Glycoprotein 1b Alpha Research Articles

Caplacizumab as an emerging treatment option for acquired thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare disease with a mortality rate of over 90% if left untreated. Therapeutic plasma exchange (PEX) is the mainstay of treatment of acquired TTP (aTTP), and with the introduction of PEX, the mortality rate declined dramatically below 20%. Although PEX together with corticosteroids are the backbone of the upfront management of patients with aTTP with successful outcomes, patients may remain refractory and/or relapse following an initial response to this treatment. There are some therapeutic options, which can be used among these patients, helping in improving outcomes of aTTP. Caplacizumab (formerly ALX-0081 or ALX-0681) is a humanized single-variable domain immunoglobulin that recognizes the human von Willebrand factor (vWF) A1 domain and inhibits the vWF–platelet glycoprotein 1b-alpha (GP1b-α) interaction. The drug was first developed for the prevention of thrombosis in high-risk patients with acute coronary syndrome undergoing percutaneous coronary intervention; however, drug development for this indication has been discontinued. Recently, caplacizumab received its first approval following Phase II TITAN and Phase III HERCULES trials in the European Union (EU) for the treatment of acute episode of aTTP in adult patients, in addition to PEX and immunosuppression. This review focuses on the use of caplacizumab as an emerging treatment option in patients with aTTP.

Determinants of Platelet-Leukocyte Aggregation and Platelet Activation in Stroke

Background: Platelet-leukocyte aggregation (PLA) and platelet activation are found to be on the higher side in ischemic stroke patients. The correlation of PLA with clinical features has not been intensively investigated and the influence of genetic factors on PLA is still unexplored. The interaction of platelets with leukocytes is mainly determined by the proteins encoded by six genes: P-Selectin (SELP encodes CD62P) on the thrombocyte binding to P-Selectin-Glycoprotein-Ligand-1 (PSGL1) on the leukocyte, intracellular-adhesion-molecule 2 (ICAM2) interacting with Integrin alpha M (ITGAM) and Glycoprotein 1b-alpha (GP1BA) binding to Integrin alpha L (ITGAL). Methods: Seventy-nine patients with acute ischemic stroke and 151 controls without vascular disease from a single German center were enrolled. A neurologist and a neuroradiologist ascertained clinical and radiological features. PLA and platelet activation were analyzed using flow cytometry with various antibodies. Coding as well as tagging SNPs in six genes determining PLA were genotyped. Three groups of parameters were correlated with each other: (i) clinical and radiological parameters, (ii) laboratory parameters, (iii) genetic parameters. For the comparisons, robust nonparametric statistical tests were applicable. Results: PLA and platelet activation were higher in ischemic stroke patients compared to controls. Both, anticoagulant and antiplatelet treatment in the patient group affected platelet activation but not PLA. PLA correlated weakly with measures of stroke severity but not with thrombus length or stroke etiology. The association of SNP rs2228315 in the P-Selectin Glycoprotein Ligand-1-gene (PSGL1) with ischemic stroke and platelet activation was significant before correction for multiple testing while a trend was observed for the association with PLA. Regression analysis revealed that (i) platelet activation was an independent determinant of stroke, (ii) that PLA correlated with stroke, sex, age and platelet activation and (iii) that platelet activation correlated only with stroke. None of the SNPs survived in the regression analysis for stroke, PLA or platelet activation as dependent variables. Conclusions: The most important result of our study is that PLA and platelet activation are independent of other vascular risk factors correlated with stroke in our sample. In addition, we identified the missense SNP rs2228315 in the PSGL1-gene as a candidate polymorphism for ischemic stroke-related PLA. Association between this SNP and stroke as well as coronary artery disease has also been shown by two other studies.

Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis

Abstract 491Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors. Disclosures:No relevant conflicts of interest to declare.

The R1306W Type 2B Natural Mutation of Von Willebrand Factor Dramatically Enhances the Multimer Sensitivity to Shear Stress

AbstractAbstract 3306 Background.Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods.In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results.The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values <10dyn/cm2 the R1306W mutant, at variance with WT vWF, already showed an initial self-aggregation with formation of a network characterized by a roughness significantly higher than that of WT vWF. Mechanical stretching experiments, performed using the AFM cantilever, showed that R1306W vWF needs 30% less energy per length unit to be stretcted compared to WT protein (1.6 vs 2.2×10−11J/m) Conclusions.These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb. Disclosures:No relevant conflicts of interest to declare.

Pregnancy-Induced Thrombocytopenia in Pseudo-Von Willebrand Disease

Abstract 4658Platelet-type von Willebrand disease, or pseudo-VWD, is a rare congenital platelet disorder due to a mutation in the glycoprotein 1b alpha receptor. This results in increased binding of normal VWF to platelets, leading to platelet aggregation and intermittent thrombocytopenia. Although the mechanism is similar to type IIb VWD, this gain of function mutation affects the platelet, rather than plasma VWF.We report a patient with pseudo-VWD who developed progressive thrombocytopenia during pregnancy. She was initially seen at 10 weeks gestation, when the platelet count was 143 × 109 /L (figure 1). As the pregnancy progressed, the platelet count decreased, accompanied by an inverse rise in VWF antigen and activity, factor VIII activity, and an increase in high molecular weight (HMW) multimers (figure 2). The VWF activity to antigen ratio remained abnormal at all times. At the time of delivery, the platelet count reached a nadir of 36 × 109/L. Post-partum, the platelet count normalized to 185 × 109/L with a concomitant fall in VWF antigen and activity levels. VWF multimer analysis after delivery showed an absence of high molecular weight multimers. [Display omitted] FIGURE 2Weeks GestationPlatelet count (×109 /L)VWF antigenVWF activityFactor VIIIPlatelet aggregationVWF multimers1014381%35%88%↑ ristocetin 0.6normal3654144%57%205%not donenormal+15 post-partum18555%19%49%not doneHMW multimers absentThe likely etiology of the progressive decrease in the platelet count is secondary to the physiologically stimulated rise in VWF levels during pregnancy. With rising VWF levels, the abnormal platelet GP1b alpha receptor binds VWF, resulting in platelet-VWF aggregates with subsequent clearance. Transfusion of normal platelets would be the only way to correct the abnormality. Our patient, a Jehovah's Witness, would not accept platelet transfusion if it became necessary. She underwent an uncomplicated cesarean section for obstetric indications.Our patient's family was the subject of the landmark article initially describing this condition (Weiss HJ, Meyer D, Rabinowitz R, et al. Pseudo- von Willebrand's disease: An intrinsic platelet defect with aggregation by unmodified human factor VIII/von Willebrand factor and enhanced adsorption of its high-molecular-weight multimers. N Engl J Med 1982; 306:326-333). This patient is the 2-year old who was not tested when the family was initially evaluated. To our knowledge, this is the first reported case of pregnancy-associated thrombocytopenia in a patient with pseudo-VWD. Disclosures:No relevant conflicts of interest to declare.

Expansion of Platelets for Transfusion

AbstractAbstract SCI-47The clinical need for platelets transfusion continues to grow. Platelet units have all the usual concerns with other donor-derived blood products and in addition have concerns over the need to store the platelets at room temperature and of the resulting increased risk of bacterial contamination. Several groups have been able to develop platelets from embryonic stem cells, but achieving significant numbers of pro-platelets and/or platelets from in vitro grown megakaryocytes (Megs) remains elusive. Three sites of thrombopoiesis in vivo have been described: 1) intramedullary, 2) at the medullary/vascular junction, or 3) intrapulmonary from circulating Megs. We investigated whether infusing murine Megs into mice can release a large number of functional platelets both to better understand thrombopoiesis and also to see if such an approach may be an alternative to generating platelets for clinical usage. We show that infused fetal-liver-derived Megs results in 100–200 platelets/Meg. Levels as high as 12% of the total platelets in the recipient mouse were from the infused Megs. These platelets had a delayed presentation, peaking at 90 mins. In spite of these being new platelets, their half-life was only ∼75% of infused platelets. The shortened half-life was not due to the fact that these platelets were fetal in nature in that infused adult-liver-derived Megs had a similar temporal profile. These derived platelets had normal-size distribution, were not activated, and had not been affected by ADAM17 during in vitro culture as determined by surface glycoprotein 1b alpha. Platelet functionality was shown by their incorporation into clots in arterioles and venules in a cremaster laser injury model and in a ferric chloride carotid artery injury model. Using BrdU-labeled Megs we show that most of the Megs are trapped in the lungs with a few also appearing in the splenic red pulp. None were seen in other tissues. Staining with species-specific CD41 antibodies showed that detectable Meg cytoplasm peaked in the pulmonary vasculature at 30 min, and like the nuclei, was mostly gone by 5 hrs. We estimate that only ∼1% of the available pulmonary capillary bed is affected by the infused Megs, and most studied animals survived the infusion of the Megs. These studies support the lungs as a potential site for thrombopoiesis and that in vitro differentiated Megs can be infused directly avoiding the challenge of developing ex vivo technology to release large number of non-activated, but functional platelets. Disclosures:No relevant conflicts of interest to declare.

Factor VIII and Platelets Synergistically Accelerate Cleavage of von Willebrand Factor by ADAMTS13 under Fluid Shear Stress

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2-3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of approximately 8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 x 10(3)/microl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by approximately 10-fold (to approximately 0.3 nm) compared with that in the absence of platelets ( approximately 3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1balpha IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1balpha-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.

Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

Background:β2-Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods:β2GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe Na-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced β2GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin-1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin-1, in comparison with non-reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

The c-Myc target glycoprotein1balpha links cytokinesis failure to oncogenic signal transduction pathways in cultured human cells.

An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbα), a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbα in a p53-deficient background. GpIbα was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbα.

Genetic risk factors in typical haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) is a disorder characterized by thrombotic microangiopathy, which is caused in 'typical forms' by gastrointestinal infections with Escherichia Coli species that produce verotoxins. Several studies have identified negative prognostic factors of the disease, among which prolonged oliguria, neurological involvement and increased leukocytosis have been more consistently reported. We have hypothesized that the genetic background may also predispose to the development of typical forms of HUS and may influence the clinical course of the disease. Fourteen polymorphisms, known to influence the coagulation pathway or the activity of the renin-angiotensin system, have been selected and studied in 150 Italian children with typical forms of HUS. Two hundred healthy Italian children were used as controls. The risk of developing HUS was strongly associated with the platelet glycoprotein 1balpha 145M allele (OR 3.08; CI: 1.62-5.85) (P < 0.001). A significant association was also found with polymorphisms located in the adipocyte-derived leucine aminopeptidase and factor V genes. A longer duration of dialysis was moderately associated with increased leukocytosis and with the 807T allele of the platelet glycoprotein 1a gene. High white blood cell count was also strongly associated with the risk of long-term sequelae (OR 2.91, CI: 1.21-6.98) (P < 0.02), whereas the 1166C allele of the angiotensin II type 1 receptor had a significant protective effect (OR 0.28, CI: 0.09-0.83) (P < 0.02). These results highlight the role of glycoprotein 1balpha in the physiopathology of typical forms of HUS and show that the genetic background plays a role in the susceptibility and severity of the disease.

Prothrombotic genetic variants and atherosclerosis in patients with cerebral ischemia of arterial origin

Objective Prothrombotic genetic variants associated with arterial disease probably affect arterial thrombus formation but may also promote atherosclerosis. We hypothesized that specific prothrombotic variants lead to advanced atherosclerosis in patients with cerebral ischemia of arterial origin. Methods and results We included 689 patients with nondisabling cerebral ischemia of arterial origin. Twenty-two variants in 14 genes were genotyped. None of the variants was associated with carotid intima-media thickness or younger age at the occurrence of cerebral ischemia. Factor V Leiden (mean prevalence difference 25%; 95% CI 11–40) and the glycoprotein 1b-alpha Thr145Met variant (mean prevalence difference 12%; 95% CI 2.3–22) were associated with symptomatic carotid stenosis. After accounting for multiple testing by determination of the false discovery rate, only the association between factor V Leiden and symptomatic carotid stenosis remained present. Conclusions Prothrombotic genetic variants showed no consistent association with three markers of advanced atherosclerosis in patients with cerebral ischemia of arterial origin. This study does not support the hypothesis that prothrombotic genetic variants have a direct role in the pathogenesis of atherosclerosis.

Genetics of type 2B von Willebrand Disease: “True 2B,” “tricky 2B,” or “Not 2B.” What Are the Modifiers of the Phenotype?

Type 2B von Willebrand disease (VWD) is a qualitative type of VWD with a unique feature among VWD types, resulting from an increased binding of von Willebrand factor (VWF) to its platelet receptor glycoprotein 1b-alpha (GP1BA). This heightened responsiveness takes place in vivo without endothelial injury or shear stress induction, typically resulting in loss of the hemostatically most active high-molecular-weight VWF multimers and leading to a bleeding diathesis. This process also typically leads to clearance of platelets and thus usually mild thrombocytopenia. At least this describes the classic representation of type 2B VWD (i.e., the typical picture we have come to know since the description/classification of this disorder in the early 1990s). Over more recent years, several reports have described individual cases, groups of patients, and families diagnosed with type 2B VWD where this picture was not typical in one or more aspects. This review discusses type 2B-like disorders and sheds light on potential phenotypic modifiers that might be responsible for the variation encountered with the classic picture of type 2B VWD as well as the impact on the diagnostic certainty of type 2B VWD.

Lung platelet‐endothelial interactions in high tidal volume ventilation

Platelets amplify endothelial P‐selectin expression in high tidal volume ventilation (AJRCMB, 2005, 33:549‐54) but mechanisms underlying platelet‐endothelial interactions are not known. We considered the roles of the platelet inflammatory proteins, glycoprotein 1b alpha (GP1b‐α), vWf and P‐selectin. We recovered lung endothelial cells (FLEC) from freshly excised (control), or isolated, blood‐perfused rat lungs that we ventilated at tidal volume of 12 ml/kg for 2h (HV). Confocal microscopy of immunofluorescently labelled FLEC revealed that GP1b‐α and vWf were weakly expressed for control, but strongly expressed and colocalized for HV. Lung perfusion with the P‐selectin function‐blocking mAb, RMP‐1 markedly inhibited the HV‐induced vWf expression (P&lt;0.05). In contrast to WT, HV failed to increase vWf expression in FLEC from P‐selectin knockout (P‐sel−/−) mice. This inhibitory effect was rescued by perfusing P‐sel−/− lungs with WT blood. We conclude that in HV, platelets transfer GP1b‐αto lung EC and that platelet P‐selectin determines EC vWf expression. Lung recruitment of inflammatory cells might result from platelet‐EC transfer of critical inflammatory receptors (Support: HL54157).

Allosteric Disulfide Bonds in Thrombosis and Thrombolysis.

Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfides are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) is the first haemostasis protein shown to be controlled by an allosteric disulfide, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface; a cryptic form that is inert, a coagulant form that binds factor VIIa to initiate coagulation, or a signaling form that binds VIIa and cleaves protease activated receptor 2 that functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 bond is central to the transition between the three activities of TF. The redox state of the bond appears to be controlled by protein disulphide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta3 and thrombomodulin also contain -RHStaple disulfides and there is circumstantial evidence that the function of these proteins may involve redox change of these disulfide bonds.

2B or not 2B? Differential identification of Type 2B, versus pseudo‐,von Willebrand disease

We read with interest the recent debate surrounding the diagnosis of type 2B von Willebrand disease (VWD) versus pseudo-VWD (Enayat et al, 2006; Favaloro, 2006), where the distinction between the two disorders is based on cryoprecipitate or radioimmunoprecipitation assay (RIPA)-based plasma/platelet mixing studies and genetic testing. In response to Dr Favaloro's invitation to hear of the experience of other workers, we would like to present our data at the Ipswich Hospital, UK, which serves a population of nearly half a million. From our records, we had one kindred comprising eight family members spanning over three generations who were historically diagnosed as type 2B VWD based on phenotypic data. However, we recently re-investigated this family with targeted mutation analysis of von Willebrand factor exon 28 and found this to be normal. Hence the diagnosis of type 2B VWD was questioned and so we proceeded to direct gene sequencing of the glycoprotein 1b alpha gene. We identified Gly249Val mutation, which is one of the three mutations previously described in pseudo-VWD (Miller et al, 1991) thus establishing the correct diagnosis of pseudo-VWD. For completion, we then performed RIPA-based plasma/platelet mixing studies which confirmed the diagnosis of pseudo-VWD in this family. Dr Favaloro suggests that genetic screening for pseudo-VWD is a secondary, confirmatory test that is rarely available, so the gold standard should rely on mixing studies, and he proposes that the RIPA-based mixing studies are superior to the cryoprecipitate challenge. However, as we are dealing with a rare orphan status disease, every laboratory should have the opportunity to refer samples for genetic testing at a tertiary centre (in our case Addenbrookes Hospital, Cambridge, UK). Equally it is debatable that the mixing studies are a ‘simple laboratory test’ that can be performed with local expertise, as in most cases these would also require referral elsewhere. Bearing in mind that both RIPA and cryoprecipitate-based mixing studies are cumbersome, technically complex and can yield false positive results, genetic testing remains a powerful diagnostic tool which, in the 21st century, is of prime importance in the differential identification of type 2B VWD versus pseudo-VWD.

Bernard-Soulier in Iceland. Bleeding Symptoms and Platelet Parameters in Patients, Carriers and Controls.

Bernard Soulier syndrome is a rare platelet disorder characterized by macrothrombocytopenia and absence of platelet aggregation in response to ristocetin due to inherited structural abnormalities in the glycoptein 1B complex. Due to the rarity of this disorder no systematic controlled evaluation has been reported to define the hematological abnormalities and clinical symptoms of these patients. In order to evaluate these findings associated with both Bernard Soulier syndrome and carrier status of glycoprotein 1B-alpha (GP1Bα) mutations we have conducted a nation-wide study in Iceland. All patients with Bernard Soulier syndrome diagnosed in Iceland are referred to the Landspitali University Hospital hemostasis center, a total of twelve patients. Of these, ten were available for the study along with 21 heterozygote carriers (first or second degree relatives) and 25 normal controls. All participating subjects answered a detailed questionnaire on bleeding history and had blood tests, including a complete blood count, coagulation tests, platelet aggregation, PFA-100 closure times, platelet flow cytometry, and DNA analysis to define the underlying GP1Bα mutational status.A diagnosis of Bernard Soulier syndrome was confirmed by platelet ristocetin aggregation and flow cytometry in all patients. Of these ten patients, seven were homozygous for a specific Icelandic mutation, T283 to C (Cys65 to Arg) in the conserved leucine-rich repeats within the ligand-binding region of platelet GP1Bα. Two patients had compound heterozygosity, Cys65 to Arg and a previously described Swedish mutation (Karlstad mutation), G1584 to A (Trp 498 to Stop) with both mutations in GP1Bα. One patient was compound heterozygous for the Karlstad mutation as well as an uncharacterized mutation. Of the 21 carriers, 14 had the Icelandic mutation and 6 had the Karlstad mutation and one had the uncharacterized mutation. As expected all patients had very significant macrothrombocytopenia with no expression of the GP1B complex on flow cytometry and absolute absence of ristocetin aggretion response. Compared to normal controls, the BS patients reported excessive bleeding manifested by markedly increased bruisability, prolonged small cut bleeding and increased mucosal bleeding (epistaxis, oral cavity, menstruation). Heterozygote carriers also reported moderately increased bruisability compared to normal controls, longer bleeding from small cuts and increased mucosal bleeding (epistaxis, oral cavity). On blood analysis the carriers had significantly lower platelets counts (189 vs. 266 × 109/L, p<0.0001 ) and larger platelets (9.6 fl vs. 7.8 fl, p<0.0001 ) than controls. The PFA-100 closure times (both CT c/epi and CT c/ADP) were markedly prolonged in all patients while carriers and controls had similar normal values.In this nation-wide study of Bernard Soulier syndrome we have characterized the clinical and hematological symptoms of patients and carriers. As expected the patients have significant clinical mucosal bleeding symptoms and hematological abnormalities. Interestingly we find evidence of mild platelet dysfunction in heterozygote carriers marked by symptoms of increased mucosal bleeding and lower platelet counts as well as larger platelets than controls.

Allosteric disulfide bonds in thrombosis and thrombolysis.

Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfide bonds are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) was the first hemostasis protein shown to be controlled by an allosteric disulfide bond, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface: a cryptic form that is inert, a coagulant form that rapidly binds factor VIIa to initiate coagulation, and a signaling form that binds FVIIa and cleaves protease-activated receptor 2, which functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 disulfide bond is central to the transition between the three forms of TF. The redox state of the bond appears to be controlled by protein disulfide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta(3) and thrombomodulin also contain -RHStaple disulfides, and there is circumstantial evidence that the function of these proteins may involve cleavage/formation of these disulfide bonds.

Vienna Stroke Registry. Impact of the platelet glycoprotein Ib alpha Kozak polymorphism on the risk of ischemic cerebrovascular events: a case-control study.

We performed a multicenter case-control study to evaluate the impact of the glycoprotein 1b alpha (-5)T/C Kozak polymorphism on the risk of ischemic cerebrovascular events. The genetic analysis in 1399 patients (745 men; median age, 70 years; interquartile ratio, 58-78) and 1066 control subjects (549 men; median age, 47 years; interquartile ratio, 39-59) was carried out with mutagenically separated polymerase chain reaction. Homozygous C/C genotype carriers had a 3.5-fold increased risk for ischemic cerebrovascular events (95% confidence interval, 1.5-7.9, P = 0.003) over T/T or T/C genotype carriers together. The effect was independent of well-established atherosclerotic risk factors. Our findings could be explained by the reported gene dose effect of the Kozak polymorphism on the density of the glycoprotein 1b alpha/IX/V receptors on platelets. According to our data, the (-5)C Kozak allele may represent a candidate genetic susceptibility factor for ischemic cerebrovascular events.

Physical Proximity and Functional Association of Glycoprotein 1bα and Protein-disulfide Isomerase on the Platelet Plasma Membrane

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).