Acquired Glanzmann's thrombasthenia associated with Hodgkin's lymphoma: a case report and review of the literature

BACKGROUND: Acquired Glanzmann's thrombasthenia is a rare hemorrhagic diathesis resulting from impaired adhesive function of the platelet receptor GPIIb/IIIa (alpha(IIb)beta3). Typically, this disorder develops during adulthood, with patients manifesting fluctuating clinical and laboratory findings. To date, the underlying defect of most if not all cases of acquired Glanzmann's thrombasthenia results from an autoantibody or plasma protein inhibitor directed toward a demonstrably normal GPIIb/IIIa glycoprotein. METHODS: In this report, a patient with a history of treated Hodgkin's lymphoma presented with a severe hemorrhagic diathesis characterized by mild thrombocytopenia, a prolonged bleeding time, and defective platelet aggregation. RESULTS: Examination of the patient's platelet GPIIb/IIIa by Western blot analysis revealed no abnormality. Mixing studies demonstrated a non-immunoglobulin G plasma inhibitory factor, whereas flow cytometry analysis revealed elevated platelet-associated immunoglobulin (Ig) M. After an emergency colectomy for severe hemorrhage, the patient's qualitative and quantitative platelet parameters significantly improved. Pathology of the resected colonic segment demonstrated atypical lymphoid hyperplastic lesions. CONCLUSIONS: To the authors' knowledge, this is the first reported case of acquired Glanzmann's thrombasthenia associated with a putative IgM autoantibody. Furthermore, this report verifies the association of acquired thrombasthenia with lymphoproliferative disease. Although rare, awareness of this hemorrhagic diathesis as a possible sequelae of active or treated lymphoid disorders should encourage clinical vigilance of these patients.     

Pseudo-Glanzmann thrombasthenia in the course of autoimmune thrombocytopenic purpura

INTRODUCTION: Auto-immune thrombocytopenic purpura is associated with platelet anti-glycoprotein antibodies, particularly with anti-GPIIb/IIIa complex. Persistence of these antibodies sometimes leads to acquired auto-immune thrombopathy. EXEGESIS: We report the case of a woman treated by splenectomy for auto-immune thrombocytopenic purpura, who developed 5 years later an ecchymotic syndrome despite normal platelet count. High bleeding time and platelet aggregation defect in vitro were evidenced. Following the initial thrombocytopenia, anti-glycoproteins GPIIb/IIIa with lupus anticoagulant and benign monoclonal gammapathy were noticed. Platelet controls showed that hypoaggregant activity was secondary to the persistence of anti-GPIIb/IIa antibodies. CONCLUSION: This acquired auto-immune thrombopathy simulating Glanzmann's thrombasthenia was secondary to the persistence of platelet anti-glycoproteins GPIIb/IIIa.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Microsporidiosis in a young ostrich (Struthio camelus)

Platelet membrane glycoproteins (GP) IIb/IIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann's thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and raplb is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.     

Type I Glanzmann thrombasthenia: most common subtypes in North Indians

The expression of GPIIb/IIIa on the platelet surface was assessed in 10 patients with Glanzmann thrombasthenia and their families by flow cytometry to determine the common subtype in North Indians. Glanzmann thrombasthenia was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to ADP, ADR, arachidonic acid, and collagen. Flow cytometry revealed variable GPIIb/IIIa expression by CD61 and CD41 in patients with Glanzmann thrombasthenia on the basis of CD61 levels, six patients were subtyped as type I because they had absent GPIIb/IIIa, three patients were subtyped as type II because their GPIIb/IIIa levels varied from 7.72% to 20.40%, and one patient was diagnosed as type III, because his clot retraction was 60% and GPIIb/IIIa was 46.0% of normal. Four fathers, three mothers, and five siblings were found to have GPIIb/IIIa levels less than 35% of normal. It is possible that low GPIIb/IIIa levels in family members may reflect their carrier status. It is postulated that flow cytometric estimation of GPIIb/IIIa in parents/siblings may detect carrier status in Glanzmann thrombasthenia.     

Natural antibodies against the immunoglobulin F(ab')2 fragment cause elimination of antigens recognized by the F(ab')2 from the circulation

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab')2 fragment of hC4G1 (F(ab')2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab')2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab')2 hC4G1 were directed against the C-terminal region of F(ab')2 fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homoreactive antibodies enhanced phagocytosis of platelets treated with the F(ab')2 hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab')2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab')2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.     

Role of surface glycoproteins in human platelet function

Glycoproteins present at the external surface of cells probably play specific roles in cellular function. Increasing evidence suggests that the glycoproteins span the plasma membrane with the bulk of the bound carbohydrate asymmetrically distributed on the outer surface. Micellar association of glycoproteins in membranes leads to pore formation and functional roles in transport through the membrane, while surface glycoproteins have been shown to be enzymes, to determine cell specificity and contribute to the cell surface change. The platelet plasma membrane contains 3 major glycoproteins, glycoproteins I, II and III as characterized in order of their decreasing molecular weight. Glycoprotein I appears to have the highest sialic acid content and to give rise to a platelet specific acidic macroglycopeptide on trypsin digestion. Specific glycoprotein abnormalities in the platelets of patients with Glanzmann's thrombasthenia suggest that the glycoproteins play a role in the mechanism of platelet aggregation. A much reduced content of glycoprotein I in the platelets of 2 patients with the Bernard Soulier syndrome may be associated with their defective adhesion to subendothelium and indicates a possible relationship on the platelet surface with the von Willebrand factor protein. Preliminary evidence suggests that in common with other plasma membranes the platelet membrane has a fluid structure and that the organization of the glycoproteins on the platelet surface is extremely sensitive to stimuli and susceptible to change.     

Constitutional thrombopathies: from the clinical description of rare diseases (Glanzmann thrombasthenia and Bernard-Soulier syndrome) to the development of new antithrombotic agents

The study of exceptional thrombocytopathies has led to considerable progress in the understanding of normal and pathologic haemostasis. Thus, precise structure/function relationships were able to be established. The lack or the abnormalities of the glycoprotein IIb/IIIa complex is responsible for the complete defect in platelet aggregation characterizing Glanzmann's Thrombasthenia, while the lack or the abnormalities of the glycoprotein Ib/IX/V complex is responsible for the defect in platelet adhesion to the subendothelium noted in the Bernard-Soulier syndrome. The bases for these abnormalities are now known at the molecular level. Recent data also underline the role of these glycoproteinic complexes as receptors for platelet activation; besides an involvement of the GPIIb/IIIa complex in adhesion and of the GPIb/IX/V complex in platelet aggregation, von Willebrand factor dependent, were newly evidenced. New antithrombotic agents directed against the GPIIb/IIIa complex are currently being tested in large clinical trials, especially in cardiovascular diseases. In the future, the inhibition of the GPIb-IX-V/von Willebrand factor axis should permit the development of a new class of antithrombotics, perhaps even more promising since they will act at the very early phases of primary hemostasis.     

Use of the exercise treadmill to measure baseline functional status and surgical outcome in patients with severe lumbar spinal stenosis

Glanzmann thrombasthenia (GT) is caused by a defect in either glycoprotein (GP)IIb (alphaIIb) or GPIIIa (beta3) genes and therefore screening of both genes is required for mutation identification. The beta subunit of the GPIIb/IIIa complex (beta3) forms a complex with another alpha subunit (alpha(v)) yielding the alpha(v)beta3 vitronectin receptor (VnR). GT patients with mutations in the GPIIIa gene that cause diminished synthesis of GPIIIa are deficient in both GPIIb/IIIa and VnR, whereas patients with mutations in the GPIIb gene are deficient in GPIIb/IIIa, yet express normal or increased VnR in their platelets. The presence or absence of VnR in platelet membranes of GT patients has therefore been used for distinguishing between mutations in the GPIIb gene and mutations in the GPIIIa gene. However, the method of assessing VnR in platelets is cumbersome and use of fresh platelets is indispensible. In the present work we devised a procedure for detection of the VnR in B-lymphocytes transformed by Epstein-Bar virus (EBV). The transformed lymphocytes transcribed GPIIIa mRNA but not GPIIb mRNA and expressed VnR on their surface. Using flow cytometry analysis or immuno-precipitation and western blotting VnR was found in B-lymphocytes of GT patients bearing a well characterized mutation in the GPIIb gene. In contrast, in B-lymphocytes of GT patients bearing 2 different mutations in the GPIIIa gene no VnR was detectable. Thus, for determining which gene is mutated in a GT patient, EBV-transformed B-lymphocytes are useful and can as well be used for analyses of GPIIIa mRNA and genomic DNA. Ten ml of blood are sufficient for the procedure.     

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.     

Fibrin-dependent platelet procoagulant activity requires GPIb receptors and von Willebrand factor

Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by approximately 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + alphavbeta3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from alphavbeta3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.     

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.     

C1q augments platelet activation in response to aggregated Ig

Immune complexes and aggregated IgG (agg-IgG) induce platelet aggregation and the release reaction. Immune complexes also activate the complement system and interact with the complement component C1q. Since platelets possess both Fc and C1q receptors capable of signal transduction, the present study focused on the interaction between these binding sites and platelet activation. Subaggregating doses of agg-IgG (20-400 microg/ml) were identified for washed platelets from each of 11 healthy donors, and platelet aggregation was monitored in the presence or the absence of increasing concentrations of C1q (5-100 microg/ml). C1q produced a dose-dependent potentiation of platelet alphaIIb/beta3 integrin activation, platelet aggregation, and granule secretion when combined with low doses of agg-IgG. C1q alone was without effect. Maximal enhancement of agg-IgG-induced platelet activation was noted at C1q concentrations ranging from 50 to 100 microg/ml. The observed C1q-induced potentiation of platelet aggregation in response to agg-IgG was blocked by polyclonal antibody F(ab')2 directed against platelet binding sites recognizing the collagen-like domain of C1q (cC1qR) or by mAb Fab (IV.3) directed against platelet FcgammaRII receptors. These data suggest a cooperative interaction between platelet FcgammaRII and cC1q receptors and support a potential role for platelet cC1q receptors in pathologic platelet activation by circulating immune complexes often associated with in vivo thrombosis and thrombocytopenia.     

Increase in beta-galactosidase activity in a non-isothermal bioreactor utilizing immobilized cells of Kluyveromyces fragilis: fundamentals and applications

Heparin-induced thrombocytopenia is an increasingly common side effect associated with heparin usage. In the more severe manifestation of the syndrome, patients can develop thrombosis; a 10% mortality is associated with heparin induced thrombocytopenia. To date, the therapeutic options for patients with heparin-induced thrombocytopenia are limited. Glycoprotein IIb/IIIa inhibitors have been shown to block platelet aggregation induced by a wide variety of agonists. The ability of antibody and synthetic small molecule inhibitors of glycoprotein IIb/IIIa to block in vitro activation and aggregation of platelets in response to heparin-induced thrombocytopenia positive serum/heparin was examined using flow cytometry, platelet aggregometry, and luminescence aggregometry. Abciximab, YM 337, and SR 121566A were each found to inhibit platelet microparticle formation and P-selectin expression in whole blood, in response to heparin-induced thrombocytopenia positive serum/heparin. In a platelet rich plasma system, the platelet aggregation response was inhibited by all three agents. The IC50 for inhibition of heparin-induced thrombocytopenia positive serum/heparin induced platelet aggregation by SR 121566A was 18 nM, a concentration which was 4 to 8 fold lower than that observed for collagen and arachidonic acid induced aggregation. Adenosine triphosphate release from activated platelets, as measured by luminescence aggregometry, was concentration-dependently inhibited by SR 121566A. These results suggest that glycoprotein Ilb/IIIa inhibitors may be beneficial in the management of heparin-induced thrombocytopenia and warrant further investigation.     

Diabetic ketoacidosis

Glanzmann's thrombasthenia is a bleeding disorder caused by qualitative and/or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. The disease is inherited in an autosomal recessive manner. In this paper, cDNA probes were used to study restriction fragment length polymorphisms (RFLPs) in GPIIIa gene. A Taq I polymorphism was identified and this RFLP was composed of variant bands of 6.5 Kb/4.0 and 2.5 Kb with a frequency of 0.46/0.54 in Chinese population. The Taq I polymorphism was further localized by polymerase chain reaction (PCR) method to exon VIII of the GPIIIa gene. In two Glanzmann's thrombasthenia families, the Taq I RFLP studied by both Southern blotting and PCR methods identified the defective GPIIIa gene inherited by patients, and determined the genotype of asymptomatic subjects. Analysis of this Taq I polymorphism by PCR method should be potentially useful in future for the carrier detection and prenatal diagnosis in Glanzmann's thrombasthenia families.     

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.     

Shear-induced platelet aggregation (SIPA) monitoring of hemostatic effect during platelet transfusion in a patient with Glanzmann's thrombasthenia: a comparison between SIPA and conventional platelet aggregation

Shear-induced platelet aggregation (SIPA) in a patient with Glanzmann's thrombasthenia was examined during platelet infusion therapy. Prior to platelet infusion, SIPA measured with the modified cone-and-plate type viscometer, as well as ADP-and collagen-induced platelet aggregation measured with the conventional platelet aggregometer, were absent. The patient's SIPA after multiple infusions of platelet, in parallel with the bleeding time and the clinical hemostatic effect, improved to the normal level, while ADP-and collagen-induced platelet aggregation remained abnormal. These observations imply that SIPA is physiologically more relevant than the conventional agonist-induced platelet aggregometry in this type of the disease.     

An antibody against the exosite of the cloned thrombin receptor inhibits experimental arterial thrombosis in the African green monkey

BACKGROUND: Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. METHODS AND RESULTS: The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor-derived peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of approximately 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. CONCLUSIONS: These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.     

Effects of a new platelet glycoprotein IIb/IIIa antagonist, SR121566, on platelet activation, platelet-leukocyte interaction and thrombin generation

The effects of SR121566, a new inhibitor of the glycoprotein (GP) IIb/IIIa complex on platelet activation and platelet-leukocyte interactions, as well as on thrombin generation were investigated. SR121566 dose-dependently inhibited adenosine diphosphate (ADP)-induced platelet fibrinogen binding determined either by flow cytometry analysis (IC50=50 nmol/l) or by measuring the binding of 125I-fibrinogen to activated human gel-filtered platelets (IC50=20 nmol/l). Consistent with its inhibitory effects on platelet fibrinogen binding, SR121566 demonstrated a dose-dependent inhibition of collagen-, ADP- or thrombin-induced platelet aggregation with IC50 values ranging between 20 and 60 nmol/l. SR121566, even tested at high concentrations, did not significantly affect ADP-induced platelet-leukocyte aggregate formation. The GPIIb/IIIa antagonist strongly inhibited thrombin generation in both native clotting blood and recalcified whole blood, suggesting that SR121566, by interfering with the platelet-activation events involved in facilitating thrombin generation, may also function as an anticoagulant, an effect which may contribute to its antithrombotic properties in humans.     

G-CSF after PBSC transplantation

We report a case of a patient with Glanzmann's thrombasthenia and anti-GPIIb/IIIa alloantibodies who developed life-threatening and intractable bleeding from gastrointestinal telangiectatic lesions. After a period of transfusion-dependent gastrointestinal bleeding despite tranexamic acid, oral iron, omeprazole and platelet transfusions, the use of oral norethisterone produced a significant improvement with a marked reduction in her transfusion requirements.