[Mortality after high-risk surgery in Jehovah's Witness patients]. / Letalität nach operativen Risikoeingriffen bei Zeugen Jehovas.

BACKGROUND: Jehovah's Witness (JW) patients strictly refuse allogeneic blood transfusion for religious reasons. Nevertheless, JW also wish to benefit from modern therapeutic concepts including major surgical procedures without facing an excessive risk of death. The Northwest Hospital in Frankfurt am Main Germany is a confidential clinic of JW and performs approximately 100 surgical interventions per year on this patient group. MATERIAL AND METHODS: A retrospective analysis of closed medical cases performed in the years 2008-2018 at the Northwest Hospital aimed to clarify (1) the frequency of surgical procedures in JW patients associated with a statistical allogeneic transfusion risk (presence of preoperative anemia and/or in-house transfusion probability >10%) during this time period, (2) the degree of acceptance of strategies avoiding blood transfusion by JW and (3) the anemia-related postoperative mortality rate in JW patients. RESULTS: In the 11- year observation period 123 surgical procedures with a relevant allogeneic transfusion risk were performed in 105 JW patients. Anemia according to World Health Organization (WHO) criteria was present in 44% of cases on the day of surgery. Synthetic and recombinant drugs (tranexamic acid, desmopressin, erythropoetin, rFVIIa) were generally accepted, acute normovolemic hemodilution (ANH) in 92% and cell salvage in 96%. Coagulation factor concentrates extracted from human plasma and therefore generally refused by JW so far, were accepted by 83% of patients following detailed elucidation. Out of 105 JW patients 7 (6.6%) died during the postoperative hospital stay. In 4 of the 7 fatal cases the cause of death could be traced back to severe postoperative anemia. CONCLUSION: Given optimal management JW patients can undergo major surgery without an excessive risk of death. The 6.6% in-hospital mortality observed in this institution was in the range of the 4% generally observed after surgery in Europe. The majority of JW patients accepted a variety of blood conservation strategies following appropriate elucidation. This also included coagulation factor concentrates extracted from human plasma enabling an effective treatment of even severe bleeding complications. In this analysis postoperative hemoglobin concentrations below 6 g/dl in older JW patients were associated with a high mortality risk due to anemia.

Microtubule plus-end tracking Adenopolyposis Coli negatively regulates proplatelet formation.

Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.

Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibß function.

UNLABELLED: Essentials A signaling role of glycoprotein (GP)Ibß is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbß function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbß subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbß in hemostasis and thrombosis. Methods GPIbß(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbß were transplanted into lethally irradiated GPIbß(-/-) recipient mice. Results hGPIbß transplanted into the bone marrow of GPIbß(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbß(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbß intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbß in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

Economic consequences of the demography of MRSA patients and the impact of broad-spectrum antimicrobials.

BACKGROUND: Studies have determined the societal impact of methicillin-resistant Staphylococcus aureus (MRSA) by modelling its impact on labour supply and productivity. In addition, most of the studies on the topic conclude that the problem of resistance should be counteracted on the macro level by reducing overall antibacterial consumption. OBJECTIVE: Two major questions have been raised in the present work. Firstly, is MRSA impairing labour supply and productivity? Secondly, is it the overall use of antibacterials that may be seen as crucial to the spread of MRSA infections? METHODS: The age distribution of MRSA patients is compared with the age distribution of the entire patient population at a German teaching hospital. In addition, the age distribution of MRSA patients was applied to the age distribution of the German population in the year 2050 in order to identify the effects of the double-ageing process on the spread of MRSA. Furthermore, recent epidemiological studies were reviewed on the impact of overall antibacterial consumption on MRSA infection rates. RESULTS: Based on available data, we show that patients infected or colonized with MRSA are, for the most part, beyond retirement age and thus not responsible for changes in labour supply or productivity. Application of age distribution of MRSA patients to the age distribution of the German population in the year 2050 gives a 24% increase in the number of MRSA cases to a total of 182 778 due to an ageing population. In addition, we show that a 32% reduction in the cost of MRSA to the German healthcare system could be reached if use of fluoroquinolones and third-generation cephalosporins was reduced by just 10% and, correspondingly, use of antiseptics for hand disinfection was increased by 10%. CONCLUSIONS: MRSA is a phenomenon that, to a larger degree, affects the elderly population rather than the labour force. When it comes to policy options to counteract MRSA on the macro level, most economic research on the topic is biased in assuming that the overall use of antibacterials is responsible for the spread of MRSA infections.

Inhibition of adhesive and signaling functions of the platelet GPIb-V-IX complex by a cell penetrating GPIbalpha peptide.

BACKGROUND: Interaction between the platelet glycoprotein (GP)Ib-V-IX complex and von Willebrand factor (VWF) is critical for initiating platelet-vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin alpha(IIb)beta3 activation. OBJECTIVE: A cell-penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbalpha subunit in VWF/GPIb-V-IX-dependent adhesion and activation. METHODS: Peptides of 11-13 amino acids, covering the 557-610 region, were coupled to a nine-arginine permeating tag (R9) and the effects of their cell entry on VWF-dependent responses were analyzed. RESULTS: The R9alpha557 peptide corresponding to the 557-569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9alpha557 on GPIb VWF-dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb-IX complex were incubated with R9alpha557. CONCLUSION: This study performed in intact platelets suggests a functional role of the 557-569 domain of GPIbalpha in controlling VWF-dependent adhesion and signaling.

Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.

Increased platelet glycoprotein V levels in patients with coronary and peripheral atherosclerosis--the influence of aspirin and cigarette smoking.

As platelet hyperactivity is important in atherosclerosis and smoking, we hypothesized higher levels of soluble platelet membrane glycoprotein V (gpV) in 95 patients with peripheral artery disease (PAD) and 92 with coronary artery disease (CAD) compared to 99 healthy controls, and examined the effects of aspirin and of smoking two cigarettes on soluble gpV and platelet function. Soluble gpV (ELISA) was significantly raised in, but not between, both PAD and CAD patients, compared to controls (p < 0.05). In multivariate analysis, systolic blood pressure, smoking and atherosclerosis (all p < 0.01) were significant influences on soluble gpV in the whole study cohort. There was a weak correlation between soluble gpV and another platelet marker, soluble P selectin (p = 0.048). Acute smoking in 14 subjects increased platelet aggregability and beta-thromboglobulin, but not soluble gpV: there were no changes in 11 non-smokers. Five days consumption of aspirin (325 mg daily) by 14 subjects did not influence levels of soluble gpV. Our data indicate that soluble gpV may be a useful new marker of platelet activation in atherosclerosis, but may be influenced by smoking status and blood pressure.

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)

A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry.

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.

GPV is a marker of in vivo platelet activation--study in a rat thrombosis model.

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

A general approach to the generation of monoclonal antibodies against members of the tetraspanin superfamily using recombinant GST fusion proteins.

Tetraspanins belong to a rapidly growing family of proteins characterized by the presence of four conserved transmembrane segments and are involved in such diverse functions as cellular activation, adhesion, migration and differentiation. In an effort to develop reagents against newly discovered tetraspanins, we have devised a simple method for the screening of monoclonal antibodies (mAbs) using recombinant GST fusion proteins. GST fusion proteins containing the second extracellular domain of different tetraspanins (CD9, CD63, CD53, CD81, A15 or CO-029) were produced separately. Mice were immunized with cells having a high expression of the chosen tetraspanin and the constructs were used to screen hybridomas in a solid phase ELISA. Several clones binding the fusion protein were identified for each construct tested: four anti-CD9 hybridoma clones, four anti-CD63, two anti-CD53, two anti-CD81, three anti-A15 and one anti-CO-029. All the newly developed mAbs recognized the native proteins by flow cytometry, immunofluorescence staining of cells and immunoprecipitation and bound to the denatured proteins on immunoblotting. Use of GST fusion protein constructs in a simple ELISA can facilitate screening for mAbs to members of the tetraspanin family, especially in cases where information is limited to the nucleotide sequence. The mAbs obtained by this strategy should prove to be valuable tools for functional studies of newly discovered tetraspanins.

Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA--its use to follow platelet storage in transfusion.

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.

Tetraspanin CD9 is associated with very late-acting integrins in human vascular smooth muscle cells and modulates collagen matrix reorganization.

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

Co-localization of CD9 and GPIIb-IIIa (alpha IIb beta 3 integrin) on activated platelet pseudopods and alpha-granule membranes.

CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of alpha-granule membranes. These two antigens also colocalized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from alpha-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets.

Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia.

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.

[Isosorbide dinitrate inhibits in vitro platelet aggregation at submicromolar concentrations]. / Le dinitrate d'isosorbide inhibe, in vitro, l'agrégation plaquettaire à des concentrations submicromolaires.

Nitrate derivatives have in vivo and in vitro platelet anti-aggregant properties in addition to their vasodilatory effects. The mode of action is related to increased intracytoplasmic cyclic GMP concentrations. It has been shown that isosorbide dinitrate (ISDN) has this type of platelet anti-aggregant activity but the reported results about the active concentrations and the inhibited pathways of activation are contradictory. This study was designed to determine whether ISDN has in vitro platelet anti-aggregant activity at low doses and to verify if this effect is selective by aggregation induced by ADP. Finally, a possible potentialisation of the inhibitors due to ISDN was looked for with cyclic nucleotide phosphodiesterase inhibitors and with agents simulating the effect of adenylate cyclase. The results showed that: 1) ISDN had platelet anti-aggregant activity in vitro at concentrations of about 10-7 M, 2) that this effect was not limited to the aggregation induced by ADP as the aggregation induced by PAF-acether was also inhibited by low dose ISDN, 3) of the cyclic nucleotide modulators tested, only quercetine (flavonoide) potentialised the effects of ISDN.