Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

α-Synuclein is the major platelet isoform but is dispensable for activation, secretion, and thrombosis.

Platelets play many roles in the vasculature ensuring proper hemostasis and maintaining integrity. These roles are facilitated, in part, by cargo molecules released from platelet granules via Soluble NSF Attachment Protein Receptor (SNARE) mediated membrane fusion, which is controlled by several protein-protein interactions. Chaperones have been characterized for t-SNAREs (i.e. Munc18b for Syntaxin-11), but none have been clearly identified for v-SNAREs. α-Synuclein has been proposed as a v-SNARE chaperone which may affect SNARE-complex assembly, fusion pore opening, and thus secretion. Despite its abundance and that it is the only isoform present, α-synuclein's role in platelet secretion is uncharacterized. In this study, immunofluorescence showed that α-synuclein was present on punctate structures that co-stained with markers for α-granules and lysosomes and in a cytoplasmic pool. We analyzed the phenotype of α-synuclein-/- mice and their platelets. Platelets from knockout mice had a mild, agonist-dependent secretion defect but aggregation and spreading in vitro were unaffected. Consistently, thrombosis/hemostasis were unaffected in the tail-bleeding, FeCl3 carotid injury and jugular vein puncture models. None of the platelet secretory machinery examined, e.g. the v-SNAREs, were affected by α-synuclein's loss. The results indicate that, despite its abundance, α-synuclein has only a limited role in platelet function and thrombosis.

What did we know? The N-terminus of α-Synuclein affects SNARE-complex assembly, fusion pore opening, and granule docking.Microvascular bleeding is seen in Parkinson Disease patients where α-synuclein has a pathological role.What did we discover? α-Synuclein colocalizes with P-selectin (α-granules) and LAMP-1 (lysosomes) in platelets.The loss of α-synuclein has only a mild, agonist-dependent effect on platelet secretion.The loss of α-synuclein had no effect on thrombosis/hemostasis in 3 injury models.What is the impact? Despite its abundance, α-synuclein is not required for platelet secretion.α-Synuclein is not required for hemostasis or thrombosis.

Plasma tissue-type plasminogen activator is associated with lipoprotein(a) and clinical outcomes in hospitalized patients with COVID-19.

Background: Patients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. Objectives: To assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. Methods: Endogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. Results: In hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non-COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. Conclusion: High Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.

The complementary roles of VAMP-2, -3, and -7 in platelet secretion and function.

Platelet secretion requires Soluble N-ethylmaleimide Sensitive Attachment Protein Receptors (SNAREs). Vesicle SNAREs/Vesicle-Associated Membrane Proteins (v-SNAREs/VAMPs) on granules and t-SNAREs in plasma membranes mediate granule release. Platelet VAMP heterogeneity has complicated the assessment of how/if each is used and affects hemostasis. To address the importance of VAMP-7 (V7), we analyzed mice with global deletions of V3 and V7 together or platelet-specific deletions of V2, V3, and global deletion of V7. We measured the kinetics of cargo release, and its effects on three injury models to define the context-specific roles of these VAMPs. Loss of V7 minimally affected dense and α granule release but did affect lysosomal release. V3-/-7-/- and V2Δ3Δ7-/- platelets showed partial defects in α and lysosomal release; dense granule secretion was unaffected. In vivo assays showed that loss of V2, V3, and V7 caused no bleeding or occlusive thrombosis. These data indicate a role for V7 in lysosome release that is partially compensated by V3. V7 and V3, together, contribute to α granule release, however none of these deletions affected hemostasis/thrombosis. Our results confirm the dominance of V8. When it is present, deletion of V2, V3, or V7 alone or in combination minimally affects platelet secretion and hemostasis.

What did we know? V8 is the primary VAMP isoform for platelet granule secretion, but V2 and V3 play compensatory roles.V3 is important for platelet endocytosis.V7 plays a minimal role in secretion and does not affect hemostasis.What did we discover? The loss of both V3 and V7 increases α and lysosomal secretion defects.Platelet-specific deletion of V2 and V3 with global V7-deletion causes defective α and lysosomal release.Secretion deficiencies in V3^−/−7^−/− and V2^Δ3^Δ7^−/− have no effect on hemostasis or thrombosis.What is the impact? We show that endosomal v-SNAREs (V3 and V7) play minor roles in secretion.V3^−/−7^−/− and platelet-specific V2^Δ3^Δ7^−/− mice are viable and will be valuable in in vivo studies of membrane trafficking.

Platelet glycogenolysis is important for energy production and function.

Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (i.e., activation, secretion, aggregation, and clot contraction) remains unclear. Patients with glycogen storage disease often present with increased bleeding and glycogen phosphorylase (GP) inhibitors, when used as treatments for diabetes, induce bleeding in preclinical studies suggesting some role for this form of glucose in hemostasis. In the present work, we examined how glycogen mobilization affects platelet function using GP inhibitors (CP316819 and CP91149) and a battery of ex vivo assays. Blocking GP activity increased glycogen levels in resting and thrombin-activated platelets and inhibited platelet secretion and clot contraction, with minimal effects on aggregation. Seahorse energy flux analysis and metabolite supplementation experiments suggested that glycogen is an important metabolic fuel whose role is affected by platelet activation and the availability of external glucose and other metabolic fuels. Our data shed light on the bleeding diathesis in glycogen storage disease patients and offer insights into the potential effects of hyperglycemia on platelets.

What did we know? Activated platelets transition from a low-energy-requiring, resting state to a high-energy-demanding state.Platelet glycogen is degraded upon activation.Glycogen storage disorders and glycogen phosphorylase inhibitors are associated with bleeding.What did we discover? Glycogen turnover occurs in resting platelets and its degradation is important for platelet functions.Glycogen phosphorylase inhibitors block secretion and clot contraction of which the latter can be reversed with alternative metabolic fuels.Glucose derived from glycogen may be routed through TCA/OxPhos versus aerobic glycolysis.What is the impact? Glycogen breakdown contributes to the high energy requirements of platelet function.Our work offers insights into potential energy sources in activated platelets.

Platelet-localized ST6Gal1 does not impact IgG sialylation.

The IgG antibody class forms an important basis of the humoral immune response, conferring reciprocal protection from both pathogens and autoimmunity. IgG function is determined by the IgG subclass, as defined by the heavy chain, as well as the glycan composition at N297, the conserved site of N-glycosylation within the Fc domain. For example, lack of core fucose promotes increased antibody-dependent cellular cytotoxicity, whereas α2,6-linked sialylation by the enzyme ST6Gal1 helps to drive immune quiescence. Despite the immunological significance of these carbohydrates, little is known about how IgG glycan composition is regulated. We previously reported that mice with ST6Gal1-deficient B cells have unaltered IgG sialylation. Likewise, ST6Gal1 released into the plasma by hepatocytes does not significantly impact overall IgG sialylation. Since IgG and ST6Gal1 have independently been shown to exist in platelet granules, it was possible that platelet granules could serve as a B cell-extrinsic site for IgG sialylation. To address this hypothesis, we used a platelet factor 4 (Pf4)-Cre mouse to delete ST6Gal1 in megakaryocytes and platelets alone or in combination with an albumin-Cre mouse to also remove it from hepatocytes and the plasma. The resulting mouse strains were viable and had no overt pathological phenotype. We also found that despite targeted ablation of ST6Gal1, no change in IgG sialylation was apparent. Together with our prior findings, we can conclude that in mice, neither B cells, the plasma, nor platelets have a substantial role in homeostatic IgG sialylation.

Protocol for optimizing production and quality control of infective EcoHIV virions.

EcoHIV is a model of HIV infection that recapitulates aspects of HIV-1 pathology in mice. However, there are limited published protocols to guide EcoHIV virion production. Here, we present a protocol for producing infective EcoHIV virions and essential quality controls. We describe steps for viral purification, titering, and multiple techniques to analyze infection efficacy. This protocol produces high infectivity in C57BL/6 mice which will aid investigators in generating preclinical data.

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

EcoHIV-Infected Mice Show No Signs of Platelet Activation.

Platelets express several surface receptors that could interact with different viruses. To understand the mechanisms of HIV-1's interaction with platelets, we chose the EcoHIV model. While EcoHIV is an established model for neuroAIDS, its effects on platelets are ill-defined. Our results indicate that EcoHIV behaves differently from HIV-1 and is cleared from circulation after 48 h post-infection. The EcoHIV course of infection resembles an HIV-1 infection under the effects of combined antiretroviral therapy (cART) since infected mice stayed immunocompetent and the virus was readily detected in the spleen. EcoHIV-infected mice failed to become thrombocytopenic and showed no signs of platelet activation. One explanation is that mouse platelets lack the EcoHIV receptor, murine Cationic Amino acid Transporter-1 (mCAT-1). No mCAT-1 was detected on their surface, nor was any mCAT-1 mRNA detected. Thus, mouse platelets would not bind or become activated by EcoHIV. However, impure virus preparations, generated by Polyethylene Glycol (PEG) precipitation, do activate platelets, suggesting that nonspecific PEG-precipitates may contain other platelet activators (e.g., histones and cell debris). Our data do not support the concept that platelets, through general surface proteins such as DC-SIGN or CLEC-2, have a wide recognition for different viruses and suggest that direct platelet/pathogen interactions are receptor/ligand specific.

A sensitive and adaptable method to measure platelet-fibrin clot contraction kinetics.

Background: Platelet-fibrin clot contraction is critical for wound closure and maintenance of vessel patency, yet a molecular understanding of the process has lagged because of a lack of flexible quantitative assay systems capable of assaying multiple samples simultaneously. Objectives: We devised a sensitive and inexpensive method to assess clot contraction kinetics under multiple conditions. Methods: Clot contraction was measured using time-lapse digital photography, automated image processing with customized software, and detailed kinetic analysis using available commercial programs. Results: Our system was responsive to alterations in platelet counts and calcium, fibrinogen, and thrombin concentrations, and our analysis detected and defined three phases of platelet-fibrin clot formation: initiation, contraction, and stabilization. Lag time, average contraction velocity, contraction extent, and area under the curve were readily calculated from the data. Using pharmacological agents (blebbistatin and eptifibatide), we confirmed the importance of myosin IIA and the interactions of integrin αIIbß3-fibrinogen/fibrin in clot contraction. As further proof of our system's utility, we showed how 2-deoxyglucose affects contraction, demonstrating the importance of platelet bioenergetics, specifically glycolysis. Conclusions: Our system is an adaptable platform for assessing the effects of multiple conditions and interventions on clot contraction kinetics in a regular laboratory setting, using readily available materials. The automated image processing software we developed will be made freely available for noncommercial uses. This assay system can be used to directly compare and define the effects of different treatments or genetic manipulations on platelet function and should provide a robust tool for future hemostasis/thrombosis research and therapeutic development.

Total Plasma Protein S Is a Prothrombotic Marker in People Living With HIV.

BACKGROUND: HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. METHODS: We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. RESULTS: We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. CONCLUSION: These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.

VAMP3 and VAMP8 Regulate the Development and Functionality of Parasitophorous Vacuoles Housing Leishmania amazonensis.

To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.

Platelet-HIV: interactions and their implications.

While it is clear that platelets interact with viruses, the ramifications and mechanisms of those interactions are still being defined for each type of viral infection. HIV/AIDS+ represents a potentially unique example of how viremia affects platelets since the increasing efficacy of antiretroviral therapeutics (ART) has made it a chronic disease that increases the risk of cardiovascular disease. In this opinion article, we discuss some of the open questions about how platelets interact with HIV. What happens to a virion once it binds a platelet? What is the nature of virus-induced platelet activation? Are platelets a normal part of the immune response to viremia that has been co-opted to increase the spread of HIV? The answers to these and similar questions will help define how platelet-directed therapeutics might be used in treating HIV/AIDS+ patients.

Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.

Differential Leukocyte and Platelet Profiles in Distinct Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) affects over 3 million individuals every year in the U.S. There is growing appreciation that TBI can produce systemic modifications, which are in part propagated through blood-brain barrier (BBB) dysfunction and blood-brain cell interactions. As such, platelets and leukocytes contribute to mechanisms of thromboinflammation after TBI. While these mechanisms have been investigated in experimental models of contusion brain injury, less is known regarding acute alterations following mild closed head injury. To investigate the role of platelet dynamics and bioenergetics after TBI, we employed two distinct, well-established models of TBI in mice: the controlled cortical impact (CCI) model of contusion brain injury and the closed head injury (CHI) model of mild diffuse brain injury. Hematology parameters, platelet-neutrophil aggregation, and platelet respirometry were assessed acutely after injury. CCI resulted in an early drop in blood leukocyte counts, while CHI increased blood leukocyte counts early after injury. Platelet-neutrophil aggregation was altered acutely after CCI compared to sham. Furthermore, platelet bioenergetic coupling efficiency was transiently reduced at 6 h and increased at 24 h post-CCI. After CHI, oxidative phosphorylation in intact platelets was reduced at 6 h and increased at 24 h compared to sham. Taken together, these data demonstrate that brain trauma initiates alterations in platelet-leukocyte dynamics and platelet metabolism, which may be time- and injury-dependent, providing evidence that platelets carry a peripheral signature of brain injury. The unique trend of platelet bioenergetics after two distinct types of TBI suggests the potential for utilization in prognosis.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin.

During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Platelet α-granule cargo packaging and release are affected by the luminal proteoglycan, serglycin.

BACKGROUND: Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. OBJECTIVE: To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments. METHODS: Platelets and megakaryocytes from SRGN-/- mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. RESULTS: Metabolic, 35 SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect α-granule morphology or number. Platelet α-granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN-/- megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and ß-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α-granule lumen of SRGN-/- platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. α-Granule swelling and decondensation were reduced in SRGN-/- platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN-/- platelets was modestly enhanced. Shedding of GP1b was unaffected. CONCLUSION: The polyanionic proteoglycan SRGN influences α-granule packaging, cargo release, and shedding of platelet membrane proteins.