Lack of COL6/collagen VI causes megakaryocyte dysfunction by impairing autophagy and inducing apoptosis.

Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-/- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-/- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-/- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-/- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-/-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Adipose mesenchymal stromal/stem cells expanded by a GMP compatible protocol displayed improved adhesion on cancer cells in flow conditions.

BACKGROUND: Adipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown. METHODS: In dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions. RESULTS: When compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (-33.9%±12.2% and -45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes. CONCLUSIONS: SRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures.

INTRODUCTION: Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. AIMS: We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. METHODS: We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. RESULTS: Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. DISCUSSION: MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.

Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media.

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (-11.6%). The relative abundance of ASC (CD34+CD45-CD31-) and endothelial precursors (CD34+CD45-CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

Impedance biosensor for real-time monitoring and prediction of thrombotic individual profile in flowing blood.

A new biosensor for the real-time analysis of thrombus formation is reported. The fast and accurate monitoring of the individual thrombotic risk represents a challenge in cardiovascular diagnostics and in treatment of hemostatic diseases. Thrombus volume, as representative index of the related thrombotic status, is usually estimated with confocal microscope at the end of each in vitro experiment, without providing a useful behavioral information of the biological sample such as platelets adhesion and aggregation in flowing blood. Our device has been developed to work either independently or integrated with the microscopy system; thus, images of the fluorescently labeled platelets are acquired in real-time during the whole blood perfusion, while the global electrical impedance of the blood sample is simultaneously monitored between a pair of specifically designed gold microelectrodes. Fusing optical and electrical data with a novel technique, the dynamic of thrombus formation events in flowing blood can be reconstructed in real-time, allowing an accurate extrapolation of the three-dimensional shape and the spatial distribution of platelet thrombi forming and growing within artificial capillaries. This biosensor is accurate and it has been used to discriminate different hemostatic conditions and to identify weakening and detaching platelet aggregates. The results obtained appear compatible with those quantified with the traditional optical method. With advantages in terms of small size, user-friendliness and promptness of response, it is a promising device for the fast and automatic individual health monitoring at the Point of Care (POC).

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach.

BACKGROUND: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. METHODS: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. RESULTS: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-ß1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. DISCUSSION: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.

Hepatitis B and C viruses and risk of non-Hodgkin lymphoma: a case-control study in Italy.

BACKGROUND: Hepatitis C virus (HCV) has been consistently associated to non-Hodgkin lymphoma (NHL); conversely, few studies have evaluated a comprehensive serological panel of hepatitis B virus (HBV) in NHL etiology. METHODS: We conducted a case-control study in Italy in 1999-2014, enrolling 571 incident, histologically confirmed NHLs and 1004 cancer-free matched controls. Study subjects provided serum for HCV and HBV testing and for HCV RNA. Odds ratios (ORs) and corresponding 95 % confidence intervals (CIs) were estimated by logistic regression, adjusting for potential confounders. RESULTS: Circulating HCV RNA was detected in 63 (11.1 %) NHL cases and 35 (3.5 %) controls (OR = 3.51, 95 % CI: 2.25-5.47). Chronic HBV infection (i.e., positive to HBV surface antigen - HBsAg(+)) was found in 3.7 % of cases and 1.7 % of controls (OR = 1.95, 95 % CI: 1.00-3.81); a significantly elevated OR was observed for B-cell NHL (2.11, 95 % CI: 1.07-4.15). People with serological evidence of past HCV or HBV infection, vaccination against HBV, or detectable antibodies against HBV core antigen (anti-HBc(+)) alone were not at increased NHL risk. CONCLUSIONS: Our results support a role of chronic HCV infection in NHL in Italy and suggest an involvement of HBV infection. Associations were clearest for B-cell NHL and diffuse large B-cell lymphoma. Prevention and treatment of HCV and HBV infection may diminish NHL incidence, notably in areas with high prevalence of hepatitis viruses infection.

Diagnostic value of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 pathway in transitional cell carcinoma of the bladder.

Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2-T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is-T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients' discomfort and improving compliance.

Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p<0.05) and increased retained platelets (from 72.3 ± 2.3% to 81.1 ± 1.8%, p<0.05) in the O'Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, p<0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.

Visualization of nitric oxide production by individual platelets during adhesion in flowing blood.

Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of l-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca(++) elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca(++) elevation. Simultaneous measurement of NO and Ca(++) indicated that NO production in individual platelets is preceded by Ca(++) elevations, with a lag phase of 33 ± 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca(++) elevation elicits the production of NO which, in turn, modulates thrombus size.

The importance of calcium in the regulation of megakaryocyte function.

Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.

C2362F mutation gives rise to an ADAMTS13-resistant von Willebrand factor.

von Willebrand factor (VWF) multimers result from proteolysis by the metalloprotease ADAMTS13. Since C2362F-VWF features abnormally large multimers with their triplet oligomer structure replaced by a diffuse smear, we explored the susceptibility of C2362F-VWF to ADAMTS13. VWF-enriched blood samples, obtained by cryoethanol precipitation of plasma from a patient with von Willebrand disease (VWD) homozygous for the C2362F mutation and a normal subject, were submitted to cleavage by recombinant ADAMTS13 under static conditions in the presence of urea. C2362F-VWF proved completely ADAMTS13-resistant in vitro. At any concentration of recombinant ADAMTS13 (from 0.1 µM to 1 µM), there was no evidence of the abnormally large VWF multimers of C2362F-VWF disappearing, nor any increased representation of triplet multimer bands, unlike the situation seen in normal VWF. This is due partly to a defective ADAMTS13 binding to C2362F-VWF under static conditions, as seen in both the patient's and recombinant mutated VWF proteins. These findings were associated with a significantly shorter than normal survival of C2362F-VWF after DDAVP, demonstrating that proteolysis and VWF survival may be independent phenomena. Our findings clearly demonstrate that the loss of cysteine 2362 makes VWF resistant to proteolysis by ADAMTS13, at least partly due to an impaired ADAMTS13 binding to VWF. This suggests that the B2 domain of VWF is involved in modulating ADAMTS13 binding to VWF and the consequent proteolytic process. The C2362F-VWF mutation also enables a new abnormality to be identified in the VWF-ADAMTS13 relationship, i.e. an ADAMTS13-resistant VWF.

Distinct spatio-temporal Ca2+ signaling elicited by integrin alpha2beta1 and glycoprotein VI under flow.

We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation.

Prothrombotic effects of fibronectin isoforms containing the EDA domain.

OBJECTIVE: Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+ FN) is absent from normal plasma, but elevated plasma levels of EDA+ FN are found in several pathological conditions. We hypothesized that EDA+ FN plays a special role in thrombosis. METHODS AND RESULTS: We used mouse strains constitutively including (EDA+/+) or excluding (EDA-/-) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA+ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 microm) grew faster when compared with EDA(WT/WT) (6.6+/-0.2 minutes versus 8.3+/-0.6 minutes, P<0.05) and the mean vessel occlusion time was shorter (9.9+/-0.4 minutes versus 14.6+/-1.7 minutes, P<0.05). However, the presence of EDA+ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA+/+ mice after collagen/epinephrine infusion was twice that of EDA(WT/WT) or EDA-/- mice. CONCLUSIONS: Our findings reveal that EDA+ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated.

Altered von Willebrand factor subunit proteolysis and multimer processing associated with the Cys2362Phe mutation in the B2 domain.

The normal von Willebrand factor (vWF) multimer pattern results from the ADAMTS-13 cleavage of the Tyr 1605-Met 1606 bond in the A2 domain of vWF. We identified a patient with severe von Willebrand disease (vWD) homozygously carrying a Cys to Phe mutation in position 2362 of vWF with markedly altered vWF multimers and an abnormal proteolytic pattern. The proband's phenotype was characterized by a marked drop in plasma vWF antigen and ristocetin cofactor activity, and a less pronounced decrease in FVIII. The vWF multimers lacked any triplet structure, replaced by single bands with an atypical mobility, surrounded by a smear, and abnormally large vWF multimers. Analysis of the plasma vWF subunit's composition revealed the 225 kDa mature form and a single 205 kDa fragment, but not the 176 kDa and 140 kDa fragments resulting from cleavage by ADAMTS-13. The 205 kDa fragment was distinctly visible, along with the normal vWF cleavage products, in the patient's parents who were heterozygous for the Cys2362Phe mutation. Their vWF levels were mildly decreased and vWF multimers were organized in triplets, but also demonstrated abnormally large forms and smearing. Our findings indicate that a proper conformation of the B2 domain, which depends on critical Cys residues, may be required for the normal proteolytic processing of vWF multimers.

Single particle tracking across sequences of microscopical images: application to platelet adhesion under flow.

A versatile and automated image processing technique and data extraction procedure from videomicroscopic data is presented. The motivation is a detailed quantification of blood platelet adhesion from laminar flow onto a surface. The characteristics of the system under observation (type of cells, their speed of movement, and the quality of the optical image to analyze) provided the criteria for developing a new procedure enabling tracking for long image sequences. Specific features of the novel method include: automatic segmentation methodology which removes operator bias; platelet recognition across the series of images based on a probability density function (two-dimensional, Gaussian-like) tailored to the physics of platelet motion on the surface; options to automatically tune the procedure parameters to explore different applications; integrated analysis of the results (platelet trajectories) to obtain relevant information, such as deposition and removal rates, displacement distributions, pause times and rolling velocities. Synthetic images, providing known reference conditions, are used to test the method. The algorithm operation is illustrated by application to images obtained by fluorescence microscopy of the interaction between platelets and von Willebrand factor-coated surfaces in parallel-plate flow chambers. Potentials and limits are discussed, together with evaluation of errors resulting from an inaccurate tracking.