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Traumatic brain injury-induced coagulopathy (TBI-IC) causes life-threatening secondary intracranial bleeding. Its pathogenesis differs mechanistically from that of coagulopathy arising from extracranial injuries and hemorrhagic shock, but it remains poorly understood. We report results of a study designed to test the hypothesis that von Willebrand factor (VWF) released during acute TBI is intrinsically hyperadhesive because its platelet-binding A1-domain is exposed and contributes to TBI-induced vascular leakage and consumptive coagulopathy. This hyperadhesive VWF can be selectively blocked by a VWF A2-domain protein to prevent TBI-IC and to improve neurological function with a minimal risk of bleeding. We demonstrated that A2 given through intraperitoneal injection or IV infusion reduced TBI-induced death by >50% and significantly improved the neurological function of C57BL/6J male mice subjected to severe lateral fluid percussion injury. A2 protected the endothelium from extracellular vesicle-induced injury, reducing TBI-induced platelet activation and microvesiculation, and preventing a TBI-induced hypercoagulable state. A2 achieved this therapeutic efficacy by specifically blocking the A1 domain exposed on the hyperadhesive VWF released during acute TBI. These results suggest that VWF plays a causal role in the development of TBI-IC and is a therapeutic target for this life-threatening complication of TBI.

Introduction: Transplant-associated thrombotic microangiopathy (TA-TMA) is an increasingly recognized hematologic complication after allogeneic hematopoietic cell transplantation (HCT). While few studies have reported germline association with rare variants in complement genes using targeted next generation sequencing (NGS) method, they were limited by small sample size (≤40 TMA cases) and lack of analysis of non-complement genes (PMIDs 26603840, 32131130). In the present study, we employed whole exome sequencing (WES) to assess rare variant contribution to the development of TMA in a hypothesis-driven pathway-specific approach. Methods: In the current case-control genetic association study conducted at Fred Hutchinson Cancer Research Center, we selected 100 patients with a diagnosis of TMA and pre-transplant DNA samples (case definition described previously in PMID 30940363, 33836868). We then performed incidence density sampling to randomly select 100 non-TMA controls after allogeneic HCT matching by age, sex, race, and year of HCT. WES (germline variant detection 40x) was conducted using Illumina NovaSeq. Sequence reads were mapped to hg38 reference genome followed by deduplication and base quality score recalibration. Joint-genotyping was performed to call single nucleotide polymorphism (SNPs) and insertion/deletion (indels) using the GATK v3.3 and Atlas2. Variants were filtered during quality control (QC) and variant quality score recalibration (VQSR) and annotated using ANNOVAR and Ensembl VEP. To optimize signal detection by reducing neutral background variation, we defined qualifying variants as those meeting all 3 criteria: 1) novel or rare variants with a minor allele frequency (MAF) We then focused on the exome profiles of 5 a priori selected genetic pathways: complement regulation (17 genes), VWF and coagulation (7 genes), VWF clearance (10 genes), ADAMTS13 mimics or interacting proteins (10 genes), and angiopoietin family and endothelial activation (7 genes). Pathway-based and gene-based collapsing association tests were performed using the Optimized Sequence Kernel Association (SKAT-O) test as an optimal test combining burden test and SKAT. Results: After joint variant calling, 91 TMA cases and 93 non-TMA controls passed all QC filters (Table 1). Among 1,485 variants detected in the 5 pathways after QC, 60 variants (73 total mutations) were considered as qualifying variants with MAF Conclusion: Contrary to the initial hypothesis, we did not observe pathogenic germline rare variants in the complement regulation pathway in patients with TA-TMA. Instead, we found a significant association in the VWF clearance pathway, particularly that of the LRP1 gene. In recent years, researchers have shown that VWF can bind to and activate complement proteins. Impaired VWF clearance could lead to the higher predisposition for complement activation observed in patients with TA-TMA. Future functional studies are needed to determine the impact of VWF clearance on the pathogenesis of the disease. Figure 1 Figure 1. Disclosures Sartain: Alexon Pharamaceuticals: Membership on an entity's Board of Directors or advisory committees. Lee: Incyte: Research Funding; Janssen: Other; Takeda: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding.

Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry studies in vitro. Similarly, the aggregation response of 4°C-stored PLTs to collagen was significantly increased compared to RT-stored PLTs in vitro. In line with these findings, we observed more PLCγ2 phosphorylation and Syk phosphorylation at baseline in 4°C-stored PLTs compared to RT-stored PLTs, suggesting more pre-activation downstream of GPVI. However, no differences in PLCγ2 phosphorylation or Syk-phosphorylation were found between RT and 4°C-stored PLTs after stimulation with CVX, and no significant differences in surface expression levels of GPVI were detected between RT and 4°C. Stored platelets in plasma showed superior function after 4°C-storage in aggregation and flow cytometry assays. In contrast, we found similar contractile forces of platelet aggregates when RT-stored or 4°C-stored PLTs were added to platelet-depleted fresh blood. Additionally, at the single cell level, we found a similar magnitude of platelet forces in RT-stored and 4°C-stored PLTs. Similar to human PLTs, mouse PLTs showed significantly more integrin activation, P-selectin exposure, and aggregation in 4°C-stored PLTs compared to RT. To test the recovery of function of stored mouse platelets in vivo, we transfused GFP-positive PLTs into GFP-negative wild-type mice. Contrary to our pre-transfusion results, we found a significantly lower integrin activation response to CVX in 4°C-stored platelets after transfusion, consistent with our previous results in healthy human volunteers. Summary: The in vivo recovery of function of stored PLTs is an underappreciated phenomenon in platelet storage biology, and most studies are solely based on functional in vitro data. Based on our post-transfusion results, storage temperature affects the ability to recover function in vivo significantly in human and mouse platelets. Whether these differences lead to differences in clinical outcomes needs to be investigated in clinical trials. Disclosures Sniadecki: Stasys Medical Corporation: Current equity holder in private company, Other: Co-founder; Curi Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Blood type O is associated with a lower risk of myocardial infarction. Platelets play a critical role in myocardial infarction. It is not known whether the expression of blood group antigens on platelet proteins alters platelet function; we hypothesized that platelet function would be different between donors with blood type O and those with non-O. To address this hypothesis, we perfused blood from healthy type O donors (n = 33) or non-O donors (n = 54) over pooled plasma derived von Willebrand factor (VWF) protein and purified blood type-specific VWF at arterial shear and measured platelet translocation dynamics. We demonstrate for the first time that type O platelets travel farther at greater speeds before forming stable bonds with VWF. To further characterize these findings, we used a novel analytical model of platelet interaction. Modeling revealed that the kinetics for GPIb/VWF binding rate are significantly lower for type O compared with non-O platelets. Our results demonstrate that platelets from type O donors interact less with VWF at arterial shear than non-O platelets. Our results suggest a potential mechanism for the reduced risk of myocardial infarction associated with blood type O.

Traumatic brain injury (TBI) is associated with coagulopathy, although it often lacks 2 key risk factors: severe bleeding and significant fluid resuscitation associated with hemorrhagic shock. The pathogenesis of TBI-associated coagulopathy remains poorly understood. We tested the hypothesis that brain-derived microparticles (BDMPs) released from an injured brain induce a hypercoagulable state that rapidly turns into consumptive coagulopathy. Here, we report that mice subjected to fluid percussion injury (1.9 ± 0.1 atm) developed a BDMP-dependent hypercoagulable state, with peak levels of plasma glial cell and neuronal BDMPs reaching 17 496 ± 4833/μL and 18 388 ± 3657/μL 3 hours after TBI, respectively. Uninjured mice injected with BDMPs developed a dose-dependent hyper-turned hypocoagulable state measured by a progressively prolonged clotting time, fibrinogen depletion, and microvascular fibrin deposition in multiple organs. The BDMPs were 50 to 300 nm with intact membranes, expressing neuronal or glial cell markers and procoagulant phosphatidylserine and tissue factor. Their procoagulant activity was greater than platelet microparticles and was dose-dependently blocked by lactadherin. Microparticles were produced from injured hippocampal cells, transmigrated through the disrupted endothelial barrier in a platelet-dependent manner, and activated platelets. These data define a novel mechanism of TBI-associated coagulopathy in mice, identify early predictive markers, and provide alternative therapeutic targets.

Traumatic brain injury (TBI)–induced coagulopathy is a common and well-recognized risk for poor clinical outcomes, but its pathogenesis remains poorly understood, and treatment options are limited and ineffective. We discuss the recent progress and knowledge gaps in understanding this lethal complication of TBI. We focus on (1) the disruption of the brain-blood barrier to disseminate brain injury systemically by releasing brain-derived molecules into the circulation and (2) TBI-induced hypercoagulable and hyperfibrinolytic states that result in persistent and delayed intracranial hemorrhage and systemic bleeding.

There is little data considering relationships among human RNA, demographic variables, and primary human cell physiology. The platelet RNA and expression-1 study measured platelet aggregation to arachidonic acid, ADP, protease-activated receptor (PAR) 1 activation peptide (PAR1-AP), and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. A total of 5911 uniquely mapped mRNAs and 181 miRNAs were commonly expressed and validated in a separate cohort. One hundred twenty-nine mRNAs and 15 miRNAs were differentially expressed (DE) by age, and targets of these miRNAs were over-represented among these mRNAs. Fifty-four mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship in these RNA pairs suggests miRNAs regulate mRNA levels on aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future platelet RNA association studies must account for age and gender.

Introduction: D-dimer has been well characterized as a prognostic biomarker for venous thromboembolism (VTE) in both the general population and cancer patients. However, it is unclear if D-dimer has prognostic value after hematopoietic transplantation (HCT) in the context of regimen-related toxicity, immune dysregulation, and infectious complications. In the current study, we examined the utility of biomarkers of coagulation activation and fibrinolysis (D-dimer, PAI-1, and TPA) as prognostic and diagnostic biomarkers for VTE post-transplant. Methods: We performed a prospective cohort study of adult allogeneic or autologous HCT recipients over 4 years at the Fred Hutchinson Cancer Research Center. Plasma samples were collected weekly from pre-transplant to discharge from HCT. Plasma samples were rapidly thawed and concentrations of D-dimer, PAI-1 activity, and TPA antigens were tested by commercially available immunoassays. VTE was defined as radiology confirmed pulmonary embolism (PE), lower extremity (LE) or upper extremity (UE) deep vein thrombosis (DVT) within 1 year after the date of transplant. This outcome was abstracted through a combination of ICD codes and individual patient record review. Cumulative incidence was estimated using the Kaplan Meier failure method. We used the Cox regression model to determine the association between baseline biomarkers and the development of VTE. The prediction accuracy of the model was assessed by the Harrell's C statistic. To explore the utility of D-dimer testing post-transplant, we selected all patients that had clinical suspicion for VTE, underwent radiographic evaluation (chest computed tomography or compression ultrasound), and had available sample for D-dimer testing within a two-week window. We defined the age-adjusted threshold as 500 ng/mL if at or below age 50 and 10 times the age if above age 50. We also repeated the analysis using a higher cut-off of 1000 ng/mL. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were generated from the 2x2 table. Results: We identified 112 HCT adult recipients (97 allogeneic and 15 autologous) who were enrolled in the prospective cohort study and provided baseline plasma samples for biomarker testing. Patients had a mean age of 48, were predominately male and white, and had acute myeloid leukemia (AML) as the most common diagnosis (Table 1). Approximately 8% (9 patients) had a history of prior VTE. During the 1-year follow-up period, a total of 8 patients developed VTE (3 PE, 3 LE-DVT, 2 UE-DVT) with a cumulative incidence of 11.8% (5.8-23.2) by 12 months. There was no association between baseline TPA and PAI-1 and VTE; however, baseline D-dimer was associated with VTE with a HR of 1.91 (p=0.007) in the unadjusted model and a HR of 1.95 (p=0.007) in the model adjusted for history of VTE (Table 2). The predictive model with baseline D-dimer and history of VTE had a Harrell's C statistic of 0.75 for discrimination. For the exploratory analysis of the D-dimer post-transplant, we used a subset of 21 patients that had clinical suspicion of VTE, radiology confirmation, and available plasma sample for D-dimer testing. The sensitivity and NPV for both age-adjusted cut-off and 1000 ng/mL were 100%; however, the sample size was small (Table 3). Conclusions: In this cohort study, we found that baseline D-dimer, but not PAI-1 or TPA, was associated with the development of VTE during the first-year post-transplant. A multivariable model with D-dimer and history of VTE had a modest discrimination for VTE prediction. Similar to many clinical settings, D-dimer testing post-transplant may aid clinicians with prediction and diagnosis of VTE. Larger cohort is needed for validation of the findings. Disclosures No relevant conflicts of interest to declare.

Introduction: Thrombotic microangiopathy (TMA) is a known complication of allogeneic hematopoietic cell transplantation (HCT). Though the presence of graft-versus-host disease (GVHD) predicts the development of TMA, only a small subset of patients with GVHD will develop this condition. In the current study, we examined soluble C5b-9 (sC5b9), the terminal complement complex, as a potential biomarker for the development of TMA among patients with active GVHD. Methods: We performed a nested case-control study using a prospective cohort of adult allogeneic HCT recipients transplanted during 2006-2013 at the Fred Hutchinson Cancer Research Center. Cases (TMA) with antecedent GVHD were ascertained and validated as previously described (BBMT 2019;25:570). Two controls (non-TMA) were randomly selected for each case at the time of TMA via the incidence density sampling method after matching on the onset timing and severity of prior GVHD. We tested plasma sC5b9 levels (exposure) in longitudinal samples at matched time points (pre-transplant, onset of GVHD, onset of TMA or matched time point) using a commercially available immunoassay kit. Furthermore, we reviewed patient records to determine the onset of systemic infection (including Gram negative bacteremia, invasive aspergillosis, BK viremia, HHV-6 invasive disease, CMV invasive disease, and EBV reactivation). Mean sC5b9 values and 95% confidence intervals were estimated at each time point. Conditional logistic regressions were used to estimate the association (odds ratio, OR) between the outcome (TMA vs. non-TMA) and the exposure (sC5b9 level) after accounting for matching. sC5b9 was modeled both as a continuous variable and a binary variable dichotomized at the median value. Results: Among 208 adult allogeneic patients enrolled in the prospective cohort, we identified 38 patients (13 TMA cases and 25 non-TMA controls) with antecedent GVHD of similar time of onset and clinical grading. The median time to the onset of GVHD and TMA was 21 days (IQR 14-31) and 37 days (IQR 23-80), respectively. Six out of 13 cases developed TMA by 28 days. Mean sC5b9 levels were significantly higher in the TMA group than the non-TMA group at the onset of TMA vs. matched time point (377 vs. 248 ng/mL) and GVHD (369 vs. 240 ng/mL) but less so prior to transplant (243 vs. 200 ng/mL) (Figure 1). Furthermore, mean sC5b9 levels were elevated in patients experiencing active infection (364.10 ng/mL) and the values for each type of infection were shown in Table 2. When considered as a continuous variable, every 100 ng/mL increase in sC5b9 at the onset of GVHD was associated with an OR of 4.18 for TMA (p=0.02) (Table 1). As a binary variable, sC5b9 ≧ 250 ng/mL vs. < 250 ng/mL had an OR of 5.51 for TMA (p=0.04). Conclusions: In the nested case-control study, we found that elevated sC5b9 level at the onset of acute GVHD was associated with the development of TMA after accounting for the timing and severity of GVHD. Previous studies have shown that testing of sC5b9 at day 28 was associated with TMA development; however, the prognostic value of day 28 testing was limited by the early onset of disease. Our study suggests that sC5b9 screening at the onset of GVHD may help predict which individuals would later develop TMA. It remains unclear whether complement activation is the driver or the effect of endothelial injury among GVHD patients. Finally, we caution that activation of the terminal complement pathway can also signify serious systemic infection. The interpretation of sC5b9 must be individualized according to the clinical scenario. Acknowledgement: We would like to acknowledge and thank Kypha Inc for providing us with the immunoassay kits. We performed the study design and data analysis independently without industry funding. Disclosures Schmidt: Kypha Inc: Employment, Equity Ownership. Lee:Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding.

Cardiolipin (CL) is an anionic phospholipid located exclusively in the mitochondrial inner membrane. Its presence in blood indicates mitochondrial damage and release from injured cells. Here, we report the detection of CL-exposed brain-derived mitochondrial microparticles (mtMPs) at 17 547 ± 2677/μL in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mtMPs accounted for 55.2% ± 12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. They were also released from cultured neuronal and glial cells undergoing apoptosis. The mtMPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier. The disrupted endothelial barrier allowed the release of mtMPs into the systemic circulation to promote coagulation in both traumatically injured and mtMP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition, and thrombosis. This mtMP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mtMP-bound CL was ∼1600 times as active as purified CL in promoting coagulation. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury–associated coagulopathy and identified potential pathways to block this activity.

Introduction: Thrombotic microangiopathy (TMA) is a known complication of allogeneic hematopoietic cell transplantation (HCT). An ongoing debate is whether TMA is caused by acute graft-versus-host disease (GVHD) or calcineurin inhibitor (CNI)/sirolimus. Previous studies have not examined the impact of CNI/sirolimus average trough levels while accounting for the confounding effect of GVHD. In the current study, we examined the association between time-varying levels of CNI/sirolimus and the onset of TMA among 2105 adult allogeneic HCT recipients. Methods: We performed a retrospective cohort analysis of allogeneic HCT recipients during 2006-2015 at Fred Hutchinson Cancer Research Center who received CNI/sirolimus for GVHD prophylaxis. TMA (outcome) was ascertained and validated as previously described (Blood 130;Suppl:666). Acute GVHD (confounder) was defined and graded according to established criteria. Consecutive CNI/sirolimus trough levels (exposure) were obtained and interval values between checks were imputed via linear interpolation. We followed patients from the time of hematopoietic cell infusion until the onset of TMA and censored patients at day 100 or >1 week of missing drug levels. To examine the association between the "type" of immunosuppressant and TMA, CNI/sirolimus exposure over time was characterized as a discrete categorical variable (tacrolimus alone, cyclosporine alone, or sirolimus + CNI). To examine the impact of the "level" of immunosuppressant on TMA, CNI/sirolimus exposure was both defined as an average of the previous 7-day trough levels (continuous variable) as well as a discrete time-above-peak variable (binary variable for tacrolimus >15 ng/mL, cyclosporine >450 ng/mL, or sirolimus >10 ng/mL). Extended Cox regression models were built to examine the time-varying association between CNI/sirolimus exposures and TMA after adjusting for GVHD. Results: In the current study, 2105 adult allogeneic HCT recipients contributed 173,171 person-days and 150 cases of TMA. The median follow-up time was 94 days and trough blood levels were checked on average 3x per week. Initial GVHD prophylaxis regimen for patients included 54% on tacrolimus (n=1135), 40% on cyclosporine (n=837), and 6% on a combination of sirolimus + CNI (n=133). Nearly no one received sirolimus alone without CNI. Eight percent of patients (n=162) had at least one switch in the immunosuppression regimen within 100 days. The median (IQR) trough levels for tacrolimus, cyclosporine, and sirolimus were 9 ng/mL (7-12), 283 ng/mL (206-372), and 5 ng/mL (4-7), respectively. Acute GVHD developed in 64% of patients including 53% grade 2 (n=1115), 9% grade 3 (n=185), 2% grade 4 (n=52). In the analysis adjusted for both discrete time-varying CNI/sirolimus exposure and GVHD, higher grades of GVHD had strong associations with TMA (Table 1: grade 2 HR 2.24, P=0.001; grade 3 HR 12.38, P In the analyses of individual drug levels adjusted by GVHD, higher trough levels of tacrolimus and cyclosporine (either as a linear average level or binary time-above-peak) were not associated with an increased risk of TMA (Table 2). However, higher sirolimus trough levels were associated with an appreciable risk of TMA (HR 1.44, P=0.001 for every 1 ng/mL increase in sirolimus average trough level; HR 3.23, P=0.164 for time above 10 ng/mL). Conclusion: In allogeneic HCT patients, acute GVHD was strongly associated with the onset of TMA independent of ongoing exposures of CNI/sirolimus. We did not find an association between CNI recipients (tacrolimus versus cyclosporine) or CNI levels and the risk of TMA. However, when combined with CNI, higher sirolimus trough levels were linearly associated with a higher risk for TMA. Limitations in this observational study include the inability to adjust for concomitant medications and conditions that might have altered drug levels and inability to adjust for target drug levels since recognized risk factors might have led clinicians to target higher drug levels due to higher risks of GVHD. These findings suggest that prevention and treatment of GVHD and avoidance of high sirolimus levels will decrease the risk of TMA. Disclosures Lee: Amgen: Consultancy, Research Funding; Mallinckrodt: Honoraria; Incyte: Consultancy; Pfizer: Consultancy; Onyx: Research Funding; Kadmon: Research Funding; Takeda: Research Funding. Garcia:Pfizer: Consultancy; Portola: Research Funding; Shingoi: Consultancy; Retham Technologies LLC: Consultancy; Janssen: Consultancy, Research Funding; Bristol Meyers Squibb: Consultancy; Daiichi Sankyo: Research Funding; Incyte: Research Funding; Boehringer Ingelheim: Consultancy.

A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving the folded A2 domain of von Willebrand factor (VWF). The cleavage is regulated by forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3 tridomain polypeptides by platelet glycoprotein Ibα or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5- to 80-pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, probably because of uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 seconds at 20pN, then decreased (slip) with increasing force, independent of ADAMTS-13. The time required to rupture A1A2A3 exhibited a similar catch-slip behavior when pulled by glycoprotein Ibα but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate kcat of 2.9 (± 59) seconds and a Kd of 5.6 (± 3.4) nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3 tridomain.

Introduction: Thrombotic microangiopathy (TMA) is a clinical syndrome that can develop after hematopoietic cell transplantation (HCT). Previous studies have been limited to small case series with conflicting results. In this large, single-center longitudinal TA-TMA database, we describe the incidence, etiology, treatment, and outcome of this rare but challenging clinical presentation. Methods: We performed a retrospective cohort analysis of consecutive allogeneic HCT recipient during 2006-2015 at Fred Hutchinson Cancer Research Center. TA-TMA was defined as persistent microangiopathic hemolytic anemia (MAHA) composed of red cell fragmentation, elevated lactate dehydrogenase (LDH), thrombocytopenia, anemia, and lack of coagulopathy (Table 1). We performed a primary search algorithm using "consecutive MAHA" followed by a secondary search algorithm using "suspected hemolysis" to identify all cases from date of transplant until date of death or last follow-up (Table 1). For each suspected case, we further reviewed available patient records to confirm the diagnosis, determine the etiology, treatment, and complication. The most likely etiology was determined by reviewing the clinical events in the 2-week window before the TMA onset. Since 96% of patients were receiving calcineurin inhibitor (CNI), we chose idiopathic/drug as a diagnosis of exclusion if all other causes were excluded. TMA resolution was defined as LDH Results: Among 2241 consecutive adult allogeneic HCT recipients, 209 patients (9%) met the laboratory criteria of TA-TMA (Table 2) with a median onset time of 62d (35d-93d). One hundred and nine patients (52%) had end-organ damage with either acute kidney injury (AKI) or neurologic deficit. The median follow-up was 181d (92d-478d) and 53 patients (25%) achieved hematologic resolution. Diagnostically, acute GVHD (n=96) represented the largest category and those with GVHD refractory to high dose steroid (n=61) had poor prognosis. Diffuse alveolar hemorrhage (DAH) and idiopathic pneumonia syndrome (IPS) (n=30) also frequently preceded the TMA onset. Finally, despite excluding patients with coagulopathy, non-specific causes of TMA such as disease recurrence (n=20) and systemic infection (n=33) were frequently evident. Overall survival, separated by TMA etiology is shown in Figure 1. Whereas the idiopathic/drug and non-refractory acute GVHD categories approached the survival of non-TMA post-HCT recipients, the other groups had worse prognosis. Regardless of cause, the most common treatment (32%) was CNI cessation or CNI switch (in no particular pattern). We did not observe an association between changes to CNI therapy and TMA resolution (24% vs. 26%, Fisher P 0.80) or overall survival at 6 months (37% vs. 34%, Wald P 0.75). This remained true when each diagnostic category and each CNI drug was separately analyzed. Plasma exchange was rarely performed at our center. Finally, TMA resolution often correlated with improvement of the underlying disorder. The achievement of TMA resolution was associated with significantly improved survival (HR 0.35, P Conclusion: Despite meeting the same laboratory definition for persistent MAHA, clinical review revealed heterogeneous disease groups with dissimilar prognostic implications. Our data suggest that TA-TMA is not one disease entity but rather a "final common pathway" for several disorders that likely have different pathophysiologic mechanisms. Instead of empiric treatment with CNI adjustment, we need to identify and understand the underlying causal pathway towards TA-TMA, and to determine if earlier recognition and successful treatment improves patient outcomes. Disclosures Lee: Kadmon: Other: One-time advisory board member; Amgen: Other: One-time advisory board member; Bristol-Myers-Squibb: Other: One-time advisory board member; Mallinckrodt: Honoraria. Gopal: Seattle Genetics: Consultancy, Research Funding.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Aggregometry is widely used to assess platelet function, but its use in identifying platelet hyperreactivity is poorly defined. We studied platelet aggregation in 359 healthy individuals using the agonists adenosine diphosphate (ADP), epinephrine, collagen, collagen-related peptide, and ristocetin. We also assessed the reproducibility of these assays in 27 subjects by studying them repeatedly on at least 4 separate occasions. Healthy subjects exhibited considerable interindividual variability in aggregation response to agonists, especially at concentrations lower than those typically used in clinical laboratories. For each agonist tested at these submaximal concentrations, a small proportion of individuals demonstrated an unusually robust aggregation response. Subjects who exhibited such in vitro hyperreactivity to one agonist tended to demonstrate a similar response to others, suggesting that hyperreactivity is a global characteristic of platelets. Epinephrine and collagen-related peptide were especially reliable and efficient in detecting hyperreactivity. For epinephrine, excellent reproducibility persisted for up to 3 years, and hyperreactivity was associated with female sex and higher fibrinogen levels (P < .02). We recommend these assays as appropriate candidates for future studies requiring accurate assessment of increased platelet reactivity. These include clinical studies to improve risk assessment for arterial thrombosis, as well as genetic studies to establish determinants of the hyperreactive platelet phenotype.

Glycoprotein (GP) Ibα, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibα that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibα to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibα. Because His86 is 2 nm away from the region of GP Ibα with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibα affinity for VWF allosterically.

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P.005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P.05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibα, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm2) and arterial (20 and 50 dyne/cm2) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.

Ultralarge von Willebrand factor (ULVWF) multimers have been implicated in the pathogenesis of the catastrophic microangiopathic disorder, thrombotic thrombocytopenic purpura. Spontaneous ULVWF binding to platelets has been ascribed to increased avidity due to the greatly increased number of binding sites for platelets (the A1 domain) per molecule. To address the mechanism of enhanced ULVWF binding to platelets, we used optical tweezers to study the unbinding forces from the glycoprotein Ib-IX (GP Ib-IX) complex of plasma VWF, ULVWF, and isolated A1 domain. The unbinding force was defined as the minimum force required to pull ligand-coated beads away from their attachment with GP Ib-IX–expressing cells. Beads coated with plasma VWF did not bind to the cells spontaneously, requiring the modulators ristocetin or botrocetin. The force required to break the ristocetin- and botrocetin-induced plasma VWF–GP Ib-IX bonds occurred in integer multiples of 6.5 pN and 8.8 pN, respectively, depending on the number of bonds formed. In contrast, beads coated with either ULVWF or A1 domain bound the cells in the absence of modulators, with bond strengths in integer multiples of approximately 11.4 pN for both. Thus, in the absence of shear stress, ULVWF multimers form spontaneous high-strength bonds with GP Ib-IX, while plasma VWF requires exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1 domain and greater than those of plasma VWF induced by either modulator. Therefore, we suggest that the conformational state of ULVWF multimers is more critical than their size for interaction with platelets.

In this issue of Blood , Boudreau et al report that activated platelets release respiratory-competent mitochondria either as free organelles or packed in platelet microparticles.[1][1] ![Figure][2] Schematic summary of the biological activities of platelet mitochondria. See Figure 7D in the

The platelet glycoprotein (GP) Ib–IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib–IX-V is within the N-terminal 282 residues of GP Ib, which consist of an N-terminal flanking sequence (His-1–Ile-35), 7 leucine-rich repeats (Leu-36–Ala-200), a C-terminal flank (Phe-201–Gly-268), and a sulfated tyrosine sequence (Asp-269–Glu-282). We have used mammalian cell expression of canine–human chimeras of GP Ib, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ib chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human–canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ib were identified as being critical for vWf adhesion to GP Ib–IX-V.