To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n ؍ 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P < .001) as compared with both the control and patient plasma with no detectable antiGPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P < .001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production. (Blood. 2003;102: 887-895)
The treatment of bleeding for haemophilic patients with inhibitors relies on the use of the bypassing agents, recombinant factor VIIa and factor eight inhibitor bypass activity (FEIBA). While both therapies are effective in the majority of bleeding episodes, there is a significant amount of interindividual variability when it comes to the response to therapy. As of yet, there is no reliable laboratory parameter that can predict the response to therapy in the same manner that factor VIII and factor IX levels predict response in non-inhibitor patients. Developing such a laboratory parameter is vital in order to maximize the clinical efficacy of these agents. Thromboelastography (TEG) is a device, which assesses clot formation over time in whole blood and has several characteristics which suggest it may be an effective way to monitor bypass agent therapy. We studied the ability of TEG to individualize the treatment regimens of three patients with high titre inhibitors assessing the response to recombinant factor VIIa, FEIBA, and when both were used sequentially. The TEG allowed for individualization of treatment for each of the three patients and resulted in more effective, convenient and less expensive treatment regimens. We thus believe that TEG is a promising device for monitoring of bypass agent therapy and should be studied further.
Bleeding is the major adverse reaction to anticoagulants, leading to significant morbidity and even mortality. Protamine is a specific antidote for heparin yet is only partially effective for enoxaparin, and the activated factor X inhibitor fondaparinux and the direct thrombin inhibitors argatroban and bivalirudin lack specific antidotes. We evaluated the ability of recombinant activated factor VII (rFVIIa), a general hemostatic agent, to reverse the anticoagulant effects of heparin, enoxaparin, fondaparinux, argatroban, and bivalirudin, as measured by thromboelastography. Whole-blood samples containing each test anticoagulant, with or without rFVIIa 1.5-4.5 microg/ml, were prepared ex vivo (n >or= 48, each anticoagulant) and analyzed by thromboelastography. The thromboelastography parameters of clot initiation, propagation, rigidity and elasticity were compared for the ex-vivo samples for each anticoagulant. The reversal ability of rFVIIa was also assessed using the standard clinical assay used to monitor each anticoagulant. Thromboelastography was performed on blood from eight stably anticoagulated patients, with and without exogenous rFVIIa. For each anticoagulant, rFVIIa significantly improved and, in some cases, completely normalized all thromboelastography parameters (P < 0.001). rFVIIa significantly (P < 0.01) decreased the activated partial thromboplastin time for argatroban-containing, bivalirudin-containing, or heparin-containing blood yet did not affect the anti-activated factor X levels for enoxaparin-containing or fondaparinux-containing blood. By thromboelastography, rFVIIa exerted generally similar reversal effects on the anticoagulated patient samples as on the ex-vivo samples. In conclusion, rFVIIa effectively reverses the anticoagulant effects of heparin, enoxaparin, fondaparinux, argatroban, and bivalirudin, and should be considered for patients with excessive bleeding associated with these anticoagulants.
We investigated the anticoagulant effects of argatroban, a direct thrombin inhibitor, versus heparin in extracorporeal membrane oxygenation (ECMO) circuits. Three sham circuits were prepared according to our hospital's standard practice and run for six hours simultaneously. Two circuits were anticoagulated with argatroban (one with heparin in the wet prime and one without). One circuit had heparin in the initial prime and was then anticoagulated with heparin. We measured thrombin generation (prothrombin fragment 1+2, D-dimer and thrombin-antithrombin complexes), activated clotting times (ACTs) and partial thromboplastin times (aPTTs), and monitored thrombus formation using thromboelastography. ACTs were >1000 s in each circuit throughout assessment. No clot initiation was detected by thromboelastography. Thrombin generation was decreased in circuits anticoagulated with argatroban versus heparin, despite aPTTs being less prolonged. These results suggest that argatroban may be more efficacious than heparin for anticoagulation in ECMO. Additional studies are warranted to further evaluate argatroban in this setting.
New anticoagulants, including the direct thrombin inhibitors (DTIs) and fondaparinux, are increasingly replacing unfractionated heparin and enoxaparin. We examined the effects of argatroban (n = 60), bivalirudin (n = 44), heparin (n = 14), enoxaparin (n = 22), and fondaparinux (n = 24) on clot formation utilizing thromboelastography. Blood samples containing anticoagulants at clinically relevant concentrations were prepared ex vivo and analyzed using kaolin or tissue factor activation. Thromboelastography parameters of clot initiation (R), clot propagation (K and angle), clot rigidity (maximum amplitude) and clot elasticity (G) were compared between anticoagulants. Thromboelastography was also performed on blood from eight patients receiving anticoagulants. Each anticoagulant exerted significant concentration-dependent effects on R, K and angle. Only heparin, enoxaparin, and fondaparinux significantly affected maximum amplitude and G. Significant differences existed for all parameters between heparin and each anticoagulant and between fondaparinux and each DTI (P < 0.001), and for angle, maximum amplitude, and G between enoxaparin and each DTI (P < 0.008). Thromboelastography responses in ex-vivo samples and patient samples were comparable. In conclusion, whereas argatroban, bivalirudin, heparin, enoxaparin and fondaparinux each delay clot formation, the DTIs do not alter clot rigidity or elasticity. The reduced bleeding reported with DTIs versus heparin may relate to the fact that clots form with normal rigidity and elasticity.
Summary. Background: The Platelet Function Analyzer-100 (PFA-100) is widely used to measure platelet reactivity in whole blood under high shear. Objective: To characterize the genetic component of platelet reactivity among normal individuals, using the PFA-100. Methods: We compared baseline platelet reactivity with sex, age, platelet count, hematocrit, plasma von Willebrand factor antigen (VWF:Ag), and alleles of seven candidate genes: integrin subunits a2 (ITGA2) and b3 (ITGB3), platelet glycoproteins GPIba (GP1BA) and GPVI (GP6), purinogenic receptors (P2RY1 and P2RY12) and cyclooxygenase-1 (COX1). Results: Based on linear and logistic regression models, we report an inverse correlation between baseline closure time (CT) initiated by collagen plus epinephrine (CEPI) and plasma VWF:Ag level, ITGA2 807T and P2RY1 893C, and an inverse correlation between baseline CT initiated by collagen plus adenosine diphosphate (CADP) and P2RY1 893C or GP1BA -5C. Conclusions: These results indicate that genetic polymorphisms in ITGA2 and P2RY1 combine with plasma VWF:Ag levels to modulate baseline platelet reactivity in response to collagen plus EPI, while genetic differences in P2RY1 and GP1BA significantly effect platelet responses to collagen plus ADP. Our results demonstrate that the PFA-100 can be used to evaluate the effects of genetic predictors of platelet function.
Immunoblot analysis using several antibodies raised against two major families of stress proteins, stress 70 and chaperomn 60 (cpn60), which are highly conserved in mammals, was carried out in diverse species often used in environmental research, including molluscs, annelids, crustaceans, echinoderms, and fish The study revealed surprisingly different patterns of antibody cross reactivity among species The monoclonal anti‐stress 70 antibody (mAb) C92 was the least cross reactive for all species tested The mAbs anti‐stress 70 N27, BRM‐22, and 3a3 were more broadly cross reactive, but their binding specificities to stress 70 isoforms in the diverse species tested did not correlate with one another or follow taxonomic lines The polyclonal anti‐stress 70 antibody reacted to proteins in the 70 to 74 kDa range in all fish examined and in most invertebrates When a polyclonal antibody (pAb) raised against cpn60 from a moth was used as a probe, specific binding was observed with proteins in the 60 to 64 kDa range in all fish examined and in most invertebrates However, the size and number of isoforms that reacted with the pAb were species specific These data suggest that these two major stress protein families are less highly conserved in invertebrates and fish than in mammals Therefore, to minimize misinterpretation when using antibodies in heterologous assays with species in which the stress response has not been well characterized, it is important to determine which isoforms of stress 70 react with a particular antibody and to take into account the differential regulation of each member of this multigene family
Immunoblot analysis using several antibodies raised against two major families of stress proteins, stress 70 and chaperomn 60 (cpn60), which are highly conserved in mammals, was carried out in diverse species often used in environmental research, including molluscs, annelids, crustaceans, echinoderms, and fish The study revealed surprisingly different patterns of antibody cross reactivity among species The monoclonal anti‐stress 70 antibody (mAb) C92 was the least cross reactive for all species tested The mAbs anti‐stress 70 N27, BRM‐22, and 3a3 were more broadly cross reactive, but their binding specificities to stress 70 isoforms in the diverse species tested did not correlate with one another or follow taxonomic lines The polyclonal anti‐stress 70 antibody reacted to proteins in the 70 to 74 kDa range in all fish examined and in most invertebrates When a polyclonal antibody (pAb) raised against cpn60 from a moth was used as a probe, specific binding was observed with proteins in the 60 to 64 kDa range in all fish examined and in most invertebrates However, the size and number of isoforms that reacted with the pAb were species specific These data suggest that these two major stress protein families are less highly conserved in invertebrates and fish than in mammals Therefore, to minimize misinterpretation when using antibodies in heterologous assays with species in which the stress response has not been well characterized, it is important to determine which isoforms of stress 70 react with a particular antibody and to take into account the differential regulation of each member of this multigene family
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