Agnostic identification of plasma biomarkers for postpartum hemorrhage risk.

BACKGROUND: Postpartum hemorrhage is difficult to predict, is associated with significant maternal morbidity, and is the leading cause of maternal mortality worldwide. The identification of maternal biomarkers that can predict increased postpartum hemorrhage risk would enhance clinical care and may uncover mechanisms that lead to postpartum hemorrhage. OBJECTIVE: This retrospective case-control study employed agnostic proteomic profiling of maternal plasma samples to identify differentially abundant proteins in controls and postpartum hemorrhage cases. STUDY DESIGN: Maternal plasma samples were procured from a cohort of >60,000 participants in a single institution's perinatal repository. Postpartum hemorrhage was defined as a decrease in hematocrit of ≥10% or receipt of transfusion within 24 hours after delivery. Postpartum hemorrhage cases (n=30) were matched by maternal age and delivery mode (vaginal or cesarean) with controls (n=56). Mass spectrometry was used to identify differentially abundant proteins using integrated peptide peak areas. Statistically significant differences between groups were defined as P<.05 after controlling for multiple comparisons. RESULTS: By study design, cases and controls did not differ in race, ethnicity, gestational age at delivery, blood type, or predelivery platelet count. Cases had slightly but significantly lower predelivery and postdelivery hematocrit and hemoglobin. Mass spectrometry detected 1140 proteins, including 77 proteins for which relative abundance differed significantly between cases and controls (fold change >1.15, P<.05). Of these differentially abundant plasma proteins, most had likely liver or placental origins. Gene ontology term analysis mapped to protein clusters involved in responses to wound healing, stress response, and host immune defense. Significantly differentially abundant proteins with the highest fold change (prostaglandin D2 synthase, periostin, and several serine protease inhibitors) did not correlate with predelivery hematocrit or hemoglobin but identified postpartum hemorrhage cases with logistic regression modeling revealing good-to-excellent area under the operator receiver characteristic curves (0.802-0.874). Incorporating predelivery hemoglobin with these candidate proteins further improved the identification of postpartum hemorrhage cases. CONCLUSION: Agnostic analysis of maternal plasma samples identified differentially abundant proteins in controls and postpartum hemorrhage cases. Several of these proteins are known to participate in biologically plausible pathways for postpartum hemorrhage risk and have potential value for predicting postpartum hemorrhage. These findings identify candidate protein biomarkers for future validation and mechanistic studies.

Cross-Talk between the Complement Pathway and the Contact Activation System of Coagulation: Activated Factor XI Neutralizes Complement Factor H.

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.

Platelets and tyrosine kinase inhibitors: clinical features, mechanisms of action, and effects on physiology.

Tyrosine kinase inhibitors (TKIs) have emerged as a promising class of target-directed, small molecule inhibitors used to treat hematologic malignancies, inflammatory diseases, and autoimmune disorders. Recently, TKIs have also gained interest as potential antiplatelet-directed therapeutics that could be leveraged to reduce pathologic thrombus formation and atherothrombotic complications, while minimally affecting platelet hemostatic function. This review provides a mechanistic overview and summarizes the known effects of tyrosine kinase inhibitors on platelet signaling and function, detailing prominent platelet signaling pathways downstream of the glycoprotein VI (GPVI) receptor, integrin αIIbß3, and G protein-coupled receptors (GPCRs). This review focuses on mechanistic as well as clinically relevant and emerging TKIs targeting major families of tyrosine kinases including but not limited to Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (Syk), Src family kinases (SFKs), Janus kinases (JAK), and signal transducers and activators of transcription (STAT) and evaluates their effects on platelet aggregation and adhesion, granule secretion, receptor expression and activation, and protein phosphorylation events. In summation, this review highlights current advances and knowledge on the effects of select TKIs on platelet biology and furthers insight on signaling pathways that may represent novel druggable targets coupled to specific platelet functional responses.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function.

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin αIIbß3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.

Role of platelets in regulating activated coagulation factor XI activity.

Factor XI (FXI) has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, whereas FXI is not essential for hemostasis but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in a purified system and increased coagulation Factor IX (FIX) activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by activated coagulation factor XII (FXIIa) and the activation of FXII by kallikrein (PKa). Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets, the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa [by FXIa-platelet membrane immunoprecipitation] showed that the interaction with platelets is zinc dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib. FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

New insights in Type I and II CD20 antibody mechanisms-of-action with a panel of novel CD20 antibodies.

Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.

Plasmin Generation Analysis in Patients with Bleeding Disorder of Unknown Cause.

Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after evaluation of plasma coagulation and platelet function. The underlying mechanisms are unclear, but increased fibrinolysis and abnormal clot formation may play a role. All BDUC patients (n=375) from the Vienna bleeding biobank were analyzed in comparison to healthy controls (HC, n=100) in this case-control study. Plasmin generation (PG) parameters were analyzed using calibrated fluorescence detection in citrated-plasma samples. Turbidimetric plasma clot formation/ lysis of 293 (78%) BDUC patients and confocal microscopy of clots from representative BDUC patients (n=6) and HC (n=9) were assessed. Fibrinolytic factors were measured using commercially available ELISAs. In PG analysis, BDUC patients exhibited lower velocity and peak plasmin, but a higher endogenous plasmin potential compared to HC. Peak plasmin correlated with maximum clot absorbance, but not with clot lysis time. Clot absorbance is an indicator of clot fiber density. Confocal microscopy analysis revealed that BDUC patients' clots had a tendency towards thicker fibers, which negatively correlated with peak plasmin (r=-0.561, p=0.030). Peak plasmin correlated weakly with FXIII, but not with the other fibrinolytic factors (alpha2-antiplasmin, thrombin activatable fibrinolysis inhibitor or plasminogen activator inhibitor 1) or bleeding severity. A model comprising fibrinogen and parameters of PG yielded high predictive power in discriminating BDUC patients from HC during 5-fold stratified cross validation (80% of data, mean AUC: 0.847). The same model generalized well to unseen data (20% of data, AUC: 0.856). Overall, BDUC patients exhibited counterintuitively reduced peak plasmin, potentially related to altered clot structure.

Fibrinolysis is impaired in patients with primary immune thrombocytopenia.

BACKGROUND: Patients with primary immune thrombocytopenia (ITP) have an increased risk of thrombosis, which may be due to altered fibrinolysis. OBJECTIVES: To elucidate the clinical impact of delayed fibrinolysis in ITP patients. METHODS: A turbidimetric clot formation and lysis assay and a fluorometric plasmin generation (PG) assay were performed, and levels of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), tPA-PAI-1 complexes, α2-antiplasmin, thrombin activatable fibrinolysis inhibitor, and D-dimer were assessed in 86 adult primary ITP patients and 78 healthy controls (HCs). RESULTS: ITP patients showed significantly delayed clot formation, increased clot density, and prolonged clot lysis time (CLT) compared with HCs, with a median (IQR) CLT of 28.0 (13.7-34.7) minutes in patients and 17.3 (12.0-28.0) minutes in HCs, while in the PG assay, only the lag time was prolonged. In ITP patients compared with controls, PAI-1 was higher (1.2 [0.8-2.6] vs 1.1 [0.6-2.1] U/mL) and tPA antigen and activity were lower (tPA antigen: 2.6 [1.1-4.4] vs 3.7 [3.2-4.7] ng/mL; tPA activity ≤ 0 U/mL: 26% vs 7%). TPA-PAI-1 complex levels were positively associated with CLT in multiple linear regression analysis (ß = 0.241; P = .019), whereas PG parameters were not associated with CLT. Six patients who developed thrombosis during follow-up had higher levels of tPA-PAI-1 complexes. CONCLUSION: Prolonged CLT and delayed onset of PG may indicate a hypofibrinolytic tendency in ITP patients, as also indicated by high PAI-1 and low tPA levels. No association was found between fibrinolytic potential and the bleeding phenotype, whereas higher tPA-PAI-1 complex levels were associated with prolonged CLT and increased in patients with future thrombosis.

Tissue factor pathway inhibitor is a potential modifier of bleeding risk in factor XI deficiency.

BACKGROUND: Factor (F) XI deficiency is associated with increased bleeding risk in some individuals. Neither FXI levels nor clinical clotting assays predict the bleeding risk. Compared with controls, FXI-deficient bleeders have reduced clot formation, decreased fibrin network density, and increased susceptibility to fibrinolysis. Tissue factor pathway inhibitor (TFPI) was recently implicated as a modifying factor in individuals with bleeding of unknown cause. OBJECTIVES: To determine the potential of TFPI in modifying the bleeding risk in FXI-deficient individuals. METHODS: The effects of TFPI on thrombin generation and clot formation, structure, and fibrinolysis in FXI-deficient plasma were measured in vitro in the absence or presence of inhibitory anti-TFPI antibody or exogenous recombinant TFPIα. Total plasma TFPI concentration was measured in 2 independent cohorts of controls and FXI-deficient individuals classified as bleeders or nonbleeders (cohort 1: 10 controls and 16 FXI-deficient individuals; cohort 2: 48 controls and 57 FXI-deficient individuals) and correlated with ex vivo plasma clot formation and fibrinolysis parameters associated with bleeding risk. RESULTS: In an in vitro FXI deficiency model, inhibition of TFPI enhanced thrombin generation and clot formation, increased the network density, and decreased fibrinolysis, whereas an increase in TFPI had the opposite effects. Compared with controls, plasma from FXI-deficient bleeders had higher TFPI concentration. Total plasma TFPI concentrations correlated with parameters from ex vivo clotting and fibrinolysis assays that differentiate FXI-deficient bleeders and nonbleeders. CONCLUSION: Coagulation and fibrinolysis parameters that differentiate FXI-deficient nonbleeders and bleeders were altered by plasma TFPIα. Total plasma TFPI was increased in FXI-deficient bleeders. TFPI may modify the bleeding risk in FXI-deficient individuals.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity ex vivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Ex vivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Revised model of the tissue factor pathway of thrombin generation: Role of the feedback activation of FXI.

BACKGROUND: Biochemical reaction networks are self-regulated in part due to feedback activation mechanisms. The tissue factor (TF) pathway of blood coagulation is a complex reaction network controlled by multiple feedback loops that coalesce around the serine protease thrombin. OBJECTIVES: Our goal was to evaluate the relative contribution of the feedback activation of coagulation factor XI (FXI) in TF-mediated thrombin generation using a comprehensive systems-based analysis. MATERIALS AND METHODS: We developed a systems biology model that improves the existing Hockin-Mann (HM) model through an integrative approach of mathematical modeling and in vitro experiments. Thrombin generation measured using in vitro assays revealed that the feedback activation of FXI contributes to the propagation of thrombin generation based on the initial concentrations of TF or activated coagulation factor X (FXa). We utilized experimental data to improve the robustness of the HM model to capture thrombin generation kinetics without a role for FXI before including the feedback activation of FXI by thrombin to construct the extended (ext.) HM model. RESULTS AND CONCLUSIONS: Using the ext.HM model, we predicted that the contribution of positive feedback of FXI activation by thrombin can be abolished by selectively eliminating the inhibitory function of tissue factor pathway inhibitor (TFPI), a serine protease inhibitor of FXa and TF-activated factor VII (FVIIa) complex. This prediction from the ext.HM model was experimentally validated using thrombin generation assays with function blocking antibodies against TFPI and plasmas depleted of FXI. Together, our results demonstrate the applications of combining experimental and modeling techniques in predicting complex biochemical reaction systems.

The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton's Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions.

Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors (TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin αIIbß3 activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton's-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.

Pharmacological targeting of coagulation factor XI mitigates the development of experimental atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: Human coagulation factor (F) XI deficiency, a defect of the contact activation system, protects against venous thrombosis, stroke, and heart attack, whereas FXII, plasma prekallikrein, or kininogen deficiencies are asymptomatic. FXI deficiency, inhibition of FXI production, activated FXI (FXIa) inhibitors, and antibodies to FXI that interfere with FXI/FXII interactions reduce experimental thrombosis and inflammation. FXI inhibitors are antithrombotic in patients, and FXI and FXII deficiencies are atheroprotective in apolipoprotein E-deficient mice. OBJECTIVES: Investigate the effects of pharmacological targeting of FXI in experimental models of atherogenesis and established atherosclerosis. METHODS AND RESULTS: Low-density lipoprotein receptor-knockout (Ldlr-/- ) mice were administered high-fat diet (HFD) for 8 weeks; concomitantly, FXI was targeted with anti-FXI antibody (14E11) or FXI antisense oligonucleotide (ASO). 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas when compared with controls, and 14E11 also reduced aortic sinus lesions. In an established disease model, in which therapy was given after atherosclerosis had developed, Ldlr-/- mice were fed HFD for 8 weeks and then administered 14E11 or FXI-ASO weekly until 16 weeks on HFD. In this established disease model, 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas, but not in aortic sinus. In cultures of human endothelium, FXIa exposure disrupted VE-Cadherin expression and increased endothelial lipoprotein permeability. Strikingly, we found that 14E11 prevented the disruption of VE-Cadherin expression in aortic sinus lesions observed in the atherogenesis mouse model. CONCLUSION: Pharmacological targeting of FXI reduced atherogenesis in Ldlr-/- mice. Interference with the contact activation system may safely reduce development or progression of atherosclerosis.

Effect of Pneumatic Tubing System Transport on Platelet Apheresis Units.

Platelet apheresis units are transfused into patients to mitigate or prevent bleeding. In a hospital, platelet apheresis units are transported from the transfusion service to the healthcare teams via two methods: a pneumatic tubing system (PTS) or ambulatory transport. Whether PTS transport affects the activity and utility of platelet apheresis units is unclear. We quantified the gravitational forces and transport time associated with PTS and ambulatory transport within our hospital. Washed platelets and supernatants were prepared from platelet apheresis units prior to transport as well as following ambulatory or PTS transport. For each group, we compared resting and agonist-induced platelet activity and platelet aggregate formation on collagen or von Willebrand factor (VWF) under shear, platelet VWF-receptor expression and VWF multimer levels. Subjection of platelet apheresis units to rapid acceleration/deceleration forces during PTS transport did not pre-activate platelets or their ability to activate in response to platelet agonists as compared to ambulatory transport. Platelets within platelet apheresis units transported via PTS retained their ability to adhere to surfaces of VWF and collagen under shear, although platelet aggregation on collagen and VWF was diminished as compared to ambulatory transport. VWF multimer levels and platelet GPIb receptor expression was unaffected by PTS transport as compared to ambulatory transport. Subjection of platelet apheresis units to PTS transport did not significantly affect the baseline or agonist-induced levels of platelet activation as compared to ambulatory transport. Our case study suggests that PTS transport may not significantly affect the hemostatic potential of platelets within platelet apheresis units.

Removal of the C-Terminal Domains of ADAMTS13 by Activated Coagulation Factor XI induces Platelet Adhesion on Endothelial Cells under Flow Conditions.

Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a "stringlike" conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.