inhibiting nonamer peptide raises hopes towards development of orally available small molecule inhibitors. DisclosuresNo relevant conflicts of interest to declare.