Dual, differential isotope labeling shows the preferential movement of labile plant constituents into mineral-bonded soil organic matter.

The formation and stabilization of soil organic matter (SOM) are major concerns in the context of global change for carbon sequestration and soil health. It is presently believed that lignin is not selectively preserved in soil and that chemically labile compounds bonding to minerals comprise a large fraction of the SOM. Labile plant inputs have been suggested to be the main precursor of the mineral-bonded SOM. Litter decomposition and SOM formation are expected to have temperature sensitivity varying with the lability of plant inputs. We tested this framework using dual (13) C and (15) N differentially labeled plant material to distinguish the metabolic and structural components within a single plant material. Big Bluestem (Andropogon gerardii) seedlings were grown in an enriched (13) C and (15) N environment and then prior to harvest, removed from the enriched environment and allowed to incorporate natural abundance (13) C-CO2 and (15) N fertilizer into the metabolic plant components. This enabled us to achieve a greater than one atom % difference in (13) C between the metabolic and structural components within the plant litter. This differentially labeled litter was incubated in soil at 15 and 35 °C, for 386 days with CO2 measured throughout the incubation. After 14, 28, 147, and 386 days of incubation, the soil was subsequently fractionated. There was no difference in temperature sensitivity of the metabolic and structural components with regard to how much was respired or in the amount of litter biomass stabilized. Only the metabolic litter component was found in the sand, silt, or clay fraction while the structural component was exclusively found in the light fraction. These results support the stabilization framework that labile plant components are the main precursor of mineral-associated organic matter.

Controls on soil carbon sequestration and dynamics: lessons from land-use change.

Soil carbon (C) dynamics and sequestration are controlled by interactions of chemical, physical and biological factors. These factors include biomass quantity and quality, physical environment and the biota. Management can alter these factors in ways that alter C dynamics. We have focused on a range of managed sites with documented land use change from agriculture or grassland to forest. Our results suggest that interactions of soil type, plant and environment impact soil C sequestration. Above and below ground C storage varied widely across sites. Results were related to plant type and calcium on sandy soils in our Northern sites. Predictors of sequestration were more difficult to detect over the temperature range of 12.4°C in the present study. Accrual of litter under pines in the moist Mississippi site limited C storage in a similar manner to our dry Nebraska site. Pre-planting heterogeneity of agricultural fields such as found in Illinois influences C contents. Manipulation of controls on C sequestration such as species planted or amelioration of soil quality before planting within managed sites could increase soil C to provide gains in terrestrial C storage. Cost effective management would also improve soil C pools positively affecting soil fertility and site productivity.

Experimental warming shows that decomposition temperature sensitivity increases with soil organic matter recalcitrance.

Soil C decomposition is sensitive to changes in temperature, and even small increases in temperature may prompt large releases of C from soils. But much of what we know about soil C responses to global change is based on short-term incubation data and model output that implicitly assumes soil C pools are composed of organic matter fractions with uniform temperature sensitivities. In contrast, kinetic theory based on chemical reactions suggests that older, more-resistant C fractions may be more temperature sensitive. Recent research on the subject is inconclusive, indicating that the temperature sensitivity of labile soil organic matter (OM) decomposition could either be greater than, less than, or equivalent to that of resistant soil OM. We incubated soils at constant temperature to deplete them of labile soil OM and then successively assessed the CO2-C efflux in response to warming. We found that the decomposition response to experimental warming early during soil incubation (when more labile C remained) was less than that later when labile C was depleted. These results suggest that the temperature sensitivity of resistant soil OM pools is greater than that for labile soil OM and that global change-driven soil C losses may be greater than previously estimated.

Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties.

We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase.

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Recovery of prostacyclin production by de-endothelialized rabbit aorta. Critical role of neointimal smooth muscle cells.

Prostacyclin (PGI2) synthetic capacity was assayed at the surface of aortas at various intervals after removal of endothelium with a balloon catheter. Results were correlated with morphologic changes in the vessel wall seen by light microscopy, scanning and transmission electron microscopy. To assay PGI2 synthetic capacity, we applied an incubation chamber to the luminal surface of the aortas; after arachidonic acid stimulation we assayed the PGI2 synthesized with a bioassay and radioimmunoassay. PGI2 synthesis in de-endothelialized aortas was determined immediately after balloon-catheter injury and at intervals of 1 h and 2, 4, 15, 35, and 70 d. PGI2 synthesis was low at 1 h and increased over time with levels at 35 and 70 d reaching that of normal artery. Scanning and transmission electron microscopy of de-endothelialized areas showed persistent absence of endothelium with formation of a neointima composed of smooth muscle cells. De-endothelialized aorta was covered with adherent platelets shortly after injury, however several days later only a few platelets adhered to the denuded surface. Results indicated that (a) endothelium is responsible for nearly all PGI2 production at the luminal surface of the normal aorta, (b) de-endothelialized muscular neointima synthesized increasing quantities of PGI2 with time after injury, and (c) increase of PGI2 production at the luminal surface of de-endothelialized aorta correlates with formation of a neointima and with the acquired thromboresistance of the aorta.

Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3' alternative splicing variant of acetylcholinesterase.

The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)2 vitamin D3 and 17beta-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3' terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

Multifocal vascular tumors in fowl induced by a newly isolated retrovirus.

The morphological features of a spontaneous, multifocal vascular neoplasm of chickens are described. Histologically, the tumor was characterized by areas consisting of freely anastomosing vascular channels with prominent papillary appearance and lined by bland-looking endothelial cells, which alternate with areas resembling cavernous hemangioma. Occasionally solid areas composed of plump, pleomorphic cells were also present. Although there was no clear evidence for metastatic spread, some tumors were obviously invasive. Electron microscopy and immunohistochemistry confirmed the endothelial nature of neoplastic cells, demonstrating in particular pinocytic vesicles, well developed junctional complexes, fragmented basal lamina, occasional Weibel-Palade bodies, and patchy factor VIII-related antigen immunoreactivity. The overall appearance of the tumor was that of a cavernous hemangioma with prominent papillary endothelial hyperplasia. Previously we have shown that the tumor was induced by a newly isolated strain of avian hemangioma retrovirus and in this study we demonstrated typical type C retrovirus particles in the tumor by electron microscopy. It is suggested that this retrovirus-induced avian tumor may serve as a useful model for the study of transformed endothelia and other vascular tumorigenesis.

Effects of irradiation on the release of growth factors from cultured bovine, porcine, and human endothelial cells.

The effects of radiation on the release of mitogenic factors into the media of cultured endothelial cells of bovine, porcine, and human origins were studied. Although unirradiated controls revealed a significant background activity, single doses of irradiation (20-60 Gy) resulted in a dose-related increased release of growth factor activity, measured by the mitogenic effects of the conditioned media on both 3T3 mouse fibroblasts and unirradiated endothelial cells serving as target cells. Receptor binding competition assays for the platelet-derived growth factor receptor revealed that 12-28% of the total mitogenic activity was due to platelet-derived growth factor-like mitogens. Mitogenic assays using endothelial cells and specific antibody mediated inhibition assays suggested that another component of the mitogenic activity was due to a fibroblast growth factor-like factor. Although radiation resulted in a significant increase in cell death, the enhanced growth factor activities did not appear to result from cell lysis-related leakage of intracellular stores of growth factor. Instead, our data suggest that the growth factors were synthesized de novo and secreted at elevated levels by the cells which maintained postradiation a high level of metabolic activity. Time course studies demonstrated that the growth factors accumulation in the conditioned media started within the first 24 h after radiation and reached a plateau within 72 h after treatment. Radiation-induced release of endothelial cell-derived growth factors may be involved in the pathogenesis of both early vascular damage and the late fibrosis which represents a prominent feature of late radiation damage in normal tissues.

Molecular composition of soil organic matter with land-use change along a bi-continental mean annual temperature gradient.

Soil organic matter (SOM) is critical for maintaining soil fertility and long-term agricultural sustainability. The molecular composition of SOM is likely altered due to global climate and land-use change; but rarely are these two aspects studied in tandem. Here we used molecular-level techniques to examine SOM composition along a bi-continental (from North to South America) mean annual temperature (MAT) gradient from seven native grassland/forest and cultivated/pasture sites. Biomarker methods included solvent extraction, base hydrolysis and cupric (II) oxide oxidation for the analysis of free lipids of plant and microbial origin, ester-bound lipids from cutin and suberin, and lignin-derived phenols, respectively. Solid-state 13C nuclear magnetic resonance (NMR) was used to examine the overall composition of SOM. Soil cultivation was found to increase the amount of microbial-derived compounds at warmer temperatures (up to 17% increase). The cultivated soils were characterized by much lower contributions of plant-derived SOM components compared to the native soils (up to 64% lower at the coldest site). In addition, cultivation caused an increase in lignin and cutin degradation (up to 68 and 15% increase, respectively), and an increase in the amount of suberin-derived inputs (up to 54% increase). Clear differences in the molecular composition of SOM due to soil cultivation were observed in soils of varying mineral composition and were attributed to disturbance, different vegetation inputs, soil aggregate destruction and MAT. A high organic allophanic tropical soil was characterized by its protection of carbohydrates and nitrogen-containing compounds. The conversion of native to cultivated land shows significant shifts in the degradation stage of SOM. In particular, cutin-derived compounds which are believed to be part of the stable SOM pool may undergo enhanced degradation with long-term cultivation and disruption of soil aggregates. On a per year basis, the total amount of cutin decreased only at the two forest sites that were converted to pasture, likely due to cutin degradation or to changes in vegetation and litter quality associated with land-use change. Overall, our study highlights that the implementation of different agricultural management practices enhances the degradation of recalcitrant SOM compounds that may become a source of atmospheric CO2 with increasing land-use and climate change.

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.

Thromboembolism in patients with the 'lupus'-type circulating anticoagulant.

Thirty-five patients with the lupus anticoagulant (LA) were followed up between 1975 and 1982. The most prevalent clinical manifestation occurring in these patients was thrombosis. Nineteen patients (54.3%) had a single or recurrent thrombotic episode. Fifteen patients (42.8%) had venous thrombosis, and arterial thrombosis manifested by stroke or transient ischemic attacks occurred in six patients. Bleeding occurred in only five patients, four of whom had severe thrombocytopenia, while no excessive bleeding was noted during 18 operative procedures. Various therapeutic regimens, including corticosteriods, nonsteroidal anti-inflammatory drugs, cytotoxic or immunosuppressive agents, had no effect on the presence of the LA in these patients. Anticoagulants were successful in the treatment and prevention of thrombotic episodes.

Pulmonary valve stenosis and hemophilia A. Report of three cases and discussion of a possible genetic linkage.

We treated three unrelated patients with hemophilia A and congenital pulmonary valve stenosis. In two patients, the occurrence of the cardiac malformation was sporadic and in one familial. The coexistence of hemophilia A and pulmonary valve stenosis might suggest a genetic linkage for both disorders. Review of the literature supports the hypothesis that the inheritance of pulmonary valve stenosis is dominant and X-linked.

Effect of bone marrow T lymphocytes treated with CAMPATH 1G on megakaryocyte colony formation.

Specific populations of lymphocytes play a significant role in the regulation of megakaryocyte (MK) development. CAMPATH 1G (C1G) and CAMPATH 1M (C1M) are T lymphocyte (TL)-specific monoclonal antibodies (MABs) that are routinely employed to reduce graft-vs.-host disease (GVHD) in allogeneic bone marrow transplantation (BMT). Following BMT, engraftment of the erythroid and myeloid lineages is prompt, but prolonged thrombocytopenia often prevails. We therefore studied the effect on MK colony formation of treating donor bone marrow (BM) with the CAMPATH MABs. MK colonies were grown in plasma clots using postirradiation aplastic canine serum (PICS-J) as MK colony-stimulating factor (MK-CSF). C1M, which causes TL destruction by complement-dependent lysis, had no effect on MK cloning efficiency. C1G is not lytic but causes the elimination of TL in the BMT recipients via antibody-dependent cell cytotoxicity (ADCC). Treatment of donor BM with C1G significantly enhanced the number of early burst-forming units (BFU-MK) and late colony-forming units (CFU-MK) and had no effect on granulocyte-macrophage (CFU-GM) or erythroid (BFU-E) colonies. Enhancement of MK cloning efficiency was concentration-dependent between 0.03 and 3 micrograms MAB/10(6) BM mononuclear cells (MNC). Similar results were observed when C1G-treated TL or purified CD4+ TL were co-cultured with untreated autologous BMMNC or peripheral blood (PB) MNC. Conditioned medium (CM) from C1G-treated TL and CD4+ TL contained soluble factors that, when combined with suboptimal doses of PICS-J, potentiated MK growth. C1G in combination with PICS-J also stimulated TL proliferation in a dose-dependent manner. The T cell CM did not contain elevated levels of interleukin-3 (IL-3), IL-6, IL-1 beta, or GM-CSF. Our data provide additional evidence for the involvement of activated TL, and perhaps novel soluble T cell products, in the immunomodulation of megakaryocytopoiesis.

Ex vivo expansion of megakaryocyte precursors by preincubation of marrow allografts with interleukin-3 and granulocyte-macrophage colony-stimulating factor in vitro.

Protracted thrombocytopenia and bleeding remain serious complications in bone marrow transplantation (BMT). Major progress has been made in facilitating myeloid and erythroid engraftment, but little has been made in accelerating thrombopoiesis post-BMT. We report that in vitro preincubation of T cell-depleted BM allografts with a combination of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 microgram/mL each) (n = 8), for 3 days prior to infusion, expands megakaryocyte (MK) precursors. MK-progenitor proliferation was assessed in plasma clot colony assays and liquid cultures following pre-exposure to IL-3/GM-CSF. We observed a 2.8-fold increase in the number of colony-forming units-megakaryocyte (CFU-MK) (17.3 +/- 5.2 vs. 6.1 +/- 3.4) (p = 0.001) and a two-fold increase in burst-forming units-megakaryocyte (BFU-MK) (0.2 vs. 0.1) (p = 0.01) per 2 x 10(5) cells/mL compared to control BM samples cultured for 3 days in medium alone. In secondary cultures, the continued presence of IL-3 and GM-CSF increased the number of CFU-MK by 200-fold (p < 0.0001) over controls and by 9.7-fold over fresh BM. A 33-fold increase (p < 0.0001) in the number of BFU-MK was elicited compared to controls. In addition, IL-3 plus GM-CSF supported increased cellularity within the colonies. The presence of IL-3 or GM-CSF alone resulted in fewer MK colonies and fewer cells per colony than both cytokines combined. In liquid cultures, the percentage of cells expressing platelet glycoprotein (GP) IIb/IIIa in the continued presence of IL-3 and GM-CSF increased following preincubation, yielding a total of 16.0 +/- 2.3 x 10(4) MK/2 x 10(6) cells at day 10 of culture. We propose that ex vivo preincubation with IL-3 and GM-CSF can expand the number of MK precursors and may facilitate platelet recovery post-BMT.

The role of the leech in medical therapeutics.

Leeching is considered by many to be a discredited medical relic of the past. This view is not justified, since leeches still play an important part in modern medicine, as in microsurgery and in the treatment of patients with post-phlebitic syndrome. Hirudin, the potent thrombin inhibitor of leech saliva, has been cloned and is used in the treatment of cardiological and hematological disorders. In our search for other antihemostatic factors in Hirudo medicinalis saliva, we found inhibitors of platelet aggregation induced by thrombin, collagen, adenosine 5'-diphosphate, epinephrine, platelet-activating factor and arachidonic acid. We purified apyrase (adenosine 5'-triphosphate diphosphohydrolase), which is a non-specific inhibitor of platelet aggregation by virtue of its action on adenosine 5'-diphosphate. We isolated and characterized the platelet-activating factor antagonist and also identified and recovered an inhibitor of coagulation factor Xa from leech saliva. This report summarizes our findings and those of other investigators, as well as the experience of one of us (A.E.) in leech therapy.

The changing course of thrombotic thrombocytopenic purpura and modern therapy.

Thrombotic thrombocytopenic purpura (TTP) is a multisystem disorder of unknown etiology. Pathologically, there appears to be an abnormal interaction between the vascular endothelium and platelets, but the primary event remains uncertain. While historically, TTP was a fatal disease, dramatic improvement in its outcome has occurred over the past two decades with the development of effective therapy. Plasma infusion or exchange remains the cornerstone of the treatment of TTP, along with corticosteroids, platelet inhibitor drugs, vincristine and splenectomy. This review summarizes the clinical findings, what is known of the pathogenesis and the available therapeutic modalities for TTP. In most cases, remissions can be attained and cures are now common. However, approximately half the patients will relapse. The clinical course at relapse is usually milder than the disease at presentation and less aggressive therapy may be needed. However, relapsing TTP still carries a significant mortality and preventive therapies are not always effective. Further progress may have to await an understanding of the fundamental etiology of this disease.

Unexplored territories in the nonsurgical patient: a look at pregnancy.

Venous thromboembolism (VTE) remains the most common cause of maternal death during pregnancy and the puerperium. The risk is increased in women older than 35 years and those with obesity, previous VTE, operative delivery, and underlying thrombophilia. Anticoagulant therapy is indicated for short-term treatment of VTE and as thromboprophylaxis in high-risk patients. Warfarin is contraindicated during the first trimester because of fetotoxicity; unfractionated heparin (UFH) is associated with practical disadvantages and a risk of heparin-induced thrombocytopoenia (HIT) and osteoporosis with long-term use. Low-molecular-weight heparins (LMWHs) are convenient to use, do not cross the placenta, carry a lower risk of HIT and osteoporosis, and have been shown to be safe and effective during treatment of approximately 500 pregnant women. LMHWs are increasingly replacing UFH as the anticoagulant of choice during pregnancy; further studies are required to determine optimal therapeutic and thromboprophylactic doses. Women with inherited or acquired thrombophilia are at increased risk of severe pregnancy complications, including recurrent miscarriage, pre-eclampsia, fetal growth restriction, abruptio placentae, and stillbirth; uteroplacental microvascular thrombosis caused by thrombophilia appears to be the pathophysiologic link. LMWHs have been shown to improve pregnancy outcome in women with a history of obstetric complications and confirmed thrombophilia.

A mouse model for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) occurs in 1% to 3% of patients receiving heparin and results from the development of antibodies that recognize heparin-platelet factor 4 (H-PF4) complexes that form on the surface of activated platelets and on the vascular endothelium. With the aim of studying the pathogenic importance of these anti-H-PF4 antibodies in vivo, we attempted to create an animal model of HIT. Such a model was produced by immunization of naive mice with affinity-purified IgG anti-H-PF4 antibodies from two patients with HIT. The immunized mice developed specific antibodies (anti-idiotypic) against the human anti-H-PF4 antibodies and 2 months later, anti-anti-idiotypic antibodies appeared, which functionally resembled the human HIT antibody. Indeed, when the animals bearing anti-anti-idiotypic antibodies were injected with heparin for 4 days, a significant decrease in their platelet counts was observed; however, heparin treatment was not associated with thrombosis in any of the immunized mice. Similar to the observation in HIT patients, injections of equivalent doses of low-molecular-weight (LMW) heparin to the immunized animals did not induce thrombocytopenia. The results of this study support the importance of anti-H-PF4 antibodies in the pathogenesis of HIT. The mouse HIT model may provide a convenient system for studies on the immunoregulation of anti-H-PF4 expression and for evaluation of potential therapeutic modalities.