Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from six patients with KHE and one KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, their CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from 7 patients with KHE/KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, platelet CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library. Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin. Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267). When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12. Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Deciphering the TLT-1 Fibrinogen Ligand Interaction

Background: Platelets, derived from megakaryocytes primarily play a central role in thrombosis and hemostasis, however, they extend beyond this role as immune cells that initiate and accelerate various vascular inflammatory conditions. Upon activation, platelets release TREM-Like Transcript-1 (TLT-1) from their a-granules onto their surface. Early studies by amino link columns preloaded with soluble TLT-1 followed by mass spectrometry and immunoblotting identified fibrinogen as a ligand for TLT-1. Fibrinogen is a plasma protein that is essential for clot formation, during inflammation and hypercoagulable states tissue deposition and plasma concentration of fibrinogen are increased, demonstrating a role of fibrinogen in both thrombosis and inflammation. TLT-1 binding fibrinogen was a surprising discovery since αIIbβ3, the most abundant platelet receptor, also binds fibrinogen and facilitates platelet aggregation. It is difficult to understand why there are two platelet specific receptors that have the same ligand, drawing us to question what the difference in function between the two is? Our studies suggest that although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory setting like αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Very little is known about the TLT-1-Fibrinogen interaction, further studies would set the stage for a better understanding as to why two fibrinogen ligands exist on platelets and potentially outline a novel platelet therapeutic target during hypercoagulable and/or hyperinflammatory states. We set out to determine the binding affinity and localize the binding sites for the TLT-1 fibrinogen molecular interaction. Aims: Delineate the TLT-1 fibrinogen molecular interaction and elucidate the mechanism by which this interaction drives inflammation and thrombosis-hemostasis. Methods: To confirm the TLT-1 fibrinogen ligand interaction we carried out a kinetics assay using an Octet Qk e Bio-layer Interferometry (BLI) that measures biomolecular complex formation in real time. The TLT-1 Chimera was captured onto an Anti-Human Fc Capture (AHC) Biosensor, washed in kinetics buffer to limit nonspecific binding and submerged in a 96 well plate containing varying concentrations of Fibrinogen. To localize the exact binding sites for this molecular interaction, we digested fibrinogen using trypsin and carried out an immunoprecipitation (IP) followed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). Results: The curve (Figure 1) shows that the TLT-1 fibrinogen interaction has increasing bimolecular complex formation with increases in concentration of Fibrinogen (15.625nM - 250nM), with a concentration of 250nM showing the best bicomplex formation. In the control well with HIV01 4E10 capture, reference and sensor well, no bicomplex formation is shown, highlighting the specificity of the TLT-1 fibrinogen interaction. The curve illustrates a strong association with no dissociation, suggesting a strong interaction between the proteins. We isolated and identified four potential peptides (Alpha chain: GGSTSYGTGSETESPR, GSESGIFTNTK, Beta chain: QDGSVDFGR , QGFGNVATNTDGK) that bind TLT-1. We are currently performing BLI Competitive kinetics assays using biotinylated constructs of the peptides isolated from the Immunoprecipitation/ LC-MS/MS. The BLI competitive assays using the four peptides are suggestive of an interaction between TLT-1 and the four peptides as illustrated by increasing bimolecular complex formation with increasing concentration of soluble TLT-1 for all four peptides(data not shown). Conclusions: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 fibrinogen interaction, suggesting a high affinity interaction between TLT-1 and fibrinogen. In our preliminary results from the BLI Competitive kinetics assays we obtained KD values within the nanomolar concentration range and are currently conducting experiments to optimize conditions to obtain our final bicomplex binding curve and KD values. We are currently assessing the identified peptides for potential of mediating the molecular interaction between TLT-1 and fibrinogen. Our poster will report the current state of these studies . Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Autoinhibition of Mouse Von Willebrand Factor: Addressing the Species Compatibility between VWF and Gpibα in Humans and Mice

Introduction: The usage of mouse models to investigate thrombosis and hemostasis has now spanned several decades, with the advent of genetic manipulation elucidating the roles of platelet receptors, secondary messengers, and coagulation factors to name a few. While there are many similarities between humans and mice, the vast differences in physical parameters of blood circulation and coagulation should not be ignored. The incompatibility of mouse platelet receptor glycoprotein (GP)Ibα with human von Willebrand factor (VWF) has been well documented, yet human GPIbα can interact with mouse plasma VWF under shear. In light of the recently discovered autoinhibitory module (AIM) in modulating the activity of human VWF, here we seek to address the role of the AIM in species compatibility between these two interacting proteins. Methods: Recombinant human and mouse VWF fragments containing the A1 domain and either intact or truncated AIM, followed by a poly-histidine tag, were expressed from stably transfected mammalian cells. Recombinant human and mouse GPIbα ligand-binding domain (LBD) (residues 1-290, mature protein numbering) were similarly expressed bearing a biotin acceptor sequence and poly-histidine tag. Stable cell lines co-expressing E. coli biotin ligase were used to express and biotinylate the LBD protein before purification. All proteins were purified by nickel-affinity chromatography followed by size exclusion chromatography. Bio-layer interferometry was performed on an Octet QK e using streptavidin biosensors to capture biotinylated GPIbα LBD fragments. Data was analyzed using a 2:1 heterogeneous ligand binding model for kinetic parameters, and steady state affinity (K D) was calculated either from binding responses at the end of the association phase, or from predicted equilibria from the aforementioned model. Human or mouse washed platelets were isolated by centrifugation, followed by pelleting the PRP in the presence of PGE-1. Platelets were resuspended to 150,000/ μL in modified Tyrode's buffer, and aggregation was monitored in a light transmission aggregometer. Results: Consistent with earlier reports, human AIM-A1 protein (VWF residues 1238-1493), at concentrations below 1 μM, showed no appreciable binding to human GPIbα LBD. Conversely, human truncated AIM-A1 protein (tAIM-A1, residues 1261-1472) exhibited a K D of 348 nM towards the LBD. Mouse tAIM-A1 proteins showed enhanced binding to mouse LBD (K D = 214 nM), but mouse AIM-A1 is able to bind to mouse LBD with a K D of 716 nM, which is considerably higher than the affinity of the human-human pair of tens of μM (Figure 1). Given the increased affinity of mouse AIM-A1 to mouse LBD, we reasoned that the mouse AIM may be less stable than the human counterpart. A chimeric AIM-A1 protein, in which human AIM sequences flank mouse A1 (human residues 1238-1271, mouse 1272-1458, and human 1459-1493), was generated. The chimeric AIM-A1 showed little binding to mouse LBD. However, this protein can still aggregate washed platelets from both species and bind to gain-of-function human GPIbα, indicating it still is a folded domain capable to recognizing its cognate receptor, mouse GPIbα (Figure 2). The isolated mouse AIM-A1 fragments are capable of inducing robust mouse and human platelet aggregation for washed platelets in a dose dependent manner. It had been previously demonstrated that in platelet rich plasma, ristocetin has no effect on mouse VWF. However, washed mouse platelets incubated with 60 nM of mouse AIM-A1 followed by addition of 1.5 mg/mL ristocetin was able to agglutinate the platelets (Figure 2). These results suggest that these proteins may be used to help elucidate the signaling pathways of GPIb-IX-V complex in mice. Conclusion: In agreement with previous reports, we observe that in general mouse AIM-A1 is more active than human AIM-A1 in its propensity to bind to GPIbα. On the aspect that human VWF in incompatible with mouse platelets, we observed that mouse LBD has lower affinity for human tAIM-A1 than human LBD does for mouse tAIM-A1. These findings altogether suggest that there are likely differences in activation forces for each species AIM, and that the GPIbα-A1 interactions, in terms of their mechanical and thermodynamic properties, are somewhat unique, and perhaps optimized, for each species. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Staged Management of Heparin-Induced Thrombocytopenia for Renal Cavo-Atrial Cancer Removal on Cardiopulmonary Bypass

Management of patients with acute heparin-induced thrombocytopenia (HIT) with cavo-atrial renal cancer requiring surgery on cardiopulmonary bypass (CPB) and possible deep hypothermia circulatory arrest is a challenge. A staged approach using Bivalirudin, plasmapheresis, and intravenous immunoglobulin (IVIG) was used to preoperatively de-escalate HIT guided by enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA). Intraoperatively heparin was used as the anticoagulant for CPB as DHCA was likely to be used to remove the atrio-caval tumor. Heparin is effective in preventing clots in the circuit during DHCA. To prevent HIT upon re-exposure to heparin during CPB, a bolus of a Cangrelor (reversible P2Y12 platelet receptor inhibitor) was given before heparin and during CPB whilst platelet activity was monitored using platelet reactivity units (PRU). Postoperatively, to prevent recurrence of HIT, plasmapheresis was used until SRA and optical density (OD) resulted. The patient had a successful outcome.

Clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia

The aim of the study was to assess the clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia (SS). Materials and methods. A total of 100 patients with SS were examined and followed-up for 15.3±8.3 months. The patients were divided into subgroups (SG): SG1 (n=51) – persons without events, SG2 (n=49) persons with recurrent ischemic events (hospitalization due to the development of pain syndrome, re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes), SGB (n=11) – persons with «major» recurrent ischemic events (re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes) , SGG (n=89) – persons without «major» events. The obtained survey data (general clinical, aggregometry, polymor phism of genes of platelet fibrinogen receptor ITGB3 (T1565C), platelet collagen receptor ITGA2 (C807T), ADP platelet receptor P2RY12, H1/H2 (T744C)) were analyzed using the STATISTICA 10.0 software. Results. In SG2, men predominated (χ2 =9.2; p<0.01), past MI was more common (χ2 =4.8; p<0.05), more stents were implanted (2.4±1.9 versus 1.7±1.1, p<0.05), TRAP-test values were higher (p<0.05) compared to SG1. In SGB, greater number of stents were implanted (3.1±2.2 versus 1.61±1.57, p<0.05), the carriage of the TC genotype of the ITGB3 gene was more common, (p<0.05), a combination of gene mutations ITGB3 and P2RY12 was more common, (p<0.05) compared to SGG. A logistic regression equation was constructed, including the presence of diabetes mellitus, the number of platelets in the blood test, the ASPI-test values, the carriage of the 1565C allele of the ITGB3 gene, the number of stents implanted, which makes it possible to determine the likelihood of developing «major» recurrent ischemic events with a cut-off threshold LP₀=0.0965, with sensitivity – 81.82 %, specificity – 78.48 %, overall accuracy – 78.89 %. Conclusions. The factors associated with the development of recurrent ischemic events are: male sex, previous MI, a greater number of implanted stents, and high TRAP-test values. The factors associated with the development of recurrent «major» ischemic events are: a greater number of implanted stents, carriage of the TC genotype of the ITGB3 gene, carriage of a combination of mutations of the H1/H2 polymorphic locus of the P2RY12 gene and the T1565C polymorphic locus of the ITGB3 gene, diabetes mellitus, the number of platelets in blood test, ASPI-test values.

COVID-19 as a Potential Trigger for Immune Thrombotic Thrombocytopenic Purpura and Reason for an Unusual Treatment: A Case Report

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder characterized by severely reduced activity of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) due to autoantibodies. This leads to the development of pathogenic multimers of VWF, causing a thrombotic microangiopathy with decreased number of platelets, hemolysis, and life-threatening tissue ischemia of mostly brain, heart, and kidneys. Standard treatment of iTTP involves daily plasma exchange to remove ultra large multimers of VWF, inhibitors, substituting ADAMTS13, and the accompaniment of an immunosuppressive treatment with steroids. Recently, caplacizumab was approved for iTTP. Caplacizumab is a nanobody binding the A1 domain of VWF, blocking its interaction with glycoprotein Ib–IX–V platelet receptor and therefore preventing platelet aggregation. VWF activities may serve as therapeutic drug monitoring of caplacizumab, whereas ADAMTS13 activities may be used for biomarkers to guide caplacizumab treatment modalities and overall treatment duration. Additional immunosuppressive treatment by inhibiting autoantibody formation (e.g., the use of Rituximab, a chimeric monoclonal antibody directed against the B-cell antigen CD20) is a further treatment option. Infections are well-known causes for an acute episode for patients with iTTP. The novel SARS-CoV-2 virus is mainly associated with acute respiratory distress as well as diffuse endothelial inflammation and increased coagulopathy. However, little is known about an infection with SARS-CoV-2 virus triggering iTTP relapses. We herein report the case of an acute iTTP episode accompanying a SARS-CoV-2 infection.