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BackgroundBefore the official US Food and Drug Administration approval in 2021, pediatric hematologists across the United States have used direct oral anticoagulants (DOACs) "off-label" and based on extrapolation from labeling for adults with venous thromboembolism (VTE) and interim results of pediatric-specific DOAC clinical studies.ObjectivesThe American Thrombosis and Hemostasis Network 15 (ATHN 15) study aimed to characterize the use of DOACs from 2015 to 2021 at 15 specialized pediatric hemostasis centers in the United States, with emphasis on safety and effectiveness.MethodsEligible participants were those aged 0 to 21 years who had a DOAC included as part of their anticoagulation regimen for the treatment of acute VTE or secondary prevention of VTE. Data were collected for up to 6 months after initiation of the DOAC.ResultsA total of 233 participants were enrolled, with a mean age of 16.5 years. Rivaroxaban was the most commonly prescribed DOAC (59.1%) followed by apixaban (38.8%). Thirty-one (13.8%) participants reported bleeding complications while on a DOAC. Major or clinically relevant nonmajor bleeding events occurred in 1 (0.4%) and 5 (2.2%) participants, respectively. Worsening menstrual bleeding was reported in 35.7% of females aged >12 years and occurred more frequently in those using rivaroxaban (45.6%) compared with apixaban (18.9%). The recurrent thrombosis rate was 4%.ConclusionPediatric hematologists at specialized hemostasis centers in the United States have been using DOACs for the treatment and prevention of VTEs, primarily in adolescents and young adults. Reported DOAC use showed adequate safety and effectiveness rates.

BackgroundCOVID-19–related acute illness is associated with an increased risk of venous thromboembolism (VTE).ObjectiveThese evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in decisions about the use of anticoagulation for thromboprophylaxis in patients with COVID-19 who do not have confirmed or suspected VTE.MethodsASH formed a multidisciplinary guideline panel, including 3 patient representatives, and applied strategies to minimize potential bias from conflicts of interest. The McMaster University GRADE Centre supported the guideline development process, including performing systematic evidence reviews (up to March 2021). The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The panel used the grading of recommendations assessment, development, and evaluation (GRADE) approach to assess evidence and make recommendations, which were subject to public comment.ResultsThe panel agreed on 1 additional recommendation. The panel issued a conditional recommendation against the use of outpatient anticoagulant prophylaxis in patients with COVID-19 who are discharged from the hospital and who do not have suspected or confirmed VTE or another indication for anticoagulation.ConclusionsThis recommendation was based on very low certainty in the evidence, underscoring the need for high-quality randomized controlled trials assessing the role of postdischarge thromboprophylaxis. Other key research priorities include better evidence on assessing risk of thrombosis and bleeding outcomes in patients with COVID-19 after hospital discharge.

Background: COVID-19–related critical illness is associated with an increased risk of venous thromboembolism (VTE). Objective: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in making decisions about the use of anticoagulation for thromboprophylaxis in patients with COVID-19–related critical illness who do not have confirmed or suspected VTE. Methods: ASH formed a multidisciplinary guideline panel that included 3 patient representatives and applied strategies to minimize potential bias from conflicts of interest. The McMaster University Grading of Recommendations Assessment, Development and Evaluation (GRADE) Centre supported the guideline development process by performing systematic evidence reviews (up to 5 March 2021). The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The panel used the GRADE approach to assess evidence and make recommendations, which were subject to public comment. This is an update on guidelines published in February 2021. Results: The panel agreed on 1 additional recommendation. The panel issued a conditional recommendation in favor of prophylactic-intensity over intermediate-intensity anticoagulation in patients with COVID-19–related critical illness who do not have confirmed or suspected VTE. Conclusions: This recommendation was based on low certainty in the evidence, which underscores the need for additional high-quality, randomized, controlled trials comparing different intensities of anticoagulation in critically ill patients. Other key research priorities include better evidence regarding predictors of thrombosis and bleeding risk in critically ill patients with COVID-19 and the impact of nonanticoagulant therapies (eg, antiviral agents, corticosteroids) on thrombotic risk.

We report the clinical and laboratory coagulation characteristics of 27 pediatric and young adult patients (2 months to 21 years) treated for symptomatic COVID-19 at a children’s hospital in the Bronx, New York between March 1 and May 31, 2020. D-Dimer was > 0.5 ug/mL (upper limit of normal) in 25 (93%) patients at admission; 11 (41%) developed peak D-Dimer > 5 ug/mL during admission. Seven (26%) patients developed venous thromboembolism: three with deep vein thrombosis and four with pulmonary embolism. Requirement of increased ventilatory support was a risk factor for thrombosis (p=0.006). Three of eight (38%) patients on prophylactic anticoagulation developed thrombosis, however no patients developed VTE on low molecular weight heparin prophylaxis titrated to anti-Xa level. Manifestation of COVID-19 disease was severe or critical in 16 (59%) patients. Four (15%) patients died of COVID-19 complications: all had comorbidities. Elevated D-dimer and increased VTE rate were observed in this young cohort, particularly in those with severe respiratory complications suggesting thrombotic coagulopathy. More data is needed to guide thromboprophylaxis in this age group.

Background: Coronavirus disease 2019 (COVID-19)–related critical illness and acute illness are associated with a risk of venous thromboembolism (VTE). Objective: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in decisions about the use of anticoagulation for thromboprophylaxis for patients with COVID-19–related critical illness and acute illness who do not have confirmed or suspected VTE. Methods: ASH formed a multidisciplinary guideline panel and applied strict management strategies to minimize potential bias from conflicts of interest. The panel included 3 patient representatives. The McMaster University GRADE Centre supported the guideline-development process, including performing systematic evidence reviews (up to 19 August 2020). The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including GRADE Evidence-to-Decision frameworks, to assess evidence and make recommendations, which were subject to public comment. Results: The panel agreed on 2 recommendations. The panel issued conditional recommendations in favor of prophylactic-intensity anticoagulation over intermediate-intensity or therapeutic-intensity anticoagulation for patients with COVID-19–related critical illness or acute illness who do not have confirmed or suspected VTE. Conclusions: These recommendations were based on very low certainty in the evidence, underscoring the need for high-quality, randomized controlled trials comparing different intensities of anticoagulation. They will be updated using a living recommendation approach as new evidence becomes available., Visual Abstract

New York City has emerged as one of the epicenters of the SARS-COV-2 pandemic, with the Bronx being disproportionately affected. This novel coronavirus has caused significant respiratory manifestations raising the concern for development of acute chest syndrome (ACS) in patients with sickle cell disease (SCD). We report a series of pediatric SCD SARS-COV-2-positive patients admitted with ACS. SARS-COV-2-positive SCD patients, who did not develop ACS, were the comparison group. Hydroxyurea use (P-value = .02) and lower absolute monocyte counts (P-value = .04) were noted in patients who did not develop ACS. These preliminary findings need to be further evaluated in larger cohorts.

Heavy menstrual bleeding (HMB) is a common complaint among adolescent girls. It reflects an abnormal volume of blood loss during the menstrual cycle. Abnormal uterine bleeding can manifest as HMB but includes menstrual irregularity. In many cases, immaturity of the hypothalamic-pituitary-ovarian axis or hormonal conditions like polycystic ovarian syndrome leading to anovulatory cycles are the underlying cause for heavy menses. However, in girls with HMB, especially those not responding to the usual hormonal attempts to manage HMB, an underlying bleeding disorder should be considered. Up to 62% of adolescents with HMB have a bleeding disorder, many without anemia at presentation. Evaluation for HMB in an adolescent girl should include referrals to an adolescent medicine specialist or gynecologist and pediatric hematologist. [ Pediatr Ann . 2020;49(4):e163–e169.]

Vitamin B12 (cobalamin) deficiency and its related hematological and neurological manifestations, of which pernicious and megaloblastic anemia are the most recognized entities, have puzzled the medical community for over a century. These conditions are described in all ages, race and ethnicities, continents, and socioeconomic groups. Among the etiologies identified in vitamin B12 deficiency, nutritional deficiency, including restrictive diets due to access to appropriate food items, food or oral aversion, or personal choice, is recognized in developed and developing countries. Other etiologies include inherited or genetic defect in the metabolism of vitamin B12, immune-mediated, or absorption-related defects. Understanding the pathophysiology of this phenomenon has allowed us to provide the adequate treatment to reverse the changes in the affected systems successfully.

Sickle Cell Disease (SCD) is a chronic hemolytic disorder associated with frequent pain episodes, end organ damage and a shortened lifespan. Currently there exist no disease specific targeted therapies for the treatment of acute vaso-occlusive crisis (VOC) and management with analgesics and hydration is purely supportive. Improvement in understanding of disease pathophysiology has resulted in a great interest in disease modifying novel therapies and many are being evaluated in clinical trials. Here we report the results from the pre-specified mid-point analysis of the Phase 2 study of Intravenous Gamma Globulin (IVIG) for the treatment of acute VOC in patients with SCD and lessons learned.

Coagulation disorders in the newborn are a challenge to caregivers. Abnormalities of the immature, complex coagulation system of the newborn can have life-threatening consequences. This review provides a summary of the most common newborn bleeding abnormalities faced by clinicians, as well as an approach to the diagnosis and management of these disorders.

Background: The coronavirus disease pandemic of 2019 (COVID-19) has been associated with coagulopathy and an increased rate of thrombosis in adults. Medical practitioners have been prompted to consider prophylactic anticoagulation in special populations diagnosed with COVID-19. Patients with sickle cell disease (SCD) are predisposed to a hypercoagulable state. Despite the concern for development of venous thromboembolism (VTE) in these patients, there are no standardized guidelines for routine thromboprophylaxis in either adults or children with SCD. Thus, VTE management options are often extrapolated from guidelines for the general population. Methods: A cross-sectional electronic survey was distributed to pediatric and adult hematology oncology practitioners through 7 SCD specific interest groups. Pediatric and adult practitioners were defined as those who medically manage patients 0-21 years of age and >21 years of age respectively. We examined responses to survey questions focused on routine thromboprophylaxis practices in children and adults with SCD prior to the pandemic and in patients with SCD admitted with COVID-19. Chi-square analyses or Fisher's exact tests for small samples were used to compare proportions as needed. Results: The survey was distributed to approximately 2,550 providers. Of 93 total responses, 14% (N=13) only treat patients >21yo; 38.7% (N=36) only treat patients 0-21yo and 47.3% (N=44) treat both. Nearly all adult practitioners (96.6%) would recommend pharmacologic prophylaxis, mechanical prophylaxis or both for hospitalized adults, but only 76% of pediatric treaters would recommend any prophylaxis (PPX) in hospitalized children (p21yo patients, 51% preferred to use both pharmacologic and non-pharmacologic PPX, 25% preferred pharmacologic alone, with just 5.3% preferring non-pharmacologic PPX only. Enoxaparin was the most frequently used anticoagulant among both pediatric and adult practitioners [78% vs 89% respectively] (Figure 2). Direct oral anticoagulants (DOACS) were infrequently recommended (3.8% for SCD children and 10.9% for adults); of these, rivaroxaban was used most often. The most common indication for starting thromboprophylaxis for both 0-21yo and >21yo patients was history of prior VTE [81% and 81% respectively], followed by hip replacement [57% and 75% respectively]. In patients admitted with COVID-19, prophylactic anticoagulation was recommended for adults by 94% of treaters. There was a significant increase in the use of prophylactic anticoagulation in children with COVID-19 vs. those without (84% vs. 40%; p=.0001). Figure 3 shows extended thromboprophylaxis practices upon discharge for COVID related admissions. Almost half of respondents (46%) would not recommend any thromboprophylaxis for children on discharge; 37% would recommend either 2-4 weeks or >4weeks of post-discharge PPX. The majority of adult providers would recommend discharge thromboprophylaxis for adults with 2 weeks post discharge (33%), and 2-4 weeks (30%) being the most common regimens. Conclusion: This pilot survey describes the thromboprophylaxis practices used for adult and pediatric SCD patients by specialty practitioners. In general, practitioners were likely to prescribe pharmacologic thromboprophylaxis for adults while mechanical PPX was preferred for children. However, in the high-risk setting of COVID-19 infection, pediatric practitioners would modify their practice to include pharmacologic thromboprophylaxis. Interestingly, despite the long-term availability of direct oral anticoagulants in adults, and recent completion of pediatric studies, the most commonly used pharmacologic agent pre-COVID in adults and children was enoxaparin. Of note, this survey was conducted prior to release of the International Society on Thrombosis and Haemostasis guidelines regarding discharge PPX in COVID-19. (Spyropoulos AC, J Thromb Haemost, 2020) These results highlight the influence of COVID-19 on the use of pharmacologic thromboprophylaxis, specifically in the pediatric population. Due to frequent hospitalization, studies are needed to guide decision making surrounding VTE PPX for the adult and pediatric inpatient SCD population. Disclosures Davila: Spire Learning: Speakers Bureau; ATHN: Other: Grant Funding. Minniti:Bluebird bio: Consultancy, Research Funding; TauTona: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Emmaus: Consultancy, Research Funding; CLS Bering: Consultancy; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding. Desai:Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; GBT, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ironwood Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Rockpointe Continuing Medical Education Company: Consultancy. O'Brien:Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Enoxaparin used as a form of thromboprophylaxis in children.

Background: There are currently four direct oral anticoagulants (DOACs) approved for the acute treatment and prevention of venous thromboembolism (VTE) in adults. Pediatric hematologists across the United States (US) are using DOACs for their patients based on extrapolated data from adult studies and recently published phase 3 pediatric-specific studies. (Brandao LR, et al. Blood, 2019 and Male C, et al. Lancet Haematol, 2020.) Because further data regarding DOAC use in children are needed, ATHN 15: Characterizing the Real-World Use of Direct Oral Anticoagulants in Pediatric Patients, was developed and is being sponsored by the American Thrombosis and Hemostasis Network (ATHN). The study is being conducted at ATHN-affiliated sites in the US and aims to characterize the real-world use of DOACs in children diagnosed with VTE. ATHN is a nonprofit network of over 140 federally funded hemophilia treatment centers (HTCs) that provides the infrastructure for clinical research and public health surveillance. Methods: Data being captured includes demographics, clinical characteristics, anticoagulation management, and treatment outcomes (bleeding and recurrent thrombosis) of patients Results: As of May 31, 2020, 76 patients from 9 sites have been enrolled (see Table 1 for demographics). Deep venous thrombosis (DVT) of the lower extremities or pelvis is the most prevalent VTE (n = 23, 30.26%), followed by DVT of the upper extremity or upper thorax and pulmonary embolism plus VTE (n = 22, 28.95 and n = 13, 17.1% respectively). 82.89% of patients did not have a history of prior VTE at the time of enrollment. Of the 15 patients with a radiologically confirmed anatomic anomaly, Paget Schroetter/Thoracic Outlet Syndrome and May-Turner Syndrome are the most common (n = 6, 40% and n = 4, 26.7%, respectively). Factor V Leiden heterozygosity, reported in 8 patients, is the most prevalent inherited thrombophilia. Forty-eight patients were identified as having a medical risk factor associated with their VTEs. Sixteen (33.3%) were classified as obese and another 13 (27.1%) had a hospital admission stay greater than 7 days and within 30 days prior to the VTE. At least one specific drug or environmental risk factor was reported in 44 patients. Concomitant use of hormonal contraception (currently taking/stopped within 4 weeks prior to VTE) and the presence of a central venous catheter (present at time of VTE or within 30 days prior to VTE) were the most commonly reported (n = 18, 40.9% and n = 15, 34.09%, respectively). Rivaroxaban is the most prescribed DOAC with 59.2% (n = 45) of patients using this agent. Apixaban and dabigatran use is also reported (n = 29; 38.2% and n = 2; 2.6% respectively). Of the 76 patients, 65 received a different anticoagulant prior to starting a DOAC. Enoxaparin and unfractionated heparin are most commonly prescribed prior to the institution of a DOAC regimen (n = 52, 80% and n = 17, 26.2%). Conclusions: Despite lack of an FDA-approved pediatric indication, hematologists in the US are already using DOACs for children with VTE. Most pediatric patients treated with a DOAC are older than 13 years of age, although we anticipate this will change as providers develop more comfort with these drugs. Interestingly, most of these patients were started on a different anticoagulation regimen prior to starting a DOAC. As enrollment continues, ATHN 15 will serve as a resource for pediatric hematologists to characterize the real-world use of DOACs in children. Further analysis will evaluate DOAC-specific utilization, efficacy and adverse events, including heavy menstrual bleeding. Disclosures Davila: Spire Learning: Speakers Bureau; ATHN: Other: Grant Funding. Corrales-Medina:Bayer: Consultancy; Takeda: Consultancy; Octapharma: Consultancy, Speakers Bureau. Raffini:XaTek: Other: Advisory Board; CSL Behring: Other: Advisory Board; HemaBiologics: Other: Advisory Board; Bayer: Other: Advisory Board; Roche: Other: Advisory Board. Thornburg:Sanofi Genzyme: Consultancy, Other: Data Safety Monitoring Board, Research Funding; NovoNordisk: Research Funding; Genentech: Speakers Bureau; Biomarin: Consultancy, Speakers Bureau; Bayer Pharmaceuticals: Research Funding; American Thrombosis and Hemostasis Network: Research Funding; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ironwood Pharmaceuticals: Consultancy, Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Spark Therapeutics: Consultancy. OffLabel Disclosure: Apixaban, Rivaroxaban, Dabigatran and Enoxaparin use in the pediatric population.

Thrombophilia refers to the propensity to develop thrombosis and can be a result of acquired and/or inherited conditions. Inherited conditions are usually identified by a blood test, where acquired conditions are characterized by a patient's clinical state or associated comorbidities. There has been a documented increase in the incidence of venous thromboembolism (VTE) in the pediatric population over the last two decades. (1) The incidence follows a bimodal pattern with neonates having the greatest risk of thromboembolism and a second peak in incidence during puberty and adolescence. The rise in VTE has been attributed to increase in medical availability and expertise in the care of children with fatal conditions and complex chronic disease. The incidence of idiopathic VTE is only 5% in children and

Sickle cell disease (SCD) is an inherited disorder of hemoglobin, with complex pathophysiology characterized by frequent painful episodes, hemolytic anemia, end-organ damage, and a shortened life span. The coagulation system is activated in SCD and has been evaluated as a therapeutic target in numerous clinical trials. These trials have not thus far demonstrated improvement in SCD-associated clinical outcomes such as pain events and rates, despite demonstrated improvement in biomarkers of activated hemostasis. Neurovascular endpoints are an attractive target and have not been systematically incorporated in clinical trials and have often been excluded because of the concern for bleeding complications. Venous thromboembolism is a serious complication in SCD and occurs at rates similar to those in serious thrombophilia. This complication is underrecognized and perhaps undertreated. The persistent prothrombotic state should lend consideration to further study of long-term secondary and targeted primary prophylaxis.

Background Sickle cell anemia (SCA) is characterized by abnormally shaped, adhesive RBCs that interact with white blood cells and the endothelium, leading to chronic hemolysis, vasculopathy and a prothrombotic state. About 10% of subjects with a thrombotic event in the general population will have an associated antiphospholipid (aPL) antibody. One proposed mechanism for the thrombophilic nature of aPL antibodies is the disruption of the potent anticoagulant annexin A5 or Annexin A5 resistance (A5R). We designed a pilot study assessing the presence of aPL antibodies and disruption of A5R in pediatric sickle cell subjects. Methods 39 subjects with SCA participated in this study. A5R, DRVVT, anti-β2GP1, anti-β2GP1, anti-phosphatidylserine and anti-cardiolipin antibody assays were performed. Results There was a high prevalence of abnormal A5R despite a low prevalence of antiphospholipid antibodies. Multivariate logistic regression analyses showed an association with silent infarcts (p = 0.015), lower hemoglobin (p = 0.037), older age (p = 0.047) and abnormal A5R. Conclusion We report an association between annexin A5 resistance and presence of silent infarct, low hemoglobin, and older age in a subgroup of SCA subjects. A potential role for perturbed A5R in the pathophysiology of SCA needs to be evaluated further.

Lanosterol synthase (LS) was isolated from microsomes from the plant pathogenic fungus Leptosphaeria nodorum (L. nodorum) and characterized with respect to its physical and kinetic properties. A medium throughput radiometric assay was developed for LS in order to screen potential inhibitors and to determine its kinetic properties. Quantification of LS activity relies on the separation of (±)2,3-oxidosqualene from lanosterol by reverse-phase high-pressure liquid chromatography (RP-HPLC) followed by analysis in a flow scintillation counter. LS from L. nodorum has an apparent Km (oxidosqualene) of 43 μM and an apparent native molecular weight of 150 kDa as determined by size exclusion chromatography. L. nodorum LS was compared to LS from Fusarium culmorum (F. culmorum), Fusarium graminearum (F. graminearum), and Saccharomyces cerevisiae (S. cerevisiae) for kinetic properties and inhibitory activity of a known LS inhibitor (4-bromophenyl)[2-fluoro-4-[[6-(methyl-2-propenylamino)hexyl]oxy]phenyly]-methanone (compound 1) and a derivative of this inhibitor (compound 2). While the Km (oxidosqualene) was similar for LS from all four organisms, there were differences in the activity of the two inhibitors against LS from these species. The characteristics of the assay and its use for screening molecules to find fungal inhibitors are described. To compare the biological activity of the inhibitors with their activity against LS, they were tested to determine the extent to which they suppressed the growth of L. nodorum and F. culmorum. The results suggest that the fungicidal activity of these inhibitors is related to their inhibitory effect on LS.

Most chloroplastic proteins are nuclear-encoded and must be transported into the organelle post-translationally. Proteinaceous components in the outer and inner envelope membranes of chloroplasts responsible for this import process were originally identified from pea seedlings. We sought to determine whether these proteins are conserved among different plant species other than pea and among different plastid types. We analyzed plant EST databases and found the presence of homologues to pea chloroplastic protein translocation components, Tic110 and Toc75, in both monocot and dicot species. Because these clones were obtained from various tissues, their presence in different types of plastids is proposed. Protein extracts were prepared from several plant species and from different plant tissues, and then probed with antisera raised against pea Tic110 and Toc75. The results support the idea that translocation components originally found in pea chloroplasts are conserved among different plant species and are present in various plastid types.

The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems. However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins. We provide evidence that the cyanobacterial protein is also localized in the outer membrane. From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs. synToc75 is essential, as homozygous null mutants were not recovered after directed mutagenesis. Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known. However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.

Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.