Blood Platelets Organize Pro- and Anti-Angiogenic Factors into Separate, Distinct Alpha Granules: Implications for the Regulation of Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 393-393 ◽  
Author(s):  
Joseph Italiano ◽  
Jennifer L. Richardson ◽  
Judah Folkman ◽  
Giannoula Klement
Keyword(s):  
Confocal Microscopy ◽  
Blood Platelets ◽  
Local Environment ◽  
Human Platelets ◽  
Angiogenic Factors ◽  
Regulatory Proteins ◽  
Platelet Factor ◽  
Proteinase Activated Receptors ◽  
The Many ◽  
Granule Release

Abstract In addition to contributing to hemostasis, platelets are increasingly being viewed as major contributors to wound healing and tumor growth. This can be attributed to the many angiogenesis regulatory proteins selectively taken up and stored within the alpha granules of platelets and deposited in the local environment of a tumor or wound upon platelet aggregation and adhesion. Platelets contain both pro and anti-angiogenesis regulators, and the mechanisms by which they modulate angiogenesis are unclear. To address this question, we examined the localization of both pro-and anti-angiogenic factors in platelets and megakaryocytes using immunostaining and confocal microscopy. Double immunofluorescence microscopy revealed that VEGF (an angiogenesis stimulator) and endostatin (an inhibitor), are localized in separate and distinct alpha granules. Immunofluorescence for thrombospondin 1, basic FGF, platelet factor 4, and placental growth factor subsequently confirmed the separate and distinct alpha granules. These observations, in combination with a recent report that proteinase-activated receptors (PARs) selectively modulate the release of angiogenesis regulators from platelets (Ma et al., PNAS 102:216), motivated the hypothesis that these distinct populations of alpha granules could undergo selective release as well. To investigate whether PARs facilitate differential release of alpha granules, we treated human platelets with PAR agonists or antagonists and assayed for differential alpha granule release. The selective PAR1 agonist stimulated the release of endostatin-containing alpha granules and suppressed the release of VEGF-containing alpha granules. Furthermore, confocal microscopy studies revealed that in the presence of the PAR1 antagonist (SCH79797), thrombin stimulated the release of endostatin-containing alpha granules, but not VEGF-containing granules. These observations suggest that platelets may be organizing angiogenesis regulatory proteins into pharmacologically and morphologically distinct populations of alpha granules, which are then susceptible to differential regulation upon platelet activation.

Organization and Physiologically Mediated Differential Release of Angiogenic Regulatory Proteins From Distinct Platelet Alpha Granules.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3045-3045
Author(s):  
Elisabeth Battinelli ◽  
Joseph Italiano
Keyword(s):  
Tumor Metastasis ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Angiogenic Factors ◽  
Regulatory Proteins ◽  
Angiogenic Balance ◽  
Angiogenic Proteins ◽  
Vessel Growth ◽  
Proteinase Activated Receptors ◽  
Immunofluorescence Labeling

Abstract 3045 Poster Board II-1021 In addition to their primary roles in hemostasis and thrombosis, platelets have been reported to participate in other physiological and pathological processes, including but not limited to inflammation, wound healing, and tumor metastasis. Although platelets are presumed to contribute to new blood vessel growth by providing numerous pro-and anti-angiogenic factors, the cellular and molecular basis by which platelets regulate angiogenesis is poorly understood. Previously we have shown that platelets differentially package angiogenic regulatory proteins in their alpha granules. More recently, we have further defined the organization of angiogenic proteins within platelet alpha granules using immunofluorescence and immunogold labeling experiments and have uncovered at least three distinct types of alpha granules. These granules were found to contain selective members of pro- and anti-angiogenic regulatory proteins, respectively. In addition, we have analyzed platelets from nine patients with metastatic renal cell carcinoma using immunofluorescence labeling for VEGF and endostatin. In comparison to volunteer control platelets without known malignancy, there is a significant increase in granules containing VEGF in patients with metastatic disease, strongly suggesting a shift of the angiogenic balance towards a more pro-angiogenic environment in the platelets from patients with malignancy. Previously we have shown that differentially packaged alpha granules are susceptible to selective release in response to proteinase-activated receptors. To explore whether selective release also occurred in physiologic conditions, we activated human platelets with known platelet agonists and looked for evidence of selective release. We have demonstrated that differential release of VEGF can be physiologically regulated by activation with ADP. Human platelets exposed to 50uM ADP result in release of VEGF and retention of endostatin, both by immunofluorecence labeling and by ELISA assay. This study provides mechanistic evidence to support an interplay between tumor cells and platelets, by illustrating a key role for platelets in tumor angiogenesis through the delivery of specific angiogenic factors. This work has implications beyond basic platelet biology as it provides a model for the platelet as a delivery system, with important clinical implications. Disclosures No relevant conflicts of interest to declare.

Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

1981 ◽  
Author(s):  
F Rendu ◽  
M Lebret ◽  
J P Caen
Keyword(s):  
Plasma Membranes ◽  
Blood Platelets ◽  
Human Platelets ◽  
Release Mechanism ◽  
Inherited Disorders ◽  
Platelet Factor ◽  
Functional Studies ◽  
Dense Bodies ◽  
Serotonin Uptake And Release

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.

Physiological and Pathological Agonists Induce Differential Release of Angiogenesis Regulatory Proteins From Platelet Alpha Granules Influencing the Angiogenic Response

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 649-649
Author(s):  
Elisabeth Battinelli ◽  
Joseph Italiano ◽  
Beth Markens
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Tumor Cells ◽  
Capillary Tube ◽  
Human Platelets ◽  
Tube Formation ◽  
Angiogenic Factors ◽  
Endothelial Cell Migration ◽  
Angiogenic Potential ◽  
Granule Release

Abstract Abstract 649 In addition to their primary roles in hemostasis and thrombosis, platelets participate in other physiological and pathological processes, including inflammation, wound healing, and tumor metastasis. Although platelets are presumed to contribute to angiogenesis by providing numerous pro-and anti-angiogenic factors, the cellular and molecular basis by which platelets regulate angiogenesis is poorly understood. Previously, we have shown that platelets differentially package specific angiogenesis regulatory proteins into distinct populations of alpha-granules. Using selective agonists for the PAR1μ and PAR4 receptor, we demonstrated that these distinct subpopulations of alpha-granules are susceptible to differential release upon platelet activation (Italiano et al. Blood 2008). To investigate whether other platelet receptors facilitate differential release of alpha-granules, we treated human platelets with physiological agonists and assayed for differential alpha-granule release. Previously we have shown that activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF (an angiogenesis stimulator) in vitro, but not endostatin (an angiogenesis inhibitor). We now have evidence that the agonist Thromboxane A2 (TXA2) acts in an opposite manner; leading to increased endostatin release, but not VEGF release. To more broadly establish the angiogenic potential of the platelet releasate stimulated by exposure to different platelet agonists, including ADP and TXA2, we used a number of well-established angiogenic assays including endothelial cell migration, capillary tube formation, and also an angiogenic protein array panel. All platelet releasates generated by activating platelets with ADP led to increased angiogenic potential with statistically significant increased cell migration, capillary tube formation, and an overall pro-angiogenic protein content of the releasate. Conversely, treatment with TXA2 led to decreased endothelial cell migration and inhibited the formation of capillary tube structures by human umbilical vein endothelial cells. Although it is established that tumor cells can interact with platelets, the mechanisms involved are not known. This prompted us to investigate the hypothesis that cancer cells may also have the capacity to induce differential alpha-granule release during the pathological angiogenesis associated with tumor growth. To investigate whether tumor cells facilitate differential release of pro-angiogenic alpha-granules, we treated human platelets with different tumor cell lines and assayed for differential alpha-granule release. Exposure of platelets to the breast cancer cell line, MCF-7 cells stimulated significant release of VEGF. The angiogenic potential of this releasate was increased as measured using the endothelial cell migration assay, capillary tube formation assay, and protein arrays. This study provides mechanistic evidence to support an interplay between tumor cells and platelet agonists to orchestrate differential release of angiogenic factors from platelets, and illustrates a key role for platelets in tumor angiogenesis through the delivery of specific angiogenic factors. Disclosures: No relevant conflicts of interest to declare.

The Effect of Phospholipase C on Platelet Factor 3 in Human Blood Platelets

1977 ◽  
Vol 37 (01) ◽  
pp. 029-035 ◽  
Author(s):  
A-B Otnaess ◽  
H Prydz
Keyword(s):  
Phospholipase C ◽  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Small Reduction ◽  
Platelet Factor ◽  
Human Blood Platelets ◽  
External Attack

SummaryIntact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested.The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing.External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.

Platelet α-Granules: Content and Characteristics of Secretion

1979 ◽  
Author(s):  
Karen Kaplan ◽  
Johan M. Broekman ◽  
Arthur Chernoff ◽  
Barbara Linder ◽  
DeWitt Goodman
Keyword(s):  
Dienoic Acid ◽  
Arterial Disease ◽  
Human Platelets ◽  
Dense Granule ◽  
Distinct Pattern ◽  
Platelet Factor ◽  
Alternative Pathways ◽  
Granule Release

Platelet α-granule contents and secretion were studied using specific radioimmunoas says and cell culture assays. Washed human platelets were disrupted by nitrogen cavitation and subcellular fractions were separated by ultracentrifugation through sucrose density gradients. α-Granules were found to contain platelet factor 4 (PF4), 8-thromboglobulin (8TG) fibrinogen, and the platelet-derived growth factor: their contents were distinct from those of dense granules and of lysos omes. A distinct pattern of release of the four agranule components was ob served. Release of α- and dense-granule components was induced by ADP and epinephrine; α-granule release was, however, more sensitive than dense granule to low levels of thrombin, collagen, and the endoperoxide analogue (15S)-hydroxy-IIα, 9α (epoxymethano)prosta-5Z, l3E-dienoic acid. Studies with cyclooxygenase (CO) inhibitors suggested that α-granule release in vitro can be mediated both by the CO pathway and by other mechanisms. Thus, aspirin and indomethacin inhibited collagen and thrombin induced release, but “the inhibitory effects were progressively overcome with higher levels of these agents. Studies in vivo showed that normal plasma levels of PF4 and 8TG were only slightly reduced by aspirin, whereas the elevated basal levels of these components found in patients with arterial disease were reduced by aspirin to a greater extent. The in viv data thus support the in vitro findings that both CO mediated and alternative pathways are involved in release of α-granule components.

Investigation of the Interaction of Blood Platelets with the Coagulation System at the Site of Plug Formation In Vivo in Man - Effect of Low-Dose Aspirin

1987 ◽  
Vol 57 (01) ◽  
pp. 062-066 ◽  
Author(s):  
P A Kyrle ◽  
J Westwick ◽  
M F Scully ◽  
V V Kakkar ◽  
G P Lewis
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Low Dose ◽  
Bleeding Time ◽  
Blood Platelets ◽  
Dose Aspirin ◽  
Low Dose Aspirin ◽  
Plug Formation ◽  
Granule Release

SummaryIn 7 healthy volunteers, formation of thrombin (represented by fibrinopeptide A (FPA) generation, α-granule release (represented by β-thromboglobulin [βTG] release) and the generation of thromboxane B2 (TxB2) were measured in vivo in blood emerging from a template bleeding time incision. At the site of plug formation, considerable platelet activation and thrombin generation were seen within the first minute, as indicated by a 110-fold, 50-fold and 30-fold increase of FPA, TxB2 and PTG over the corresponding plasma values. After a further increase of the markers in the subsequent 3 minutes, they reached a plateau during the fourth and fifth minute. A low-dose aspirin regimen (0.42 mg.kg-1.day-1 for 7 days) caused >90% inhibition of TxB2formation in both bleeding time blood and clotted blood. At the site of plug formation, a-granule release was substantially reduced within the first three minutes and thrombin generation was similarly inhibited. We conclude that (a) marked platelet activation and considerable thrombin generation occur in the early stages.of haemostasis, (b) α-granule release in vivo is partially dependent upon cyclo-oxygenase-controlled mechanisms and (c) thrombin generation at the site of plug formation is promoted by the activation of platelets.

Opposite Effect of Lysine on Platelet Aggregation Induced by Arachidonate and by Other Aggregants

1982 ◽  
Vol 48 (01) ◽  
pp. 078-083 ◽  
Author(s):  
C Ts'ao ◽  
S J Hart ◽  
D V Krajewski ◽  
P G Sorensen
Keyword(s):  
Platelet Aggregation ◽  
Secondary Phase ◽  
Aminocaproic Acid ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Primary Phase ◽  
High Doses ◽  
The Many ◽  
Induced Aggregation

SummaryEarlier, we found that ε-aminocaproic acid (EACA) inhibited human platelet aggregation induced by adenosine diphosphate (ADP) and collagen, but not aggregation by arachidonic acid (AA). Since EACA is structurally similar to lysine, yet these two agents exhibit vast difference in their antifibrinolytic activities, we chose to study the effect of lysine on platelet aggregation. We used L-lysine-HCl in these studies because of its high solubility in aqueous solutions while causing no change in pH when added to human plasma. With lysine, we repeatedly found inhibition of ADP-, collagen- and ristocetin-induced aggregation, but potentiation of AA-induced aggregation. Both the inhibitory and potentiation effects were dose-dependent. Low doses of lysine inhibited the secondary phase of aggregation; high doses of it also inhibited the primary phase of aggregation. Potentiation of AA-induced aggregation was accompanied by increased release of serotonin and formation of malondialdehyde. These effects were not confined to human platelets; rat platelets were similarly affected. Platelets, exposed to lysine and then washed and resuspended in an artificial medium not containing lysine, remained hypersensitive to AA, but no longer showed decreased aggregation by collagen. Comparing the effects of lysine with equimolar concentrations of sucrose, EACA, and α-amino-n-butyric acid, we attribute the potent inhibitory effect of lysine to either the excess positive charge or H+ and C1− ions. The -NH2 group on the α-carbon on lysine appears to be the determining factor for the potentiation effect; the effect seems to be exerted on the cyclooxygenase level of AA metabolism. Lysine and other chemicals with platelet-affecting properties similar to lysine may be used as a tool for the study of the many aspects of a platelet aggregation reaction.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Detection of Enhanced In Vivo Platelet α-Granule Release in Different Patient Groups - Comparison of β-Thromboglobulin, Platelet Factor 4 and Thrombospondin Assays

1984 ◽  
Vol 52 (02) ◽  
pp. 183-187 ◽  
Author(s):  
D A Lane ◽  
H Ireland ◽  
S Wolff ◽  
E Ranasinghe ◽  
J Dawes
Keyword(s):  
Platelet Factor 4 ◽  
Renal Elimination ◽  
Elevated Plasma ◽  
Platelet Factor ◽  
Release Reaction ◽  
Heparin Anticoagulation ◽  
Patient Groups ◽  
Granule Release ◽  
Sensitive Marker

SummaryDuring the platelet release reaction β-thromboglobulin (βTG), platelet factor 4 (PF4) and thrombospondin (TSP) are released from the platelet into plasma and assays of these proteins can be used to monitor in vivo platelet activation. We have assessed their relative merits as markers of the in vivo platelet α-granule release reaction in a number of patient groups which have previously been shown to have elevated plasma βTG and/or PF4 levels. It is concluded that in diseases or conditions not complicated by its reduced clearance, βTG is the most sensitive marker of in vivo platelet α-granule release. However, the TSP assay may be the least ambiguous when monitoring the platelet α-granule release reaction in patients with renal failure who are undergoing haemodialysis with heparin anticoagulation. Under these circumstances plasma βTG, but not PF4 or TSP, levels are elevated because of impaired renal catabolism, and the presence of a heparin-releasable reservoir of PF4 on the endothelium complicates the use of the PF4 assay. In liver failure none of these assays may accurately reflect platelet α-granule release because of impaired hepatic or renal elimination of the proteins.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

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