Factors associated with pre-slaughter mortality in turkeys and end of lay hens.

Pre-slaughter transportation may affect poultry welfare and mortality rates. A retrospective analysis was conducted to examine the effect of environmental, management and individual factors on the percentage of dead birds during pre-slaughter transportation (dead-on-arrival, DOA). The variables accounted for in the analyses included: environmental temperature, travel duration, genetic line, gender, crate type and crate stocking density. Among the 41 452 loads of turkeys (34 696 388 birds) and 3241 of end of lay hens (21 788 124 birds) transported to three large abattoirs in northern Italy in a 3-year period, the median DOA was 0.14% in turkeys, and 0.38% in hens. In turkeys, travel duration longer than 30 min, temperature higher than 26°C and high in-crate densities were associated with increased DOA. In winter (⩽2°C), high stocking densities did not reduce the mortality risk from cold stress; on the contrary, for stocking densities either near to or just above the maximum density in EC Reg. 1/2005, the DOA risk was greater than for loads with densities of 10 kg/m2 less than the EC maximum. Male birds and specific genetic lines also showed a higher DOA. In hens, transportation lasting longer than 2 h and the brown-feathered breed were associated with higher DOA. Dead-on-arrival progressively increased with travel duration, remaining constant between 4 and 6 h and peaking at 8 h (median: 0.57%). The maximum DOA increase was detected during winter. These results show that several species-specific factors may lead to increased risk of mortality.

Clinical-grade quality platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelet concentrates promoted expansion of mesenchymal stromal cells.

BACKGROUND AND OBJECTIVES: The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelets as a source of growth factors for the expansion of mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. MATERIALS AND METHODS: PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl2 -activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). RESULTS: PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. CONCLUSIONS: The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs.

Metformin improves putative longevity effectors in peripheral mononuclear cells from subjects with prediabetes. A randomized controlled trial.

BACKGROUND AND AIMS: Prediabetes increases cardiovascular risk and is associated with excess mortality. In preclinical models, metformin has been shown to exert anti-ageing effects. In this study, we sought to assess whether metformin modulates putative effector longevity programs in prediabetic subjects. METHODS AND RESULTS: In a randomized, single-blind, placebo-controlled trial, 38 prediabetic subjects received metformin (1500 mg/day) or placebo for 2 months. At baseline and after treatment, we collected anthropometric and metabolic parameters. Gene and protein levels of SIRT1, mTOR, p53, p66Shc, SIRT1 activity, AMPK activation, telomere length, and SIRT1 promoter chromatin accessibility were determined in peripheral blood mononuclear cells (PBMCs). Plasma N-glycans, non-invasive surrogate markers of ageing, were also analysed. Compared to baseline, metformin significantly improved metabolic parameters and insulin sensitivity, increased SIRT1 gene/protein expression and SIRT1 promoter chromatin accessibility, elevated mTOR gene expression with concomitant reduction in p70S6K phosphorylation in subjects' PBMCs, and modified the plasma N-glycan profile. Compared to placebo, metformin increased SIRT1 protein expression and reduced p70S6K phosphorylation (a proxy of mTOR activity). Plasma N-glycans were also favourably modified by metformin compared to placebo. CONCLUSION: In individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. ClinicalTrials. gov identifier: NCT01765946 - January 2013.

Retrospective space-time analysis methods to support West Nile virus surveillance activities.

The steep increase in human West Nile virus (WNV) infections in 2011-2012 in north-eastern Italy prompted a refinement of the surveillance plan. Data from the 2010-2012 surveillance activities on mosquitoes, equines, and humans were analysed through Bernoulli space-time scan statistics, to detect the presence of recurrent WNV infection hotspots. Linear models were fit to detect the possible relationships between WNV occurrence in humans and its activity in mosquitoes. Clusters were detected for all of the hosts, defining a limited area on which to focus surveillance and promptly identify WNV reactivation. Positive relationships were identified between WNV in humans and in mosquitoes; although it was not possible to define precise spatial and temporal scales at which entomological surveillance could predict the increasing risk of human infections. This stresses the necessity to improve entomological surveillance by increasing both the density of trapping sites and the frequency of captures.

Growth factor release from platelet concentrates: analytic quantification and characterization for clinical applications.

BACKGROUND AND OBJECTIVES: Clinical use of plasma rich in growth factors requires biochemical product control. We aimed to measure and modulate concentrations of growth factors in solutions deriving from platelet apheresis or whole blood. MATERIALS AND METHODS: Growth factor concentrations were measured 5', 10', 20', 30', 60' after CaCl2 was added at 40°C to platelet-apheresis products (n = 39) or after 60' in platelet concentrates from whole blood (n = 13). Growth factor release was also obtained in platelet apheresis a) by incubation at 22°C or 40°C for 10' or 30' (n = 4); b) by repeated freeze-thaw (n = 9). RESULTS: Fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) isoforms AA and AB and transforming growth factor beta (TGF-ß) concentrations (pg/10(9 ) plt) were 25-60% higher in growth factors solutions from whole blood compared to platelet apheresis. Vascular endothelial growth factor (VEGF), TGF-ß and PDGF isoforms were released early (5-10') during incubation: TGF-ß concentration increased also at 30'. FGF and epidermal growth factor (EGF) were released only after 30'. Incubation at 40°C/10' increased VEGF (+70%) and decreased EGF (-30%) and PDGF-BB (-50%) versus 22°C/30'. Shock significantly increased TGF-ß (1.6-fold), EGF (1.5-fold), FGF (4.5-fold) and lowered PDGF isoforms (0.2- to 0.5-fold) versus prolonged incubation at 40°C. CONCLUSION: Platelets from platelet apheresis and whole-blood release all investigated growth factors. The release can be regulated controlling incubation time and/or temperature and performing cell lysis.

Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness.

A new freezing and storage procedure improves safety and viability of haematopoietic stem cells and neutrophil engraftment: a single institution experience.

BACKGROUND AND OBJECTIVES: Autologous peripheral blood stem cell transplantation has recently become a standard therapeutic approach to virus-related or infected haematological malignancies. Collection, manipulation, storage and thawing of leukapheresis products in this subset of patients require strict monitoring to prevent infection risk for operators and risk of contamination for other stored bags. MATERIALS AND METHODS: This is a non-randomized retrospective observational study. In the 2000-2002 period, a single bag freezing procedure was used for autologous peripheral blood stem cell transplantation. Bags were stored in tanks containing liquid and gas phase nitrogen. In 2002, the processing procedure was revised, and a second additional safety bag and a new storage tank containing jacketed liquid nitrogen have been used. RESULTS: A total of 524 bags were thawed, of which 121 processed with the single bag method and 403 with the double bag method. Forty-nine and 109 patients were infused respectively. The observed rupture rate with the single bag in liquid and gas phase nitrogen was 17 and 2.5%, respectively, against a rupture rate as little as 0.24% with the new methodology. Viability revealed levels of 84.4% +/- 6.1% and 96.9% +/- 2.4% for the single and double-bag respectively. This statistically significant (P < 0.0001) difference correlated with better neutrophil engraftment. CONCLUSIONS: The new proposed method, based on a double bag and storage freezer without liquid or gas phase nitrogen into a cryogenic chamber, significantly reduces bag rupture and bio-hazard and improves stem cell viability and neutrophil engraftment remarkably.

Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor.

BACKGROUND AND OBJECTIVES: Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma. MATERIALS AND METHODS: Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations. RESULTS: Total nucleated cells and CD34+ cells increased 59.5 and 4.0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit-granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion. CONCLUSIONS: We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.

Dentists' perceptions, attitudes, knowledge, and experience about child abuse and neglect in northeast Italy.

OBJECTIVE: The aim of this study was to analyze dentists' perceptions, attitudes, knowledge and experience about child abuse and neglect (CAN) in an area of northeast Italy and the factors affecting the recognition and reporting of CAN cases. MATERIAL AND METHODS: One hundred six dentists working in both public and private sectors in the provinces of Padua and Treviso were interviewed by a single operator. Descriptive and assessing association analyses were carried out. RESULTS: Dentists' perceptions about CAN is low, and these professionals have a poor attitude toward confronting it according to the code of conduct and laws. Available information and education are also poor Education affects the detection and the reporting of CAN cases in a relevant way. Female gender is another factor that affects the attitude and the perception of CAN. CONCLUSIONS: The results, which are consistent with other studies, show that there is a general lack of knowledge about CAN that prevents dentists from detecting and identifying suspected cases. Despite its frequent occurrence among dental patients, neglect is the least known and identified type of abuse. Education is the critical element in enhancing the ability of professionals to detect cases.

Blood clotting in space.

We describe herein a novel in vitro approach that can be used effectively to obtain valuable insights into the role of platelets, various coagulation proteins as well as proteins of the subendothelial extracellular matrix involved in the hemostatic and thrombotic processes occurring under microgravity. At difference with other experimental approaches proposed in the past our device operates in a closed system and under different shear forces, which better mimics flow conditions occurring in vessels. Furthermore our device by allowing real time monitoring of the thrombotic process and its underlying mechanisms can be regarded as a reliable system for the precise assessment of platelet function.

On the mechanism of the spermine-exerted inhibition on alpha-thrombin-induced platelet activation.

Previous reports have shown that various amines inhibited platelet activation, but no definitive conclusions on their action mechanism were drawn. We have further investigated the action of spermine on platelet responses evoked by alpha-thrombin and other agonists. Spermine inhibited in a concentration-dependent manner (1-10 mM), and more efficiently than spermidine and putrescine, the alpha-thrombin-induced (1.5 nM) platelet activation. Spermine added at a concentration that inhibited completely aggregation only partially affected the thrombin-induced increase in cytosolic Ca(2+) concentration, protein phosphorylation, and ATP secretion. The polyamine had little effect on the morphology of resting platelets, as measured by electron microscopy, thrombin hydrolytic activity, and fibrinogen clotting capacity but decreased the thrombin binding to platelets and isolated glycocalicin. Spermine partially inhibited the aggregation elicited by ADP, vasopressin, platelet-activating factor, thrombin receptor-activating peptide, fluoroaluminate, ionomycin, and dioctanoylglycerol but did not affect the cytosolic Ca(2+) increase induced by these agonists. The polyamine bound to both glycocalicin and platelets, and it inhibited the fibrinogen binding to stimulated platelets. The amount of 14C-spermine bound to resting cells decreased in the presence of the glycoprotein GPIb-antibody LJIB1, whereas the polyamine bound to activated platelets, which was higher than that tied to resting cells, was markedly reduced by LJCP8 or decorsin, a GPIIb/IIIa antibody and antagonist-peptide, respectively. These results indicate that spermine specifically inhibits the thrombin binding to GPIb of resting platelets and the fibrinogen binding to GPIIb/IIIa (integrin alpha(IIb)beta(3)) of activated platelets.

Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow.

We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.

Characterization of the initial alpha-thrombin interaction with glycoprotein Ib alpha in relation to platelet activation.

We have evaluated the properties of alpha-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37 degrees C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha-thrombin interaction site on glycoprotein (GP) Ib alpha. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib alpha antibody. Results were similar at room temperature (22-25 degrees C), whereas the initial characteristics of alpha-thrombin interaction with platelets were preserved for at least 20 min at 4 degrees C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib alpha, completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10(-8) M) and relatively high capacity, rather than "high" affinity (kd on the order of 10(-10) M) and low capacity as currently thought. The parameters of alpha-thrombin binding to intact GP Ib alpha on platelets at 4 degrees C corresponded closely to those measured with isolated GP Ib alpha fragments regardless of temperature. Blocking the alpha-thrombin-GP Ib alpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib alpha contributes to the thrombogenic potential of alpha-thrombin.

Thrombin interaction with platelet GpIB: role of the heparin binding domain.

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Frequency and functional relevance of genetic threonine145/methionine145 dimorphism in platelet glycoprotein Ib alpha in an Italian population.

BACKGROUND: Threonine145/methionine145 dimorphism in platelet glycoprotein (GP) Ib alpha defines the human platelet antigen (HPA)-2 system that has been implicated in refractoriness to HLA-matched platelet transfusion and in neonatal immune thrombocytopenic purpura. STUDY DESIGN AND METHODS: The occurrence of this amino acid dimorphism was investigated in 379 Italian blood donors by studying their genomic DNA. Two oligonucleotide primers, Ib alpha-3 (5'-GGACGTCTCCTTCAACCGGC-3') and Ib alpha-4 (5'-GCTTTGGTGGGGAACTTGAC-3'), were used in a polymerase chain reaction to generate a 591-base pair fragment that was digested with the restriction enzyme Acy I. To investigate whether this dimorphism is involved in the binding of von Willebrand factor (vWF) to GPlb, the binding of vWF to the GPlb/IX complex was measured in two Met145/Met145 and two Thr145/Thr145 subjects. RESULTS: The genotypic frequencies are 78.9% for Thr/Thr, 19.8% for Thr/Met), and 1.3% for Met/Met; the allelic frequencies are 88.8% for Thr145 and 11.2% for Met145. Estimates for binding of subunit molecules per platelet at saturation and inhibition constant in mol per L, respectively, follow. In the presence of ristocetin (0.5 mg/mL), they are 11,460 +/- 2,040 and 1.26 +/- 0.44 x 10(-8) for normals and 11,230 +/- 2,330 and 1.29 +/- 0.48 x 10(-8) for patients. In the presence of botrocetin (2.5 micrograms/mL), they are 64,260 +/- 7,760 and 2.99 +/- 0.96 x 10(-8) for normals and 65,770 +/- 11,570 and 2.47 +/- 0.22 x 10(-8) for patients. Platelet aggregation responses obtained using platelet-rich plasma from donors with Met145/Met145 or Thr145/Thr145 genotype were within normal limits. CONCLUSION: Genotypic and phenotypic frequencies in the HPA-2 system in this population are consistent with those reported among the white population. Furthermore, the HPA-2 system is not involved in the binding of vWF to GPlb.

Porcine von Willebrand factor binding to human platelet GPIb induces transmembrane calcium influx.

Porcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) Ib and, upon stirring (1500 rpm/min) at 37 degrees C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP Ib, obtained with anti GP Ib monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP Ib monoclonal antibody which does not inhibit the vWF binding to GP Ib. An anti GP IIb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP Ib occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP Ib, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxygenase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

Identification of three tyrosine residues of glycoprotein Ib alpha with distinct roles in von Willebrand factor and alpha-thrombin binding.

The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of alpha-thrombin. There are two distinct but partially overlapping regions of GP Ib alpha thought to be involved in interacting with vWF (residues 251-279) and alpha-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J.A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ib alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr-->Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of alpha-thrombin. GP Ib alpha fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind alpha-thrombin and, in addition, had markedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277, a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structural features of GP Ib alpha selectively involved in supporting the interaction with vWF or alpha-thrombin.

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.

Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology.