Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.

This report describes studies into the pathophysiology of heparin- induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin- induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule.

Summary. Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet-associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two proteinspecific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti-GP Ib/IX autoantibodies in 76 patient samples. The protein-specific assays were able to differentiate immune from non-immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.

This report describes studies into the pathophysiology of heparin- induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin- induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule.

Multimerin 1 (MMRN1) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family. In vivo, MMRN1 is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRN1 binding to activated platelets and endothelial cells, we investigated the identity of the major MMRN1 receptors on these cells using wild-type and RGE-forms of recombinant MMRN1. Ligand capture, cell adhesion, ELISA and flow cytometry analyses of platelet-MMRN1 binding, indicated that MMRN1 binds to integrins alphaIIbbeta3 and alphavbeta3. Endothelial cell binding to MMRN1 was predominantly mediated by alphavbeta3 and did not require the MMRN1 RGD site or cellular activation. Like many other alphavbeta3 ligands, MMRN1 had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRN1 processing, indicated that MMRN1 adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRN1 attachment to cells and indicate that MMRN1 is a ligand for alphaIIbbeta3 and alphavbeta3.

Treatment options for patients with chronic refractory immune thrombocytopenic purpura (ITP) are limited. Because combination immunosuppressant therapy appeared to be effective in ITP and other disorders, we used this approach in patients with particularly severe and refractory ITP. In this retrospective, observational study, we determined the response (platelet count above 30 ؋ 10 9 /L and doubling of baseline) among 19 refractory ITP patients. Treatment consisted of azathioprine, mycophenolate mofetil, and cyclosporine. The patients had failed a median of 6 prior treatments, including splenectomy (in all except 1 IntroductionImmune thrombocytopenic purpura (ITP) is an acquired bleeding disorder characterized by autoantibody-mediated platelet destruction and impaired platelet production. Patients with chronic refractory ITP have the highest risk of death and disease-related or therapy-related complications. 1,2 Treatment options include aggressive immunosuppressant therapy, and most recently thrombopoietin (TPO) receptor agonists. 3,4 Single-agent immunosuppressant drugs such as azathioprine and cyclosporine have been used to treat refractory patients with moderate success 5 ; however dose escalation can cause morbidity, and other options are needed.Over the past several decades, physicians have noted that greater efficacy can be achieved using a combination of unrelated but synergistic medications. 6,7 In this report, we describe our experience using a combination of azathioprine, mycophenolate mofetil, and cyclosporine to treat patients with particularly severe and refractory ITP. MethodsPatients in this report had a platelet count less than 20 ϫ 10 9 /L that persisted for at least 12 months with an inadequate or transient response to multiple therapies. The senior author (J.G.K.) offered the option of a combination of immunosuppressant therapy. Patients with comorbidities such as liver failure or uncontrolled hypertension were not offered this treatment. Institutional Review Board approval from McMaster University was obtained to retrospectively review the medical charts of all patients with ITP treated in our clinic; this report describes only those patients treated with combination immunosuppressant therapy. Institutional Review Board approval was not required for the administration of the combination of immunosuppressant agents (each on its own an accepted therapy for ITP 8 ), which was given per clinical need.Medical records of each patient were reviewed by 3 independent assessors and data were abstracted in triplicate and verified for consistency. Platelet count measurements and follow-up visits were done as per routine care and mean monthly platelet counts were calculated. Target doses of immunosuppressant medications were azathioprine 2 mg/kg per day; mycophenolate mofetil 1 to 2 g/d; and cyclosporine 2 mg/kg per day. Low-dose cyclosporine was chosen to minimize toxicity and avoid the need for drug level monitoring.We defined overall response as a platelet count level of 30 ϫ 10 9 /L or higher and do...

We describe a prospective study comparing four different assays for PAIgG. Platelets from patients with a variety of thrombocytopenic disorders were collected into ACD, washed, and the PAIgG then measured using three assays for surface PAIgG. These included: (a) a direct binding assay using 125I-monoclonal anti-IgG (MoAb); (b) a direct binding assay using 125I-staphylococcal protein A (SPA); and (c) a two-stage assay. PAIgG also was measured using an assay for 'total' PAIgG following platelet lysis. The mean +/- SD number of molecules of IgG per platelet on washed platelets from 29 healthy, non-thrombocytopenic controls was: 86 +/- 80 (125I-MoAb); 94 +/- 96 (125I-SPA); 3520 +/- 1890 (two-stage surface assay); and 10,850 +/- 3720 (total PAIgG). A total of 62 different patients with idiopathic thrombocytopenic purpura or thrombocytopenia complicating systematic lupus erythematosus, and 73 different patients with 'non-immune' thrombocytopenia, were tested using each of the four assays. These 'non-immune' thrombocytopenic patients included patients with carcinoma, septicaemia, pre-eclampsia, chronic leukaemia, thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome, acute leukaemia and myelodysplasia. All four assays gave similar results for both the immune and non-immune thrombocytopenic patients. The sensitivity of the assays for the most severely thrombocytopenic patients with immune thrombocytopenia was: MoAb 60%; SPA 88%; two-stage 82%; and 'total' PAIgG 88%. The specificity of the four assays in the non-immune thrombocytopenic patients was 57% 'total' PAIgG; 63% two-stage surface; 25% SPA; 38% MoAb.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive and specific test was used to identify a platelet-agglutinating factor in sera from patients with thrombotic thrombocytopenic purpura. Serum from patients plus a preparation rich in large multimers of factor VIII: von Willebrand factor were added to target platelets, and agglutination occurred in 41 of 48 samples. Edetic acid, heparin, or heating, but not aspirin, monomeric IgG, or dansylarginine N-(3-ethyl-1,5-pentanediyl)amide inhibited the platelet-agglutinating factor. In-vitro agglutination requires the presence of a platelet-agglutinating factor and large multimers of von Willebrand factor. High concentrations of either component lowers the amount of the other required for platelet agglutination. Some patients may be more susceptible to the agglutinating factor because of a congenital or acquired abnormality in processing unusually large multimers of von Willebrand factor or because of infections or inflammatory disorders that lead to increased synthesis of large multimers of von Willebrand factor.