Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

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.     

Impaired oxygenation of gastric mucosa in portal hypertension

Increased susceptibility to mucosal damage is a prominent feature of portal hypertensive gastropathy. Since the portal hypertensive gastric mucosa has extensive microvascular changes, we postulated that the increased sensitivity to mucosal damage could have an ischemic basis. We measured distribution of gastric serosal and mucosal oxygenation in a group of portal hypertensive and sham-operated rats, and then studied the effects of intragastric aspirin. In the basal state, gastric mucosa of portal hypertensive rats had significantly reduced oxygenation compared to controls (24±5 vs 45±7 mm Hg PO ₂,P < 0.02), while serosal oxygenation was similar between the two groups. Intragastric aspirin produced significantly greater mucosal damage to portal hypertensive rats and mucosal oxygenation was almost one third that of sham-operated controls. Systemic arterial pressures and oxygenation were similar between the two groups. We conclude that there is impairment of gastric mucosal oxygenation and increased mucosal damage by aspirin in portal hypertensive rats compared with sham-operated controls. These results support our hypothesis that the increased sensitivity of the portal hypertensive mucosa to damage is a consequence of impaired mucosal oxygenation.This study was supported by the Veterans Administration Research Service.This work was presented, in part, at the Plenary Session of the American Association for the Study of Liver Diseases, May 1988, New Orleans, Louisiana.     

Novel erythropoiesis stimulating protein (darbepoetin alfa) alleviates anemia associated with chronic inflammatory disease in a rodent model

OBJECTIVE: We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy. METHODS: Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly. RESULTS: Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing. CONCLUSION: ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.     

Autologous bone marrow transplantation for patients with relapsed Hodgkin's disease.

PURPOSE: High-dose therapy and autologous bone marrow transplantation (ABMT) are being increasingly utilized for the management of patients with relapsed Hodgkin's disease. Because patients with relapsed Hodgkin's disease often initially respond to salvage chemotherapy regimens, ABMT is frequently delayed until late in the course of the disease. The optimal timing for ABMT has not been identified. The purpose of this study was to determine the value of ABMT earlier in the course of Hodgkin's disease. PATIENTS AND METHODS: We treated 70 patients between October 1984 and October 1988 with high-dose cyclophosphamide, carmustine, and etoposide, followed by infusion of previously cryopreserved autologous bone marrow, and analyzed the results to determine the impact of timing of ABMT on treatment outcome. One (17 patients), two (24 patients), or three or more (29 patients) chemotherapy regimens had failed in patients before ABMT. RESULTS: The results for all 70 patients included a complete remission rate of 59%, an early death rate of 11%, a 4-year survival of 47%, and 27% of all treated patients alive and in complete remission at 4 years. The median follow-up for patients remaining in complete remission is 56 months (range 26 to 73 months). The frequency of achieving a complete remission was higher in patients in whom fewer regimens had failed before ABMT (i.e., 82% versus 58% versus 45%, p = 0.02), as was the 4-year disease-free survival (i.e., 44% versus 33% versus 21%, p = 0.04). CONCLUSION: ABMT is a more effective therapy when used early for patients with relapsed Hodgkin's disease.     

Role of kidney in the catabolic clearance of human platelet antiheparin proteins from rat circulation

Stimulated platelets release at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) from which beta-thromboglobulin (beta TG) is derived. We have found previously marked elevation of LA-PF4/beta TG antigen in platelet poor plasma of patients with chronic renal failure, whereas levels of PF4 remained normal. Therefore, we examined the role of the kidneys in the metabolic clearance of LA-PF4/beta TG and PF4. The supernates of aggregates of thrombin-stimulated human platelets were injected into sham operated control rats, nephrectomized rats, and into rats with acute ureteral ligation. The disappearance of human LA-PF4/beta TG antigen and PF4 in rat plasma determined by specific radioimmunoassays followed biphasic exponential curves. The half-lives (t1/2) for the fast and slow components of LA-PF4 in control rats were 6.4 and 68.4 min. Nephrectomy significantly increased these times to 9.7 and 144 min, while ureteral ligation resulted in no significant change. Comparison of the level of LA-PF4/beta TG antigen and of creatinine in aorta and in renal vein showed 25%-30% extraction of these compounds by the kidney. Less than 0.1% of the total LA-PF4 antigen injected was recovered in the urine of control rats. In contrast to these results, the clearance of PF4 was not affected by nephrectomy. In conclusion: (1) functional renal tissue is necessary for normal clearance of LA- PF4/beta TG, but renal excretion does not play a major role in its elimination suggesting that the protein is catabolized by the kidney; and (2) catabolic clearance of PF4 does not depend on functioning kidney tissue.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Inferior Vena Caval and Right Atrial Thrombosis Complicating an Amebic Hepatic Abscess

We describe a 50-yr-old black laborer who presented with right lower chest pain, weight loss, and pedal edema. Ultrasonography and computed tomograms showed a large abscess cavity in the right lobe of the liver which extended very close to the inferior vena cava. The lumen of the adjacent inferior vena cava was partially occluded by thrombus, which could be traced up into the cavity of the right atrium. The hepatic veins were normally patent. Sterile blood-stained pus was aspirated from the abscess. Antibodies against Entamoeba histolytica were present in high titer in the patient's serum. Although propagation of hepatocellular carcinoma into the inferior vena cava and even up into the right atrium is well recognized, inferior vena caval thrombosis extending up into the right atrium has not hitherto been reported as a complication of amebic hepatic abscess.     

Factor V deficiency in Philadelphia-positive chronic myelogenous leukemia

Factor V deficiency has been identified in 8 of 8 patients 7--20 yr of age, with Philadelphia-positive (Ph1+) chronic myelogenous leukemia (CML). In these 8 patients, factor V deficiency was not due to hepatic dysfunction, factor V inhibitors, or disseminated intravascular coagulation. In 3 patients, factor V activity rose 10%--12% (0.10--0.12 U/ml) after the infusion of 28--31 ml/kg body weight of fresh frozen plasma (FFP). The rise persisted less than 14 hr. The mean measured postinfusion rise in factor V was 18% of the expected rise calculated from the volume of FFP infused in the patients' plasma volume. In 4 patients, a small transient rise in factor V activity occurred after splenectomy or plateletpheresis. Factor V deficiency was completely corrected after a marked reduction in bone marrow cellularity in 2 patients with Ph1+ CML treated with extensive chemotherapy, total body irradiation, and bone marrow transplantation. Factor V deficiency was retrospectively observed in 6 of 20 patients, ages 20--80 yr, with Ph1+ CML and 3 of 6 patients with other myeloproliferative disorders. The factor V deficiency appears to be associated with the large myeloid- megakaryocytic cell mass characteristic of CML and other myeloproliferative disorders.