Characterization of CD41+ cells in the lymph node.

Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

Increased B4GALT1 expression is associated with platelet surface galactosylation and thrombopoietin plasma levels in MPNs.

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure ß4-N-acetyllactosamine (Galß1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding ß-1,4-galactosyltransferase 1 (ß4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify ß4GalT1 as a potential actionable target for therapy.

ß4GALT1 controls ß1 integrin function to govern thrombopoiesis and hematopoietic stem cell homeostasis.

Glycosylation is critical to megakaryocyte (MK) and thrombopoiesis in the context of gene mutations that affect sialylation and galactosylation. Here, we identify the conserved B4galt1 gene as a critical regulator of thrombopoiesis in MKs. ß4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances ß1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking ß4GalT1 adhere avidly to ß1 integrin ligands laminin, fibronectin, and collagen, while other platelet functions are normal. Impaired thrombopoiesis leads to increased plasma thrombopoietin (TPO) levels and perturbed hematopoietic stem cells (HSCs). Remarkably, ß1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate ß4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for ß4GalT1 in the regulation of ß1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of ß4GalT1 further affect HSC homeostasis.

Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation.

UNLABELLED: Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. SIGNIFICANCE: The mechanism by which CALR mutations induce MPN remains unknown. In this report, we show that the positive charge of the CALR mutant C-terminus is necessary to transform hematopoietic cells by enabling binding between mutant CALR and the thrombopoietin receptor MPL.

Regulating billions of blood platelets: glycans and beyond.

The human body produces and removes 10(11) platelets daily to maintain a normal steady state platelet count. Platelet production must be regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet production and removal in physiological and pathological conditions. This review will focus on different mechanisms of platelet senescence and clearance with specific emphasis on the role of posttranslational modifications. It will also briefly address platelet transfusion and the role of glycans in the clearance of stored platelets.

Novel mechanisms of platelet clearance and thrombopoietin regulation.

PURPOSE OF REVIEW: The human body produces and removes 10 platelets daily to maintain a normal steady-state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet removal and production in physiological and pathological conditions. This review will focus on different mechanisms of platelet clearance, with focus on the biological significance of platelet glycans. RECENT FINDINGS: The Ashwell-Morrell receptor (AMR) recognizes senescent, desialylated platelets under steady state conditions. Desialylated platelets and the AMR are the physiological ligand-receptor pair regulating hepatic thrombopoietin (TPO) mRNA production, resolving the longstanding mystery of steady state TPO regulation. The AMR-mediated removal of desialylated platelets regulates TPO synthesis in the liver by recruiting JAK2 and STAT3 to increase thrombopoiesis. SUMMARY: Inhibition of TPO production downstream of the hepatic AMR-JAK2 signaling cascade could additionally contribute to the thrombocytopenia associated with JAK1/2 treatment, which is clinically used in myeloproliferative neoplasms.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice.

Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2(fl/fl) Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2(fl/fl) Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver-derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

In vivo platelet activation and platelet hyperreactivity in abacavir-treated HIV-infected patients.

Abacavir (ABC) has been associated with ischaemic cardiovascular events in HIV-infected patients, but the pathogenic mechanisms are unknown. Aim of our study was to assess whether ABC induces in vivo platelet activation and ex vivo platelet hyper-reactivity. In a retrospective, case-control study, in vivo platelet activation markers were measured in 69 HIV-infected patients, before starting therapy and after 6-12 months of either ABC (n=35) or tenofovir (TDF) (n=34), and compared with those from 20 untreated HIV-infected patients. A subgroup of patients was restudied after 28-34 months for ex vivo platelet reactivity. In vivo platelet activation markers were assessed by ELISA or flow cytometry, ex vivo platelet reactivity by light transmission aggregometry (LTA) and PFA-100®. Thein vitro effects of the ABC metabolite, carbovir triphosphate, on aggregation and intra-platelet cGMP were also studied. sPLA2, sPsel and sGPV increased significantly 6-12 months after the beginning of ABC, but not of TDF or of no treatment. Ex vivo platelet function studies showed enhanced LTA, shorter PFA-100® C/ADP closure time and enhanced platelet expression of P-sel and CD40L in the ABC group. The intake of ABC blunted the increase of intraplatelet cGMP induced by nitric oxide (NO) and acutely enhanced collagen-induced aggregation. Preincubation of control platelets with carbovir triphosphate in vitro enhanced platelet aggregation and blunted NO-induced cGMP elevation. In conclusion, treatment with ABC enhances in vivo platelet activation and induces platelet hyperreactivity by blunting the inhibitory effects of NO on platelets. These effects may lead to an increase of ischaemic cardiovascular events.

Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake.

Activated platelets express CD40L on their plasma membrane and release the soluble fragment sCD40L. The interaction between platelet surface CD40L and endothelial cell CD40 leads to the activation of endothelium contributing to atherothrombosis. Few studies have directly demonstrated an increased expression of platelet CD40L in conditions of in vivo platelet activation in humans, and no data are available on its relevance for endothelial activation. We aimed to assess whether platelets activated in vivo at a localized site of vascular injury in humans express CD40L and release sCD40L, whether the level of platelet CD40L expression attained in vivo is sufficient to induce endothelial activation, and whether platelet CD40L expression is inhibited by aspirin intake. We used the skin-bleeding-time test as a model to study the interaction between platelets and a damaged vessel wall by measuring CD40L in the blood emerging from a skin wound in vivo in healthy volunteers. In some experiments, shed blood was analyzed before and 1 h after the intake of 500 mg of aspirin. Platelets from the bleeding-time blood express CD40L and release soluble sCD40L, in a time-dependent way. In vivo platelet CD40L expression was mild but sufficient to induce VCAM-1 expression and IL-8 secretion in coincubation experiments with cultured human endothelial cells. Moreover, platelets recovered from the bleeding-time blood activated endothelial cells; an anti-CD40L antibody blocked this effect. On the contrary, the amount of sCD40L released by activated platelets at a localized site of vascular injury did not reach the concentrations required to induce endothelial cell activation. Soluble monocyte chemoattractant protein-1, a marker of endothelium activation, was increased in shed blood and correlated with platelet CD40L expression. Aspirin intake did not inhibit CD40L expression by platelets in vivo. We concluded that CD40L expressed by platelets in vivo in humans upon contact with a damaged vessel wall activates endothelium; aspirin treatment does not inhibit this mechanism.

Hyperglycemia-induced platelet activation in type 2 diabetes is resistant to aspirin but not to a nitric oxide-donating agent.

OBJECTIVE: Acute, short-term hyperglycemia enhances high shear stress-induced platelet activation in type 2 diabetes. Several observations suggest that platelets in type 2 diabetes are resistant to inhibition by aspirin. Our aim was to assess comparatively the effect of aspirin, a nitric oxide-donating agent (NCX 4016), their combination, or placebo on platelet activation induced by acute hyperglycemia in type 2 diabetes. RESEARCH DESIGN AND METHODS: In a double-blind, placebo-controlled, randomized trial, 40 type 2 diabetic patients were allocated to 100 mg aspirin once daily, 800 mg NCX 4016 b.i.d., both of them, or placebo for 15 days. On day 15, 1 h after the morning dose, a 4-h hyperglycemic clamp (plasma glucose 13.9 mmol/l) was performed, and blood samples were collected before and immediately after it for platelet activation and cyclooxygenase-1 (COX-1) inhibition studies. RESULTS Acute hyperglycemia enhanced shear stress-induced platelet activation in placebo-treated patients (basal closure time 63 +/- 7.1 s, after hyperglycemia 49.5 +/- 1.4 s, -13.5 +/- 6.3 s, P < 0.048). Pretreatment with aspirin, despite full inhibition of platelet COX-1, did not prevent it (-12.7 +/- 6.9 s, NS vs. placebo). On the contrary, pretreatment with the NO donor NCX 4016, alone or in combination with aspirin, suppressed platelet activation induced by acute hyperglycemia (NCX 4016 +10.5 +/- 8.3 s; NCX 4016 plus aspirin: +12.0 +/- 10.7 s, P < 0.05 vs. placebo for both). Other parameters of shear stress-dependent platelet activation were also more inhibited by NCX 4016 than by aspirin, despite lesser inhibition of COX-1. CONCLUSIONS: Acute hyperglycemia-induced enhancement of platelet activation is resistant to aspirin; a NO-donating agent suppresses it. Therapeutic approaches aiming at a wider platelet inhibitory action than that exerted by aspirin may prove useful in patients with type 2 diabetes.

Diagnosis of platelet-type von Willebrand disease by flow cytometry.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder which is due to a mutation in the gene encoding for platelet glycoprotein Ibalpha (GPIbalpha) resulting in enhanced affinity for von Willebrand factor (VWF). PT-VWD is often mistakenly diagnosed as type 2B VWD for the similarities between these two conditions. We characterized a new case of PT-VWD and evaluated the usefulness of a flow cytometric assay in the differential diagnosis between PT-VWD (n=1) and type 2B VWD (n=4). The flow cytometric assay was able to highlight the increased affinity of VWF for GPIbalpha as much as did RIPA and to differentiate the two diseases through mixing tests. Genetic analysis revealed a heterozygous point mutation in codon 239 of the GPIbalpha gene leading to a methionine to valine substitution (M239V). Flow cytometry represents a useful tool for the diagnosis of PT-VWD.

Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo.

Platelet activation at a site of vascular injury is essential for the arrest of bleeding; however, excessive platelet activation at a site of arterial damage can result in the unwarranted formation of arterial thrombi, precipitating acute myocardial infarction, or ischemic stroke. Activation of platelets beyond the purpose of hemostasis may occur when substances facilitating thrombus growth and stability accumulate. Human platelets contain matrix metalloproteinase 2 (MMP-2) and release it upon activation. Active MMP-2 amplifies the platelet aggregation response to several agonists by potentiating phosphatidylinositol 3-kinase activation. Using several in vivo thrombosis models, we show that the inactivation of the MMP-2 gene prevented thrombosis induced by weak, but not strong, stimuli in mice but produced only a moderate prolongation of the bleeding time. Moreover, using cross-transfusion experiments and wild-type/MMP-2(-/-) chimeric mice, we show that it is platelet-derived MMP-2 that facilitates thrombus formation. Finally, we show that platelets activated by a mild vascular damage induce thrombus formation at a downstream arterial injury site by releasing MMP-2. Thus, platelet-derived MMP-2 plays a crucial role in thrombus formation by amplifying the response of platelets to weak activating stimuli. These findings open new possibilities for the prevention of thrombosis by the development of MMP-2 inhibitors.

Dominant inheritance of a novel integrin beta3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families.

BACKGROUND: Defects of integrin alpha(IIb)beta(3) are typical of Glanzmann's thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann's thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease. DESIGN AND METHODS: We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin beta(3)-gene (ITGB3) mutation. RESULTS: The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface alpha(Ib) beta(3), impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated alpha(IIb)beta(3) upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective alpha(IIb)beta(3)-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647-686 of the betaTD ectodomain of integrin beta(3). Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome. CONCLUSIONS: This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin beta(3), and its betaTD domain, in platelet formation and function.

HIV type 1 infection, and not short-term HAART, induces endothelial dysfunction.

OBJECTIVE: Ischemic cardiovascular events increasingly occur during long-lasting HIV infection and are attributed either to the infection itself or to the use of HAART. Endothelial dysfunction and platelet activation are markers of atherosclerosis. Our aim was to assess whether patients with chronic HIV infection present endothelial dysfunction and whether this is the consequence of infection or of HAART. DESIGN: Fifty-six HIV-infected patients were studied in a retrospective cohort study before and 3, 6, 12 and 24 months after starting HAART with protease inhibitors (n = 28) or nonnucleoside reverse transcriptase inhibitors (n = 28), and compared with 28 age-matched and sex-matched healthy controls, and with 10 naive HIV-infected patients studied at diagnosis and after 12 months of untreated infection. METHODS: Soluble endothelial and platelet activation markers were measured in plasma by flow cytometry. RESULTS: Soluble P-selectin, soluble vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 and von Willebrand factor were significantly higher in HIV-infected patients than in healthy controls, whereas soluble CD40 ligand and tissue type plasminogen activator were within normal range. During follow-up, soluble vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 and von Willebrand factor but not soluble P-selectin decreased progressively, without significant differences between protease inhibitors and nonnucleoside reverse transcriptase inhibitors treatment. In naive, untreated patients, increased plasma markers of endothelial dysfunction were confirmed at diagnosis, with no changes upon follow-up. CONCLUSION: Chronic HIV infection, and not its pharmacological treatment, induces alterations of markers of endothelial function. Short-term treatment with HAART reduces some markers of endothelial dysfunction, with no differences between protease inhibitors and nonnucleoside reverse transcriptase inhibitors.

Allergen induces the migration of platelets to lung tissue in allergic asthma.

RATIONALE: Platelets are essential for pulmonary leukocyte recruitment, airway hyperresponsiveness, and bronchial remodeling in animals with allergic inflammation and can be found in bronchoalveolar lavage of sensitized animals. No studies, however, have explored the direct migration of platelets to lungs. OBJECTIVES: To assess whether platelets migrate into lung parenchyma in response to inhaled allergen in ovalbumin-sensitized mice; to assess the role of the FcepsilonRI receptor in this phenomenon; and to evaluate whether platelets from patients with asthma, or from sensitized mice, undergo chemotaxis in vitro in response to relevant antigens. METHODS: Ovalbumin-sensitized wild-type (WT) mice, or FcRgamma(-/-) mice lacking the FcepsilonRIgamma, were challenged with aerosolized allergen and lungs analyzed by platelet-specific immunohistochemistry. In some experiments, mice were depleted of platelets and cross-transfused with either WT or FcRgamma(-/-) platelets to assess the role of platelet FcRgamma(-/-). Chemotaxis of platelets from patients with asthma or from sensitized mice was studied in vitro. MEASUREMENTS AND MAIN RESULTS: Histology of lungs revealed isolated platelets, migrating out of vessels and localizing underneath the airways after allergen challenge in WT but not in FcRgamma(-/-) mice. Platelets from patients with asthma and from sensitized WT mice, but not from sensitized FcRgamma(-/-) mice, migrated in vitro toward the relevant allergen or an anti-IgE. Platelets from normal mice were found to express FcepsilonRIgamma and platelet-bound IgEs were increased in sensitized mice. CONCLUSIONS: Platelets migrate extravascularly in response to a sensitizing allergen via a mechanism dependent on the interaction among allergen, allergen-specific IgE, and the FcepsilonRI, and this may allow them to participate directly in allergic tissue inflammation.

Laboratory diagnosis and monitoring of desmopressin treatment of von Willebrand's disease by flow cytometry.

BACKGROUND AND OBJECTIVES: von Willebrand's disease (VWD) is a heterogeneous bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The diagnosis of VWD requires several laboratory tests. The aim of our study was to validate a flow cytometric test for the diagnosis of VWD and for monitoring the effects of desmopressin therapy. DESIGN AND METHODS: Flow cytometric analysis of ristocetin-induced VWF binding to platelets was performed in platelet-rich plasma (PRP) samples from patients with VWD and from control subjects and in samples of formalin-fixed platelets in the presence of plasma from patients or controls. In 12 VWD patients the test was conducted before and 1 hour after desmopressin infusion. Results were compared with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA-100 and the skin bleeding time. RESULTS: Ristocetin-induced VWF binding to platelets, evaluated by both flow cytometry-based assays, was significantly reduced in patients with type1, 2A and 2M VWD as compared with that in healthy subjects. Patients with type 2B VWD showed reduced binding of VWF to formalin-fixed platelets, but increased binding to autologous platelets in PRP, similar to RIPA. VWF binding to platelets assessed by both flow cytometric assays correlated significantly with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA100 and bleeding time. VWF binding to platelets increased after desmopressin infusion. INTERPRETATION AND CONCLUSIONS: The measurement of ristocetin-induced binding of VWF to platelets by flow cytometry is a sensitive, simple and rapid test for the diagnosis of VWD and for the monitoring of the effects of desmopressin therapy. The flow cytometric assay performed with autologous platelets is useful in the identification of type 2B VWD patients.

Platelets release active matrix metalloproteinase-2 in vivo in humans at a site of vascular injury: lack of inhibition by aspirin.

When stimulated in vitro, human platelets release matrix metalloproteinase-2 (MMP-2) that, in turn, potentiates platelet activation. The present study investigated if MMP-2 is released from activated platelets in vivo in humans and whether aspirin inhibits this release. MMP-2 levels were measured by zymography, immunoblotting, flow-cytometry and an activity assay system, in plasma prepared from blood emerging from a skin wound inflicted for the measurement of the bleeding time (shed blood) and simultaneously from venous blood in 27 healthy human volunteers. In a subgroup, the same measurements were carried out before and 1 h after aspirin intake. MMP-2 was significantly higher in shed blood than in venous blood and increased progressively, consistent with ongoing platelet activation. A significant correlation was evident between platelet number and MMP-2 in shed blood; platelet MMP-2 content in shed blood was lower than that of platelets in venous blood. The level of active MMP-2 released by activated platelets in vivo was within the range of concentrations that potentiate platelet activation. Aspirin did not reduce MMP-2 release in vivo. In conclusion, MMP-2 is released from platelets in vivo in humans at a localised site of vessel wall damage in amounts sufficient to potentiate platelet aggregation; aspirin does not reduce this release.

Selective cytochrome c displacement by phosphate and Ca(2+) in brain mitochondria.

In brain mitochondria, phosphate- and Ca(2+)-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca(2+). Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2-[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC(12)-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC(12)-HPC liposomes by either phosphate or Ca(2+), but only Ca(2+) produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Deltapsi(m)) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca(2+) caused inhibition of complex IV activity and impairment of Deltapsi(m). In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.