RBCs regulate platelet function and hemostasis under shear conditions through biophysical and biochemical means.

ABSTRACT: Red blood cells (RBCs) have been hypothesized to support hemostasis by facilitating platelet margination and releasing platelet-activating factors such as adenosine 5'-diphosphate (ADP). Significant knowledge gaps remain regarding how RBCs influence platelet function, especially in (patho)physiologically relevant hemodynamic conditions. Here, we present results showing how RBCs affect platelet function and hemostasis in conditions of anemia, thrombocytopenia, and pancytopenia and how the biochemical and biophysical properties of RBCs regulate platelet function at the blood and vessel wall interface and in the fluid phase under flow conditions. We found that RBCs promoted platelet deposition to collagen under flow conditions in moderate (50 × 103/µL) but not severe (10 × 103/µL) thrombocytopenia in vitro. Reduction in hematocrit by 45% increased bleeding in mice with hemolytic anemia. In contrast, bleeding diathesis was observed in mice with a 90% but not with a 60% reduction in platelet counts. RBC transfusion improved hemostasis by enhancing fibrin clot formation at the site of vascular injury in mice with severe pancytopenia induced by total body irradiation. Altering membrane deformability changed the ability of RBCs to promote shear-induced platelet aggregation. RBC-derived ADP contributed to platelet activation and aggregation in vitro under pathologically high shear stresses, as observed in patients supported by left ventricular assist devices. These findings demonstrate that RBCs support platelet function and hemostasis through multiple mechanisms, both at the blood and vessel wall interface and in the fluidic phase of circulation.

Anticoagulation targeting membrane-bound anionic phospholipids improves outcomes of traumatic brain injury in mice.

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI in mouse models combined with in vitro experiments. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V (ANV) with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. The phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared with EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Conformation-dependent blockage of activated VWF improves outcomes of traumatic brain injury in mice.

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.

von Willebrand factor enhances microvesicle-induced vascular leakage and coagulopathy in mice with traumatic brain injury.

von Willebrand factor (VWF) is an adhesive ligand, and its activity is proteolytically regulated by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat 13). An elevated level of plasma VWF has been widely considered a marker for endothelial cell activation in trauma and inflammation, but its causal role in these pathological conditions remains poorly defined. Using a fluid percussion injury mouse model, we demonstrated that VWF released during acute traumatic brain injury (TBI) was activated and became microvesicle-bound. The VWF-bound microvesicles promoted vascular leakage and systemic coagulation. Recombinant ADAMTS-13 given either before or after TBI reduced the VWF reactivity with minimal influence on VWF secretion. rADAMTS-13 protected the integrity of endothelial cell barriers and prevented TBI-induced coagulopathy by enhancing VWF cleavage without impairing basal hemostasis. Promoting microvesicle clearance by lactadherin had efficacy similar to that of rADAMTS-13. This study uncovers a novel synergistic action between VWF and cellular microvesicles in TBI-induced vascular leakage and coagulopathy and demonstrates protective effects of rADAMTS-13.

Placenta-derived extracellular vesicles induce preeclampsia in mouse models.

Preeclampsia is a pregnancy-induced condition that impairs the mother's health and results in pregnancy termination or premature delivery. Elevated levels of placenta-derived extracellular vesicles (pcEV) in the circulation have been consistently associated with preeclampsia, but whether these vesicles induce preeclampsia or are the product of preeclampsia is not known. Guided by a small cohort study of preeclamptic patients, we examined the impact of pcEV on the pathogenesis of preeclampsia in mouse models. We detected pcEV in pregnant C56BL/6J mice with a peak level of 3.8±0.9×107/mL at 17-18 days post-coitum. However, these pregnant mice developed hypertension and proteinuria only after being infused with vesicles purified from injured placenta. These extracellular vesicles released from injured placenta disrupted endothelial integrity and induced vasoconstriction. Enhancing the clearance of extracellular vesicles prevented the development of the extracellular vesicle-induced preeclampsia in mice. Our results demonstrate a causal role of pcEV in preeclampsia and identify microvesicle clearance as a new therapeutic strategy for the treatment of this pregnancy-associated complication.

Brain-Derived Extracellular Vesicles Induce Vasoconstriction and Reduce Cerebral Blood Flow in Mice.

Traumatic brain injury (TBI) impairs cerebrovascular autoregulation and reduces cerebral blood flow (CBF), leading to ischemic secondary injuries. We have shown that injured brains release brain-derived extracellular vesicles (BDEVs) into circulation, where they cause a systemic hypercoagulable state that rapidly turns into consumptive coagulopathy. The BDEVs induce endothelial injury and permeability, leading to the hypothesis that they contribute to TBI-induced cerebrovascular dysregulation. In a study designed to test this hypothesis, we detected circulating BDEVs in C57BL/6J mice subjected to severe TBI, reaching peak levels of 3 × 104/µL at 3 h post-injury (71.2 ± 21.5% of total annexin V-binding EVs). We further showed in an adaptive transfer model that 41.7 ± 5.8% of non-injured mice died within 6 h after being infused with 3 × 104/µL of BDEVs. The BDEVs transmigrated through the vessel walls, induced rapid vasoconstriction by inducing calcium influx in vascular smooth muscle cells, and reduced CBF by 93.8 ± 5.6% within 30 min after infusion. The CBF suppression was persistent in mice that eventually died, but it recovered quickly in surviving mice. It was prevented by the calcium channel blocker nimodipine. When being separated, neither protein nor phospholipid components from the lethal number of BDEVs induced vasoconstriction, reduced CBF, and caused death. These results demonstrate a novel vasoconstrictive activity of BDEVs that depends on the structure of BDEVs and contributes to TBI-induced disseminated cerebral ischemia and sudden death.

Hyperadhesive von Willebrand Factor Promotes Extracellular Vesicle-Induced Angiogenesis: Implication for LVAD-Induced Bleeding.

Bleeding associated with left ventricular assist device (LVAD) implantation has been attributed to the loss of large von Willebrand factor (VWF) multimers to excessive cleavage by ADAMTS-13, but this mechanism is not fully supported by the current evidence. We analyzed VWF reactivity in longitudinal samples from LVAD patients and studied normal VWF and platelets exposed to high shear stress to show that VWF became hyperadhesive in LVAD patients to induce platelet microvesiculation. Platelet microvesicles activated endothelial cells, induced vascular permeability, and promoted angiogenesis in a VWF-dependent manner. Our findings suggest that LVAD-driven high shear stress primarily activates VWF, rather than inducing cleavage in the majority of patients.

Prognostic Biomarkers for Thrombotic Microangiopathy after Acute Graft-versus-Host Disease: A Nested Case-Control Study.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a complication of allogeneic hematopoietic cell transplantation (HCT) that often occurs following the development of acute graft-versus-host disease (aGVHD). In this study, we aimed to identify early TMA biomarkers among patients with aGVHD. We performed a nested-case-control study from a prospective cohort of allogeneic HCT recipients, matching on the timing and severity of antecedent aGVHD. We identified 13 TMA cases and 25 non-TMA controls from 208 patients in the cohort. Using multivariable conditional logistic regression, the odds ratio for TMA compared with non-TMA was 2.65 (95% confidence interval [CI], 1.00 to 7.04) for every 100 ng/mL increase in terminal complement complex sC5b9 and 2.62 (95% CI, 1.56 to 4.38) for every 1000 pg/mL increase in angiopoietin-2 (ANG2) at the onset of aGVHD. ADAMTS13 and von Willebrand factor (VWF) antigens were not appreciably associated with TMA. Using a Cox regression model incorporating sC5b9 >300 ng/mL and ANG2 >3000 pg/mL at the onset of aGVHD, the adjusted hazard ratio for mortality was 5.33 (95% CI, 1.57 to 18.03) for the high-risk group (both elevated) and 4.40 (95% CI, 1.60 to 12.07) for the intermediate-risk group (one elevated) compared with the low-risk group (neither elevated). In conclusion, we found that elevated sC5b9 and ANG2 levels at the onset of aGVHD were associated with the development of TMA and possibly mortality after accounting for the timing and severity of aGVHD. The results suggest important roles of complement activation and endothelial dysfunction in the pathogenesis of TMA. Measurement of these biomarkers at the onset of aGVHD may inform prognostic enrichment for preventive trials and improve clinical care.

Storage temperature determines platelet GPVI levels and function in mice and humans.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature-stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

Physical proximity and functional cooperation of glycoprotein 130 and glycoprotein VI in platelet membrane lipid rafts.

OBJECTIVE: Clinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)-6 forms a complex with soluble IL-6 receptor α (sIL-6Rα) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase Cγ2 (PLCγ2) in collagen-induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3. METHODS AND RESULTS: We conducted complementary in vitro experiments to show that the IL-6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL-6-sIL-6Rα complex to activate the kinase Syk and the substrate PLCγ2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130-Janus tyrosine kinase (JAK)-STAT3 signaling abolished the cross-activation and reduced platelet reactivity to collagen. CONCLUSION: These results demonstrate cross-talk between collagen and IL-6 signal pathways. This cross-talk could potentially provide a novel mechanism for inflammation-induced platelet hyperactivity, so the IL-6-GP130-JAK-STAT3 pathway has been identified as a potential target to block this hyperactivity.

Cancer cell-derived von Willebrand factor enhanced metastasis of gastric adenocarcinoma.

Cancer prognosis is poor for patients with blood-borne metastasis. Platelets are known to assist cancer cells in transmigrating through the endothelium, but ligands for the platelet-mediated cancer metastasis remain poorly defined. von Willebrand factor (vWF) is a major platelet ligand that has been widely used as a biomarker in cancer and associated inflammation. However, its functional role in cancer growth and metastasis is largely unknown. Here we report that gastric cancer cells from patients and cells from two well-established gastric cancer lines express vWF and secrete it into the circulation, upon which it rapidly becomes cell-bound to mediate cancer-cell aggregation and interaction with platelets and endothelial cells. The vWF-mediated homotypic and heterotypic cell-cell interactions promote the pulmonary graft of vWF-overexpressing gastric cancer BGC823 cells in a mouse model. The metastasis-promoting activity of vWF was blocked by antibodies against vWF and its platelet receptor GP Ibα. It was also reduced by an inhibitory siRNA that suppresses vWF expression. These findings demonstrate a causal role of cancer-cell-derived vWF in mediating gastric cancer metastasis and identify vWF as a new therapeutic target.

Association of Single Nucleotide Polymorphisms in the ST3GAL4 Gene with VWF Antigen and Factor VIII Activity.

VWF is extensively glycosylated with biantennary core fucosylated glycans. Most N-linked and O-linked glycans on VWF are sialylated. FVIII is also glycosylated, with a glycan structure similar to that of VWF. ST3GAL sialyltransferases catalyze the transfer of sialic acids in the α2,3 linkage to termini of N- and O-glycans. This sialic acid modification is critical for VWF synthesis and activity. We analyzed genetic and phenotypic data from the Atherosclerosis Risk in Communities (ARIC) study for the association of single nucleotide polymorphisms (SNPs) in the ST3GAL4 gene with plasma VWF levels and FVIII activity in 12,117 subjects. We also analyzed ST3GAL4 SNPs found in 2,535 subjects of 26 ethnicities from the 1000 Genomes (1000G) project for ethnic diversity, SNP imputation, and ST3GAL4 haplotypes. We identified 14 and 1,714 ST3GAL4 variants in the ARIC GWAS and 1000G databases respectively, with 46% being ethnically diverse in their allele frequencies. Among the 14 ST3GAL4 SNPs found in ARIC GWAS, the intronic rs2186717, rs7928391, and rs11220465 were associated with VWF levels and with FVIII activity after adjustment for age, BMI, hypertension, diabetes, ever-smoking status, and ABO. This study illustrates the power of next-generation sequencing in the discovery of new genetic variants and a significant ethnic diversity in the ST3GAL4 gene. We discuss potential mechanisms through which these intronic SNPs regulate ST3GAL4 biosynthesis and the activity that affects VWF and FVIII.

Quantitative Influence of ABO Blood Groups on Factor VIII and Its Ratio to von Willebrand Factor, Novel Observations from an ARIC Study of 11,673 Subjects.

ABO blood groups are known to influence the plasma level of von Willebrand factor (VWF), but little is known about the relationship between ABO and coagulation factor VIII (FVIII). We analyzed the influence of ABO genotypes on VWF antigen, FVIII activity, and their quantitative relationship in 11,673 participants in the Atherosclerosis Risk in Communities (ARIC) study. VWF, FVIII, and FVIII/VWF levels varied significantly among O, A (A1 and A2), B and AB subjects, and the extent of which varied between Americans of European (EA) and African (AA) descent. We validated a strong influence of ABO blood type on VWF levels (15.2%), but also detected a direct ABO influence on FVIII activity (0.6%) and FVIII/VWF ratio (3.8%) after adjustment for VWF. We determined that FVIII activity changed 0.54% for every 1% change in VWF antigen level. This VWF-FVIII relationship differed between subjects with O and B blood types in EA, AA, and in male, but not female subjects. Variations in FVIII activity were primarily detected at low VWF levels. These new quantitative influences on VWF, FVIII and the FVIII/VWF ratio help understand how ABO genotypes differentially influence VWF, FVIII and their ratio, particularly in racial and gender specific manners.

Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species.

BACKGROUND: Piperlongumine (PL) is a compound isolated from the piper longum plant. It possesses anti-cancer activities through blocking the transcription factor STAT3 and by inducing reactive oxygen species (ROS) in cancer, but not normal cells. It also inhibits platelet aggregation induced by collagen, but the underlying mechanism is not known. OBJECTIVE: We conducted in vitro experiments to test the hypothesis that PL regulates a non-transcriptional activity of STAT3 to specifically reduce the reactivity of human platelets to collagen. RESULTS: PL dose-dependently blocked collagen-induced platelet aggregation, calcium influx, CD62p expression and thrombus formation on collagen with a maximal inhibition at 100 µM. It reduced platelet microvesiculation induced by collagen. PL blocked the activation of JAK2 and STAT3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets pretreated with a STAT3 inhibitor. Although PL induced ROS production in platelets; quenching ROS using excessive reducing agents: 20 µM GSH and 0.5 mM L-Cysteine, did not block the inhibitory effects. The NADPH oxidase inhibitor Apocynin also had no effect. CONCLUSIONS: PL inhibited collagen-induced platelet reactivity by targeting the JAK2-STAT3 pathway. We also provide experimental evidence that PL and collagen induce different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants in redox signals.